2.1.3. Novel approach for modulation of TAM behavior by CuNG

The first study that reveals CuNG as strong immunomodulator is associated with almost complete regression of drug-resistant tumor [21, 22]. CuNG treatment induces gradual reversal of immunosuppression as evident by restoration of lymphoproliferative response in drugresistant tumor-bearing animal model. CuNG treatment in vivo increased the number of IFNγ producing CD4<sup>+</sup> and CD8<sup>+</sup> cells but decreased the number of T regulatory marker-expressing T cells in tumor sites. Although no direct cell-mediated cytotoxicity was observed, robust expression of apoptogenic cytokines viz. IFN-γ and TNF-α at tumor site as well as peripheral and spleen mononuclear cells were sufficient enough to resolve many drug- and radiationresistant tumors [21, 22]. This is particularly substantial as local cytokine milieu in tumor microenvironment profoundly affects the functional plasticity of macrophages, which plays a key role in skewing suppressive Th2 response toward Th1 type [11, 23]. The evocation of Th1 response largely depends on the critical level of the two regulatory cytokines, IL-10 and IL-12. IL-10 inhibits important aspects of cell-mediated immunity, whereas IL-12 induces type 1 cytokine production and effective antitumor cell-mediated response [3, 6].

Numerous therapeutic strategies involving TAM relies on reeducation or polarization of TAM suggesting that it could be possible to convert them toward nonsuppressive and antitumorigenic type by creating appropriate cytokine microenvironment [23]. Previously it was shown that in the presence of IL-12, TAMs rapidly alter their functional phenotype from tumor supportive and immunosuppressive to antitumorigenic type [24]. Our study in this direction showed that CuNG owns the potential to alter the immunosuppressive phenotype of TAMs toward proinflammatory by evoking a robust IL-12 and decreased IL-10 and TGF-β production by TAMs and thereby polarize its functional phenotype toward inflammatory both in vitro and in vivo [25]. CuNG-mediated cytokine alteration in TAMs is associated with the establishment of beneficial cytokine in tumor microenvironment that skewed the unresponsive CD4<sup>+</sup> T-cell population toward Th1 type in contact in an independent manner. The critical balance between elevated IL-12 and reduced IL-10 in CuNG treated TAMs is significant; because in one hand presence of IL-12 results in hugely elevated levels of IFN-γ [3], which limits T-cell survival and shortens Th1 response [26], on the other hand small amount of IL-10 limits the self-killing mechanism of Th1 cells and thereby prolonged its persistence [27]. Interestingly, CuNG-treated TAMs maintained a stable balance between IL-10 and IL-12 production, where IL-12 levels were higher than IL-10. This critical balance between these two cytokines was sufficient enough to induce Th1 response. Further study disclosed that in vitro treatment of CuNG significantly reduces the immunosuppressive cytokines and augments IL-12 generation in the blood monocytes of patients with metastatic cancers. Therefore, abrogation of immune suppression in tumor microenvironment through CuNG is principally due to the reprogramming of TAM in terms of their cytokine profile [25].

To decipher the underlying mechanisms of phenotypic conversion of TAM immunosuppressive (IL-12low, TGF-βhigh) to proimmunogenic (IL-12high, TGF-βlow) type, our study in this direction reveals that CuNG induces ROS generation by TAMs, which is associated with the activation of two mitogen-activated protein kinases (MAPKs) (p38MAPK and ERK1/2), and also causes upregulation of intracellular GSH in TAMs [28]. Earlier reports showed that LPSinduced IL-12 production in normal macrophages is regulated by the activation of p38MAPK signaling [29], and activation of ERK1/2 in macrophages is associated with the production of IL-10 and TGF-β and negatively regulates IL-12 production [30–32]. CuNGmediated ROS generation leads to the activation of p38MAPK that upregulated the initial IL-12 production and to the activation of ERK1/2 pathway along with GSH upregulation separately, found responsible for IFN-γ production by TAMs. It is important to note that GSH/oxidized GSH plays a crucial role in regulating IFN-γ-mediated augmentation of IL-12 production by macrophages and DCs. CuNG-mediated activation of ERK1/2 signaling and GSH upregulation are independently associated with IFN-γ augmentation in TAMs. This IFN-γ, further, increased the GSH production that, in turn, prolonged IL-12 production and downregulated TGF-β production, thereby plays the decisive role in CuNG-mediated reprogramming of regulatory cytokine production by TAMs. Although ROS-mediated cytokine modulation in TAMs toward proimmunogenic (IL-12high, TGF-βlow) type has also been found by application of other Schiff base metal, e.g., ZnNG and FeNG [28], unlike CuNG, their effect were found to be temporary. Therefore, inclusion of copper within NG scaffold provides the compound with a unique characteristic to skew and maintain a sustained proimmunogenic phenotype in TAM.

