Section 2 Treatment Targets

#### **Chapter 8**

## A Role for Cardiac Glycosides in GBM Therapy

*Yuchen Du, Xiao-Nan Li, Peiying Yang and Robert A. Newman*

#### **Abstract**

There is a pressing need for new effective therapeutic strategies to treat glioblastoma (GBM). Cardiac glycoside compounds consisting of both cardenolides and bufadienolides have been shown to possess potent activity against GBM cell lines and in vivo GBM tumors. In addition, recent research has shown that certain cardiac glycoside compounds contribute to an additive and even synergistic manner with the standard of care GBM treatments such as radiotherapy and chemotherapy. Finally, the finding that cardiac glycosides may offer a unique role in the control of GBM stem cells offers hope for better therapeutic outcomes in treating this deadly form of brain cancer.

**Keywords:** cardenolides, bufadienolides, digoxin, oleandrin, *Nerium oleander*, PBI-05204, glioblastoma, radiotherapy, stem cells, Na,K-ATPase

#### **1. Introduction**

While basic and clinical research has led to better diagnostic techniques and therapeutics for the treatment of glioblastoma, unfortunately, these have translated into only a modest improvement in median survival for this disease due to a high rate of recurrence [1]. As median survival for most GBM patients from time of diagnosis is less than 15 months, the need for new therapeutic approaches is clear [1–3]. Recent studies have shown that both cardenolide and bufadienolides compounds may offer a new therapeutic strategy for the treatment of GBM either as standalone compounds or in combination with other therapeutic modalities such as radiotherapy or standard of care drugs such as temozolomide. One cardenolide compound, oleandrin, derived from *N. oleander*, has shown particular promise against human GBM tumors both in vitro and in vivo. Oleandrin is a good addition to radiotherapy and certain chemotherapeutic agents such as temozolomide. In addition, this molecule and extracts containing it, such as PBI-05204, have now been shown to provide valuable activity against GBM stem cells which, in large part, account for the treatment of resistance and disease recurrence.

#### **2. Cardiac glycosides and GBM**

Chemically, cardiac glycosides can be divided into two groups: cardenolides and bufadienolides. Bufadienolides include bufalin, gammabufotalin, marinobufagenin, and proscillaridin while common cardenolides include digoxin, digitoxin, ouabain, lanatoside C, and oleandrin [4, 5]. The action of members of both classes of compounds are known to have a role as therapeutic compounds for the treatment of congestive heart failure and, over the past decade, many of these compounds have also been reported to have the potential to treat a variety of human malignant diseases [6–9]. While high doses of cardenolides are frequently associated with cardiotoxicity, lower doses are still used for the treatment of congestive heart failure [10, 11]. In addition, low concentrations of selected cardenolides, such as oleandrin, are known to activate a precise signaling pathway or signalosome involving α-subunits of Na,K-ATPase and acting via Src-EGFR-Ras–Raf-extracellular signal-regulated kinase (ERK), Akt/Protein kinase (PK)B, and phosphoinositide 3-kinase (PI3K) to inhibit cell proliferation and survival [12, 13]. Proteomic profiling reveals upregulated PI3K-Akt–mTOR signaling across brain metastasis histology [14]. Given these are pivotal oncogenic factors for various malignancies, this represents an important new approach to the treatment of cancer. While the majority of published studies cite in vitro activity against key cancer cell lines, some, such as PBI-05204 (an extract of *N. oleander* containing oleandrin as a key active ingredient), have advanced to Phase I and II clinical trials for the treatment of patients with cancer [15, 16]. Importantly, constituents of both classes of cardiac glycoside compounds have shown promise as potential novel therapeutic agents for the treatment of GBM [17–28]. This is further supported by the result of our systematic repurposed drug screening to discover an effective therapeutic approach for the treatment of medulloblastoma. By applying a systemic biological approach including driver signaling network identification and drug functional network-based drug repositioning, we screened more than 1300 drug candidates. Among the 100 drugs predicted to be the most effective for the treatment of group 3 and 4 medulloblastoma, five cardiac glycosides including both cardenolides and bufadienolides were identified as having great potential to inhibit the growth of Group 3 and 4 medulloblastoma, which augments the therapeutic potential of cardiac glycosides in GBM [29].

#### **2.1 Bufadienolides and GBM**

Chansu is a traditional Chinese medicine and has been used for many years as a treatment for cancer. Bufalin, an active component of Chansu, is a naturally occurring compound classified as a bufadienolides and has been recognized as a specific inhibitor of Na, K-ATPase [21]. This compound has been shown to have antitumor activity against various cancers, such as liver, lung, intestinal, gastric, gynecological, and pancreatic [30]. Lan et al. point out that the sodium pump α-1 subunit of Na, K-ATPase regulates bufalin sensitivity of human glioblastoma cells through the p53 signaling pathway [20]. A novel observation by these researchers indicated that bufalin inhibits glioblastoma growth by promoting proteasomal degradation of the Na, K-ATPase α-1 subunit [31]. Additional mechanistic studies confirmed the important roles of Src and p53 signaling in mediating apoptosis. Importantly, bufalin inhibited the growth of glioma xenografts. The authors concluded that therapies targeting specific Na+ , K+ -ATPase α subunits such as α-1 and p53 signaling-mitochondrial apoptotic pathways may have the potential to treat gliomas [31].

A related bufadienolides compound, gamabufotalin, is another component of the traditional Chinese medicine Chansu and its pharmaceutical formula known as Huachansu. Yuan et al. have shown that gamabufotalin exhibited selective cytocidal effects against intractable cancer cells including glioblastoma, but minimal effects on

#### *A Role for Cardiac Glycosides in GBM Therapy DOI: http://dx.doi.org/10.5772/intechopen.105022*

normal peripheral blood mononuclear cells prepared from healthy volunteers [22]. Additionally, they also reported that gamabufotalin efficiently downregulated the percentage of CD4+ CD25+ Foxp3+ regulatory T (Treg) cells, which have been considered to play a critical role in limiting antitumor immune response in the body and promoting immunological "ignorance' of cancer cells [32]. Recent research by Yuan et al. have shown that treatment of the human glioblastoma cell line U-87 with gamabufotalin produced downregulation of the expression of uPA, CA9, and upregulated the expression of TIMP3, all of which are associated with invasion/metastasis. They conclude that this molecule exhibits significant cytotoxicity against cancerous glial cells with high potency and selectivity through multiple cytotoxic signaling pathways [22].

A related bufadienolide, marinobufagenin (MBG), has also been reported to be able to inhibit glioma growth through its ability to bind to the sodium pump α-1 unit and interaction with the ERK signaling mediated mitochondrial apoptotic (MAPK/ERK) pathway [33]. MBG treatment of U87MG and U251 cells markedly inhibited α-1 subunit expression. The effect of MBG to inhibit U251 xenograft subcutaneous growth was also assessed. Mice were treated with MBG for 9 days after which tumor volume and weights were assessed. These determinations showed significant inhibition of tumor growth resulting from MBG treatment. In addition, immunohistochemical analysis of tumor tissue demonstrated a significant decrease in the activated form of p65. Taken together, the authors stated that their results indicate that MBG effectively inhibits glioma growth through ERK-mediated mitochondrial apoptotic pathways [33]. Furthermore, MBG was observed to inhibit activation of NF-κβ and expression of other proinflammatory mediators including iNOS, COX-2, TNF-α, and IL-6 suggesting anti-inflammatory activity.

Proscillaridin is a cardiac glycoside that is derived from plants of the genus Scilla and *Drimia maritima*. Denicolai et al. [28] used two human primary GBM stem cell lines (GSCs), GBM6 and GBM9 in addition to the regular GBM cells to investigate the relative potential of proscillaridin to inhibit cell growth both in vitro and in vivo. The chosen cell lines are interesting in that GBM6 cells are highly malignant whereas GBM9 cells exhibit a much lower migratory capability yet have a higher proliferation rate [28]. Proscillaridin A exerted both anti-proliferative and anti-migratory activities in these cell lines at a concentration of 0.05 μM. The authors stated that proscillaridin was more active than temozolomide which in their study did not affect the migration or proliferation rate of either GBM6 or GBM9 cells. Exploring likely mechanisms of action for proscillaridin, the authors reported that this compound induced concentration-dependent cytotoxicity through both an increase in GBM cell death and a G2/M cell phase arrest thereby impairing a GBM stem self-renewal capacity.

#### **2.2 Cardenolides and GBM**

#### *2.2.1 Digoxin*

Of all the known cardenolide compounds the most widely studied is digoxin which in the recent past was widely used for the treatment of atrial fibrillation. Its potential activity as a repurposed drug for the control of GBM, however, is less well-known. Papale et al. [34] have examined the potential role of digoxin in the control of adverse effects of GSCs. They hypothesized that GSCs express receptors that can bind alarmins released during necrosis, an event favoring GSCs migration. Alarmins are endogenous molecules that are constitutively available and released upon tissue damage and activate the immune system. Uncontrolled and excessive release of alarmins is

believed to contribute to dysregulated processes seen in many inflammatory conditions such as tumorigenesis and tumor metastasis [35]. To investigate this hypothesis, GSC cell lines were kept under hypoxic conditions to determine the expression of hypoxic markers as well as receptors for advanced glycation end products. The authors reported that necrotic extracts increased migration, invasion, and cellular adhesion. Importantly, HIF-1α inhibition by digoxin prevented the response of GSCs to hypoxia. They concluded that inhibition of hypoxic pathways may represent a target for preventing brain invasion by glioblastoma stem cells [34]. Hypoxia and necrosis, with subsequent microenvironment inflammation, can be considered as two main features of growing GBM tumors and thus are believed to play a major role in determining the metastatic potential of GSCs in a tumor. The potential role of a cardenolide such as digoxin as an inhibitor of HIF-1α is intriguing as it may represent a novel means of inhibiting this master regulator in the complicated process of cellular adaptation to tumor microenvironments.

A related study of the role of hypoxia with regard to its potential to increase the expression of stem cell markers and promotion of clonogenicity of glioblastoma neurospheres was undertaken by Bar et al. [36]. They examined the effect of hypoxia on stem-like cells in glioblastoma using GBM-derived neurosphere cultures. When these were grown in 1% oxygen, HIF-1α protein levels increased dramatically as did mRNA encoding other hypoxic response genes, such as hypoxia-inducible gene-2, lysyl oxidase, and vascular endothelial growth factor. The rise in the stem-like fraction in GBM following hypoxia was paralleled by a two-fold increase in clonogenicity. The authors examined the potential of digoxin to prevent hypoxic-related events. They observed that this cardenolide suppressed HIF-1α protein expression, HIF-1α downstream targets, and slowed tumor growth in vivo. In addition, their data demonstrated that pretreatment with digoxin reduced GBM flank xenograft growth of hypoxic GBM cells. Daily intraperitoneal injections of digoxin were reported to have significantly inhibited the growth of established xenografts and suppressed the expression of vascular endothelial growth factors [36].

As stated earlier, we have shown that systemic in vivo treatment of patient-derived orthotopic xenograft (PDOX or orthotopic PDX) models of groups 3 (ICb-2555 MB) medulloblastoma that harbors c-Myc amplification and group 4 (ICb-1078 MB, that harbors an n-MYC amplification) medulloblastoma with digoxin, a member of cardiac glycoside approved for the treatment of heart failure, significantly prolonged animal survival times at plasma concentrations known to be tolerated in human. The antitumor effect of digoxin in medulloblastoma appears to be mediated by the down regulation of the Erk and Akt signaling pathway [29].

#### *2.2.2 Digitoxin*

Digitoxin is a cardiac glycoside similar in structure and effects to digoxin, though the effects are longer-lasting. This drug has been used to treat pain and inflammation associated with various diseases such as arthritis, AIDS, and atherosclerosis [23, 37, 38]. Studies have also shown that digitoxin induces growth inhibition and/or apoptosis of a variety of human cancer cells in vitro and in vivo [29]. Lee et al. examined the potential sensitizing effects of digitoxin and tumor necrosis factor-related ligand (TRAIL) mediated apoptosis in human glioma cells [23]. TRAIL, a member of the tumor necrosis factor family, can bind to death receptors (DR4 or DR5) leading to oligomerization of the receptor's intracellular death domains and then to the recruitment of the adaptor molecule. Fas-associated death domain protein, and activation of caspases 3

#### *A Role for Cardiac Glycosides in GBM Therapy DOI: http://dx.doi.org/10.5772/intechopen.105022*

and 8 [23]. However, an obstacle to effective therapy is the development of resistance to TRAIL by brain tumors. The research conducted by Lee et al. presented evidence that a combination of non-apoptosis inducing concentrations of digitoxin and TRAIL led to apoptosis of human glioma cells. Furthermore, they showed that the upregulation of DR5 expression and downregulation of the expression of survivin synergistically enhanced TRAIL-induced apoptosis by digitoxin in human glioma cells [23].

In a more recent article, researchers examined the sensitizing effects of digitoxin to TRAIL-induced apoptosis in GSCs cultured in vitro. They reported that the combination of TRAIL and digitoxin led to apoptosis of GSCs and an upregulation of DR5 expression in addition to down-regulation of surviving expression [24].

#### *2.2.3 Ouabain*

Ouabain, also known as g-strophanthin, is a plant-derived cardenolide that has in the past been used as an arrow poison in Africa. However, it has also been more traditionally used to treat hypotension, congestive heart failure, and some arrhythmias [39]. Interestingly, ouabain is also an endogenous molecule found in animals and humans during normal conditions and increases in concentration in response to high salt intake [40]. It has been reported that ouabain can activate multiple protein kinases such as MAPK, PKC, and phosphoinositide 3-kinase (PI3k)/Akt by binding to Na,K-ATPase [25] and that this is part of the anticancer mechanisms of this molecule. Yan et al. noted that some of these pathways are involved in p66hc phosphorylation [25] suggesting to them that ouabain-induced reactive oxygen species (ROS) was involved.

They examined the intracellular changes induced by ouabain in human glioblastoma cells and noted that prior reports of ouabain-induced mitochondrial membrane loss and elevated ROS production were associated with human cancer cell apoptosis. In a set of interesting experiments, these investigators showed that ROS was increased in glioblastoma cells exposed to ouabain, however, this was not due to calcium overload. Rather, it appears to be the result of p66Shc phosphorylation as part of the Src/Ras/ERK signal pathway [25].

Yang et al. also explored mechanisms of ouabain-mediated cell death of glioblastoma cells. Compared to untreated U-87MG cells, ouabain suppressed survival and attenuated cell motility in a concentration-dependent manner. In addition, they observed downregulation of p-Akt, mTOR, p-mTOR, and HIF-1α at low concentrations of ouabain. The authors suggest that these results indicate that ouabain exerted suppressive effects on tumor cell growth and motility, leading to cell death via regulating the intracellular Akt/mTOR signaling pathway and inhibiting the expression of HIF-1α in glioma cells [41].

#### *2.2.4 Lanatoside C*

Lanatoside C is an antiarrhythmic agent, a naturally occurring compound extracted from *Digitalis lanata*. Badr et al. reported that this cardenolide is a sensitizer of GBM cells to TRAIL-induced cell death partly by upregulation of the death receptor 5. This was evident in GBM cells in culture as well as in a GBM xenograft model in vivo [26]. Cells treated with lanatoside C showed necrotic cell morphology with the absence of caspase activation, low mitochondrial potential, and early intracellular ATP depletion. This suggests mitigation of apoptosis resistance by inducing an alternate cell death pathway. The combined treatment was highly effective as a

low dose of lanatoside C sensitized GBM cells to TRAIL in culture killing over 90% of U87GBM cells, while it had no significant effect on primary fibroblasts [26]. The authors pointed out that to use the suggested combination of TRAIL with lanatoside C in vivo, there would have to be a means of delivering TRAIL intracerebrally.

In follow-up articles, the lab of Bakhos Tannous, PhD (Massachusetts General Hospital) used an adeno-associated virus (AAV) vector specifically designed for intracranial delivery of secreted, soluble tumor necrosis factor-related apoptosisinducing ligand (sTRAIL) to GBM tumors in mice. They combined the AAV delivery vehicle with the TRAIL-sensitizing cardenolide, lanatoside C. This unique combination was applied to two different GBM models using human U87 glioma cells, primary patient-derived GBM neural spheres in culture and orthotopic GBM xenograft models in mice. The authors correctly point out that a major pitfall in testing new GBM therapeutics is the use of animal models that do not accurately recapitulate a phenocopy of the human tumor [42, 43]. Typical cell lines such as U87 form local tumors that do not invade the brain per se. Therefore, the investigators tested the AAV-sTRAIL and lanatoside C therapy using primary cells dissociated from GBM patient specimens and grown as stem-like neural spheres which invade the mouse brain upon intracranial injection which replicates that occurring in the original tumor [43]. Despite the ingenuity of this therapeutic approach both the single and multi-injection approach of sTRAIL combined with lanatoside C showed only a modest survival benefit with animals eventually succumbing to the disease.

#### *2.2.5 UNBS1450*

UNBS1450 is a hemi-synthetic cardenolide belonging to the cardiac steroid glycoside family. The molecule has been shown to induce apoptotic cell death in malignant cells. It inhibits NF-kβ transactivation and triggers apoptosis by cleavage of pro-caspases 8, 9, and 3/7, by decreasing expression of anti-apoptotic Mcl-1, and by recruitment of pro-apoptotic Bak and Bax proteins [44]. UNBS1450 has been tested in 58 distinct human cancer cell lines and displays antitumor effects similar to Taxol [45]. It has also been reported to be active on Taxol-resistance cell lines. Of particular interest was the observation that this semi-synthetic cardenolide demonstrated antiproliferative effects against three glioblastoma cell lines with a level of activity similar to vincristine but much greater than those displayed by temozolomide, tamoxifen, hydroxy-tamoxifen, lomustine, procarbazine, and carmustine [46, 47]. The ability of UNBS1450 to be especially effective against glioblastoma cell lines can be explained, in part, by the fact that U373-MG GBM cells express a higher level of Na, K-ATP α-1 subunits than normal cells which is a particular target for this molecule. Similar to other cardenolides, UNBS1450 also decreases the intracellular ATP concentration more markedly in glioblastoma cells than in normal cells [47]. The advanced feature of this compound is that it can inhibit three isoforms (α3β1, α2β1, and α1β1) with relatively higher efficiency (~6 to >200 times) than ouabain and digoxin [47]. While UNBS1450 was tested in clinical trials using a dose-intensification study to find the MTD, toxicity, and pharmacokinetic parameters of the molecule in patients with lymphoma, the clinical trials were unfortunately closed by the sponsor before reaching the MTD in patients [5].

#### *2.2.6 Oleandrin*

Oleandrin is a highly lipid-soluble cardenolide isolated from the plant *N. oleander* and has been used as a traditional herbal medicine due to its excellent

#### *A Role for Cardiac Glycosides in GBM Therapy DOI: http://dx.doi.org/10.5772/intechopen.105022*

pharmacological properties [38]. Like other cardenolides, oleandrin has been used for the treatment of congestive heart failure; however, more recently oleandrin has attracted attention due to its extensive anti-cancer and novel anti-viral effects. In vitro and in vivo investigations have shown that oleandrin possesses anticancer properties against several cancers including melanoma, leukemia, sarcoma, prostate, lung, pancreatic, and brain cancers [5–7, 12, 13, 17, 38]. Mechanisms underlying the anticancer activity of oleandrin include cell cycle arrest [48], altered membrane fluidity [49], modulation of cell signaling pathways (NF-kβ, JNK) [50], elevated Ca2+ and Na+ levels, decreased K+ levels inside the cell [51, 52], oxidative and mitochondrial stress [53], altered IL-8 levels [54], reduced expression of Rad51 [55], and decreased activation of fibroblast growth factor-2 [56]. Defined extracts of *N. oleander* containing this molecule (i.e., Anvirzel™ and PBI-05204) have undergone clinical trials in cancer patients where both the relative safety and pharmacokinetics of oleandrin were determined [15, 16, 57].

