**2.5 Prostate cancer**

Human bone marrow mesenchymal stem cell (MSC) derived EVs are involved in the modulation of cell signaling, cellular differentiation, and proliferation—and this is seen across multiple disease paradigms. These regenerative EVs have been shown to reverse the malignant phenotype in prostate and colorectal cancer, recovering function in a murine model of AKI, as well as mitigating radiation damage to marrow [36]. In models of prostate cancers, the reversal of taxane resistance and tumorigenic phenotype in a human prostate carcinoma cell line (as well as human explants) can be accomplished by treatment with healthy MSC-derived EVs [61].

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*Stem Cells and Extracellular Vesicles: Biological Regulators of Physiology and Disease*

Other populations of "therapeutic" EVs (outside of the bone marrow) have also been isolated and applied: EVs isolated from normal prostate cells acquired via patient biopsy reverse the resistance of malignant prostate cells to various drugs. On the contrary we have shown that EVs derived from cancerous cells can drive cancer progression and enhance resistance to certain chemotherapies, which again highlights the specificity, plurality, the yin/yang of EV functionality [61]. Panagopoulos et al. confirmed much of this work, showing that vesicles from both *in vivo* prostate cancer cell and explant cultured prostate cancer cells can induce cellular changes that produce a neoplastic phenotype in normal prostate cell lines [65]. These results were also reproduced using vesicles from patients with other malignancies, namely

Our group has developed a unique biomarker system focusing on EVs isolated from the saliva of patients who have experienced mild traumatic brain injury (mTBI) [66]. Rather than conventional human serum isolation, this has been a novel protocol allowing for the easily accessible collection of saliva laden with EVs that have freely trafficked from injured brain parenchyma into the saliva—allowing for a representative sample which captures the shift of various EV populations and cargo following brain trauma. EVs are membrane bound, and thus are not subject to the same degradation that conventional serum biomarkers face, making them ideal biomarker candidates. Salivary EVs in particular can be isolated based on tissue specificity and have well established roles in the detection of numerous other disease states, including oral squamous cell carcinoma [79]. Bolstering their utility as a unique biomarker, upon analysis and characterization of patients that had sustained mTBI it became apparent that EVs isolated from saliva had numerous neural markers on them, confirming their origin from brain parenchyma [66]. Following analysis of the expression of Alzheimer disease (AD) genes in patients who had suffered mTBI vs. healthy controls, multiple important AD specific genes were significantly upregulated in patients that had suffered mTBI when compared to healthy controls; allowing for the identification of mTBI-specific genetic profiles derived from neural derived EVs. The potential characterization of early mTBI biomarker genes, including (but not limited to) CTSD, CDC2 and casein kinase (CSNK1A1) is being explored [66]. Longitudinal analysis of these patients coupled with further analysis of the identified surrogate markers allows for possible prognostication of mTBI patients in regard to severity of post-TBI concussion symptoms, chronicity of symptomology and potential recovery. This is all made possible by the ubiquitous

EVs secreted by blood borne hematologic cancer have modulating affects impacting a variety of cancer hallmarks. EVs have a direct effect on phenotypic and genotypic changes, highlighting the central role of EVs in the progressions and

Pathways involved in angiogenesis have been shown to modulate cancer progressions and chemotherapeutic evasion in multiple models [61]. Vesicles shed by chronic lymphocytic and myelogenous leukemia (CLL, CML) transmit cargo

*DOI: http://dx.doi.org/10.5772/intechopen.86845*

**2.6 Neural-derived EVs in traumatic brain injury**

prostate, and lung [59].

and specific nature of EVs.

**2.7 Hematologic malignancies**

reversal of hematologic malignancies.

*2.7.1 Impact on various leukemias*

*Stem Cells and Extracellular Vesicles: Biological Regulators of Physiology and Disease DOI: http://dx.doi.org/10.5772/intechopen.86845*

Other populations of "therapeutic" EVs (outside of the bone marrow) have also been isolated and applied: EVs isolated from normal prostate cells acquired via patient biopsy reverse the resistance of malignant prostate cells to various drugs. On the contrary we have shown that EVs derived from cancerous cells can drive cancer progression and enhance resistance to certain chemotherapies, which again highlights the specificity, plurality, the yin/yang of EV functionality [61]. Panagopoulos et al. confirmed much of this work, showing that vesicles from both *in vivo* prostate cancer cell and explant cultured prostate cancer cells can induce cellular changes that produce a neoplastic phenotype in normal prostate cell lines [65]. These results were also reproduced using vesicles from patients with other malignancies, namely prostate, and lung [59].