## 2.2. Role of myeloid-derived suppressor cells (MDSCs) in immune regulation in cancer

### 2.2.1. History, origin, and phenotype

antitumorigenic type by creating appropriate cytokine microenvironment [23]. Previously it was shown that in the presence of IL-12, TAMs rapidly alter their functional phenotype from tumor supportive and immunosuppressive to antitumorigenic type [24]. Our study in this direction showed that CuNG owns the potential to alter the immunosuppressive phenotype of TAMs toward proinflammatory by evoking a robust IL-12 and decreased IL-10 and TGF-β production by TAMs and thereby polarize its functional phenotype toward inflammatory both in vitro and in vivo [25]. CuNG-mediated cytokine alteration in TAMs is associated with the establishment of beneficial cytokine in tumor microenvironment that skewed the unresponsive CD4<sup>+</sup> T-cell population toward Th1 type in contact in an independent manner. The critical balance between elevated IL-12 and reduced IL-10 in CuNG treated TAMs is significant; because in one hand presence of IL-12 results in hugely elevated levels of IFN-γ [3], which limits T-cell survival and shortens Th1 response [26], on the other hand small amount of IL-10 limits the self-killing mechanism of Th1 cells and thereby prolonged its persistence [27]. Interestingly, CuNG-treated TAMs maintained a stable balance between IL-10 and IL-12 production, where IL-12 levels were higher than IL-10. This critical balance between these two cytokines was sufficient enough to induce Th1 response. Further study disclosed that in vitro treatment of CuNG significantly reduces the immunosuppressive cytokines and augments IL-12 generation in the blood monocytes of patients with metastatic cancers. Therefore, abrogation of immune suppression in tumor microenvironment through CuNG is principally due to the reprogramming of TAM in terms of their

To decipher the underlying mechanisms of phenotypic conversion of TAM immunosuppressive (IL-12low, TGF-βhigh) to proimmunogenic (IL-12high, TGF-βlow) type, our study in this direction reveals that CuNG induces ROS generation by TAMs, which is associated with the activation of two mitogen-activated protein kinases (MAPKs) (p38MAPK and ERK1/2), and also causes upregulation of intracellular GSH in TAMs [28]. Earlier reports showed that LPSinduced IL-12 production in normal macrophages is regulated by the activation of p38MAPK signaling [29], and activation of ERK1/2 in macrophages is associated with the production of IL-10 and TGF-β and negatively regulates IL-12 production [30–32]. CuNGmediated ROS generation leads to the activation of p38MAPK that upregulated the initial IL-12 production and to the activation of ERK1/2 pathway along with GSH upregulation separately, found responsible for IFN-γ production by TAMs. It is important to note that GSH/oxidized GSH plays a crucial role in regulating IFN-γ-mediated augmentation of IL-12 production by macrophages and DCs. CuNG-mediated activation of ERK1/2 signaling and GSH upregulation are independently associated with IFN-γ augmentation in TAMs. This IFN-γ, further, increased the GSH production that, in turn, prolonged IL-12 production and downregulated TGF-β production, thereby plays the decisive role in CuNG-mediated reprogramming of regulatory cytokine production by TAMs. Although ROS-mediated cytokine modulation in TAMs toward proimmunogenic (IL-12high, TGF-βlow) type has also been found by application of other Schiff base metal, e.g., ZnNG and FeNG [28], unlike CuNG, their effect were found to be temporary. Therefore, inclusion of copper within NG scaffold provides the compound with a unique characteristic to skew and maintain a sustained

cytokine profile [25].