Of particular interest is the ability of oleandrin to act as a chemosensitizer for both chemotherapeutic and radiotherapeutic strategies. The development of resistance to drugs and radiotherapy is a major hurdle toward the effective treatment of cancer [58]. Oleandrin has been shown to reduce radiotherapy resistance in triple-negative breast cancer cells [59] and was also shown to sensitize human prostate cancer cells to radiotherapy [60]. It has also been indicated to exhibit significant antitumor effects in radiotherapy resistant MDA-MB231 cells which was reported to be due to inhibition of phosphor-STAT3, reduced levels of OCT3/4, β-catenin, and decreased MMP-9 activity [59]. These results have been suggested as important with respect to breast cancer invasion. Additionally, various studies have shown that oleandrin decreases tumor size and tumor development and inhibits cellular proliferation in human or murine glioma cells by increasing brain-derived neurotrophic factor (BDNF) levels, decreasing tumor infiltration, and reducing angiogenesis. It was also concluded that oleandrin can be used in adjuvant therapy with currently available chemotherapeutics such as temozolomide.

Oleandrin and extracts that contain this molecule may have unique abilities for the effective treatment of GBM. Digoxin is actively excluded from the brain via P-glycoprotein, yet oleandrin efficiently crosses the blood–brain barrier and inhibits P-glycoprotein expression [17, 61]. Lin et al. investigated 12 human tumor cell lines to explore pathways of tumor cell sensitivity to cardenolide compounds [62]. In vitro models of human glioma included HF U251 cells as well as native and modified melanoma BRO cells. A study by Lefranc and Kiss suggested that high expression of Na,K-ATPase α1 isoform in the presence of low α3 expression was associated with relative sensitivity to cardiac glycosides such as oleandrin, ouabain, and bufalin [63]. Other investigators, however, have found that the higher the Na, K-ATPase α3/α1 ratio, the greater the sensitivity to oleandrin [64].

Garofolo et al. examined the effects of oleandrin on glioma models in vivo [13]. They inoculated human glioma cells into mice and investigated the antitumor efficacy of oleandrin. Administration of this cardenolide reduced glioma growth and lowered cell proliferation. Furthermore, in a recent review of the potential of oleandrin to treat glioblastoma [17], the authors point out that oleandrin increases the cerebral levels of brain-derived neurotrophic factor (BDNF), decreases both microglia/ macrophage infiltration and CD68 immunoreactivity in tumors, lowers astrogliosis in the tumoral penumbra, and attenuates glioma infiltration into healthy parenchymal tissue. In BDNF-knock out mice (bdnftm1Jae/J) and Trk-silenced glioma cells, the efficacy of oleandrin was diminished indicating a key role for BDNF in oleandrin's

antitumor efficacy. Garofalo et al. [65] had previously shown that BDNF inhibited the chemotaxis of glioma cells by blocking the small G-protein RhoA through the truncated TrkB.T1 receptor and that BDNF infusion reduced glioma volume in mice. Additionally, oleandrin was also shown to enhance survival in glioma-implanted mice increasing the efficacy of temozolomide [13].

Colapietro et al. recently reported the efficacy of PBI-05204 (a defined extract of *N. oleander* containing oleandrin as a principle active ingredient) in inhibiting the growth of human glioblastoma. Their studies were designed to investigate the antitumor efficacy of this botanical drug against glioblastoma using both in vitro and in vivo cancer models as well as exploring its efficacy against glioblastoma stem cells. They reported that three human GBM cell lines, U87MG, U251, and T98G were inhibited by PBI-05204 in a concentration-dependent manner that was characterized by induction of apoptosis as evidenced by increased ANNEXIN V staining and caspase activities [66]. An important clue to the mechanisms of anti-glioma growth was the finding that the expression of proteins associated with both Akt and mTOR pathways was suppressed by PBI-05204 in all three cell lines. PBI-05204 significantly suppressed U87 spheroid formation and the expression of important stem cell markers such as SOX2, CD44, and CXCR4. Oral administration of PBI-05204 to nude mice resulted in a dose-dependent inhibition of U87MG, U251, and T98G xenograft growth. Additionally, PBI-05204 treated mice carrying U87-Luc cells as an orthotopic model exhibited significantly delayed onset of tumor progression and significantly increased overall survival. Immunohistochemical staining of xenograft tumor sections revealed declines in Ki67 and CD31 positively stained cells but increased TUNEL staining. Given the fact that PBI-05204 has already been in phase I and II clinical trials for cancer patients, the authors concluded that further examination of the role of PBI-05204 in GBM patients should be considered [66].

#### **2.3 Combination therapies with cardiac glycoside compounds**

Cardiac glycosides represent a class of compounds that work well together with both drugs and radiotherapy in models of GBM. This effective combination of therapeutic strategies has been shown for both bufadienolides and cardenolide compounds. With respect to bufalin, for example, Zhang et al. investigated the response of U251 and U87MG glioblastoma cell lines. Bufalin reduces cell proliferation in both cell lines and induced a G2/M cell cycle arrest [67]. They also observed that bufalin disrupted the mitochondrial membrane potential leading to reduced oxygen consumption and ATP production. In addition, homologous recombination efficacy, a measure of DNA repair, was reduced by ~40%. This was associated with increased γH2AX expression, a marker for the presence of DNA double-strand breaks. Bufalin was additive with radiation in the treatment of GBM cells in vitro. Cell death increased significantly under combination treatment compared to radiation treatment alone [67].

In a recent article, Colapietro et al. explored the role of PBI-05204 in models of human glioblastoma when combined with radiotherapy [58]. This study demonstrated that PBI-05204 treatment led to an increase *in vitro* the sensitivity of GBM cells to radiation in which the main mechanisms were the transition from autophagy to apoptosis, enhanced DNA damage, and reduced DNA repair after radiotherapy administration. The combination of PBI-05204 with radiotherapy was associated with reduced tumor progression as evidenced in both subcutaneous as well as orthotopic implanted GBM tumors. The authors state that, collectively, their results reveal that

#### *A Role for Cardiac Glycosides in GBM Therapy DOI: http://dx.doi.org/10.5772/intechopen.105022*

PBI-05204 enhances antitumor activity of radiotherapy in preclinical/murine models of human GBM and again call for further exploration of the use of this botanical drug in combination therapies in clinical trials.

Cardiac glycoside compounds have also been reported to be able to add to the antitumor efficacy of chemotherapeutic compounds used to treat GBM. Gamabufotalin has been reported to promote temozolomide sensitivity in glioblastoma cells [68]. Both in vitro and in vivo studies were undertaken to examine mechanisms to explain gamabufotalin's ability to increase sensitivity of GBM to temozolomide. Studies revealed a negative feedback loop between ATPA3 (α3 subunit of Na,K-ATPase) and AQP4 (aquaporin 4, a 'water channel' protein molecule), which were predicted by molecular modeling and docking studies to interact with gamabufotalin. The role of AQP4 in GBM growth and proliferation is an interesting finding in light of other studies showing that AQP4 knock out could play a role in several neurodegenerative diseases. Lan et al. reported that AQP4 suppression could significantly promote temozolomide sensitivity with the result that gamabufotalin might mediate inhibition of GBM via regulation of the ATP1A3-AQP4 signaling pathway [69].

In unpublished studies, we have also explored the in vitro and in vivo effects of oleandrin when combined with both radiotherapy and temozolomide in human glioblastoma cell models. Our studies extend a potential role of oleandrin and extracts that contain this molecule (e.g., PBI-05204) in combination with radiotherapy [67]. As radiotherapy and temozolomide are considered 'standard of care' treatment for GBM, any extension of their relative efficacy and success in clinical outcomes is indeed welcomed. Our preliminary studies have indicated again that the combined use of oleandrin with radiotherapy and temozolomide inhibited autophagy in favor of apoptotic pathways, reduced expression of NF-κβ, and reduced cell survival mechanisms while inducing DNA damage by suppression of Rad51. The combined treatments led to an increase in disease-free survival in mice with orthotopically implanted GBM tumors compared to either temozolomide or oleandrin treatment alone. Additional confirmatory studies are needed and are presently underway.

Furthermore, to enhance the translational potential of the therapeutic activity of oleandrin and extracts containing this compound (PBI-05204) in GBM, we evaluated the anti-proliferative effect of oleandrin in primary GBM cells isolated from human GBM-derived intra-cerebral (IC) orthotopic PDX models. We treated the three primary human GBM cell lines, IC-3704, IC-4687, and IC-3752 with different doses of oleandrin (1–100 or 1–1000 nM) and tested cell viability at 72 hrs after treatment. As shown in **Figure 1**, oleandrin exposure significantly inhibited the growth of all three GBM cells in a dose-dependent manner, with comparable low median inhibitory concentrations (IC50) of 8.57, 9.73, and 6.02 nM for IC-3704 (**Figure 1A**), IC-4687 (**Figure 1B**) and IC-3752 (**Figure 1C**), respectively. To further test the antitumor efficacy of oleandrin-containing extract (PBI-05204) on human GBM tumors, we evaluated the overall survival of mice bearing the human GBM-derived IC orthotopic PDX tumor. The IC-1406 GBM was established through direct injection of surgical tumor specimens into mouse cerebrum areas [70]. The tumor was collected from a patient with a diagnosis of Turcort's syndrome carrying a c.137G > T (p.546 l) in the PMS2 gene and this mutation was present in orthotopic tumors. The IC-1406 GBM cell orthotopic model was developed by injecting these particular cells (1 × 105 ) into the right cerebrum. Treatment with PBI-05204 (25 mg/kg, qd × 5 days) was started at 2 weeks post-tumor cell injection. Analysis of median survival times of mice bearing IC-1406 GBM tumor was significantly

#### **Figure 1.**

*Growth curves of human GBM cells derived from a human orthotopic PDX model. Primary cultured cells from IC-3704GBM (a), IC-4687GBM (b) and IC-3752GBM (c) cells (6 × 103 ) were plated and allowed to attach for overnight. They were then treated with oleandrin (1–100 nM) for 72 hrs. Cell proliferation was assessed by MTT assays. Data are presented as mean ± SD.*

increased from 90 days in the control group to 122 days in the PBI-05204 treated group (p < 0.006) (**Figure 2A**), suggesting oleandrin and PBI-05204 exert strong antitumor efficacy in PDX derived GBM cells and their orthotopic model. While we had reported previously that PBI-05204 enhanced the antitumor efficacy of radiotherapy using established human GBM cell lines, such as U87MG, U251, and TG98 cell line mouse xenograft models, we then examined the possibility that PBI-05204 may have significant sensitizing effects on radiotherapy using a patientderived orthotopic GBM PDX model IC-1128GBM [71]. As shown in **Figure 2B**, the combination of radiotherapy (XRT, 2 Gy × 5) and PBI-05204 resulted in a significant enhancement of overall survival compared to control or either single treatment modality alone. For example, the average overall survival of mice treated with PBI-05204 plus XRT was 158 days which was significantly longer than that in control mice (116 days, p < 0.001), PBI-05204 treated mice (118 days, p < 0.001), or XRT treated mice (144 days, p = 0.022), again suggesting PBI-05204 can enhance the antitumor efficacy of radiotherapy in GBM.

To understand the potential mechanisms involved in PBI-05204-elicited antitumor effects in the PDX derived GBM tumor, we examined the expression of cell cycle and apoptosis regulators as well as cell signaling proteins in tumor tissues collected from the IC-1406 PDX orthotopic model using Reverse Phase Proteomic *A Role for Cardiac Glycosides in GBM Therapy DOI: http://dx.doi.org/10.5772/intechopen.105022*

#### **Figure 2.**

*Antitumor efficacy of PBI-05204 alone or in combination with radiotherapy in human GBM-derived intracerebral (IC) orthotopic PDX models. (a) Kaplan Meyer curves of mice bearing orthotopic PDX model of IC-1406 GBM treated with PBI-05204. (b) Kaplan Meyer curves of IC-1128 GBM derived PDX model at passage 8 (rVIII) after treatment with PBI-05204 (25 mg/kg), radiotherapy (XRT, 2 Gy/day × 5 days), or a combination of PBI-05204 and XRT.*

Array (RPPA) analysis as performed by the Functional Proteomics Core Facility at The University of Texas MD Anderson Cancer Center. As shown in the Heatmap (**Figure 3A**), PBI-05204 treatment led to altered expression of several proteins associated with cell cycle, apoptosis, and oncogenic signaling pathway in the IC-1406 PDX model. Among these proteins, PBI-05204 BT significantly down-regulated SOX2 by 41%, an important stem cell marker presented in various cancer stem cells including GBM. Intriguingly, the abundance of tumor suppressor and negative regulator of PI3K/Akt pathway PTEN was significantly increased by PBI-05204 treatment by almost 2-fold. Consistent with this finding, the activity of a downstream target of PI3k/Akt pathways Ribosomal protein S6 was notably decreased by PBI-05204 evidenced by the phosphorylation of this protein was reduced in PBI-05204 treated tumor tissues compared to that of control mice (**Figure 3B**). These findings suggest that PBI-05204 can potentially inhibit the growth of GBM by upregulating PTEN and consequently downregulating the PI3K/Akt pathway and affecting cancer stem cells which were consistent with our previous study using the established human GBM cells.

#### **Figure 3.**

*Proteomic analysis of tumor tissues derived from IC1406 PDX models by reverse phase proteomic Array (RPPA). (a) Heatmap of cell cycle regulating proteins and cell signaling proteins in PBI-05204 treated tumor tissues by RPPA. (b) Expression of cell cycle regulating proteins and cell signaling proteins showing about 20% changes following PBI-05204 treatment. Data are presented as mean ± SD. \* p < 0.05 versus control.*

#### **3. Cardiac glycosides and glioblastoma stem cells**

Conventional treatment of GBM promotes a transient elimination of the tumor and, unfortunately, is almost always followed by tumor recurrence due to an increase in glioblastoma stem cell (GSC) populations [72]. It is believed that GSCs are the primary driving force behind GBM relapses. GSCs are typically resistant to further chemotherapeutic efforts and are typically resistant to additional radiotherapy [72]. To effectively eliminate GSCs, it is critical to target their essential functions and metabolism before effective strategies can be developed against them. While no single therapeutic modality has yet been shown to be completely effective against a heterogenous GSC population, recent studies have shown that cardiac glycosides may prove to have effective activity against GSCs and offer insights as to how they inhibit this specific cell population.

One important target that has been suggested as important for GSC proliferation is the hypoxia-inducible factor (HIF) family of transcriptional factors as they are master regulators of diverse cellular responses to hypoxic conditions. Among these, HIF1α plays a pivotal role in GBM survival, resistance, and invasion [73]. Nigim et al. [74] have reported a new orthotopic model of glioblastoma that recapitulates the hypoxic tumor environment of GBM tumors. This model is based on stem-like GBM cells that were isolated from a recurrent GBM. Their research demonstrates that digoxin is an effective inhibitor of HIF-1α expression and angiogenesis in vivo and provides survival benefits. Using the MGG123 model, the authors have shown that digoxin potently inhibits HIF-1α protein expression even after its induction with hypoxic conditions in vitro. Importantly, they also demonstrated digoxin-mediated HIF-1α silencing in orthotopic GBM xenografts. A related series of studies reported by Bar et al. demonstrated that digoxin also inhibits the growth of cultured GBM cells and flank GBM xenografts with concomitant reduction of HIF-1α and CD133 levels [36]. Thus, digoxin, and perhaps related cardiac glycosides, may effectively target HIF-1α, an important target against GSCs.

Proscillaridin A was shown to have cytotoxic and exhibit anti-migratory properties on GBM cell lines including stem-like cells, but not on healthy neural cells [28]. Berges et al. disclosed a novel pathway by which proscillaridin A and digoxin modulate microtubule network functioning in GBM and stem-like cells [27]. They found that at low concentrations proscillaridin A induced an alteration of microtubule dynamic instability. This was the result of GSK3β activation following the binding of proscillaridin binding to Na, K-ATPase, leading, in turn, to EB1 phosphorylation and subsequent inhibition of cell migration. They conclude that cardiac glycosides at low concentrations mimic the anti-migratory and cytotoxic effects of microtubule inhibiting drugs although they bind to Na, K-ATPase, and not directly to tubulin. As such, cardiac glycosides may represent an alternative treatment strategy and potent candidates for drug repositioning.

Many articles have cited the importance of cardiac glycosides targeting certain alpha subunits (e.g., α1 and/or α3) of Na,K-ATPase to combat the proliferation of glioblastoma cells, Li et al. [75] have indicated that targeting the β2 subunit of Na,K-ATPase represents a new approach to induce glioblastoma cell apoptosis through elevation of intracellular Ca2+. The β-subunit is a glycoprotein involved in the structural maturation of Na,K-ATPase, and regulates enzyme stability, α-subunit activity, and cell adhesion processes. They point out that selectively targeting the β2 subunit that is not expressed in the heart might avoid cardiotoxicity. In contrast, the β2 subunit is more highly expressed in glioblastoma stem-like cells than in GBM cells. Its down-regulation selectively induces apoptosis in GSCs and is associated with significant inhibition of tumor growth in vivo.

Our own research has recently reported the effect of a defined extract of *Nerium oleander* containing oleandrin (PBI-05204) against human glioblastoma models and its ability to modulate GSC cell-renewal properties [67]. Three human GBM cell lines, U87MG, U251, and T98 associated with Akt and mTOR pathways were inhibited by PBI-05204 in a concentration-dependent manner that was characterized by induction of apoptosis as evidenced by increased ANNEXIN V staining and caspase activities. PBI-05204 significantly suppressed U87 spheroid formation and the expression of important stem cell markers such as SOX2, CD44, and CXCR4. Additionally, we also reported that when PBI-05204 was added to the irradiated GBM cells, it enhanced the antitumor efficacy of radiation in both GBM cells and their relevant animal models as well as significantly reducing the stemness of GBM cells. This was believed due to the down-regulation of CD44 and stro-1, an important mesenchymal stem cell marker in U87MG cells [58].

#### **4. Conclusions**

Cardenolide and bufadienolides compounds as well as semi-synthetic cardiac glycoside compounds such as UNBS1450 have now been shown to have potent activity against GBM cell lines as well as established in vivo tumor models. These compounds have been reported to have multiple mechanisms of action which are, in many cases, unique from those of conventional chemotherapeutic agents already approved as the standard of care drugs for GBM. It would thus appear that the combination of cardenolide or bufadienolides compounds with approved radiotherapy and chemotherapy (i.e., temozolomide) approaches to the treatment of GBM is an option worth exploring. Additionally, some cardenolides, such as oleandrin, are capable of crossing the blood–brain barrier and residing there in the brain (up to 24 hrs) longer than that in plasma providing an advantage of these compounds for the treatment of GBM. Finally, considering cognitive impairment is one of the major toxicities of radiotherapy and that some of these compounds, including neriifolin, oleandrin, and others, have been shown to exert neuroprotective effects [76], these compounds might not only be able to slow down the growth of GBM, but also provide a benefit assisting in the repair of radiation-induced damage to injured neurons. Some cardenolide compounds such as PBI-05204 containing oleandrin have already been through both Phase I and II clinical trials in cancer patients. Exciting new research has now clearly shown that this class of compounds also has potent activity in effectively reducing GBM stem cell populations known to be an important reason for the progression of disease after initial surgery and other therapeutic strategies have been performed.

#### **Acknowledgements**

The authors are grateful for the financial support of Phoenix Biotechnology, Inc.