### **2.6 Neural-derived EVs in traumatic brain injury**

Our group has developed a unique biomarker system focusing on EVs isolated from the saliva of patients who have experienced mild traumatic brain injury (mTBI) [66]. Rather than conventional human serum isolation, this has been a novel protocol allowing for the easily accessible collection of saliva laden with EVs that have freely trafficked from injured brain parenchyma into the saliva—allowing for a representative sample which captures the shift of various EV populations and cargo following brain trauma. EVs are membrane bound, and thus are not subject to the same degradation that conventional serum biomarkers face, making them ideal biomarker candidates. Salivary EVs in particular can be isolated based on tissue specificity and have well established roles in the detection of numerous other disease states, including oral squamous cell carcinoma [79]. Bolstering their utility as a unique biomarker, upon analysis and characterization of patients that had sustained mTBI it became apparent that EVs isolated from saliva had numerous neural markers on them, confirming their origin from brain parenchyma [66]. Following analysis of the expression of Alzheimer disease (AD) genes in patients who had suffered mTBI vs. healthy controls, multiple important AD specific genes were significantly upregulated in patients that had suffered mTBI when compared to healthy controls; allowing for the identification of mTBI-specific genetic profiles derived from neural derived EVs. The potential characterization of early mTBI biomarker genes, including (but not limited to) CTSD, CDC2 and casein kinase (CSNK1A1) is being explored [66]. Longitudinal analysis of these patients coupled with further analysis of the identified surrogate markers allows for possible prognostication of mTBI patients in regard to severity of post-TBI concussion symptoms, chronicity of symptomology and potential recovery. This is all made possible by the ubiquitous and specific nature of EVs.

### **2.7 Hematologic malignancies**

EVs secreted by blood borne hematologic cancer have modulating affects impacting a variety of cancer hallmarks. EVs have a direct effect on phenotypic and genotypic changes, highlighting the central role of EVs in the progressions and reversal of hematologic malignancies.

### *2.7.1 Impact on various leukemias*

Pathways involved in angiogenesis have been shown to modulate cancer progressions and chemotherapeutic evasion in multiple models [61]. Vesicles shed by chronic lymphocytic and myelogenous leukemia (CLL, CML) transmit cargo

*Innovations in Cell Research and Therapy*

cellular origin and physiological state [48].

across numerous solid and hematological cancers.

**2.4 Breast**

(phagocytosis/endocytosis) of vesicle contents into the recipient cells. Effector molecules (e.g., mRNA), non-coding regulatory RNAs (e.g., microRNAs or miRNAs), proteins, and transcription factors can all be delivered, each having short- and longterm implications on effector cell phenotype and function [58, 59]. As discussed, various other studies have also highlighted the ability of EVs to directly transfer relatively larger molecules such as cellular receptors, major histocompatibility complex (MHC) molecules, antigens, as well as entire organelles, some containing fully intact mitochondria, lysosomes, Golgi and intermediate filaments [60].

EV studies relating to both breast and prostate cancer highlight many of the salient principles observed in lung cancer studies, and also exhibit the promising roles that EV play in evolving chemoresistance. As we will come to see across a variety of cancer models and disease states, EV function carries great pluralityexhibiting multiple, and often times contradictory effects depending on their

