**2.3. MSCs in tumor microenvironments can modulate the immune response**

*Immune response in tumor microenvironments*: In addition to protecting the host from external invaders, the immune system recognizes tumor antigens and eliminates malignant tumors [58]. Therefore, tumor growth, invasion, and metastasis are important aspects of the tumor's immune escape mechanism [59, 60]. During tumor initiation, TAMs and MSCs migrate to the tumor microenvironments. TAMs act as the main inflammatory component of the tumor microenvironment [61, 62]. In contrast, TAMs can show antitumor activities linked to the M1 phenotype via IFN-γ, TNF-α, TGF-β, PGE2, and IL-10 [72, 77–82]. Also, M1 TAMs secrete free oxygen radicals, nitrogen radicals, and pro-inflammatory interleukins (e.g., IL-1β, IL-6, IL-12, IL-23, and TNF-β) facilitating the killing of tumoral cells. The MSCs can be activated by the pro-inflammatory cytokines IFN-γ, TNF-α, or IL-1βn in tumor microenvironments [30, 52, 69, 83, 84]; additionally, the tumor cells and M2 produce immunomodulatory molecules, such as IDO, PGE2, IL-6, IL-10, HLA-G5, and NO [64, 65]. IDO is the critical rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway, resulting in tryptophan depletion and halting the growth of various cells. In tumor microenvironments, MSCs can be activated by pro-inflammatory cytokines IFN-γ, TNF-α, or IL-1β [66, 67]. Within the immunomodulatory molecules secreted by MSCs, Prostaglandin E2 (PGE2) has a multifunctional role in pathological processes including the regulation of inflammation and cancer. The production of PGE2 by MSCs increases after stimulation with TNF-α or IFN-γ. In addition, PGE2 increases the level of expression of IL-10 and decreases the expression of TNF-α, IFN-γ, and IL-12 in cells of the developing immune system and of macrophages [68, 69]. PGE2 regulates the secretion of IFN-γ and IL-4 in Th1 and Th2 cells, respectively, and promotes proliferation of Treg cells [19]. It has been reported that IL-6 secreted by MSCs inhibits monocyte differentiation toward CD and decreases the activation capacity of CD to T cells [70, 71]. In addition, IL-6 secreted by MSCs resulted in a delay in apoptosis of lymphocytes and neutrophils [72, 73]. Another important molecule in the moderation of the immune response is nitric oxide (NO). NO is produced by inducible NO synthase (iNOS) through stimulation by inflammatory factors such as IL-1, IFN-γ, and TNF- α [72, 74] and also inhibits the functions of T cells [75]. In contrast to the reported evidence that MSCs can suppress the immune response, Ohlsson et al. reported that administration of tumor cells and MSCs simultaneously caused an increase in the inflammatory component in the stroma, mainly composed of granulocytes and monocytes, whereas when administered separately, this was not observed [75]. In a rat-induced colon cancer model, it was observed that the colon tumor cells inoculated in a gelatin matrix, when implanted subcutaneously, developed larger tumors than animals that surgically received colon cancer cells combined with MSCs. MSCs inhibited rat colon carcinoma by increasing the leukocyte infiltrate [75]. It was observed that the increase in infiltrations of both granulocytes and macrophages was much higher in rats co-injected with tumor cell lines and MSCs than in rats injected with tumors without MSCs. These data suggested that MSCs had pro-inflammatory effects in this model. In this same work, a greater degree of infiltration of granulocytes and macrophages was observed, but to a lesser extent, when only MSCs were added to the gelatin. [75].

tumor size was observed along with the appearance of stromal cells of the implant; MSCs

Multipotent Stromal Cells in a Tumor Microenvironment http://dx.doi.org/10.5772/intechopen.77345 69

The innate tropism of MSCs to injured sites, including established tumors, has been widely reported, although the mechanism behind it has not yet been fully elucidated that the proinflammatory cytokines secreted by the reactive stroma are involved [24]. The most accepted explanation to date is that the tumors behave as unresolved wounds since their stroma closely resemble the healing granulation tissue and produce cytokines, chemokines, and other chemotactic agents [27] and the chemotactic properties of MSC are similar to those of leukocytes [87, 88]. The tropism of MSCs for tumors has been widely studied and exploited with very good results for the supply of antitumor drugs in animal models of lung and breast cancer, melanoma, and glioma [88].

Like any other cell in culture, when long-term MSCs are manipulated in vitro, they can have chromosomal aberrations and produce tumors in healthy animals because they undergo cell crisis [89]; this has been observed mainly in mouse cells, which require extensive cultures to produce a significant number of MSCs free of hematopoiesis [90]. For example, it has been demonstrated that the intravenous administration of MSCs derived from bone marrow in NOC/SCID mice generates cellular aggregates that are retained in the pulmonary capillaries, forming emboli when they are injected in large quantities. Once lodged in the capillaries, they expand and invade the lung parenchyma and form tumor nodules [90]. These lesions rarely contain lung epithelial cells, but have the characteristics of cartilage and immature bone that resembles a well differentiated osteosarcoma. However, until now, no type of transformation has been demonstrated by human MSCs adequately expanded ex vivo for cell therapy (no more than five passages) [90]. The Canadian Trial Critical Care Trials Group recently reported a meta-analysis of randomized, nonrandomized, controlled, and uncontrolled clinical trials, phase I and phase II, where they found no reports associating the administration of autologous or allogeneic MSCs and tumor formation in 36 clinical studies [91]. However, a longer

follow-up is necessary to evaluate the tumorigenic potential of human MSCs.