#### **Conflict of interest**

Robert A. Newman is the Chief Science Officer for Phoenix Biotechnology, Inc.; Peiying Yang is a consultant for Phoenix Biotechnology, Inc. All other authors claim no conflict of interest.

*A Role for Cardiac Glycosides in GBM Therapy DOI: http://dx.doi.org/10.5772/intechopen.105022*

#### **Author details**

Yuchen Du1 , Xiao-Nan Li1 , Peiying Yang2 and Robert A. Newman3 \*

1 Northwestern University Feinberg School of Medicine and Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA

2 University of Texas MD Anderson Cancer Center, Houston, TX, USA

3 Phoenix Biotechnology, Inc., San Antonio, TX, USA

\*Address all correspondence to: newmanscientificconsulting@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 9**

## Potential Role of Cancer Stem Cells in Glioblastoma: A Therapeutic Aspect

*Meenakshi Tiwari, Lokendra Kumar Sharma and Ajit Kumar Saxena*

#### **Abstract**

High-grade glioma (HGG) such as glioblastoma multiforme (GBM) is an aggressive brain tumor that is still associated with poor prognosis. With the discovery and advancement in understanding of cancer stem cells (CSC) in glioma, these cells have emerged as seed cells for tumor growth and recurrence and appear as a potential target for therapeutics. Glioma stem cells (GSCs) demonstrate capacity of selfrenewal, proliferation, and differentiation into multiple cell types and can contribute to tumor heterogeneity. Their role is established in tumorigenesis, metastasis, chemoand radio-resistance and appears as a major cause for tumor recurrence. Thus, targeting GSCs by various therapeutics may improve effectiveness of the drugs in use alone or in combination to significantly improve patient survival outcome in GBM cases. In this chapter, we have discussed various mechanisms that drive GSC including signaling pathways and tumor microenvironment. We have also discussed the mechanism behind resistance of GSCs toward therapeutics and the pathways that can be targeted to improve the outcome of the patients.

**Keywords:** Glioblastoma multiforme, cancer stem cells, glioma stem cells, signaling pathways, chemotherapeutics, tumor microenvironment, resistance to therapy

#### **1. Introduction**

Glioblastoma multiforme (GBM), classified as grade IV glioma, is highly invasive, heterogeneous, and malignant primary brain tumor. It accounts for ~57% of all gliomas and ~ 48% of all primary malignant central nervous system (CNS) tumors [1, 2]. Such tumors are associated with poor quality of life of the patient due to progressive decline in neurologic function, thus making a huge impact on the patients, care givers, and their families. The standard treatment includes multimodal approach involving maximal surgical resection followed by radiotherapy, systemic therapy (chemotherapy, targeted therapy), and supportive care; however, long-term survival is exceptional. Despite the treatment, these tumors regrow and that too with aggressive phenotype, which worsen the symptoms leading to prognosis with average overall survival time < 14.6 months for primary GBM patients and < 6.9 months for recurrent

GBM patients [3]. Understanding the molecular mechanism involved in therapeutic resistance and tumor regrowth despite standard treatment is imperative.

In this regard, researchers have identified existence of cancer stem cells (CSCs) in a variety of cancers that play crucial role in tumor initiation, maintenance, resistance to therapy, recurrence, metastasis, and generation of more aggressive phenotype [4]. These properties of CSC are manifested by their potential to self-renew, proliferate, ability to differentiate in multiple phenotypes, plasticity, quiescence, and dormancy. It is suggested that these CSCs originate either from normal stem cells that were already present in tissue or can be generated from dedifferentiation of somatic cells from bulk of tumor. Based on the properties of CSCs, they pose not only a barrier for anticancer therapy but also are responsible for recurrence into more aggressive phenotype. Various researchers have shown that CSC escape anticancer therapy due to their ability to enter dormancy, plasticity, renewal, and regrowth into heterogeneous group of tumor cells. Of interest, recent evidences have suggested that these CSCs are further enriched in response to standard radio-chemotherapy, which may be responsible for tumor regrowth and aggressive phenotype. These enriched CSCs might be result from the existing population of CSCs that evades the therapy or as per recent evidences, can be generated from non-CSCs from the bulk of tumor in response to therapy. Of note, CSCs have been identified in HGG cases also known as glioma stem cells (GSCs) that contribute to tumor heterogeneity and resistance to therapies, thus a major contributor of tumor recurrence. These GSCs are considered as a potential therapeutic target, therefore understanding the molecular pathways that drive GSCs becomes imperative [5]. In this book chapter, we have discussed about the properties of cancer stem cells, cell surface markers, signaling pathways, and mechanism of resistance to therapies and ways by which these pathways can be targeted using different chemotherapeutic agents.

#### **2. Biology of cancer stem cells**

Stem cells are specialized cells present in our body that possess properties such as capacity to self-renew, proliferate, and differentiate into multiple cell types. This quality of self-renewal along with associated signaling pathways is shared between both stem cells and cancer stem cells with added feature of oncogenicity in CSCs. The most common pathways that drive multipotency and self-renewal of stem cells include the Notch, Sonic hedgehog (Shh), and Wnt signaling pathways [4]. Due to activation of oncogenic pathways, CSCs can give rise to tumor mass consisting of heterogeneous cell population. Initially, Bonnet and Dick characterized CSCs in acute myeloid leukemia as leukemia-initiating cell that possessed properties of leukemia stem cell [6]. Later, such cells were also identified in a variety of solid tumors, including prostate [7], colon [8], lung [9], ovarian [10], and brain [11] tumors. It is hypothesized that CSCs are the seed of a tumor that are responsible for tumorigenesis by initiation, maintenance, propagation, resistance to therapy, recurrence as well as progression of the tumor [12].

#### **3. Glioma stem cells**

In brain tumors, presence of CSC has been identified and characterized by various groups and are defined as GSCs or glioma initiating cells [11]. When cultured, these

*Potential Role of Cancer Stem Cells in Glioblastoma: A Therapeutic Aspect DOI: http://dx.doi.org/10.5772/intechopen.106332*

cells grown into neurospheres that constitute of cells that express SC markers including Nestin and CD133. When these cells are injected into nude mice, they lead to tumor formation due to their SC properties [13]. To add further, various groups have utilized properties of stem cells that are present in brain predominantly in subventricular zone (SVZ) to initiate tumor by exposure to chemicals (ethylnitrosourea) or viruses (avian sarcoma virus) in animals that strongly support the importance of stem cells in tumor formation [7, 14, 15]. These cells contribute to tumor heterogeneity and plasticity and have shown resistance to therapies and thus have emerged as a major contributor of tumor recurrence [5, 16, 17]. These CSCs are also influenced by micro environmental conditions such as nutrient deprivation, hypoxia, pH, vasculature, radiation, and chemotherapeutic treatment (explained in detail in coming sections) [16–19].

Several putative GSC surface markers, such as CD133, CD15, and CD44, and GSC transcription factors, such as SRY-box transcription factor 2 (SOX2), octamerbinding transcription factor 4 (OCT4), and NANOG, have been discovered [20, 21]. However, before its clinical implication, higher sensitivity and specificity of these GSC markers need to be established [21, 22].

#### **4. Major signaling pathways that drive glioma stem cells**

In order to maintain stemness properties, GSCs depend upon number of signaling pathways that also support them to sustain under adverse conditions during tumorigenesis [23–25]. To understand the process of stemness in GSC, the signaling pathways that are also a part of normal neuronal stem cells are discussed. These pathways mainly include Notch, bone morphogenic proteins (BMPs), NF-*κ*B, Wnt, epidermal growth factor (EGF), and Shh that determine the property of stemness.

#### **4.1 Notch signaling**

Notch signaling pathway is crucial in developmental process and plays a major role during embryonic development. This pathway regulates cellular proliferation, differentiation, apoptosis, and cell lineage decisions. In GSCs, Notch signaling pathways are highly active, which in turn maintain stemness by inhibiting differentiation. Notch signaling is also involved in oncogenic transformation. It has been identified that inhibition of Notch signaling decreases oncogenic potential of GSCs [26, 27].

#### **4.2 Bone morphogenetic proteins (BMPs)**

BMP group of molecules belongs to the transforming growth factor-β (TGF-β) superfamily of proteins. BMPs plays role during embryogenesis, development as well as adult tissue homeostasis. It interacts with different signaling molecules including Wnt/*β*-catenin, basic helix-loop-helix (bHLH), and hypoxia-inducible factor-1*α* (HIF-1*α*) to regulate different processes in all the body organs [28, 29]. BMPs have been identified to regulate the niche as well as stem cells residing within. Besides normal functions, BMPs are also involved in tumorigenesis where BMP2 and BMP4 have emerged as key players. It is identified that dysregulation of the BMP pathway results in sustained cell transformation in stem cells and their niche. BMP signaling pathways are also involved in regulation of cellular proliferation, differentiation, and apoptosis in NSCs. NSCs are differentiated to astroglial lineage via Wnt-mediated

BMP signaling [30] and antagonist of BMP can inhibit differentiation of GSCs and maintains its self-renewal and tumorigenic potential [31]. Further, it was demonstrated that delivery of BMP4 can inhibit brain tumor growth in in vivo system and decreased the rate of mortality [32].

#### **4.3 Wnt/β-catenin signaling**

Wnt/β-catenin signaling is a highly conserved pathway that regulates cellular proliferation, differentiation, migration, genetic stability, apoptosis, and stem cell renewal. This pathway also regulates NSC expansion and promotes astroglial lineage differentiation during neural development [33, 34]. In GSC, *β*-catenin regulates proliferation and differentiation and dysregulated Wnt signaling leads to tumor growth [35–37]. *β*-Catenin interacts with FoxM1 to regulate the transcription of various oncogeneic genes such as c-Myc that leads to gliomagenesis [38, 39].

#### **4.4 Epidermal growth factor receptor (EGFR) signaling**

The EGFR pathway is one of the most crucial pathways involved in cellular processes including proliferation, differentiation, migration, and apoptosis in a variety of cells including stem cells. Dysregulation of this pathway has been linked to cancer. Critical role of EGFR has been identified in NSCs as well [40–42]. In GSC EGFR works through activation of *β*-catenin pathway and promotes self-renewal capacity of GSC and induction of tumorigenic potential [43].

#### **4.5 Sonic hedgehog (Shh) signaling**

The Shh signaling pathway is crucial for proper embryonic development as it governs tissue polarity, patterning maintenance, cellular proliferation, intercellular communication, and differentiation [44, 45]. Persistent Shh pathway signaling has been observed in the subventricular zone of adult brain where it plays a critical role in regional specification and maintenance of NSCs [46]. Aberrant regulation of the Shh pathway due to mutation has been identified to cause tumorigenesis in a wide variety of cancer tissues including gliomas and GSCs. This pathway is highly active in GSCs where it regulates stemness genes and thus maintains self-renewal of GSC and promotes tumorigenesis and inhibition of Shh signaling reduces both stemness as well as *in vivo* tumorigenicity by induction of autophagic cell death [47].

#### **5. Pathways that contributing to resistance of glioma stem cells toward therapies that lead to tumor regrowth**

Resistance of CSCs toward therapies resulting in their enrichment and regrowth of tumor due to proliferation of these cells has been suggested by various researchers [48–50]. In HGG, despite the effectiveness of TMZ in removing the bulk tumor cells, regrowth with a more aggressive phenotype is inevitable, and researchers have identified critical role of CSCs in such regrowth. For instance, in HGG, treatment with therapeutic doses of temozolomide (TMZ) leads to expansion of GSCs pool in both patient-derived and established glioma cell lines. Such expansions are reported not only due to enrichment and proliferation of existing CSCs but also due to interconversion between differentiated tumor cells and GSCs [18]. Similarly,

*Potential Role of Cancer Stem Cells in Glioblastoma: A Therapeutic Aspect DOI: http://dx.doi.org/10.5772/intechopen.106332*

bevacizumab (VEGF antibody) although reduces GBM tumor growth, it is followed by tumor regrowth where the role of autocrine signaling through the VEGF-VEGFR2- Neuropilin-1 (NRP1) axis leads to enrichment of active VEGFR2 GSC subset in human GBM cells [51]. It is evident that the therapeutics evoke enrichment of CSCs involving multiple mechanisms. Thus, understanding various ways by which CSCs escape the radio- and chemotherapy, more effective treatment modalities can be developed. Broadly, in CSCs various different mechanism such as epithelial-mesenchymal transition (EMT), multiple drug resistance (MDR) dormancy, tumor environment contribute to resistance toward therapeutics and other adverse conditions faced by them in tumor microenvironment and are discussed as follows.

#### **5.1 DNA repair systems**

GSCs possess better DNA repair capacity as compared with bulk tumor cells [52]. These cells express higher levels of DNA repair enzymes such as O6-methylguaninemethyltransferase (MGMT), which are responsible for therapy resistance against DNA alkylating agents such as TMZ [53–56]. However, there are contradictory studies that also suggest that TMZ resistance in GSCs is independent of MGMT status and alternate pathways might be involved [57, 58]. Further, preferential expression of DNA checkpoint kinases 1 (Chk1) and 2 (Chk2) lead to more efficient repair of DNA damage in CD133-positive glioma cells than CD133-negative glioma cells [54]. Other transcriptional regulators such as BMI, DNA-PK, poly (ADP-ribose) polymerase-1, hnRNP U, and histone H1, which play a role in DNA double-strand break repair, are highly expressed in CD133-positive GBM cells and play pivotal role in GSCs' functions [59, 60].

#### **5.2 Epithelial-mesenchymal transition (EMT)**

EMT involves phenotypic changes in cells from epithelial to mesenchymal type involving high expression of markers such as N-cadherin and vimentin under various physiological as well as pathological conditions including cancer [61]. Interestingly, CSCs also share the EMT-like cell features [62], and it is believed that the link between EMT and CSCs might be responsible for cancer drug resistance acquisition and plasticity resulting in cancer cells transformation into the malignant cells and *vice versa* [63]. Circulating tumor cells from patients with metastasis co-express markers of EMT as well as CSCs. Further, induction of EMT confers stem-like features in cancer cells [64, 65]. Various regulators of EMT have been identified that regulate stemness. ZEB1 is one such regulator of EMT that regulates stemness and chemoresistance induction by regulating MGMT via miR-200c and c-MYB in malignant glioma [66]. Therefore, a strong association of EMT and CSCs has been identified that provides not only resistance but also promotes metastasis [67].

#### **5.3 Dormancy of CSCs**

As the understanding of CSC biology has improved, it has been identified that CSCs can exist in proliferative or dormant state. Dormant CSCs maintain a low metabolic activity, however, show similarities with the normal proliferative counterpart in terms of stemness and other signaling pathways. For instance, dormant stem cells are low in metabolic activity that preferentially utilize the glycolytic pathway and produce low levels of levels of reactive oxygen species (ROS) [68]. However, these

dormant/quiescent cells demonstrate high plasticity and can be reactivated to reenter proliferative stage and lead to tumor formation. Such dormant cells are also chemoresistant due to their dormant nature; interestingly, proliferative CSC can also enter dormancy in response to chemotherapeutics agents. In GBM, existence of a relatively quiescent subset of GSCs has been observed, which is responsible for maintaining the long-term tumor growth and responsible for recurrence by entering into highly proliferative cells upon receiving proper signals [69].

#### **5.4 Anti-apoptosis**

Various anti-apoptotic protein such as B-cell lymphoma-2 (BCL-2), BCL2 like 1 (BCL2L1), myeloid cell leukemia-1, MCL1 and BCL-xL are highly expressed in GSCs than differentiated bulk tumor cells. These proteins not only play role in GSCs maintenance but also provide survival advantage to these cells against various chemotherapeutic agents [70]. Other mediator of GSCs resistance includes BMI1, a GSC-enriched protein that inhibits p53-mediated apoptosis against TMZ [71]. Inhibition of such anti-apoptotic pathways can increase sensitivity of GSCs against different therapeutic agents.

#### **5.5 Multidrug resistance**

Stem cells express higher levels of several ATP-binding cassette (ABC) transporters resulting in efflux ability for various antineoplastic drugs [72]. In GSCs, increased *ABCG1* expression has been documented in the side population cells in flow cytometry that present the GSC phenotype [73]. Further, multidrug resistance 1 (*MDR1*) overexpression was reported higher in CD133+ GSCs than CD133− bulk tumor cells [74]. *ABCG2*/*BCRP* and *ABCB1*/*MDR1* overexpression in GSCs has also been correlated with resistance of GSCs to chemotherapeutic drugs and use of an ABC transporter inhibitor, such as verapamil, can help in increasing sensitivity of GSCs toward chemotherapeutic agents such as temozolomide, doxorubicin, and mitoxantrone in GSCs [75]. Similarly, methylation of ABC transporter *ABCG2/BCRP* promoter by melatonin (*N*-acetyl-5-methoxytryptamine) promotes toxic effect of TMZ on GSCs [75]. Interestingly, treatment with chemotherapeutic agents can further increase expression of these MDR proteins conferring resistance to these cells against chemotherapeutic agents [76]. Thus, inhibition of drug efflux proteins such as MDR proteins appears as a potential target for increasing sensitivity of GSCs toward various chemotherapeutic agents [75, 77].

#### **5.6 Metabolism**

GSCs show metabolic adaptations to survive adverse conditions of tumor microenvironment that includes low pH, hypoxia, and low nutrient supply; at the same time they proliferate at a high rate to maintain their stemness [16, 17]. Majority of GSCs rely on glucose uptake via high-affinity glucose transporter 3 (GLUT3) to provide carbon source for nucleotide biosynthesis for rapid proliferating cells along with high energy demands [78–80]. These cells also highly express glutamine synthetase as compared with differentiated glioma cells for higher glutamine uptake, which acts as preferential source for *de-novo* purine biosynthesis [81]. Further studies demonstrate that in therapy-resistant GSCs expression of glucose uptake associated genes is downregulated, and they preferentially use fatty acids as a major ATP source [82].

Additionally, slow-cycling GSCs rely on oxidative phosphorylation and lipid metabolism than fast-cycling GSCs which prefer glycolysis [83]. These anabolic advantages of GSCs may contribute to their chemoresistant phenotype and can be targeted to improve sensitivity of GBM treatment.

#### **5.7 Autophagy**

Autophagy is a catabolic pathway which is a cellular stress response under physiological as well as pathological conditions. This pathways acts by removal of damaged macromolecules such as proteins, nucleic acid, and lipids and recycles them for cellular processes and thus promotes cell survival; however, defect or dysregulation of such pathway may lead to cell death [84]. Role of autophagy is well established in a variety of cancers including GBM where it can play a role in cell survival or cell death [84, 85]. Autophagy also contributes to the maintenance of stemness characteristics of GSCs as well as provides chemoresistance to CSC against therapeutic agents [19, 86]. Inhibition of autophagy sensitizes GSCs towards a variety of therapeutic agents [19, 87–89]. Interestingly, other studies demonstrated that induction of autophagy by mammalian target of rapamycin (mTOR) inhibitors as well as curcumin-induced autophagy shows anti proliferative effect, induces differentiation and also improves sensitivity of GSC towards DNA damaging agents [90–92]. Together, these results suggest that GSCs require a balanced level of autophagy, too much or too little can significantly affect their stemness potential and resistance toward therapeutics. Further, role of autophagy has also been shown to support motility/migration capacity of GSCs [93]. However, role of autophagy in suppression of the self-renewal ability and tumorigenicity of GSCs has also been demonstrated where autophagy mediates Notch1 degradation [94]. Thus, role of autophagy in GSCs is crucial for maintenance of stemness as well as chemotherapeutic agents; targeting such pathway appears as a p These cells also highly express glutamine synthetase otential strategy to make the existing treatment more effective.