In breast cancer, while healthy mammary epithelial cells within the breast stroma secrete EVs that prevent the release of breast cancer derived EVs, the EVs shed by the disease cells promote the opposite, imparting an immense impact on chemoresistance. Cancer derived EVs are known to shuttle pro-oncogenic proteins and nucleic acids from diseases cells to surrounding healthy stroma and connective tissue [61]. Zhou et al. reported that breast cancer secreted exosomes are enriched in particular RNA species, such as miR-105, which destroys the vascular endothelial barrier, allowing cancer to enter the circulation and spread [62]. Studies employing fluorescently labeled miRNA-loaded EVs showed that tamoxifen resistant breast cancer cells *in vitro* can carry multiple miRNA profiles. EVs packed with fluorescently-tagged miR-221/222 can also shuttle their cargo to sensitive cells of the same type, thereby transferring resistance RNAs which effectively reduced gene expression of P27 and estrogen receptor-α (ERα) in target cell. The loss of p27 has been linked to drug resistance, as it is able to take a cell that is arrested in its cell cycle and stimulate its reentry back into active cycling [63]. However, as discussed, healthy stromal cells counteract the effects of oncogenic vesicles. This competition between "good" and "bad" vesicles is a fine balance; a yin/yang that loses equilibrium as cancer overwhelms healthy stoma. When stromal cells are outcompeted and significantly influenced by oncogenic EV signaling, the now altered stroma in turn activates STAT1 and NOTCH3 signaling in breast cancer cells, promoting cancer initiating cell populations responsible for drug resistance and nascent tumor formation [64]. This is a common theme in EV-mediated cancer progression which we will see is universal

Human bone marrow mesenchymal stem cell (MSC) derived EVs are involved in the modulation of cell signaling, cellular differentiation, and proliferation—and this is seen across multiple disease paradigms. These regenerative EVs have been shown to reverse the malignant phenotype in prostate and colorectal cancer, recovering function in a murine model of AKI, as well as mitigating radiation damage to marrow [36]. In models of prostate cancers, the reversal of taxane resistance and tumorigenic phenotype in a human prostate carcinoma cell line (as well as human explants) can be accomplished by treatment with healthy MSC-derived EVs [61].

**134**

**2.5 Prostate cancer**

containing a myriad of cancer-inducing factors, such as rapamycin/p70S6K/ hypoxia-inducible factor-1α axis. Similar to lung cancer-derived EVS, these CMLderived vesicles have been shown to bolster the survival of CML B-cells via the establishment and proliferation of vascular endothelial growth factor within the forming leukemic bone marrow stromal cells [67, 69]. In multiple myeloma (MM) models, bone marrow stromal cell-derived exosomes, mediate cellular communication by transferring mRNAs, miRNAs, and proteins important in proliferation, survival, and chemoresistance [77]. Experiments utilizing *in vivo* mouse K562 CML cells showed that neogenic angiogenesis can be induced by immortalized myelogenous leukemia cell line K562 exosomes, as well as neogenic changes in human umbilical endothelial cells [69].

Other hematologic malignancies show similar cancer induction potential. CMLderived EVs given to rat models can induce CML-like characteristics via the transfer of their oncogenic cargo [67]. Bone marrow stromal cells respond to this influence by producing interleukin (IL)-8 (mRNA and protein), a potent pro-angiogenic factor that modulates both *in vitro* and *in vivo* the leukemia cell malignant phenotype [67]. In our own established acute myeloid leukemia (AML) model, we explored the potential of human bone marrow MSC-derived EVs as a direct adjunct therapy for AML. Our studies indicated that the killing potential of cytarabine, at even relatively low doses, is potentiated by the addition of healthy MSC-derived EVs. We believe EVs can also alter a cancer cell's sensitivity to chemotherapy via EV guided horizontal information transfer. This has implications directly on the cell itself but likely also impacts the surrounding stroma in order to further promote oncogenic growth and drug resistance of leukemia cells [68]. In models of MM when marrow MSC-derived exosomes are cultured with cancerous MM cells, there is a significant increase in multiple anti-apoptotic pathways which promoted MM cell viability. These exosomes, derived from stromal cells within a microenvironment amidst developing active cancer, were also able to induce drug resistance to the proteasome inhibitor bortezomib via activation of several survival relevant pathways, including c-Jun N-terminal kinase, p38, p53, and Akt [97].