174 Anti-cancer Drugs - Nature, Synthesis and Cell

proimmunogenic phenotype in TAM.

The descriptions of myeloid suppressor cells were first reported more than 20 years back in normal and tumor-bearing mice as well as in patients with cancer. Initially they were known as natural suppressor (NS) cells due to induction of tolerance to foreign antigens by inhibiting various activities of the immune system [33]. Recent evidence has disclosed that these suppressive cell populations significantly differ from normal myeloid precursors. In healthy individuals, normal hematopoiesis give rise to common myeloid progenitor cells in bone marrow, which, in turn, convert to immature myeloid cells (IMC) that ultimately differentiated into mature granulocytes, macrophages, or dendritic cells (DCs) [34]. However, in pathological conditions such as cancer, sepsis, trauma, and various infectious diseases, the differentiation of IMCs are partially blocked and thus resulting in the expansion of MDSCs [34]. Based on the origin and biological function, the term MDSCs has been suggested [35].

MDSCs are a heterogeneous group of myeloid population comprising granulocyte, monocyte, and dendritic cells, which have been prevented from fully differentiating into mature cells and are capable of suppressing the immune response [34]. They do not express the cell-surface markers that are exclusive for monocytes, macrophages, or DCs and are comprised of a mixture of myeloid cells with a morphology similar to both granulocytes and monocytes. In healthy mice, around 20–30 of cells represent this phenotype and approximately 2–4% of cells are present in the spleen, although the frequency of these cells in tumor-bearing mice is largely enhanced. In mice, MDSCs are characterized by the co-expression of the myeloid lineage differentiation antigens Gr1 and CD11b (also known as αM-integrin) [34]. Since Gr-1 antigen consists of two separate epitopes, Ly6G and Ly6C, the establishment of Ly-6C- and Ly-6Gspecific mAbs has led to the identification of two MDSC subsets in the spleens of tumorbearing mice: CD11b+ Ly-6ClowLy-6G+ MDSCs with granulocytic morphology (PMN-MDSCs) and CD11b<sup>+</sup> Ly-6ChighLy-6G<sup>−</sup> MDSCs with a monocytic phenotype (MO-MDSCs) [36]. The Ly6G molecule is expressed primarily by granulocytes, whereas Ly6C is highly expressed by monocytes. Although the pattern of PMN-MDSC and MO-MDSC subsets differs between tumors and organs, over 80% of MDSCs are PMN-MDSCs, whereas less than 10% of MDSCs are MO-MDSCs in most of experimental models [36]. However, it is difficult to discriminate PMN-MDSCs from neutrophils as neutrophils also express both CD11b and Ly6G.

In humans, identification of MDSCs is very difficult due to the absence of a good marker such as Gr-1 and fewer opportunities to obtain samples. However, several studies have identified PMN-MDSCs (CD11b<sup>+</sup> CD14- CD15<sup>+</sup> cells with a PMN morphology) and MO-MDSCs (being CD11b+ CD14<sup>+</sup> HLA-DRlow/<sup>−</sup> and secreting TGF-β) [34] in the peripheral blood of patients with cancer. Other studies have shown that PMN-MDSCs are usually defined as CD14<sup>−</sup> CD11b<sup>+</sup> or, more narrowly, as cells expressing the common myeloid marker CD33, but lack expression of markers of mature myeloid and lymphoid cells and of the MHC class II molecule HLA-DR [34, 37]. MO-MDSCs have also been identified within a CD15<sup>+</sup> population in human peripheral blood [34]. In healthy individuals, IMCs represents about 0.5% of peripheral blood mononuclear cells [37]. Nevertheless, information on each human MDSC subset is much less specific than it is for mice and warrants further investigations.