#### **5.8 Extrinsic chemoresistance**

Besides the signaling pathways and genetic signature of GSCs, extracellular environment also called as microenvironment in which these cells resides also plays crucial role in its functions and determines response towards therapeutic agents [95]. It has been identified that GSCs reside in inner tumor mass where rapid growth and high energy requirement of these cells along with neovasculature result in hypoxic conditions as well as low pH [29, 96]. These adverse conditions further promote expression of GSC markers and associated phenotype [97]. Various hypoxia and acidic pH-induced genes such as hypoxia-inducible factor (HIF) 1 and 2alpha and vascular endothelial growth factor (VEGF) are highly expressed in GSCs that contribute to GSC functions [98, 99]. It has been shown that in GSCs Notch signaling and MGMT expression are also regulated by HIF-1α, resulting in GSC stemness and also resistance toward TMZ [100, 101]. Further, hypoxic GSCs release extracellular vesicles that deliver HIF-1α induced miR-30b-3p that further activates STAT3 pathway and promotes TMZ resistance [102]. Further, it has been identified that TMZ increases the GSC pool in non-GSC subpopulations, indicating that non-GSC shows plasticity and can be converted to GSCs that might be responsible for resistance as well as regrowth of the tumor [18, 19]. Together, these findings suggest that *stemness* of GSCs may be regulated by tumor microenvironment as well as cellular plasticity; TMZ can

#### *Glioblastoma - Current Evidence*

stimulate the dedifferentiation of non-GSCs, which might contribute to resistance and recurrence after therapy [18, 19].

#### **5.9 Role of Notch and sonic hedgehog pathways in mediating chemoresistance**

Various signaling pathways such as Notch and SHH are active in GSCs compared with bulk tumor cells [103]. Further, in response to TMZ treatment of GSCs from primary GBM cells resulted in upregulation of *NOTCH 1*, *NCOR2*, *HES1*, *HES5*, and *GLI1* genes, suggesting resistance of these cells and increase in the population of such stem cells in glioma, which could be reversed by inhibitors of Notch or SHH inhibitors [104]. Epithelial-mesenchymal transition (EMT) mediates GBM chemoresistance. Another fact contributing to resistance of GSCs is the potential of epithelial-mesenchymal transition. It has been shown that EMT mediator gene *ZEB1 can* regulate *stemness* and *SOX2* and OLIG2 in gliomas [105].

#### **6. Strategies targeting glioma stem cells**

Despite extensive research in oncology, the target is being missed leading to recurrence in a variety of high-grade tumors including malignant gliomas. With advancement in understanding of GSCs and its capacity of initiation, progression, resistance as well as recurrence of tumor, they appear as most promising target to treat cancers such as HGG. The drugs that can target GSC are being developed using multiple strategist including molecular targeting, autophagy inhibition, drug repositioning, and indirect targeting of GSC niches [17, 21, 106].

#### **6.1 Targeting GSC markers and related signaling pathways**

GSCs are regulated by various pathways involving differential expression of epidermal growth factor receptor (EGFR), Notch1, sonic hedgehog (Shh), and STAT3, as well as related signaling pathways.

As discussed earlier, CD133 is the most well-characterized cell surface marker for GSCs, which has become a potential target for antibody-based therapy. Different immunotherapeutic approaches such use of synthetic monoclonal antibody, dualantigen T cell engager, and chimeric antigen receptor (CAR) T cell have been utilized to target CD133+ GSCs. RW03 (anti-CD133 antibody) targets self-renewal ability of GSCs without effecting its proliferative capacity and could be a promising strategy in targeting GSCs [107]. Further, photothermal therapy has also shown selective efficacy in diminishing CD133-positive cells both *in-vitro* and *in-vivo* [108].

EGFR, a receptor tyrosine kinase, which is highly expressed in GSCs, is crucial for its survival, self-renewal, and tumorigenicity. Of importance, EGFR variant III (EGFRvIII) mutation is most commonly detectable (25–33%) in GBM cases [109]. Thus, EGFR inhibition becomes a potential target to inhibit GSCs proliferation, selfrenewal, and induction of apoptosis [110]. EGFR inhibition in fact enhanced chemoand radio-sensitivity of human glioma CSCs. Various reversible and irreversible inhibits of EGFR are available that can bind EGFR alone or along with its co-receptor HER2 [111, 112]. First-generation EGFR TKIs include gefitinib and erlotinib that can reversibly bind EGFR along with HER2; however, less than 20% of patients presented a response to these treatments [112, 113]. Irreversible inhibitor of EGFR, osimertinib, has shown efficiency in crossing the blood-brain barrier (BBB) and significantly

#### *Potential Role of Cancer Stem Cells in Glioblastoma: A Therapeutic Aspect DOI: http://dx.doi.org/10.5772/intechopen.106332*

inhibits GBM tumorigenesis *in-vivo* [114]. It has also entered phase II clinical studies [115, 116], however, has shown to be marginally effective, which could be due to heterogeneity of GBM [117, 118]. Further, combined treatment of antibodies against EGFRvIII and CD133 showed higher effectivity in elimination of GSCs compared with the antibody against either EGFRvIII or CD133 [111].

Various signaling pathways such as Notch and SHH are active in GSCs compared with bulk tumor cells [114]. Further, in response to TMZ treatment of GSCs from primary GBM cells resulted in upregulation of *NOTCH 1*, *NCOR2*, *HES1*, *HES5*, and *GLI1* genes, suggesting resistance of these cells and increase in the population of such stem cells in glioma, which could be reversed by inhibitors of Notch or SHH inhibitors [118]. Notch1 signaling that contributes to regulation of GSC can be blocked by γ-secretase inhibitor [119]. RO4929097, a γ-secretase inhibitor, reduces the viability of GSCs [120]. Further, Notch1 also regulates VEGF activity in GSCs, and co-inhibition of Notch1 and VEGF have shown synergistic effects in GBM [121]; however, their combined inhibition (RO4929097 with bevacizumab) in phase I clinical trial did not show much improvement in overall survival (OS) and progression-free survival (PFS) in GBM cases [122]. Other studies also identify the role of γ-secretase inhibitor, N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) in improving TMZ sensitivity [123].

Shh/Gli signaling that regulates GSCs cell proliferation, stem cell fate determination, and differentiation has also appeared as potential target for GSCs therapeutics [124]. Inhibition of hedgehog pathway by LDE225 induces autophagic cell death in GSCs with higher sensitivity of CD133-positive cells than CD133-negative cells [125]. LDE225 inhibits expression and nuclear translocation of Gli proteins, a transcriptional effectors of the Shh signaling pathway [126]. Casein kinase 2 (CK2) is another target to inhibit Shh/Gli signaling via transcriptional activation of β-catenin [127] and inhibition of CK2 by, CX-4945 (silmitasertib), reduces MGMT expression and sensitized tumor cells to TMZ [128].

Signal transducer and activator of transcription 3 (STAT3) that regulates multiple processes such as cell cycle and survival, regulation, immune response, and differentiation, tumorigenic transformation has also been implicated in GSC maintenance [129, 130]. Resveratrol (RV), a polyphenol present in grapes, a tumor preventive agent targets STAT3 signaling. In glioma, RV has shown antineoplastic actions by apoptosis induction and improving radio sensitivity of GSCs CD133+ cell population along with reducing of tumorigenic potential. Furthermore, RV also modulates Wnt signaling pathway and EMT activators, thereby regulating stemness of GSCs and reducing cellular motility [131, 132]. Another molecule that inhibits STAT pathway is WP1066, which is an analog of the natural product caffeic acid benzyl ester and targets GSCs. This molecule has shown promising results in clinical trial for patients with recurrent malignant glioma [133]. Other STAT3 inhibitors, STX-0119 and WP1066 have shown ability to suppress GSC proliferation *in-vitro*; however, inhibition of tumor growth in subcutaneous xenograft model of GSCs was shown only by STX-0119. STX-0119 further demonstrated ability to downregulate expression of GSCs markers [134]. Another small-molecule STAT3 inhibitor, ODZ10117, also decreased the stem cell properties of GSCs and reduced tumor growth in vivo [130].

#### **6.2 Targeting tumor microenvironment**

GSCs are localized in specific niches, which have been identified as protective microenvironments in GBM. Five types of GSC niches have been identified where different cell types exists and have specific singling pathways: peri-vascular, peri-arteriolar, peri-hypoxic, peri-immune, and extracellular matrix out of which peri-vascular niche is the most frequently described GSC [135]. GSC microenvironment lacks organizations and has compromised BBB, higher levels of hypoxia, and excessive angiogenesis making it a target for anti-angiogenic therapy [136, 137].

#### **6.3 Drugs targeting metabolic pathways**

Drug repositioning also called as repurpose drugs is a growing concept that explores pre-existing a well-established drug to treat diseases aside from the intended ones. This concept results in lowering the overall developmental cost, time and risk assessments, as the efficacy and safety of the original drug have already been well accessed and approved by regulatory authorities [138]. In case of GSCs, repurpose drugs are being tested and have shown encouraging results. Especially, anti-diabetes drugs have been most well studied with promising results in GSCs targeting. Metformin, successfully used for type 2 diabetes mellitus, has entered phase I clinical trial for GBM in combination with TMZ [139]. Metformin preferentially acts in GSCs by inhibiting Akt activation and also induces conversion on GSCs to non-GSCs [140, 141]. Similarly glimepiride, another anti-diabetes drug, impairs GSCs by targeting glycolytic flux and increases its radio sensitivity to GBM [142]. Further, more repurpose drugs need to be identified that can effectively target GSCs along with its associated mechanism before it can be used in clinical application [138].

#### **6.4 Targeting autophagy pathways**

Autophagy is a cellular stress response, which can either promote survival or cell death. Our laboratory along with others has identified that autophagy is required for maintenance of GSCs and also plays a role in resistance of GSCs toward chemotherapy [19, 142, 143]. Targeting autophagy by commonly used agent chloroquine (CQ ), which blocks the fusion of autophagosomes with lysosomes, has been shown to inhibit GSCs as well as sensitized them toward chemotherapeutic agents [19, 144]. This drug has also entered multiple clinical trials as an adjuvant treatment for GBM; where its antitumor effects of CQ are not limited to GSCs [145]. Further, combination of autophagy inhibitors with radiation effectively induced apoptosis and inhibited tumorosphere formation in GSCs [146, 147]. More selective autophagy inhibitor NSC185058, antagonist of autophagy-related 4B, inhibits tumorigenic potential of GSCs and enhances GBM sensitivity to radiotherapy in xenograft mouse models [148].

#### **7. Conclusions and future directions**

Treatment of HGG remains challenging. With identification of GSCs and their properties to resist treatment and repopulate the original tumor, a big momentum has been created in the development of novel therapeutics. Such therapeutic will involve a combination of drugs that controls the bulk tumor mass along with CSCs-directed agents. It has been identified that GSCs are responsible for tumorigenesis, therapeutic resistance, and tumor recurrence, and thus GSC-targeting drugs are being developed for improvement of treatment regime. These GSCs can survive cancer treatment by activating multiple mechanisms such as EMT, signaling pathways to regulate selfrenewal, its interaction with tumor microenvironment, higher expression of drug

#### *Potential Role of Cancer Stem Cells in Glioblastoma: A Therapeutic Aspect DOI: http://dx.doi.org/10.5772/intechopen.106332*

transporters or detoxification proteins, plasticity, autophagy induction, anti-apoptotic mechanism, induction of dormant phenotype, and many others to overcome the toxic effects of therapeutics. With knowledge of these pathways, anticancer therapeutics are targeted against GSCs, which includes directing specific and pathways that regulate GSCs and protect them from therapeutic stress. Such GSC-directed drugs can be combined with agents that are currently in use to achieve better survival rates of cancer patients.

Identification of bioactive products and their molecular mechanisms that can modulate GSCs needs to be incorporated in treatment regime of HGG patients. With recent advancements in the field of high-throughput screening and genetic and epigenetic signatures, specific targeted drugs that can target bulk tumor with minimal generation of induced GSCs along with combination of drug that can target GSCs can be developed. Furthermore, tumor microenvironment that significantly regulates GSCs is also a potential target to prevent rate of dedifferentiation. It is important to consider that current therapeutic can result in conversion of non-GSC to GSCs; therefore, newer drugs or combinations need to be developed that can prevent this detrimental conversion. More stringent strategies involving GSC-targeted therapy along with glioma molecular subtypes need to be designed for selective and effective clinical trials.

However, most therapeutic agents have failed to be approved for clinical application or during clinical trials due to lack of understanding of the underlying mechanisms or failure to consider individual characteristics of the tumor. Further investigation of the molecular pathways that drive GSCs and make them resistant to therapies along with subtype-specific pathways of GSCs is required. Such studies will significantly improve not only the understanding of disease but will also direct the development of highly specific drugs with minimal side effects along with improved patient outcome.

#### **Acknowledgements**

The authors would like to acknowledge the funding support from Department of Biotechnology Bio-CARe grant, Govt. of India (BT/P19357/BIC/101/927/2016 to M.T.) and Intramural Research Grant by SGPGIMS (PGI/DIR/RC/36/2021) to LKS.

#### **Conflict of interest**

The authors declare that they have no conflict of interest.

*Glioblastoma - Current Evidence*

#### **Author details**

Meenakshi Tiwari1 \*, Lokendra Kumar Sharma2 and Ajit Kumar Saxena1

1 Department of Pathology/Lab Medicine, All India Institute of Medical Sciences-Patna, Patna, Bihar, India

2 Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

\*Address all correspondence to: meenakshimani79@yahoo.co.in; drmeenakshit@aiimspatna.org

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Potential Role of Cancer Stem Cells in Glioblastoma: A Therapeutic Aspect DOI: http://dx.doi.org/10.5772/intechopen.106332*

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#### **Chapter 10**

## A Story of Immunization with Autologous IFN-γ Secreting Glioma Cells in Patients with Glioblastoma Multiforme is Safe and Prolongs Both Overall and Progress Free Survival

*Leif G. Salford, Peter Siesjö, Gunnar Skagerberg, Anna Rydelius, Catharina Blennow, Åsa Lilja, Bertil Rolf Ragnar Persson, Susanne Strömblad, Edward Visse and Bengt Widegren*

#### **Abstract**

The study was a non-randomized controlled phase I-II trial to study were to ascertain the safety, feasibility and efficacy of immunotherapy with autologous IFN-γ transfected tumour cells in patients with glioblastoma multiforme. Autologous tumour cells harvested during surgery were cultured and transduced with the human IFN-γ gene. Irradiated cells were administered as intradermal immunizations every third week. Endpoints for safety were records of toxicity and adverse events, for feasibility the per cent of treated patients out of eligible patients and time to treatment and for clinical efficacy overall survival (OS) and progress free survival (PFS). Eight eligible patients, between 50 and 69 years, were immunized between 8 and 14 times after treatment with surgery and radiotherapy without adverse events or toxicity. Neurological status and quality of life were unchanged during immunotherapy. The immunized patients had a significantly (*p* < 0.05) longer median overall survival (488 days, 16.1 months than a matched control group of nine patients treated with only surgery and radiotherapy (271 days, 9.0 months). The prolongation of survival was also significant compared to all GBM treated at the same institution during the same period and published control groups within the same age cohort.

**Keywords:** brain tumour, clinical trial, interferon-gamma, immunotherapy, translational

#### **1. Introduction**

**157** The most aggressive primary brain tumour, glioblastoma multiforme (GBM) [1], is the most therapy-resistant human tumour. The mean survival time after diagnosis for

patients with GBM had been approximately a year for more than 30 years when our study was performed, despite advances in surgery and radiotherapy. Consequently, very few patients survive the disease [2].

By the use of combined radiotherapy and chemotherapy with temozolomide, Stupp et al. demonstrated a small but significant increase in mean survival time from 12.1 to 14.3 months [3]. Unlike most other tumours, there is a considerable age-related impact on the survival of patients with GBM, where patients under the age of 50 years have more prolonged survival than those over the age of 50 [4, 5]. The mechanisms behind this are not precise, and both diverse biologies of the tumours in patients of different ages and senescence of the immune system have been proposed [6, 7]. The importance of immune reactivity against tumours has been highlighted by several reports, demonstrating a clear correlation between the numbers of tumour infiltrating lymphocytes and the prognosis of survival in patients with various neoplastic diseases [8, 9].

Glioblastomas induce profound immune suppression by several proposed mechanisms, such as releasing immunosuppressive substances, such as prostaglandin E2 (PGE2) and interleukin-10 (IL-10). It also releases growth factor-beta (TGF-α) [10, 11], which up-regulate apoptotic ligands such as programmed death receptor 1-ligand (PD1-L) [12] and induction of regulatory T cells [13].

Experimental intracranial tumour models report successful immunotherapy results [14, 15]. In addition, several investigators have reported promising preliminary results of clinical immunotherapy in patients with glioblastoma multiforme [16–18]. However, the results have been difficult to interpret due to heterogeneity of patients regarding age, the extent of resection and additional therapy.

We have previously reported successful immunotherapy against rodent brain tumours using autologous tumour cells secreting the cytokines IFN-γ, IL-7, nor expressing the adhesion molecule B7-1, where immunizations with IFN-γ secreting tumour cells were the most potent treatment [19, 20]. In our models, the proportion of CD8+ T-cells and NK cells of tumour-infiltrating leukocytes from immunized animals was larger than in tumours from control immunized animals [21].

Here we report the result of those experiments translated into a clinical trial of patients diagnosed with GBM aged 50–69 years. The study's goal was to ascertain whether immunization with transduced autologous tumour cells secreting IFN-γ; was feasible, safe for the patients and could show any evidence of clinical responses.

#### **2. Material and methods**

#### **2.1 Study design**

We designed this study as a phase I-II, non-randomized, therapeutic, exploratory, controlled study. Endpoints for feasibility were the number of treated patients out of eligible patients and the time from surgery to the start of immunizations. Endpoints for safety were records of toxicity and adverse reactions. Immune responses became monitored with immunohistochemistry of skin biopsies from the vaccination sites. Overall survival (OS) and progression-free survival (PFS) set the endpoints for clinical responses.

*A Story of Immunization with Autologous IFN-γ Secreting Glioma Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.105202*

#### **2.2 Patients**

The study was performed with the permission of the Swedish Medical Products Agency and with the acceptance of the Local Ethical Board of the University of Lund. All patients gave their written consent to participate in the study. The patients were recruited from glioma cases referred to the Department of Neurosurgery at Lund University Hospital during 2000–2004. It is to be noted that temozolomide or other chemotherapeutic drugs were not included in the normal therapy in this age cohort at the time of the study. All patients were recruited before the inclusion of temozolomide in the regular treatment of glioma.

#### **A. Inclusion criteria:**


#### **B. Exclusion criteria:**


#### **C. Patient recruitment**


#### **Table 1.**

*Criteria of inclusion, exclusion, and recruitment of patients.*

#### **2.3 Provisional and definite inclusion of patients**

Patients with a confirmed diagnosis of GBM according to WHO-criteria [1] and whose first postoperative MRI revealed the resection to comprise 80% or more of the preoperative tumour volume were provisionally included in the study. The tentatively included patients whose tumour cells did not exhibit in vitro growth sufficient enough for transduction and immunization or where the cells could not be transfected appropriately constituted the control group. **Table 1** show criteria for inclusion (A), exclusion (B) and patient recruitment (C).

#### **2.4 Preoperative investigations**

Preoperatively the patients were examined with MRI, including diffusion- and perfusion sequences [22]. In addition, preoperatively and postoperatively, the patients were also evaluated by neurological (NIHSS) and quality of life (QOL) assessments (SF 36).