### *2.7.2 Impact on chemo-resistance*

Cancer derived EVs play a central role in facilitating the escape of cell death, by cancer cell. Proteins such as BCL-2, MCL-1, BCL-X, and BAX as well as other cell death-related proteins were shown to be more concentrated in the EV of apoptosisresistant primary AML blasts than EVs from more sensitive AML cells [70]. Via confocal-microscopy-based colocalization studies, the direct transfer of EVs from resistant to sensitive cells has been observed. Leukemia derived EV harbor multiple bioactive lipids, proteins and miRNAS important in chemoresistance. Ibrutinib is a drug used clinically to combat leukemia. Analysis of plasma samples collected from CLL patients showed exosomes bearing unique micro-RNA prolife, including miR-29 family, miR-150, miR-155, and miR-223, showed a different exosome profile from what is seen when disease is suppressed with ibrutinib treatment—perhaps, indicating the potential pathophysiology by which cancerous EVs impart resistance, as well as creating a potential for biomarker identification [75]. EVs packed with miR-221/222, from tamoxifen resistant MCF-7 breast cancer cells, can shuttle their cargo to sensitive cells of the same type, thereby transferring resistance.

### *2.7.3 Impact on the cancer microenvironment*

Healthy bone marrow stroma likely functions to maintain and protect healthy bone marrow stroma from nascent cancer. At first detection of threat, the bone

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*Stem Cells and Extracellular Vesicles: Biological Regulators of Physiology and Disease*

synergistic adjunct to conventional AML therapies, such as cytarabine.

marrow microenvironment and residing cells, such as MSCs, combat early cancer much of this is likely EV mediated. Our established leukemic cell model has established this hypothesis, indicating that MSC-EVs impart a robust anti-proliferative and pro-apoptotic effect on leukemic cells *in vitro*. We also have preliminary data using EVs toward clinically relevant endpoints, and have showed they serve as a

As discussed, the bone marrow stroma can be recapitulated by active cancer via multiple EV-dependent mechanisms. Leukemic models have shown the net effect of EV-modulation translated to a phenotypic change of the bone marrow stromal cells toward a more inflammatory signature that resembles the phenotype of cancer-associated fibroblasts (CAFs) [72]. CAFs show enhanced proliferation, migration, and secretion of inflammatory cytokines, all contributing to a tumor-supportive niche [72]. As a result, stromal cells exposed to a leukemic EVs are not killed but "reprogrammed" to be pro-oncogenic and support tumor growth. As discussed, EV populations change depending on disease state [48]. In the case of CLL, as leukemic cancer cells progress varying EV populations establish control within the microenvironment. CLL-derived EVs rapidly deliver their biologic cargo to the surrounding stromal cells, and promoting CAF phenotypes with enhanced proliferative and migratory properties [72]. CLL models have shown that CAF-derived factors may also have an immunogenic effect on the T and myeloid cells, altering their phenotypes into immunosuppressive and tumor-promoting Th2/M2-like cells, respectively. These modifications lead to defective T-cell and myeloid cell immune responses and an inflammatory milieu

Leukemic EVs impart genotypic and phenotypic effects on all components of the leukemic microenvironment. The bony endosteal compartment of the bone marrow niche, composed of osteocytes/osteoblasts/osteoclasts, is reprogrammed by AML derived EVs toward inflammatory myelofibrotic cells. These cells support leukemic growth and support BM fibrosis, a well-established risk factor for leukemia [75]. Metastasis is crucial to cancer survival. Leukemia derived EVs have also been shown to disturb the architecture of multiple tight junction proteins in cells of the basement membrane, allowing cancer to detach, mobilize, and metastasize beyond in situ disease. Leukemic EVs can also bolster angiogenesis [75]. *In vitro* studies, first reported by Umezu et al., clearly showed leukemic cell to endothelial cell communication via exosomal miRNAs by fluorophore signaling localization, allowing for the creation of new blood vessels to feed cancer growing in newly seeded microenvironmental niches [76]. Microenvironment stromal cells have been shown to directly take up cargo from EVs fluorescently labeled with GFP. As we've seen in lung and breast cancer cells, Boelens et al. showed that this cross talk is reciprocal and that when stromal cells are influenced by oncogenic EV signaling the stromal cells themselves in turn activate STAT1 and NOTCH3 signaling in developing cancer cells. This cell signaling in turn leads to cell populations responsible for drug resistance and nascent tumor formation [64, 78]. Schepers et al. have shown that AML cells (likely via an EV directed mechanism) cause numerous chromosomal anomalies and genetic mutations within the surrounding stroma, thereby altering the biology of the stem cell continuum away from normal hematopoiesis, and toward transforming bone marrow stem cells toward immature progenitors that will subsequently develop into leukemic blasts or altered cancer-stem cells capable of supporting a pro-leukemic environment [75]. The CAF phenotype promoted by tumor-derived EVs, has, itself, secondary effects on endothelial cells, increasing angiogenesis. The sum and synergy of all of these EV-directed microenvironment modulations means the leukemia-modified stroma favors leukemic blast prolifera-