Along with the increasing number of cancer metastasis mechanisms being discovered, it has been reported that MSCs can induce metastasis in vitro and in vivo [78, 83, 92, 93]. Previous studies showed when human breast cancer cells were co-incubated with MSCs, the gene expression of onco and proto-oncogenes in breast cancer cells was upregulated [48]. These molecular and morphological alterations were accompanied by a metastatic phenotype. Breast cancer cells induce the motility of tumor cells through the secretion of CCL5, increasing invasiveness and metastatic potentials [83]. The invasion mediated by CCL5/RANTES is also

On the contrary, it has been shown that the increase in metastatic capacity when MSCs are coinjected with tumor cells is reversed when the MSCs are injected in a different site from the tumor and this anti-metastatic effect by the MSCs remains independent of tumor distance [83]. Other mechanisms, such as the induction of EMT, the regulation of CSC, and the displacement of mesenchymal niches are also implicated in tumor metastasis [95]. Breast cancer cells cocultured with MSCs derived from human bone marrow (ratio 1:1) upregulate the expression of

closely related to the increased activity of matrix metalloproteinase 9 (MMP-9) [94].

**2.5. MSCs might promote metastasis**

derived from adipose tissue may promote tumor growth [86].

#### **2.4. MSCs may promote tumor growth**

The tumor microenvironment, is composed of cancer cells, noncancerous cells, and stromal cells, all this as a whole influences the growth of the tumor [28]. The tumor stroma hosts many types of cells, as well as MEC. These cells include different types of immune cells, fibroblasts, endothelial cells, and myofibroblasts [28]. MSCs perform homing at tumor sites and then integrate into the tumor stroma [76, 77]. These cells interact with each other and with cancer cells, resulting in the promotion of tumor growth. The ability of MSCs to promote tumor growth and metastasis was demonstrated in murine models of breast cancer with similar results from cancer cells co-implanted with MSCs [24, 78, 79]. In turn, it was observed that allogenic mice transplanted with B16 melanoma cells did not in the development of tumors when B16 cells were co-injected with MSCs [80]. This finding indicates that MSCs exert essential immunosuppressive and antitumor effects at the onset of the tumor. Human bone marrow-derived MSCs have increased the growth of estrogen receptor-alpha (Erα) positive breast cancer cell lines: T47D, BT474, and ZR-75-1, in an in vitro three-dimensional tumor environment assay, in contrast, have had no effect on the ERα negative cell line MDA-MB-231 [81]. Nonetheless, the growth rate of (another ERα negative cell line) was high in the presence of human MSCs [81]. Another study showed both human fetal MSCs transplanted subcutaneously into BALB/c-nu/nu mice with human adipose-derived MSCs alone or together with cell lines F6 (human mesenchymal stem cells F6) or SW480 (human colon adenocarcinoma cell line) in a ratio 1:1 or 1:10, favoring the growth of these tumor cell lines [79]. Other authors reported that tumor cells procured from primary breast cancer were grown in the presence of human bone marrow-derived MSCs (ratio 1:1). Additionally, this was tested on secondary mice, where a greater tumor-producing ability compared with the cells obtained from primary tumors and grown in the absence of MSCs was observed [82]. In addition, tumor incidence and/or size [83, 84] as well as tumor vascularization [30] increased when breast, lung, colon, or prostate tumor cells were co-injected with MSCs independent of the source of origin from the same. Similar results were observed with MSCs derived from adipose tissue or human bone marrow. The same was demonstrated with tumor cells of osteosarcoma, melanoma, and glioma [85]. Another interesting observation relates to adipose tissue adjacent to the tumor implant (e.g., lung cancer models or to Kaposi's sarcoma xenografts), where a substantial increase in tumor size was observed along with the appearance of stromal cells of the implant; MSCs derived from adipose tissue may promote tumor growth [86].

The innate tropism of MSCs to injured sites, including established tumors, has been widely reported, although the mechanism behind it has not yet been fully elucidated that the proinflammatory cytokines secreted by the reactive stroma are involved [24]. The most accepted explanation to date is that the tumors behave as unresolved wounds since their stroma closely resemble the healing granulation tissue and produce cytokines, chemokines, and other chemotactic agents [27] and the chemotactic properties of MSC are similar to those of leukocytes [87, 88]. The tropism of MSCs for tumors has been widely studied and exploited with very good results for the supply of antitumor drugs in animal models of lung and breast cancer, melanoma, and glioma [88].

Like any other cell in culture, when long-term MSCs are manipulated in vitro, they can have chromosomal aberrations and produce tumors in healthy animals because they undergo cell crisis [89]; this has been observed mainly in mouse cells, which require extensive cultures to produce a significant number of MSCs free of hematopoiesis [90]. For example, it has been demonstrated that the intravenous administration of MSCs derived from bone marrow in NOC/SCID mice generates cellular aggregates that are retained in the pulmonary capillaries, forming emboli when they are injected in large quantities. Once lodged in the capillaries, they expand and invade the lung parenchyma and form tumor nodules [90]. These lesions rarely contain lung epithelial cells, but have the characteristics of cartilage and immature bone that resembles a well differentiated osteosarcoma. However, until now, no type of transformation has been demonstrated by human MSCs adequately expanded ex vivo for cell therapy (no more than five passages) [90]. The Canadian Trial Critical Care Trials Group recently reported a meta-analysis of randomized, nonrandomized, controlled, and uncontrolled clinical trials, phase I and phase II, where they found no reports associating the administration of autologous or allogeneic MSCs and tumor formation in 36 clinical studies [91]. However, a longer follow-up is necessary to evaluate the tumorigenic potential of human MSCs.