#### **2.5 Surgical treatment**

Temozolomide or other chemotherapeutic drugs were not included in the normal therapy in this age cohort at the time of the study. Tumour resection was performed using standard neurosurgical techniques, frequently applying neuro-navigation and ultrasonic aspiration. Viable tumour tissue was harvested for histopathological diagnosis and for culturing in vitro.

Based on clinical experience and judgment, repeated surgery was considered and performed as needed for diagnostic or palliative purposes throughout the study.

#### **2.6 Postoperative treatment**

All patients received irradiation treatment of the brain (58 Gy in 29 fractions) commencing within five weeks from surgery. Steroids were administered when symptoms occurred after tumour recurrence. No patients received steroids during the immunization period.

#### **2.7 Postoperative investigations**

Postoperative MRI as above was performed within 48 hours after surgery before immunization was started and at every second immunization. At the same time, patients were evaluated preoperatively with *National Institutes of Health Stroke Scale* (NIHSS ) and *Short-Form Health Survey* (SF-36).

#### **2.8 Cell culture**

Tumour tissue obtained at surgery was cultured in vitro and regularly karyotyped until the cells exhibited an abnormal karyotype. Then the cultivated cells were transduced using an adenoviral vector carrying the IFN-γ Human gene as well as the gene for the Green Fluorescent Protein and subsequently irradiated with 100 Gy to prevent a further growth in vivo [23, 24].

*A Story of Immunization with Autologous IFN-γ Secreting Glioma Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.105202*

**Figure 1.** *Timeline of immunization and monitoring procedures.*

#### **2.9 Immunization procedure**

The patients included in the study received intradermal injections of their own irradiated and transfected cultured tumour cells at five sites in the upper arm at alternating sides every third week. The immunizations were repeated up to fourteen times or until the patient deteriorated and required steroids to alleviate symptoms. **Figure 1** shows the immunization and monitoring procedures.

#### **2.10 Histopathological studies**

Besides establishing the WHO diagnosis at the first surgery, further histopathological investigations were performed in some surgical specimens and in material obtained at autopsy in some patients [25].

#### **3. Results**

#### **3.1 Immunotherapy of GBM with autologous IFN-γ transfected tumour cells is safe**

There could be a potential danger of evoking immune responses against normal CNS cells or inducing an inflammatory response that might spread to the regular brain after immunotherapy utilizing autologous tumour cells. However, we did not observe any major side effects or toxicity after immunizations.

The induction of autoimmune responses would most definitively have influenced the patients' neurologic status. Neurological and cognitive grades were evaluated with the NIH Stroke Scale (NHSS) and no deterioration before tumour recurrence were recorded during immunizations in any patient (data not shown). Neither did postoperative MRI investigations show any inflammatory changes around the resection cavity nor in surrounding brain tissue [22].

Apart from achieving prolonged survival, a novel therapy also aims at maintaining or improving the quality of life (QOL) of the treated patients. Assessment of QOL

by SF-36 did not reveal any deterioration during immunizations. However, there was a tendency to short-term memory deficits in some of the treated patients (data not shown). Nevertheless, overall the patients in the study experienced an increase in QOL during period of the treatment.

#### **3.2 Immunotherapy of GBM with autologous IFN-γ transfected tumour cells is feasible in 45% of the eligible patients**

In total 28 patients were provisionally included in the study before surgery. After surgery, only 17 patients fulfilled all criteria of inclusion in the study (**Table 1**).

Eleven patients were excluded - due to another diagnosis than GBM, (5/11), insufficient tumour resection (5/11) or major psychiatric illness (1/11) (**Table 1**).

In nine of the 17 included patients, malignant cells were successfully cultured in vitro and became transduced as described above. One of these patients underwent six immunizations but was excluded from the study due to incomplete resection at review (**Table 1**). Thus finally, the treatment group in the study consisted of eight immunized patients with detailed information shown in **Table 2**. It was possible to vaccinate 8/17(45%) of eligible patients with GBM and 8/23(35%) of patients diagnosed with GBM.

The control group consisted of the remaining nine patients (**Table 2**). In the treatment group, patients became vaccinated between 8 and 14 times. Additional immunizations


#### **Table 2.**

*Individual patient data of treated and control groups.*


*A Story of Immunization with Autologous IFN-γ Secreting Glioma Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.105202*

*Other ctrl, all patients with the diagnosis of GBM between 50 and 70 years of age treated during 2000–2003 (2 years) at our institution except the treated and matched control patients involved in the study; OS, overall survival; PFS, progress free survival*

*\*Months*

*# Patients surviving less than 30 days postoperatively were excluded due to presumed surgical mortality.*

**Table 3.**

*Group data of treated and matched control of the study and as well as other controls.*

were given depending on the availability of cells and patient status, although the protocol stipulated a minimum of four immunizations (**Table 3**). One patient received four additional immunizations after special approval from the Medical Products Agency. In conclusion, the immunization procedure was feasible in 45% of eligible patients.

#### **3.3 Immunotherapy of GBM with autologous IFN-γ transfected tumour cells prolongs survival**

The eight treated patients had a significantly prolonged overall median survival (488 days, 16.3 months) compared to the control group (288 days, 9.0 months) (**Figure 2** and **Table 3**). There was also a significantly longer progress-free survival in the treated group (**Table 3**). No noteworthy differences between the groups appeared regarding age, gender, or repeated surgery (**Table 3**).

#### **Figure 2.**

*Kaplan-Maier graph showing overall survival of immunized matched and non-matched control patients. The survival was analysed with the logrank test, the p value depicted refers to comparison between immunized and matched control patients.*

#### **Figure 3.**

*Representative MRI (T1 with gadolinium) images from nonresponding and responding patients preoperatively, postoperatively and at the sixth immunization. The postoperative image of the non-responding patient shows a dense area, which constituted a haemorrhage also seen on non-gadolinium enhanced images (not shown).*

*A Story of Immunization with Autologous IFN-γ Secreting Glioma Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.105202*

Serial MR examinations showed no or stable contrast-enhancing areas in the responding patients and progressing towards contrast-enhancing areas in nonresponding patients during immunizations (**Figure 3**). To rule out selection bias, we compared the matched control group with all patients treated at the same institution (all patients 50–69 years during 2000–2003 minus treated and matched control groups, *n* = 91). The data given in **Table 3** show no significant differences between the survival times of the matched control group and the other control group, which indicate no apparent selection bias.

There was also a clear indication that age was a prognostic factor apart from immunotherapy. Non-immunized patients aged 50–59 years survived 12.2 months, and immunized patients survived 22.2 months while non-immunized patients in the group aged 60–69 years survived 7.7 months, and vaccinated patients survived 14.3 months. Of the non-immunized patients, 0/9 survived >18 months, while 4/8 of the vaccinated patients survived >18 months and 2/8 >24 months. However, the study and control groups were too small to conduct more detailed statistics as COX regression analysis. In summary, the immunized group of patients had a prolonged overall survival (7.3 months compared to matched controls and 9.9 months compared to unmatched controls) that was not previously reported for patients with GBM over 50 years.

**Figure 4** shows the survival results from nine vaccinated patients and 11 patients treated with surgery only, and subsequent radiotherapy presented at the World Federation of Neuro-Oncology and the European Neuro-Oncology Association in Edinburgh, Scotland. Post-diagnosis survival in nine glioma patients treated with vaccination was 14.3 months, which is significant (*P* < 0.02) longer compared to the 9.6 months of 11 patients normally treated with surgery only, and subsequent radiotherapy [26, 27].

#### **Figure 4.**

*Post-diagnosis survival in nine glioma patients treated with vaccination and 11 patients normally treated with surgery and subsequent radiotherapy alone. Regression equations: Survival vaccinated (month) = 62(±18) – 0.75(±0.29)⋅Age(a) Survival normally treated (month) = 46(±12) – 0.64(±0.23)⋅Age(a) (dashed line).*

#### **4. Discussion**

Based on our experimental results, we have treated eight patients with the diagnosis of GBM using immunizations with autologous tumour cells transfected with the human IFN-γ gene and compared them to 9 untreated but otherwise identically treated patients. Immunotherapy of malignant primary CNS tumours is no novelty, and the different therapeutic modalities attempted in general immunotherapy has also been utilized in trials of immunotherapy of these tumours with limited results [28, 29].

Promising results have been reported from several clinical trials based on immune therapy against high-grade gliomas:


We chose to select the patients within a defined age cohort and within an outlined resection volume to rule out confounding factors. Although under discussion, recent reports have indicated that the extent of resection of high-grade gliomas is a prognostic factor and therefore, we excluded patients with a resection less than 80% of the preoperative volume [34]. Other reports of immunotherapy of high-grade gliomas have claimed a higher success rate of a culture of explanted tumour biopsies than we have found [18, 32, 33] Although several putative tumour markers for glioblastoma multiforme have been proposed [35], there are no ubiquitous ones that can be used for the identification of tumour cells in culture.

Unlike other investigators, who have used panels of associated tumour markers, we have utilized karyotyping to detect tumour cells in cultures to avoid contamination of non-tumour cells [24]. This procedure may have excluded tumour cells with a near-normal karyotype, but it has reduced the probability of including contaminating non-tumour cells in the vaccine. The prognosis for patients with malignant primary CNS tumours varies depending on grade and type of tumour, age, performance status at diagnosis, and expression pattern of different proteins and genes [34, 36, 37]. Even within the entity of GBM, the survival range is extensive, and a major impact of age and performance status at diagnosis has been demonstrated. This makes the interpretation of results from clinical studies difficult when patients of different ages and grades of tumour are included.

In some of the studies published on immunotherapy of patients with primary malignant brain tumours, younger patients and also patients with the diagnosis of anaplastic astrocytoma have been included. The latter group has a substantially longer expected survival than patients with GBM, and therefore, it is hard to evaluate the actual effect of immunotherapy in some of these studies [18, 33, 38] The reported mean survival rates for treated patients with GBM in these studies were 700, 462 and 931 days with mean ages 49, 50, and 44 years respectively. In the report by Steiner

*A Story of Immunization with Autologous IFN-γ Secreting Glioma Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.105202*

et al., the survivals of individual patients were stated and the median survival of patients 50–69 was 500 days (range 252–868). Although the current patient group is too small for statistical sub-analysis, both age and immunotherapy were strongly indicated as independent predictors of increased survival (data not shown).

Additional patients have received immunizations after adjuvant temozolomide and radiotherapy followed by 4–6 cycles of temozolomide, and one other patient, not included in this study, aged 57, who received this therapy had an overall survival of 24,5 months. This is a preliminary indication that immunizations might be feasible in this setting, and another case of concurrent immunotherapy and administration of temozolomide has been reported [39]. DTH reactions in the skin at the immunization sites were recorded in all patients, but there was no correlation with overall survival (data not shown).

Analysis of peripheral blood, before and during the vaccinations, has shown signs of immune activation. Recombinant antibody micro-array technology [40] has been used to perform differential plasma protein profiling of the non-immunized and immunized GMB patients and of age-matched healthy controls from this study [41]. We have previously reported that in one patient who was re-operated on during immunizations and in the patients re-operated on after the cessation of immunizations, a transient influx of T cells into the tumour tissue could be observed [25]. This indicates that the same pattern of a lymphocyte influx as observed in our experimental model indeed occurs after clinical immunotherapy. However, whether there is a specific pattern in responders compared to serial biopsies of tumour tissue can only study non-responders and controls immunotherapy.

As reported previously, there were no signs of inflammation or oedema in the tumour tissue or the surrounding brain as judged by magnetic resonance tomography (MRT) after immunotherapy [22] which has been reported after treatment of high-grade gliomas with oncolytic viruses. This could be explained by inappropriate methods to detect an inflammatory reaction or by the minimal tumour volumes during immunizations in most patients. An alternative explanation is that the current immunotherapy does not induce a recordable inflammatory reaction that can be demonstrated with MRT. Immunotherapy has a potential risk of inducing autoimmune reactions that could damage normal tissue. In the CNS, these reactions could be deleterious and possibly life-threatening due to cerebral inflammation and oedema induction.

We have not recorded any such adverse reactions during immunizations. This agrees with additional immunotherapy trials of CNS tumours and is somewhat surprising as strong immune responses are evoked against antigens that might be shared with normal CNS resident cells. The reasons for this are unknown but could depend on the immune privilege of the normal CNS or the absence of shared antigens. GBM is, with anecdotal exceptions, an incurable disease in adults. Therapies that aim to cure the disease will realistically first prolong survival with gradual improvements in treating other tumours. It is now generally accepted that treatments that aim to lengthen survival should also strive to maintain or improve the quality of life. The treated patients in this study did not experience a diminished QOL during the immunizations, but further studies will have to confirm this. Neither do we know whether maintained QOL was related to the direct nor indirect effects of the immunizations.

The treated group had a statistically increased overall survival compared to both a matched control group and another control group encompassing all patients with GBM over 50 years of age, treated in our institution during the same period. There was no difference in survival between the matched control group and the

non-matched control group. Furthermore, when considering RTOG-RPA classes (both treated and control patients belong to class IV-V) the expected overall survival in these groups (8.9 and 11.1 months) matches that of the overall survival of both control groups [41, 42].

#### **5. Conclusion**

In conclusion, this is the first study to show a significant prolongation of survival after immunotherapy of patients with GBM in the age group over 50 years. Taken into account that age is a predictor for survival of patients with glioblastoma multiforme; treatment of younger patients might result in longer periods of survival with unchanged or improved quality of life.

### **Acknowledgements**

Dedicated to Hans and Märit Rausing who during 1996–2010 generously supported the research project "BRIGTT" (Brain Immuno Gene Tumour Therapy) which was performed at the Rausing Laboratory during 1996–2010.

We thank professor Hans-Olov Sjögren for his fruitful advice and discussions. We also thank research nurses Anna Evaldsson, Anita Nilsson and Charlotte Orre for coordination of pre and post-operative examinations.

The Hedvig Foundation, the Hans and Märit Rausing Charitable Foundation, the Hans and Märit Rausing Charitable Trust UK, the Swedish Cancer Foundation, the Lund University Hospital Funds, the Lund University funds and the County Council (ALF and Region Skåne) funds supported this project.

*A Story of Immunization with Autologous IFN-γ Secreting Glioma Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.105202*

#### **Author details**

Leif G. Salford1,2\*, Peter Siesjö1,2, Gunnar Skagerberg1,2, Anna Rydelius2,3, Catharina Blennow1,2, Åsa Lilja1,4,5, Bertil Rolf Ragnar Persson2,4,5, Susanne Strömblad1,2, Edward Visse1,2 and Bengt Widegren2,6

1 Department of Neurosurgery, Lund University, Lund, Sweden

2 Department of Genetics, The Rausing Laboratory, Lund University, Lund, Sweden

3 Department of Neurology, Lund University, Lund, Sweden

4 Department of Psychology, Lund University, Lund, Sweden

5 Medical Radiation Physics, Lund University, Lund, Sweden

6 Medical Radiation Genetics, Lund University, Lund, Sweden

\*Address all correspondence to: leif.salford@med.lu.se

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*A Story of Immunization with Autologous IFN-γ Secreting Glioma Cells in Patients… DOI: http://dx.doi.org/10.5772/intechopen.105202*

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#### **Chapter 11**

## Potential of Lipid Based Nanodrug Carriers for Targeted Treatment of Glioblastoma: Recent Progress and Challenges Ahead

*Bhabani Sankar Satpathy, Binapani Barik, Ladi Alik Kumar and Sangram Biswal*

#### **Abstract**

Malignant brain tumor at its fourth stage (glioblastoma) is the most dangerous and an unsolved medical challenge till today. Present therapeutic strategies including chemo treatment, radiation along with surgery all together have not succeeded to control the progression of glioblastoma. Challenges in the early detection, unavailability of specific therapeutic strategy and severe cytotoxicity of available chemotherapeutics are the some of the prime causes of treatment failure. Especially presence of blood-brain barrier (BBB) highly limits pharmacological effect of conventional chemotherapy. In lieu of this, lipid based nanodrug carriers (LNCs) have now been evolved with great potential in improving the drug efficacy for the treatment of glioma. Further, LNCs engineered with specific targeting ligand might significantly reduce the dosage regimen, increase specificity, improve bioavailability and reduce off-target distribution. Such modified LNCs possess sufficient ability to cross BBB to deliver the loaded cargo(s) at target location inside the brain; thereby ensuring improved treatment outcome with less side effects than conventional treatment. This review primarily focuses on recent advancements in various engineered LNCs for the treatment of brain cancer. Also, the existing impediments for nanomedicines associated with their effective large scale synthesis or sufficient clinical application have also been highlighted.

**Keywords:** lipid based nanodrug carriers, glioblastoma, advancements, challenges

#### **1. Introduction**

Brain tumor at its malignant stage is the toughest challenge to treat. Glioma is the commonest form of malignant brain tumors and silently progresses to its fourth and most aggressive stage; called gliobalstoma. In fact, modern medical science in spite of cutting age technological advancements is yet to find specific answers for advanced malignant brain tumor.

#### *Glioblastoma - Current Evidence*

An uncontrolled growth of cells beyond the cellular regulation inside the brain environment eventually leads to benign and/or malignant cancers [1]. The most common site for the development of tumor inside the brain is glial cells. Further, tumors as per their growth and location inside the brain are further classified from grade I (low grade) to grade IV (highly metastatic) type tumors [2]. Grade I stage of tumor (mostly goes unnoticed) can progress to the malignant stage more often and throws a tough challenge for treatment. Also, secondary metastatic brain tumors can be developed in adults from primary lungs/breast cancer [3]. Among the various grades of brain tumor, grade IV glioma, also called glioblastoma multiforme has been recognized as the severest and highly metastatic type brain tumor [4]. A vast majority of patients across the globe diagnoses with de novo or primary glioblastoma in recent years. Progression of brain tumors are often associated with typical increase in intracranial pressure, altered consciousness, occasional seizures along with severe headaches, vomiting, fever, gastric disturbances etc. [5]. However, these problems are highly variable from patient to patient and thus cannot be generalized prognosis parameters. Thus, primary stage of glioma often goes unnoticed. Aetiological causes related to the development of brain cancer are yet to be unravelled, which further makes the treatment extremely challenging. Classical subtype of glioma is assumed to be associated with amplification of chromosome 7 along with loss of chromosome 10. Coupled with these, over-expression of epidermal growth factor (EGFR) receptor and mutations are other proposed aetiologies of glioblastoma [6]. Mesenchymal glioblastoma has been shown to maintain a higher expression of CH13L1, MET, and genes associated with tumour necrosis factor, nuclear factor-κB, along with deletions of NF1. Mutations in IDH1, TP53 and modification of platelet-derived growth factor receptor A are also associated with secondary glioblastoma or lower-grade gliomas [7]. Though, neural glioblastomas at initial diagnosis shows similar characteristics to normal brain tissue; however, there is overexpression EGFR to several folds than normal.

At present, glioblastoma has been identified as the most complex, metastatic and treatment-resistant type of cancers with alarming prevalence around the globe. In 2020, more than 13,000 Americans have been diagnosed with GBM, which accounts for more than 48 percent of all malignant brain tumor cases [8]. Till now, average length of survival for patients with glioblastoma has been estimated to be only 1 to 1.5 years while the five-year survival rate has been roughly estimated as 6–7% only [9]. Over the past decade, mortality and survival rate of glioblastoma patients has not been improved as such in the developed nations. Even, uncontrollable prevalence of the disease is being witnessed in developing and under-developed countries. India has now become the new epi-centre for all cancer related deaths in recent years among which glioblastoma-related death cases occupies second lead position after breast cancer.