*DOI: http://dx.doi.org/10.5772/intechopen.86845*

characteristic of CLL promotion [75].

tion while stymieing normal hematopoiesis [69, 76].

### *Stem Cells and Extracellular Vesicles: Biological Regulators of Physiology and Disease DOI: http://dx.doi.org/10.5772/intechopen.86845*

marrow microenvironment and residing cells, such as MSCs, combat early cancer much of this is likely EV mediated. Our established leukemic cell model has established this hypothesis, indicating that MSC-EVs impart a robust anti-proliferative and pro-apoptotic effect on leukemic cells *in vitro*. We also have preliminary data using EVs toward clinically relevant endpoints, and have showed they serve as a synergistic adjunct to conventional AML therapies, such as cytarabine.

As discussed, the bone marrow stroma can be recapitulated by active cancer via multiple EV-dependent mechanisms. Leukemic models have shown the net effect of EV-modulation translated to a phenotypic change of the bone marrow stromal cells toward a more inflammatory signature that resembles the phenotype of cancer-associated fibroblasts (CAFs) [72]. CAFs show enhanced proliferation, migration, and secretion of inflammatory cytokines, all contributing to a tumor-supportive niche [72]. As a result, stromal cells exposed to a leukemic EVs are not killed but "reprogrammed" to be pro-oncogenic and support tumor growth. As discussed, EV populations change depending on disease state [48]. In the case of CLL, as leukemic cancer cells progress varying EV populations establish control within the microenvironment. CLL-derived EVs rapidly deliver their biologic cargo to the surrounding stromal cells, and promoting CAF phenotypes with enhanced proliferative and migratory properties [72]. CLL models have shown that CAF-derived factors may also have an immunogenic effect on the T and myeloid cells, altering their phenotypes into immunosuppressive and tumor-promoting Th2/M2-like cells, respectively. These modifications lead to defective T-cell and myeloid cell immune responses and an inflammatory milieu characteristic of CLL promotion [75].

Leukemic EVs impart genotypic and phenotypic effects on all components of the leukemic microenvironment. The bony endosteal compartment of the bone marrow niche, composed of osteocytes/osteoblasts/osteoclasts, is reprogrammed by AML derived EVs toward inflammatory myelofibrotic cells. These cells support leukemic growth and support BM fibrosis, a well-established risk factor for leukemia [75]. Metastasis is crucial to cancer survival. Leukemia derived EVs have also been shown to disturb the architecture of multiple tight junction proteins in cells of the basement membrane, allowing cancer to detach, mobilize, and metastasize beyond in situ disease. Leukemic EVs can also bolster angiogenesis [75]. *In vitro* studies, first reported by Umezu et al., clearly showed leukemic cell to endothelial cell communication via exosomal miRNAs by fluorophore signaling localization, allowing for the creation of new blood vessels to feed cancer growing in newly seeded microenvironmental niches [76]. Microenvironment stromal cells have been shown to directly take up cargo from EVs fluorescently labeled with GFP. As we've seen in lung and breast cancer cells, Boelens et al. showed that this cross talk is reciprocal and that when stromal cells are influenced by oncogenic EV signaling the stromal cells themselves in turn activate STAT1 and NOTCH3 signaling in developing cancer cells. This cell signaling in turn leads to cell populations responsible for drug resistance and nascent tumor formation [64, 78]. Schepers et al. have shown that AML cells (likely via an EV directed mechanism) cause numerous chromosomal anomalies and genetic mutations within the surrounding stroma, thereby altering the biology of the stem cell continuum away from normal hematopoiesis, and toward transforming bone marrow stem cells toward immature progenitors that will subsequently develop into leukemic blasts or altered cancer-stem cells capable of supporting a pro-leukemic environment [75]. The CAF phenotype promoted by tumor-derived EVs, has, itself, secondary effects on endothelial cells, increasing angiogenesis. The sum and synergy of all of these EV-directed microenvironment modulations means the leukemia-modified stroma favors leukemic blast proliferation while stymieing normal hematopoiesis [69, 76].