#### 2.2.2. MDSC-mediated immunosuppression in cancer

MDSC suppresses immunity by perturbing both innate and adaptive immune responses. MDSC exerts its suppressive activity against T cells through diverse mechanisms [34]. One of such mechanisms is associated with L-arginine metabolism. Expression of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) in MDSCs is dependent on the substrate Larginine. Arg1 and reactive oxygen species (ROS) are upregulated in activated PMN-MDSCs [36, 38], whereas Arg1 and iNOS are highly expressed in activated MO-MDSCs [39]. The upregulation of either Arg1 or iNOS results in L-arginine shortage from the tumor microenvironment, leading to consequent inhibition of T-cell proliferation through multiple mechanisms such as reduction of CD3 ζ-chain expression and IFN-γ/IL-2 secretion by T cells [40]. High levels of ROS in PMN-MDSCs can induce nitrosylation of the T-cell receptor (TCR) during direct cell-to-cell communication, which contributes to the inhibition of antigen-specific T-cell activation [34]. ROS production by PMN-MDSCs is known to be induced by several tumorderived factors such as TGF-β, IL-6, IL-10, and GM-CSF [41]. The suppressive function of PMN-MDSCs depends on Arg1 and ROS [36, 38], whereas that of MO-MDSCs requires a signal transducer and activator of transcription 1 (STAT1) and iNOS [38]. In activated PMN-MDSCs, STAT3 is highly activated, which results in increased expression levels of ROS through the upregulation of NADPH oxidase (NOX2) but not NO production [38, 39]. On the other hand, STAT1 and iNOS are highly upregulated in MO-MDSCs, resulting in increased levels of NO but not ROS production [34]. In addition, STAT6 signaling pathway is involved in the upregulation of Arg1 and TGF-β through activation of IL-4 and IL-13, leading to immunosuppressive activity [40]. However, the immunosuppressive mechanisms overlap between G-MDSCs and M-MDSCs in human cancers. iNOS is also upregulated in PMN-MDSCs in a variety of human cancers [42–44]. CD14<sup>+</sup> HLA-DR−/low MO-MDSCs express NADPH oxidase component gp91 (phox) and produce high level of ROS in human non-small cell lung cancers [45]. These MO-MDSCs inhibit T-cell proliferation and IFN-γ secretion in a cell-contact-dependent manner.

Another mechanism of MDSC-mediated T-cell suppression is associated with cysteine deprivation from local environment [46]. Cysteine is the essential component required for T-cell activation, differentiation, and proliferation, which they cannot synthesize; rather dependent on antigen-presenting cells. Dendritic cells and macrophages can deliver cysteine to T cells by converting methionine and cystine to cysteine [47]. Like the APCs, MDSCs also import extracellular cystine for converting it to cysteine, but unlike APCs, they do not export cysteine, leading to lack of cystine from local environment for dendritic cells and macrophage [46].

The potential suppressive property of MDSCs can also be reflected on the innate immunity. Studies have shown that MDSCs impair NK-cell development, IFN-γ production, and cytotoxicity against tumor cells. This suppression is mediated by membrane-bound TGF-β1 and through downregulation of NKG2D (the primary activating receptor for NK cells) [48]. Cytotoxic activity of NK cell and their apoptogenic cytokine secretion is also disrupted by CD14+ HLA-DR−/lo MO-MDSCs secretion in a cell-contact-dependent manner in human hepatocellular carcinoma [48]. The inhibition of NK cells is independent on arginase activity. The similar inhibitory mechanism of NK-cell development and functions by IL-1β-induced PMN-MDSCs have been also demonstrated in a mouse model [49]. Furthermore, MDSCs can skew macrophage-derived cytokine profiles from type 1 to type 2 putatively through Toll-like receptor 4 signaling pathway [49]. This effect is amplified by macrophages that increase the MDSC production of IL-10. Indeed, increased MDSC levels in peripheral blood and tumor are closely associated with the infiltration of CD163+ M2 macrophages in human esophageal cancer [50].