Along with extremely poor prognosis associated with glioblastoma, there is too serious dearth of promising therapeutic options. Much of the available treatment strategies alone or in combinations have been failed measurably over the past years to meet the treatment expectations. Usually, combination of various strategies like surgery, radiation, chemotherapy, non-chemodrug therapy etc. are employed to control the progression of tumor cells to other parts of the brain or to be metastatic [10, 11]. Surgery followed by radiation therapy is applied as the first line of treatment in the initial phases of glioma. Surgery is employed to remove maximum possible mass of tumor tissue from the brain, while radiation therapy is employed

#### *Potential of Lipid Based Nanodrug Carriers for Targeted Treatment of Glioblastoma: Recent… DOI: http://dx.doi.org/10.5772/intechopen.108419*

to circumvent tumor mass *via* precise, focused high energy beams [12]. However, in many cases, effective application of surgery and radiation are extremely constrained as majority of brain tumors are usually detected at the advanced stages, i.e., at stage III or at stage IV. Additionally, highly sensitive nature of brain tissue and presence of delicate nervous network across the brain hemispheres with all major control systems of perception, mood, behaviour, cognition etc. further limits surgical procedures and effective radiation therapy [13, 14]. Hence, chemotherapy remains as the inevitable option to check the progression of tumor cells through cytotoxic anticancer drugs. Non-chemotherapeutic drugs are also used during treatment period to control tumorassociated headache/pain and epileptic seizures [15]. However, conventional chemodrug treatment faces the usual problem just likes other conventional dosage forms such as failure to discriminate in between cancerous tissue and normal healthy tissue or lack of targetability. As a result, off-target biodistribution of cytotoxic anticancer drugs across all vital organs inside the body occurs, which in turn aggravates a wide range of adverse drug effects including alopecia, gastric disturbances, bone marrow depression, heart problems, kidney damage, immunity suppression and many other associated complications in cancer patients [16]. It has now been an accepted fact that the presently available clinical options all together have neither succeeded in extending cancer patient lives just beyond a few extra months nor been able to improve their quality of life after chemo-treatment cycles. In a nutshell, extremely poor prognosis, highly sensitive micro-environment of brain coupled with failure of conventional treatment options has made glioblastoma as a life-threatening disease. At present, it is too one of the most expensive cancers to treat, often leaving patients and families with major financial hardship during the treatments and in turn deteriorates socioeconomic burden of the society as well [17]. In the lieu of which, advanced treatment options are being investigated heavily over the past years to improve the treatment outcomes and simultaneously to minimize the dose-related toxic effects on the body.

Moving from the initial treatment options like surgery and radiation, which have their inherent limitations; anti-cancer drug therapy through modified nanocarriers with improved targeting features is being explored as alternative option to improve overall treatment outcomes in cancer patients. In view of the presence of BBB as the major obstacle in brain-drug targeting, especially, lipid based nanocarrier based delivery systems have been recognized as hopeful options in glioblastoma owing to their highly lipophilic, ultra-small size, tuneable surface features. The cytotoxic anticancer drugs can be loaded into such nanocarrier vehicles and thus can be effectively surpass the BBB to get into the brain. Additionally, such carriers are now being manipulated at their surface with specific targeting ligands like antibodies, aptamers, small molecules, peptides etc. to enhance their targetability and reduce off-target distribution [18]. These engineered LNCs have been emerged as the prime research area in nanomedicine mediated brain cancer therapy now-a-days.

LNCs have the capability to bypass the BBB without disrupting its normal functionalization [19, 20]. Furthermore, LNCs in lieu of their architectural uniqueness provide requisite criteria of lipophilicity and sustained release of drug from their core/matrix. Attachment of tumor-specific ligands further makes them more specific and helps to mitigate peripheral toxicities [21]. After crossing the BBB, LNCs are endocytosed by endothelial cells and release the drug inside the cell [22]. There is too a growing interest to improve the *in vivo* performance of nanocarriers *via* conjugating them with thiolated and preactivated polymers to efficiently inhibit the P-glycoprotein (P-gp) efflux at brain luminal side [22, 23]. Glioblastoma possesses

a leaky vasculature, and thus may be amenable to LNC-based drug delivery systems that lead to enhanced drug deposition while limiting systemic drug exposure. Various types of LNCs have been investigated over the last decade to enhance therapeutic efficacy of anticancer drugs for the treatment of advanced stage glioma. In the present topic, we want to cover recent advancements in LNCs based drug targeting strategies for glioma. Specifically, we will restrict our discussion mostly on nanoliposomal vesicles and solid-lipid nanocarriers, which have been reported over the recent years for glioma/glioblastoma treatment. Side by side, some lights have been thrown on the challenges faced by such targeted LNCs for their successful clinical translation, regulatory hurdles along with scale-up issues for industrial production.

#### **2. Blood-brain barrier: the prime culprit against effective drug therapy in glioblastoma**

Brain, the controlling system of the whole body is undoubtedly the most complex, mysterious structure, which controls a multitude of crucial functions of the body including cognition, information processing, homeostasis, perception, motor control, mood, as well as learning and behaviour [24]. Such important functions are mediated by uncountable nervous networks which are present across the cerebellum. BBB is the main check-gate, which actively protects brain neural tissues from the influx of toxins and other compounds, including therapeutic molecules [25]. In fact, presence of BBB strictly restricts the success of chemotherapy as majority of anticancer drugs fails to permeate sufficiently across BBB, thus results in a sub-therapeutic concentration associated with low clinical outcome.

BBB is characterized by the presence of tight intercellular junctions along with lack of fenestrations. Main components of BBB are tightly placed brain endothelial cells, basal membranes, pericytes embedded in the basal membrane, along with astrocytic end feet [25]. All these structures are so uniquely placed close to each other that they collectively form a strong barrier on the way of every component having higher molecular weight or large size to pass from blood to brain. Only essential components like glucose and essential amino acids can get access inside the brain. Exogenous compounds including drugs having nano-size range or lipophilic property may cross the BBB by passive diffusion. Alternatively, some therapeutics can also cross the BBB through carrier-mediated active transport. Along with the strong barrier system like BBB, the efflux transporter systems present at the luminal side of brain also play crucial role in preventing therapeutic molecules to attain their pharmacological concentration [25, 26].

Similarly, in terms of molecule permeability, it has been found that molecules larger than 400 Da are very unlikely to cross the BBB (especially if highly water soluble) unless a suitable specific transporter is present. However, as mentioned earlier, highly lipophilic molecules tend to have better permeability than neutral or hydrophilic molecules owing to the high lipophilicity of the BBB. Temozolomide is an example of the poorly water-soluble drug with a molecular weight of 194.154 g/ mol, which can readily cross the BBB. Similarly drugs like carmustine, lomustine etc. also have reasonable BBB permeation ability owing to their molecular cut-off range and lipophilic nature and have already been recommended for glioma therapy. These, along with few other drugs *viz*. capecitabine, paclitaxel etc. are presently some of the widely used chemotherapy drugs recommended in glioblastoma [27]. However, many lipophilic drugs in their native form/conventional formulation too fail to achieve

#### *Potential of Lipid Based Nanodrug Carriers for Targeted Treatment of Glioblastoma: Recent… DOI: http://dx.doi.org/10.5772/intechopen.108419*

required therapeutic concentration at the brain tissue owing to their molecular size, in vivo stability issue, low half-life or affected by efflux transporter systems across BBB. Drugs bound to plasma proteins are also unavailable for crossing the BBB, since most of the proteins require specific transporters for BBB permeation. This phenomenon was demonstrated using Evans blue (an albumin-binding dye), which is completely unable to permeate across the intact BBB [28].

Dose-related adverse reactions are also obvious phenomena with conventional drugs, which further limit their chemo treatment cycle [29, 30]. Hence, it must be taken into account that merely a high degree of lipophilicity or delivery in conventional dosage forms does not either guarantee sufficient availability of drug inside the brain nor ensures its decreased off-target distribution throughout the healthy tissues.

In this context, there always felt an age-old need for an ideal delivery system that has to transport a drug with high efficiency to target brain cells, with minimal healthy tissue toxicity or off-target distribution. To achieve this, delivery of drugs/chemotherapeutics through the LNC based platforms has been attempted over the past few years by the pharma researchers and formulation scientists across the globe.

#### **2.1 Lipid based nanocarriers: effective drug targeting platforms to brain**

LCNs have been heavily investigated in recent years to improve the drug delivery at brain tissues owing to their lipophilic nature and ultra-small size. The key features of LNCs primarily involve their desirable size range, surface properties, and also ease of surface manipulation with targeting ligands [31]. The development of a broad range of LCNs with varying size, composition, and functionality has provided a significant resource for nanomedicine based glioblastoma therapy.

However, requirements for LCNs fabrication for effective glioma therapy also depend on tumor characteristics, its location and complicacy. Although LCNs avoid renal clearance preferably within the range of 10–50 nm, but they tend to accumulate heavily in the reticulo-endothelial system (RES), which is also another major setback for their sufficient brain bioavailability. Further, LCNs like other nanodrug carriers below the size of 10 nm possess the risk of higher glomerular filtration followed by renal clearance [32, 33]. All such problems are now being addressed successfully by the advanced formulation technologies, adaptation of cutting age research instruments and effective surface manipulation and employment of novel polymers (natural/synthetic). For example, problem associated with higher RES uptake can be subsided by surface coating/shielding of the LNCs with specific hydrophilic polymers like polyethylene glycol (PEG). Presence of PEG over the surface of LNCs renders hydrophilicity with subsequent reduction in RES uptake and enhancement in plasma half-life [34]. Similarly, by optimizing critical in-process parameters during formulation development such as polymer:drug ratio, amount of drug, sonication time, speed of centrifugation, filtration/separation technique, surface conjugation etc., desired size range of LCNs can be attained (preferably within 10–50 nm) for effective BBB permeation.

Likewise, the off-target bio distribution of the nanodrug carriers can be effectively reduced by surface conjugation with tumor-specific ligands. Several ligands like aptamers, antibodies, small molecules, peptides, sugar moiety etc., can be attached to LCNs to make them more specific with enhanced brain targetability [35]. Such engineered LCNs can effectively reduce healthy tissue toxicity along with chemoresistance of cancer cells, since they promote higher brain uptake of cytotoxic drugs around the tumor area with considerable decrease in drug efflux, thereby enhancing therapeutic outcome as well as (**Figure 1**).

#### **Figure 1.**

*A representative diagram of blood-brain barrier showing permeation of ultra-small size lipophilic drug carriers, whereas inability of macromolecular drug/ carriers to cross the barrier.*

#### **2.2 Types of lipid nanocarriers employed for drug targeting to brain**

Lipid based nanocarriers are categorized into mainly three types, *viz*. nanoliposomes, solid-lipid nanoparticles, nanostructured lipid carriers. In our study, we would mostly restrict the discussion on these lipid based nanodrug carriers for glioblastoma therapy, excluding other organic/inorganic nanoparticles or other novel carriers.

#### *2.2.1 Nanoliposomes*

This is the first generation of novel drug delivery system, developed in 1960. It is prepared to resemble to the cell membrane compositions mainly by using fats, phospholipids, and cholesterol [36]. Due to its high flexibility, low toxicity, better stability, and biocompatibility, specifically targeting character with highly versatile nature, it has got immense attention in glioblastoma therapy [37, 38].

Liposomes are colloidal nano carriers, comprised in a vesicle. It can be uni-lamellar or multi lamellar i.e. comprising of more than one number of lipid bilayers encapsulating hydrophilic core or aqueous core. Due to unique structural features, both hydrophilic and lipophilic drugs can be delivered through nanoliposomes. By applying various in vitro techniques, the surface of liposomes can be easily modified with surfactants (e.g. tween 80, tween 20) bile salts, or tumor-specific targeting ligands [39, 40]. However, one of the major limitations related to liposome is their earlier uptake by phagocytic cells leading to shorter in circulation half-life. To avoid this PEG is functionalized over the conventional liposomes to keep it safe from the eyes of macrophages and to extend blood circulation profile [41].

#### *2.2.2 Solid lipid nano carrier (SLNs)*

This the first generation of solid-lipid based nano carrier was developed in 1991. It is usually spherical in shape having the diameter about 50–100 nm, dispersed in water

#### *Potential of Lipid Based Nanodrug Carriers for Targeted Treatment of Glioblastoma: Recent… DOI: http://dx.doi.org/10.5772/intechopen.108419*

or in an aqueous surfactant phase [42]. SLNs have advantages like better stability, low melting point, nontoxic, ease to preparation, higher plasma pharmacokinetics, better bioavailability across BBB, good biocompatibility, bio degradability, very low cytotoxicity along with cost effective method of production [43]. It is an oil in water (o/w) system, in which the oil phase/liquid-lipid is replaced with the solid lipid to make it solid in both room and body temperature. The main ingredients used for the production of SLNs includes monostearates, stearyl alcohol, stearic acid, glycerol, cetyl palmitate etc. including stabilizers like tween 80, poloxamer 188, and dimethyl dioctadecyl ammonium bromine. The variation of ratio occurs in between the range of solid lipid (4:1) to the liquid lipid (1:4), surfactant concentration (0.25 to 6% w/v) to the total lipid concentration (1–30% w/v) [44]. However, it has also got few limitations like moderately drug loading capacity and expulsion of drug due to crystallization during under long-term storage condition.

#### **2.3 Targeting strategies adopted by lipid nanodrug carriers for brain delivery**

LNCs with their loaded cargo can be directly targeted to the brain owing to their ultra-small size and lipophilicity, as discussed previously. Since, most of the LNCs constitute phospholipid, sphingo lipid, cholesterol-based structures, they usually possess a cell-mimicking property, for which once get inside the cell, they tend to retain there with subsequent release of loaded cargo. In such cases, no artificial surface manipulation is done, and thus it does not guarantee glioma cell-specific drug targeting also.

Tumor vasculature usually shows abnormal architecture with highly permeable capillaries. Along with that the tumor mass too possesses a poor lymphatic drainage system, which thus allows accumulation of micromolecules having molecular cut-off size ≤40 kDa. LCNs mediated drug targeting actually utilizes this unique feature along with its lipophilic nature to invade inside the tumor tissue. The phenomenon popularly known as the enhanced permeability and retention (EPR) effect is taken as the prime mechanism in passive targeting of nanodrug carriers [45, 46]. Passive method of targeting the chemotherapeutics does not involve targeting to any specific receptor/protein expressed over tumor cell surface. It, thus primarily depends on the size and physicochemical properties of the nanocarriers. The ideal size range to benefit from the EPR effect is usually between 10 and 100 nm. But for successful BBB permeation of LNCs, an average hydrodynamic diameter around 10–50 is now preferably investigated. Outside this range, smaller particles usually clear by the kidney, preventing accumulation within the tumor site, while larger size particles fail to adequately penetrate through the glioma vasculature [46, 47].

In lieu of problems associated with passive targeting, surface engineering of nanocarriers with tumor cell-specific ligands have been investigated widely in past few years. The development of a broad range of LCNs with varying size, composition, and functionality has actually provided a significant revolution in glioblastoma therapy. While, passive targeting utilizes unique internal architecture of tumor tissue to target nano size delivery vehicles, active targeting is primarily based on surface engineering of nanodrug carriers with specific targeting ligands to make them more precise. Though, the leaky tumor vasculature coupled with weak lymphatic drainage of tumor provides a golden opportunity for direct targeting of nanosize drug carriers even without any surface manipulation [48], however, the chances of healthy tissue accumulation still remain there. Thus, surface engineering of LNCs has been emerged as hopeful alternative to decrease drug uptake in normal tissue and to increase accumulation in glioma to elicit better therapeutic outcome.

Active targeting in glioblastoma involves targeting surface membrane proteins that are upregulated in cancer cells [49]. Targeting molecules can be monoclonal antibodies or their fragments, aptamers, small molecules, oligopeptides etc. LNCs attached with surface ligands can be preferably localized to tumor tissue, expressing the associated receptors or antigens and can deliver the loaded drug *via* ligand-receptor interaction [50]. Some ligand receptor interactions also facilitate receptor-mediated endocytosis, which in turn enhances payload delivery inside the tumor cell.

#### **2.4 Major types of targeting ligands in glioblastoma**

#### *2.4.1 Monoclonal antibodies (mAb)*

Biocompatible mAb has been utilized from a decade as the first line of targeting ligand owing to their highly specific nature in various cancer treatments including malignant brain tumors. Many tumors up-regulate growth factor receptors, such as HER2/ neu in certain breast cancers, which can be targeted with anti-HER2/ neu surface antibodies [51]. Similar mAb mediated targeting strategy has now been investigated for glioblastoma. Though, unlike breast or prostate cancer, the specific receptors/ proteins having higher expression in case of brain tumor are very limited, but some of the recently reported research has provided evidence of improved treatment efficacy with mAb-engineered LNCs in malignant brain tumor as compared to conventional chemo-treatment. One recent example of such mAb is CD 133. This pentaspan transmembrane glycoprotein family member is also known as prominin-1 and has been found closely associated with glioblastoma. Research finding has identified CD133 as a major hallmark of glioblastoma stem cells [52]. Recent reports have further shown that CD133 antigen has elevated expression in glioblastoma, medulloblastomas, along with other brain cancers [53]. Thus, it could serve as a prognostic indicator of tumor recurrence or malignant progression.

#### *2.4.2 Aptamers*

Aptamers have recently emerged as effective ligands for their higher specificity, safer in vivo application with lesser chances of immunogenicity. They are basically folded single stranded oligonucleotides (25–100 nucleotides) that bind to specific molecular targets [54]. Aptamer-conjugated nanoparticles *in vitro* have displayed increased cytotoxicity and decreased volume of xenografts compared with non-targeted nanoparticles [55]. Aptamers possess many unique characteristics which make them an ideal imaging and targeting agent for the treatment of glioblastoma. Owing to their higher sensitivity, selective nature, ease of fabrication aptamers are presently lucrative drug-delivery platforms in glioblastoma [56, 57]. Although mAbs have been long history of use as potent therapeutic tool, however, their therapeutic application for glioblastoma including other neurodegenerative diseases has been limited, thanks to the presence of BBB, which checks effective entry of traditional antibodies. As compared to conventional mAbs, aptamers are more stable, smaller size and also easily accessible to chemical modifications. Adverse effects associated with aptamers are also rare. They can be physically/ chemically conjugated to a wide range of probes and therapeutic agents, which make them promising entity for imaging and detection in brain cancer. Successful application of aptamers for the diagnosis or treatment of glioblastoma has been reported in many recent researches. Recent research identified A40s, a novel aptamer that was internalized effectively in GBM stem cells and

#### *Potential of Lipid Based Nanodrug Carriers for Targeted Treatment of Glioblastoma: Recent… DOI: http://dx.doi.org/10.5772/intechopen.108419*

successfully delivered miR-34c and anti-miR10b to the stem cell population. The data demonstrated that A40s crossed the BBB to reach the tumor location and selectively attached with the EphA2 receptor, which in turn led to inhibition in tumor growth and reduction in tumor relapse [58].

#### *2.4.3 Folic acid (FA)*

FA is essential for DNA synthesis, DNA repair, and methylation of DNA and is therefore necessary for cell survival and proliferation. The human folate receptor (FR), a glycosyl phosphatidyl inositol anchored membrane protein of 38 kDa, which shows high affinity for FA. At present, FR is considered an essential marker component in most of the cancers including glioblastoma. FR expression is very low or almost undetectable in most of the normal cells/tissues, but its expression is much higher in ovarian, breast, brain, lung, colorectal cancers [59]. FR-mediated liposomal delivery has been shown to enhance the antitumor efficacy of doxorubicin both in vitro and in vivo, and to overcome P-glycoprotein-mediated multi-drug resistance. Using folate as a targeting ligand, FR-targeting nanodrug delivery systems have been developed to target in situ glioma tumors [60].