*Innovations in Cell Research and Therapy*

umbilical endothelial cells [69].

c-Jun N-terminal kinase, p38, p53, and Akt [97].

*2.7.3 Impact on the cancer microenvironment*

*2.7.2 Impact on chemo-resistance*

containing a myriad of cancer-inducing factors, such as rapamycin/p70S6K/ hypoxia-inducible factor-1α axis. Similar to lung cancer-derived EVS, these CMLderived vesicles have been shown to bolster the survival of CML B-cells via the establishment and proliferation of vascular endothelial growth factor within the forming leukemic bone marrow stromal cells [67, 69]. In multiple myeloma (MM) models, bone marrow stromal cell-derived exosomes, mediate cellular communication by transferring mRNAs, miRNAs, and proteins important in proliferation, survival, and chemoresistance [77]. Experiments utilizing *in vivo* mouse K562 CML cells showed that neogenic angiogenesis can be induced by immortalized myelogenous leukemia cell line K562 exosomes, as well as neogenic changes in human

Other hematologic malignancies show similar cancer induction potential. CMLderived EVs given to rat models can induce CML-like characteristics via the transfer of their oncogenic cargo [67]. Bone marrow stromal cells respond to this influence by producing interleukin (IL)-8 (mRNA and protein), a potent pro-angiogenic factor that modulates both *in vitro* and *in vivo* the leukemia cell malignant phenotype [67]. In our own established acute myeloid leukemia (AML) model, we explored the potential of human bone marrow MSC-derived EVs as a direct adjunct therapy for AML. Our studies indicated that the killing potential of cytarabine, at even relatively low doses, is potentiated by the addition of healthy MSC-derived EVs. We believe EVs can also alter a cancer cell's sensitivity to chemotherapy via EV guided horizontal information transfer. This has implications directly on the cell itself but likely also impacts the surrounding stroma in order to further promote oncogenic growth and drug resistance of leukemia cells [68]. In models of MM when marrow MSC-derived exosomes are cultured with cancerous MM cells, there is a significant increase in multiple anti-apoptotic pathways which promoted MM cell viability. These exosomes, derived from stromal cells within a microenvironment amidst developing active cancer, were also able to induce drug resistance to the proteasome inhibitor bortezomib via activation of several survival relevant pathways, including

Cancer derived EVs play a central role in facilitating the escape of cell death, by cancer cell. Proteins such as BCL-2, MCL-1, BCL-X, and BAX as well as other cell death-related proteins were shown to be more concentrated in the EV of apoptosisresistant primary AML blasts than EVs from more sensitive AML cells [70]. Via confocal-microscopy-based colocalization studies, the direct transfer of EVs from resistant to sensitive cells has been observed. Leukemia derived EV harbor multiple bioactive lipids, proteins and miRNAS important in chemoresistance. Ibrutinib is a drug used clinically to combat leukemia. Analysis of plasma samples collected from CLL patients showed exosomes bearing unique micro-RNA prolife, including miR-29 family, miR-150, miR-155, and miR-223, showed a different exosome profile from what is seen when disease is suppressed with ibrutinib treatment—perhaps, indicating the potential pathophysiology by which cancerous EVs impart resistance, as well as creating a potential for biomarker identification [75]. EVs packed with miR-221/222, from tamoxifen resistant MCF-7 breast cancer cells, can shuttle their

cargo to sensitive cells of the same type, thereby transferring resistance.

Healthy bone marrow stroma likely functions to maintain and protect healthy bone marrow stroma from nascent cancer. At first detection of threat, the bone

**136**