#### *2.4.4 Transferrin (Tf)*

Tf receptor has been evolved as another important target for receptor-mediated transcytosis across the BBB. Owing to its higher expression on BBB endothelium, Tf-conjugation to the LNCs could be used as an effective active targeting strategy to enhance therapeutic outcomes in glioblastoma. Tf is basically a single chain irontransporting glycoprotein that supplies iron into cells via receptor-mediated endocytosis [61]. Though, expression of Tf receptor remains very low in most of the normal tissues but its expression increases drastically in case of brain cancer. The binding affinity of Tf to its receptors on the external surface of tumor endothelial cells has been found 10 to 100 times more than in normal endothelial cells [62]. LNCs can take advantage of this feature through surface conjugation with Tf, which will be then actively transported into the tumor cells. Tf modified liposomes, nanoparticles and dendrimers have been widely investigated in recent years.

#### *2.4.5 Oligopeptides*

Oligopeptides are another class of emerging targeting ligands, which are now heavily investigated for glioma-specific drug targeting [63, 64]. The Arg-Gly-Asp (RGD) oligopeptide is a component of the extracellular matrix protein fibronectin, which is involved in the cell adhesion, migration and proliferation [64]. RGD is known to serve as a recognition motif in multiple ligands for several different integrin receptors. RGD-containing peptide can be internalized into cells by integrin-mediated endocytosis.

#### **3. Advancements in lipid nanocarrier based drug delivery research in glioblastoma**

LNCs in view of their architectural uniqueness and preferable in vitro characteristics have become leading choice of delivery vehicle in glioblastoma research [33].

Many recent studies have depicted superiority of the LNCs in successful drug targeting to brain as compared to conventional formulations. S D. Hettiarachchi and his coresearchers developed a nano drug formulation of triple conjugated delivery system which included conjugation of two drugs to achieve synergistic effect in glioma. The triple conjugated delivery system comprised of transferrin, epirubicin and temozolomide. The in vitro results showed higher anticancer effect for transferrin conjugated samples. MTT assay depicted dramatically reduced cell viability in case of targeted nanocarriers as compared to non-transferrin conjugated carriers. The triple system of transferrin conjugated samples was significantly more cytotoxic to glioblastoma cell lines and was more effective than their equivalent single agents [65].

Another new strategy reported potentiality of aptamer-based immunoliposomes in modifying PD-1-silencing T cells. PD-1 gene was knocked out from CD8+ T cells using CRISPR/Cas9 system to liberate T cell activity from immunosuppression. The work involved stimulation of PD-1− T cells followed by functional modification of tumor-specific nanoliposomes (hEnd-Apt/CD3-Lipo) to generate FC/PD-1− CTLs. The activation and proliferation of the modified FC/PD-1− CTLs were then measured [66]. The anticancer potential of experimental CTLs against HepG2-tumors was evaluated in xenograft mice. Results indicated that the modification of hEnd-Apt/ CD3-Lipo nanocomposites on the FC/PD-1− CTLs had a more substantial synergetic effect in inhibiting tumor growth and prolonging animal survival, rather than other control liposomes [66]. Though, the study was not directed towards glioblastoma therapy, but the active targeting of immunoliposomes towards PD-1 receptor could be taken an attractive strategy for futuristic potential application in glioblastoma. Seeing the over-expression of PD-1 in many brain/CNS disorders including glioma, the outcome of the study could be used as an important input for further research of LNCs based PD-1 targeting to glioblastoma.

The therapeutic potential of hyaluronic acid (HA) as a targeting ligand for glioblastoma was investigated in a study by Stephen L et al. Anticancer effect of HA-conjugated doxorubicin loaded LNCs was reported in cortical astrocytes, MG, and A172 cells. In the study, three different glioblastoma cell lines were employed *viz.* invasive/non-tumorigenic (A172 cells), non-invasive/slightly tumorigenic (U251), and invasive/ highly tumorigenic (U87MG). A 24-hour potency assay demonstrated that the LC50 of experimental LNCs on A172 cells was nearly 5 folds lower than the corresponding LC50 for the cortical astrocytes and nearly 3 folds lower than that for MG cells [67]. The study thus highlighted potential application of HA in promoting preferential tumor cell uptake, with significant enhancement in chemotherapeutic potency in glioblastoma cells as compared to astrocytes.

Application of monoclonal antibodies as glioma-specific ligands through nanoliposomal vesicular carriers has already been reported. A recent liposomal delivery study has suggested conjugation of CD133 antibodies as a suitable method for targeting glioblastoma [52]. The study reported brain targeted delivery of gemcitabine, a widely used anticancer drug for cancers. However, being a BCS class III category of drug, it has higher water solubility with low permeability. Hence, to meet the challenge of sufficient brain uptake, gemcitabine was loaded in nanoliposome and the surface of the gemcitabine loaded liposome was functionalized with CD 133. The experimental CD 133 modified nanolipsomes was then tested for their in vitro and in vivo performance in glioblastoma cells. The in vitro study showed that conjugation of CD133 significantly enhanced the cytotoxicity of gemcitabine through endocytosis of CD133 surface markers overexpressed on glioblastoma cells [52]. The anti-tumor effect of CD133-modified nanoliposome was 15 times higher than that of free drug.

#### *Potential of Lipid Based Nanodrug Carriers for Targeted Treatment of Glioblastoma: Recent… DOI: http://dx.doi.org/10.5772/intechopen.108419*

The formulation also showed enhanced in vivo stability and cytotoxicity through in glioma bearing xenograft models. Moreover, monitoring of body weight changes showed that the use of targeted nanoliposomes significantly reduced the toxicity of gemicitabine.

Compared to single anticancer drug based chemotherapy, a combination of gene and drug therapy is being investigated in recent studies to achieve breakthrough in glioma treatment. It was expected that therapeutic genes and chemical drugs could act on different targeting sites with different mechanisms and could achieve synergistic therapeutic efficacy. The study explored the potential application of angiopep-2 through paclitaxel loaded cationic nanoliposomes. Angiopep-2 possesses the ability to target the low-density lipoprotein receptor-related protein, which is over-expressed on the BBB and glioma cells [68]. In a study, angiopep-2 modified cationic liposome was developed (ANG-CLP) for effective co-delivery of a therapeutic gene and an anticancer drug. The gene encoding the human tumour necrosis factor-related apoptosis-inducing ligand (pEGFP-hTRAIL) was used along with paclitaxel as the drug of choice for targeted delivery to glioma through LNCs. The dual targeting co-delivery system improved cellular uptake and gene expression in U87 MG human glioblastoma cells and also in the infiltrating margin of intracranial U87 MG glioma-bearing models [69]. The dual targeting LNCs selectively induced apoptosis in U87 MG cells while reducing toxicity to BCECs. Results of the pharmacodynamics studies showed that the apoptosis of glioma cells in *in vitro* BBB models and in U87 MG glioma-bearing mice treated by the experimental LNCs was more apparent and widespread than that treated by single medication systems and unmodified co-delivery system. Along with that, the median survival time of brain tumour-bearing mice group treated with angiopep-2-targetd LNCs was 69.5 days, which was significantly longer than that of conventional nanolipsome and standard drug treated groups. The treatment groups received commercial temozolomide showed median survival time of 47 days only [69].

Receptor-mediated endocytosis is one of the major mechanisms which can be effectively employed as active targeting approach to deliver the conventional chemotherapeutic agents to permeate across BBB. The receptors for insulin, transferrin, endothelial growth factors, amino acids, follic acid along with various metabolic nutrients are expressed on BBB, which thus can be taken as an opportunity to modify the surface of nanocarriers with relevant targeting moiety to make them brain specific. Dual-targeting doxorubicin encapsulated nanoliposomes were produced by conjugating the experimental liposomes with both folate and Tf, which were then tested for their effectiveness in glioma model [70]. The nanoliposomes were characterized by particle size, drug entrapment efficiency, and in vitro drug release profile. Drug accumulation, P-gp expression, and drug transport across the BBB in the dual-targeting nanoliposomes were examined by using bEnd3 BBB models. In vivo studies demonstrated that the dual-targeted nanoliposomes could successfully transport doxorubicin across the BBB and mainly distributed in the brain glioma. The anti-tumor effect of the dual-targeting liposome was also found significantly higher as compared to plain liposomes and free drug in terms of increased survival time and decreased tumor volume [70].

From our laboratory, we also carried out few works related to the brain delivery or BBB permeation ability of anticancer drugs through LNCs based strategy. Though our works were mostly based on passive targeting approach where we have mostly utilized the lipophilic nature and nanosize property of our developed liposomal vesicles to target the anticancer drug to brain, but the outcomes of the

work was quite impressive, which has compelled us for their further clinical translational studies. One of the recent studies from our laboratory reported the successful delivery of lomustine in glioma cells via lipid nanovesicular constructs [71]. Experimental LNCs were developed by modified lipid layer hydration technique and evaluated for different *in vitro* characteristics. Anticancer potential of selected lomustine loaded LNCs was tested on C6 glioma cell line *in vitro*. The experimental LNCs were within a size of less than 50 nm along with 8.8% drug loading capacity. Confocal microscopy revealed reasonable internalization of the selected LNCs in C6 cells. Experimental formulations were found more cytotoxic than free lomustine and blank LNCs as depicted from MTT assay. A clear improvement in pharmacokinetic profile both in blood and brain in the experimental mice models was observed for drug loaded LNCs than free drug. The formulations showed negligible haemolysis in mice blood cells, which further justified their safer in vivo applications.

Another similar study by Satapathy et al., reported delivery of docetaxel successfully to the rat brain through DSPE-modified nanoliposomes. In the work, the researchers simply aggravated the passive targeting strategy by utilizing DSPE, a sphingolipid, which has abundant presence the in brain and CNS. In the work, they developed a DSPE incorporated LNCs encapsulating docetaxel and investigated its BBB crossing potential, both qualitatively and quantitatively, in vivo [72]. Pharmacokinetic and biodistribution data showed an enhanced residence time of the docetaxel in the blood and efficient permeation of the drug from the docetaxel loaded LNCs through the BBB, as compared to free drug. The technetium-99 m labeled experimental LNCs effectively crossed the BBB and accumulated in the brain tissue in a time dependant manner as depicted from single photon emission tomography data [72]. At 4 h experimental time period, radiolabelled-LNCs were clearly tracked in the rat brain, whereas the same signal was absent in case of radiolabelled-free drug, which thus clearly confirmed that the sphingolipid modified LNCs possessed the necessary potential for BBB permeation and could be effective for the treatment of glioblastoma. Similar study from another research group in same department revealed successful delivery of docetaxel to rat brain through experimental nanoliposomes. Anti-proliferative effect of the experimental docetaxel loaded LNCs was conducted on C6 rat glioma cells. MTT assay showed that IC50 values of docetaxel from experimental nanoliposomes (9.5 ± 0.8 nM) was significantly less in comparison to freedrug (IC50 value, 70.8 ± 0.1 nM) and marketed Taxotere (IC50 value, 86.5 ± 0.3 nM) [73]. Flow cytometric analysis of C6 glioma cells incubated with fluorescein isothiocyanate (FITC)-labelled docetaxel loaded LNCs indicated about 18 and 23% enhancement of cellular uptake at 0.5 h at 0.5 h and 6 h of treatments in comparison to untreated cells.

Triggered drug delivery now-a-days has been merged as an interesting active targeting option for improved delivery of drugs through nanocarriers for the treatment of glioblastoma. A recent study showed that repeated pulsed high-intensity focused ultrasound can be used to improve the delivery of doxorubicin loaded nanocarriers to brain [74]. Atherosclerotic plaque-specific peptide-1 (AP-1) was used as the targeting ligand over the surface of doxorubicin loaded LNCs to selectively target glioblastoma cells. Compared with the control group, the animals treated with AP-1-conjugated nanoliposomes (5 mg/kg) showed significantly enhanced accumulation of drug at the sonicated tumor site and also a significantly elevated tumor-to-normal brain drug ratio (p = 0.001) (**Table 1**).

*Potential of Lipid Based Nanodrug Carriers for Targeted Treatment of Glioblastoma: Recent… DOI: http://dx.doi.org/10.5772/intechopen.108419*

#### **4. Challenges ahead**

It is a fact that nanomedicine has revolutionized the field of medical diagnostics and treatment and significantly improved the therapeutic and pharmacokinetic profile of conventional chemotherapy for effective targeting at brain. However, in spite of all eye-catching progress in nanocarrier based drug targeting, lots of challenges still remain, which in fact need serious insight analysis. Common obstacles with the use of LNCs for successful treatment of glioblastoma yet remain unaddressed largely in the form of the RES uptake, opsonisation, *in vivo* stability etc. [85].

Another issue is the cell/tissue accumulation and toxicity concern of engineered LNCs. Ultra-small size and brain specific delivery through targeting ligands though helpful for increased cellular uptake and diminished off-target toxicity, but accumulation of such engineered nanodrug systems in healthy organ cannot be fully ruled out. Such *in vivo* studies related to the toxicological concern of engineered nanodrug carriers are too highly lacking. Since, the toxic effects upon long-term accumulation of nanodrug carriers largely depend on various physico-chemical factors including shape, size, composition, biocompatibility, route of administration, degradation mechanism, drug-tissue interaction, protein binding etc., these factors thus need to be vividly analysed from case to case basis. The safety and pharmacological effect of engineered LNCs can be influenced by minor variations in multiple parameters and need to be carefully examined in preclinical and clinical studies. Systematic impact analysis of the possible acute/chronic toxicity effects of novel LNCs on humans and environment is the need of the hour.

Oral administration of LNCs is still not a feasible strategy due to stability and liver metabolism issues. Even, after intravenous administration, it is still unclear, how the properties of engineered LNCs change in brain microenvironments, or their effect on complement activation, blood coagulation, etc. Thus, many such important factors related to the *in vivo* behaviour engineered LNCs and their post treatment effect on normal brain cells need thorough investigation.

There is still dearth of ample pre-clinical research outcome of engineered LNCs on glioblastoma. Most of the studies related to glioblastoma are confined to *in vitro* cell line studies. Though experiments on *in vivo* efficacy of LNCs in brain tumor bearing xenograft model is there, but results of such research are highly variable with lack *of in vitro-in vivo* correlation data. Due to reliable *in vitro-in vivo* correlation related studies with variable research outcomes, such engineered LNCs face serious hurdle in clearing requisite regulatory approval for clinical trials [85]. The insufficiency of specific regulatory guidelines for the development, evaluation, *in vivo* testing of engineered LNCs is also another crucial factor in clinical translation. The leading pharma houses or pharma-research and development laboratories are still in confusion, whether to rely on the clinical efficacy of engineered nanodrug carriers for the treatment of glioblastoma on large scale basis. To find a sponsor for clinical trial of engineered nanodrug carriers still remains a tough task.

For anticancer drug loaded LNCs, dose ranges need to be correctly defined along with sufficient blood and brain pharmacokinetics data. Since, clinical testing of nanodrug carriers intended for the treatment of glioblastoma starts from phase II stage, i.e. subsiding phase I clinical trial on healthy volunteers, therefore establishment of proper in vivo safety, pharmacokinetic and dose-range data are highly crucial. In case of in vivo experiments, concerns are also being raised by some formulation scientists and medical experts on the rationality of *in vivo* experiments using xenograft


*Potential of Lipid Based Nanodrug Carriers for Targeted Treatment of Glioblastoma: Recent… DOI: http://dx.doi.org/10.5772/intechopen.108419*


#### **Table 1.**

*Research outcomes on lipid nanocarrier based drug delivery systems, targeting strategy adopted in metastatic glioma.*

mice/rat model bearing brain tumor. As such animal systems are usually athymic or immune-compromised; data derived out of these animal experiments cannot be fully relied on to carry out direct clinical testing on human subjects. In view of the significant anatomical/ physiological differences between immune-compromised laboratory animal model and human subjects in the development and progression of glioblastoma, it has been a point of long argument that whether these animal models could really mimic the human brain micro environment or whether such pre-clinical safety/ dose-range data can be reciprocated in clinical settings. It is a fact that laboratory rodents employed for the study do not suffer from glioblastoma or any other brain/ CNS cancers frequently as normal humans. Furthermore, immune response, cellular reaction, metabolism profile between laboratory animals and human subjects vary significantly differently. In a lay man language the material, which behave nontoxic to animals may show severe toxicity to humans or vice versa. Again till now, exact mechanism behind development/progression of glioblastoma in humans is largely unclear just as other cancer types. We seriously lack sufficient knowledge or well characterized data on specific biochemical factors, diseased conditions or antigens/ proteins responsible for development of glioblastoma. Thus, how much it will be rational to trust on the animal experiment data involving artificial/forcefully develop glioblastoma in nude/athymic animal models. Whether the use of such genetically modified animal models could really serve the purpose of successful clinical translation of LNCs? The budding scientists and medical/pharmacy/clinical professionals have to find specific answer for these unsolved questions in order to convince the manufacturers/sponsors to go ahead for large scale production.

Moving from the regulatory or clinical application problems towards large scale production at industrial scale, there is too lots of challenges remain unaddressed. Many pharmaceutical companies are still hesitant to invest directly in the large scale production of LNCs based delivery platforms. Batch to batch variation, problems with scale up, high cost of raw materials, availability of standardized unique protocol for

manufacturing and testing, stability issues, low drug carrying capacity are some of the major issues associated with LNCs. As a result, maximum research outcomes are confined in academic or small scale research laboratories and cannot able to reach from bench to bed side. To simplify the approval process for LNC based drug delivery system, a closer cooperation among various regulatory agencies is also warranted. Government of various countries too have ample responsibility with regard to develop advanced/simplified protocols that must be genuine, less tedious, yet sufficiently rigorous to address any safety concerns in a timely manner.

#### **5. Conclusion**

Glioblastoma still remains an area of unmet medical challenge despite remarkable progress in understanding its genesis and propagation. With advancements in molecular biology, biotechnology and interdisciplinary research horizon covering nanotechnology, computational biology, genetic engineering etc., successful treatment strategies are highly expected in near future. Continuous research by formulation scientists have led to development of novel lipid nanocarrier based formulations, which are showing promise in glioblastoma both *in vitro* and *in vivo* rodent models of the disease. Few of the nanodrug carriers have already seen day light with successful clinical applications in brain cancer patients. However, number of such advanced engineered nanocarrier system at clinical trial stage is still very limited. Stringent regulatory procedure coupled with lack of sponsors/industrial collaborators are being the major hurdles in successful clinical translation of the nanodrug carriers from laboratory to bed side. Active targeting strategies with tumor-specific ligands though emerged as hopeful approach in elevating treatment outcomes and to reduce chemo-induced side effects in glioblastoma, but in reality, lots of challenges are need to be focused. Recent studies have introduced MRI and near infrared imaging to the administration of dual-targeted nanodrug carriers, enabling targeting to be imaged with these new theranostics. Although the engineered LNCs could be plausible option for treating glioblastoma, detailed in depth analysis is highly essential to bring out desired outcomes in patients. In vivo performances of engineered LNCs are yet highly variable and *in vitro*-*in vivo* correlation data is seriously lacking. Till now, the leading pharma manufactures in India hesitate to go ahead for the large-scale production of targeted nanodrug carriers. Data are also scarce and dissatisfactory for targeted nanomedicnes to show improved clinical outcomes or improved quality of life post treatment in glioblastoma. Despite these daunting facts there is still hope. Personalized cancer planning, advance diagnosis, ample pre-clinical research, continuous research idea exchange between industry and academia are some of the highly focused area, which could finally make this goal a reality. With the growing global trend, the future of modern multimodal, multi-centered treatment approach of LNCs for regular clinical application in glioblastoma looks feasible.

*Potential of Lipid Based Nanodrug Carriers for Targeted Treatment of Glioblastoma: Recent… DOI: http://dx.doi.org/10.5772/intechopen.108419*

#### **Author details**

Bhabani Sankar Satpathy1 \*, Binapani Barik<sup>2</sup> , Ladi Alik Kumar2 and Sangram Biswal1

1 School of Pharmaceutical Sciences, Siksha 'O' Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India

2 Centurion University of Technology and Management, R. Sitapur, Odisha, India

\*Address all correspondence to: bhabanisatapathy@soa.ac.in; bbhabanisatapathy@yahoo.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 12**

## Glycan and Glycosylation as a Target for Treatment of Glioblastoma

*Atit Silsirivanit*

#### **Abstract**

Glycosylation is an important post-translational modification regulating many cellular processes. In cancer, aberrant glycosylation leads to the expression of tumorassociated glycans that are possibly used as therapeutic targets or biomarkers for diagnosis, monitoring, and prognostic prediction. The cumulative evidence suggested the significance of alteration of glycosylation in glioblastoma (GBM). Aberrant glycosylation presents truncated or uncommon glycans on glycoproteins, glycolipids, and other glycoconjugates. These aberrant glycans consequently promote the tumor development, metastasis, and therapeutic resistance. The glycosylation changes occurred in either cancer cells or the tumor microenvironment. GBM-associated glycans and their corresponding enzymes are proposed to be a target for GBM treatment. Several tools, such as lectin and inhibitors, are possibly applied to target the tumorassociated glycans and glycosylation for the treatment of GBM. This chapter provides information insight into glycosylation changes and their roles in the development and progression of GBM. The perspectives on targeting glycans and glycosylation for the treatment of GBM are enclosed.

**Keywords:** glioma, glioblastoma, glycosylation, glycan, lectin

#### **1. Introduction**

Glycosylation is a critical process to maturate the glycoproteins and glycolipids. Many factors were demonstrated to regulate this process, including 1) nucleotide sugar donors, 2) glycosyltransferase enzymes, and 3) glycosidase enzymes. The activated nucleotide sugars, synthesized through the hexosamine biosynthesis pathway, are served as sugar donors for the glycosylation process. More than 200 glycosyltransferase (GT) and glycosidase (GA) enzymes, residing in the endoplasmic reticulum (ER) or Golgi apparatus, are responsible for the addition and removal of sugar onto the glycoconjugates [1]. There are two major types of protein core-glycosylation, including N-linked and O-linked glycosylation (**Figure 1**). Both N-linked and O-linked glycans are generally terminal-modified with sialic acid and fucose *via* sialylation and fucosylation, respectively. Glycosylation is a sensitive process that could be influenced by several stimulants and cellular stresses. Many studies

#### **Figure 1.**

*Protein glycosylation. After transcription and translation, the proteins undergo N-linked glycosylation in endoplasmic reticulum or O-linked glycosylation in Golgi apparatus. Both N-linked glycans and O-linked glycans undergo peripheral modifications, fucosylation, and sialylation in the Golgi apparatus.*

showed that altered glycosylation is associated with the carcinogenesis and progression of cancers [2, 3]. Defects in glycosylation are possibly caused by the alteration of nucleotide sugar synthesis or the imbalanced expression of glycosyltransferases or glycosidases [4]. Aberrant glycosylation in cancer cells causes the glycan truncation or the expression of uncommon glycans. These aberrantly expressed glycans are possibly used as a biomarker or a target for the treatment of cancers. Many tumor-associated glycans were demonstrated to play essential roles in tumor development, progression, and therapeutic resistance [2, 5].

Recent evidence suggests the alteration of glycosylation in glioblastoma (GBM) [3, 4, 6, 7]. GBM-associated glycans and glycoconjugates, such as the cluster of differentiation 44 (CD44), CD133, and ephrin-A1, were discovered to play important roles in tumor progression, leading to the poor prognosis of patients [8, 9]. Defects of glycosylation in GBM tumors were found in glycoproteins, glycolipids, glycosaminoglycans, or proteoglycans. The alteration of protein glycosylation occurred in both N-linked and O-linked glycosylation. Besides, aberrant terminal glycan modification of sialylation or fucosylation was also observed in GBM [3]. Moreover, the glycans and glycosylation also exhibited the functional significance in glioma stem-like cells (GSC) by regulating the stem cell-related phenotypes [10, 11].

Not only in cancer cells, the tumor microenvironment (TME) was also presented with aberrant glycosylation [12, 13]. Glycosylation changes in TME were found to promote tumor progression, immunosuppression, and therapeutic resistance [14]. Therefore, it is proposed that glycosylation changes of TME might be an alternative target for the treatment of GBM.

This chapter collectively summarizes the recent information on glycan and glycosylation changes and their roles in GBM progression and therapeutic resistance. The information provided here may fulfill our understanding of the roles of glycosylation and its potential to be a target for the treatment of GBM.

### **2. N-linked glycosylation**

The N-linked glycosylation transfers the oligosaccharide chain to the target polypeptide by forming the linkage between the *N*-acetyl glucosamine (GlcNAc) residue and the amide side chain of the asparagine residue. The process starts in ER; an oligosaccharide is firstly synthesized on the dolichol phosphate carrier and transferred to the protein acceptor by the oligosaccharyltransferase enzyme. The premature glycan chain of N-linked glycoprotein is subsequently modified by the sequential reactions of sugar addition or removal, controlled by several GTs and GAs. The final steps, sialylation and fucosylation, are accomplished in the Golgi apparatus. Many studies demonstrated the alteration of *N*-linked glycosylation and its related enzymes in GBM (**Table 1**).


*β1,3-N-acetylglucosaminyltransferase-8, B3GnT8; β1,4-Galactosyltransferase 5, B4GalT5; mannosylglycoprotein β-N-acetylglucosaminyltransferase-1, MGAT1; mannosylglycoprotein β-N-acetylglucosaminyltransferase-5, MGAT5; polypeptide GalNAc transferase-2, GALNT-2; polypeptide GalNAc transferase-12, GALNT-12; Fucosyltransferase-8, FUT-8;* α*2,3-sialyltransferase-3, ST3Gal-3.*

#### **Table 1.**

*Glycosyltransferases involved in the progression of GBM.*

An increase of bi-, tri-, and tetra-anternary N-linked glycans was found to be associated with the progression of GBM [15–17, 26]. The α1,6-mannosylglycoproteinβ-*N*-acetylglucosaminyltransferase-5 (MGAT), an enzyme responsible for the synthesis of biantennary *N*-linked oligosaccharide, was found to promote the invasiveness of GSC [16]. N-linked glycosylation of the receptor protein tyrosine phosphatase type mu (RPTPmu) controlled by MGAT5 was demonstrated to suppress its function and consequently enhance the migration ability of GBM cells through phospholipase C (PLC)/protein kinase C (PKC) pathway [17]. In addition, the MGAT1 (a member of the N-linked associated *N*-acetylglucosaminyltransferase group) was highly detected in GBM and plays an essential role in promoting the proliferation and invasion of cancer cells [15, 17].

A new subclass of *N*-glycosylation called-Paucimannosylation, producing a truncated N-glycan (Man3GlcNAc2Fuc), was found to elevate in GBM compared with non-tumor tissues [27, 28]. The glycan was found to be involved in the proliferation, migration, and invasion of cancer cells [27].

Another N-link-associated enzyme, a β1,3-*N*-acetylglucosaminyltransferase-8 (B3GnT8), an enzyme that controls the formation of polylactosamine on β1–6 branched N-glycans, was found to regulate the proliferation and metastatic ability of cancer cells [22]. The β1,4-galactosyltransferase-5 (B4Gal-5) producing highly branched N-glycans was found to regulate the sensitivity of cancer cells to anticancer drugs-etoposide and arsenic trioxide. Suppression of B4GalT-5 could enhance the apoptosis induction effects of these drugs in cancer cells, suggesting its potential to improve the therapeutic efficiency for malignant glioma [19, 20]. Moreover, the B4GalT-5 was also found to regulate self-renewal and tumorigenicity of glioma stemlike cells [20].

Inhibition of N-glycan synthesis by the specific siRNA or inhibitors significantly suppresses tumor growth, metastasis, and radioresistance of GBM [15–18, 29–31]. This information suggested the potential of N-glycosylation to be a target for the treatment of GBM.

#### **3. O-linked glycosylation**

Golgi-resident glycosyltransferases are responsible for the synthesis of O-glycans *via* O-linked glycosylation. A particular serine (Ser) and threonine (Thr) residues can be O-glycosidic linked with various kinds of oligosaccharides. This chapter focuses on the mucin-type O-glycosylation or O-GalNAcylation, an O-linked modification of Ser/Thr by *N*-acetylgalactosamine (GalNAc), followed by the formation of complex oligosaccharide structure. There are 20 isoforms of polypeptide GalNAc transferase (ppGalNAcT or GALNT) identified in humans; the enzymes catalyze the transferring of GalNAc from activated nucleotide sugar donor to initially modify the Ser or Thr residues of a specific glycoprotein [32]. Alteration of O-linked, especially O-GalNAc, glycosylation was observed in many types of cancer [1, 5, 32]. Truncated O-glycans and their associated mucin glycoproteins were applicable as a marker for diagnosis, monitoring, and prognostic prediction of cancer [1].

In GBM, the alteration of O-linked glycosylation played a significant role in the tumor progression and therapeutic resistance [23, 24]. The significance of GALNT enzymes in the progression of GBM has been revealed, suggesting their possibility of being a new target for GBM treatment (**Table 1**). GALNT-2 was demonstrated to *Glycan and Glycosylation as a Target for Treatment of Glioblastoma DOI: http://dx.doi.org/10.5772/intechopen.106044*

promote the migration and invasion of cancer cells [23]. Expression of GALNT-12 was associated with poor prognosis of GBM patients as it promotes cancer cell proliferation, migration, and invasion *via* PI3K/Akt/mTOR cascade [24]. The tumor-associated truncated O-linked glycan and its receptor, macrophage galactose-type lectins, were found to modulate the function of tumor-associated macrophages and microglia in GBM [33]. Using lectin from *Dolichos biflorus*, the GalNAc-associated glycan was highly detected in GSC compared with its differentiated form, suggesting its potential to be a GSC marker (**Table 2**) [11]. This information suggested the possibility of using GALNTs as a biomarker and a therapeutic target for GBM.

#### **4. Fucosylation**

The terminal glycan modification by fucose, called "Fucosylation," is controlled by the fucosyltransferase (FUT) enzymes. In human, 13 FUTs are classified according to their activities into 1) α1,2-FUTs (FUT-1 and FUT-2), 2) α1,3-FUTs (FUT-3, FUT-4, FUT-5, FUT-6, FUT-7, FUT-9, FUT-10, and FUT-11), 3) α1,4-FUTs (FUT-3 and FUT-5), 4) α1,6-FUTs (FUT-8), and 5) O-FUTs (Pofut-1 and Pofut-2) [34]. Altered expression of FUTs and the fucosylated-glycans were found to associate with tumor development and progression [5, 35]. In GBM, the aggressiveness and malignant phenotypes GBM were associated with fucosylated Lewis antigens' expression [36]. The enzyme FUT-8, responsible for α-1,6-fucosylation of N-glycans, was discovered to promote the growth, migration, and invasion of GBM cells [25]. Inhibition of fucosylation by the inhibitor-2F-peracetyl-fucose could sensitize the effect of temozolomide (TMZ), suggesting the potential of FUT-8 to be a target for GBM treatment [25].

#### **5. Sialylation**

Sialylation is a modification of glycoproteins and glycolipids by sialic acid (Sia). There are 20 sialyltransferase enzymes (STs) responsible for three types of sialylations: 1) α2,3-sialylation, 2) α2,6-sialylation, and 3) α2,8-sialylation (**Table 2**) [37, 38].


#### **Table 2.**

*Sialylation and the associated enzymes and glycan structures.*

Sialylation was demonstrated to involve in the stemness maintenance of GSC and tumor progression, suggesting its possibility to be a promising target for the treatment of GBM [39, 40]. An α-2,3 sialylation was found to promote the progression, while α-2,6 sialylation suppresses the GBM. Inhibition of α-2,3 or enhancement of α-2,6 sialylation significantly suppresses the metastatic ability of GBM cells [41–43]. Using lectin from *Maackia amurensis*, α-2,3 sialylation was found to be enhanced in GSC and play an essential role in stemness maintenance [39]. Suppression of sialylation using ST inhibitor or sialidase leads to the apoptosis of GSC [39]. The mechanism by which α-2,3 sialylation regulates stemness of GSC is probably explained by its role in the stabilization of surface CD133, an important functional GSC marker [43]. Moreover, the lectin *M. amurensis* lectin-II (MAL-II) could significantly induce the apoptosis of GSC, suggesting its potential for GBM treatment [39]. In addition, suppression of sialylation by a specific inhibitor was found to enhance the sensitivity of GBM cells to the general chemo-drugs—cisplatin and 5-fluorouracil [39]. This collective evidence suggested the potential of α-2,3 sialylation as a target for the treatment of GBM.

In addition, sialidases or neuraminidases (NEU), the enzymes that remove terminal Sia from the oligosaccharide chain of glycoproteins and glycolipids, were also altered in GBM. The overexpression of NEU3 significantly suppresses cancer cells' migration and invasion ability by promoting focal adhesions through calpaindependent proteolysis [44]. NEU4 was found to be upregulated in GSC, and suppression of NEU4 significantly reduces cell survival and stemness properties of the cells [45].

#### **6. Gangliosides, glycosaminoglycans, and proteoglycans**

Altered syntheses of gangliosides, glycosaminoglycans, and proteoglycans were observed to play significant roles in GBM [46–52]. The GD3-gangliosides, heparan sulfate (HS) glycosaminoglycans, and their responsible enzymes were found to be altered in GBM and proposed as a potential GBM marker [46–48]. Glycosaminoglycans played essential roles in the communication between GBM cells and their TME. Alteration of HS synthesis by ablation of heparanase (HPSE) results in the significant reduction of tumor cell adhesion and invasion [48]. This information implied that HS is an important factor in promoting GBM invasion; it is therefore possibly proposed as a therapeutic target for GBM.

Alteration of proteoglycan synthesis was found to associate with the development and progression of GBM [49–52]. Expression of tumor-associated proteoglycans and their related enzymes were found to facilitate the tyrosine kinase signaling pathway, which benefits the progression of GBM, suggesting their potential as a promising prognostic marker and target for GBM treatment [49]. The elevation of neuro-glial proteoglycan-NG2 was associated with the invasiveness of GBM [50]. NG2 was found to control the vascular morphology and functions, suggesting its role in facilitating metastasis *via* tumor vascularization of GBM [51]. Targeting NG2, in combination with GD3A (a GBM-associated ganglioside), could significantly reduce the viability of GBM cells [53]. This information suggested the significance of NG2 in the progression of GBM and its possibility of being a target for treatment. Moreover, chondroitin sulfate proteoglycans (CSPGs) play important roles in organizing the tumor microenvironment to prevent tumor invasion. CSPGs were drastically decreased in a diffusely infiltrating tumor of GBM [52].

#### **7. Conclusion and perspectives**

Alteration of glycosylation was predominantly observed in either cancer cells or TME in GBM. Both core-glycosylation and peripheral glycan modifications were important factors in regulating the tumor development, progression, and therapeutic resistance. Several strategies have been proposed to target glycans and glycosylation for the treatment of GBM.

Suppression of glycosylation using specific interferences or inhibitors is a potential strategy to target glycosylation [54, 55]. However, there is a limitation to using the broad-spectrum glycosylation inhibitors for cancer treatment as they also affect the neighboring non-tumor cells. Targeting glycosylation of a particular glycoprotein or glycoconjugate is a possible strategy for cancer treatment. In GBM, interference of hyaluronic acid synthesis by methylumbelliferone (4-MU), an inhibitor of hyaluronic acid synthase capable of crossing the blood-brain barrier (BBB), was found to significantly inhibit the proliferation of GBM [56].

The short peptide is recently applicable for targeting or suppressing the specific glycoform of a particular glycoprotein in cancer cells. The deglycosylated form of brevican (dg-Bcan), an ECM-associated glycoprotein upregulated in GBM, was explicitly bound by a small 8-amino acid dg-Bcan-Targeting Peptide (BTP). The radiolabeled-BTP could be internalized into the cancer cell, suggesting its potential to be used as an imaging agent to detect GBM [57]. Further studies to apply this peptide for the treatment of GBM by conjugating it with chemo-drugs or other substances are noteworthy.

Based on the sugar preferential of lectins, the plant lectins were widely used to determine the expression of GBM-associated glycans as well as the functional analyses either *in vitro* or *in vivo* model (**Table 3**) [11, 18, 19, 21, 26, 59, 60]. Using the lectin as a therapeutic agent for GBM is another approach, either combined with other chemo-drugs or as a single agent.

The *Phaseolus vulgaris* erythroagglutinin (PHA-E) was used to detect the β1,4- GlcNAc-containing N-glycans. It strongly inhibits the migration ability of GBM cells, suggesting its potential to be used for the treatment of GBM [18]. In addition, PHA-E was also found to inhibit the functions of the epidermal growth factor receptor (EGF-R) and a drug efflux pump-P-glycoprotein on GBM [59, 61]. This information suggested the involvement of β1,4-GlcNAc in cancer cells' growth and drug resistance. Moreover, the potential of PHA-E as a chemosensitizing agent for GBM was also reported [61]. MAL-II is another lectin that can suppress the stemness maintenance and induce apoptosis of GSCs, suggesting its application as a therapeutic agent for GBM [39]. Lectin from *Griffonia simplicifolia* I (GSL-I) was used to identify the GBM-specific cell surface glycobiomarkers compared with the low-grade glioma. The identified markers may be applicable for diagnosis and possibly used as a target for the treatment of GBM [58]. With another type of brain tumor, the lectin from *Canavalia brasiliensis* seeds (ConBr) was found to suppress the ERK1/2 and Akt signaling pathways, consequently inhibiting the migration ability of rat neuroblastoma cells [62]. Besides the lectins, monoclonal antibodies against the specific glycans have been established and used to detect cancer-associated glycans. The antibodies can also suppress or activate the functions of glycans in cancer cells; this information suggests the possibility of using a glycan-specific antibody to treat the GBM patients [63, 64].

In conclusion, glycans and glycosylation have been identified to play significant roles in GBM progression and therapeutic resistance. Targeting glycans and glycosylation is possibly an alternative strategy for the treatment of GBM; however, further studies to target specific glycosylation of a particular glycoconjugate are still needed. In addition,


#### **Table 3.**

*Lectins used in GBM studies.*

the clinical studies or trials on the potential of using glycans and glycosylation as a target for GBM treatment are still a large gap that needs to be further evaluated.

### **Acknowledgements**

The author would like to thank the supports from the National Research Council of Thailand and Khon Kaen University, Thailand.

#### **Conflict of interest**

The author declares no conflict of interest.

### **Author details**

Atit Silsirivanit Faculty of Medicine, Department of Biochemistry, Khon Kaen University, Khon Kaen, Thailand

\*Address all correspondence to: atitsil@kku.ac.th

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Amit Agrawal and Daulat Singh Kunwar*

Glioblastoma (GBM) is a common and aggressive brain cancer with features of necrosis and endothelial proliferation in the histopathologic examination. Its presentation and management depend on tumor location, size, grade, and underlying histopathological characteristics. GBM tumors have clinical features of increased intracranial pressure, focal neurological deficits, or seizures (generalized or partial) with rapid progression. This book discusses GBM and its diagnosis, treatment, and management.

Published in London, UK © 2023 IntechOpen © dzika\_mrowka / iStock

Glioblastoma - Current Evidence

Glioblastoma

Current Evidence

*Edited by Amit Agrawal* 

*and Daulat Singh Kunwar*