**2.5. MSCs might promote metastasis**

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].

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

**2.4. MSCs may promote tumor growth**

68 Stromal Cells - Structure, Function, and Therapeutic Implications

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

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

**Figure 3.** Interaction of tumor cells with MSCs during cancer progression. MSCs can interact with tumor cells at the primary site of the tumor and during metastasis by promoting cancer progression and invasion. One of the mechanisms involved in these processes is that MSCs induce EMT in tumor cells through close cell-cell contact, which could be due in part to the secretion of TGF-β [38, 82]. Studies have shown that secretion of osteopontin (OPN) by tumor cells, induces MSCs to secrete chemokine (motif CC) ligand 5 (CCL5) by stimulating the metastasis of the cancer cell through interaction with its specific chemokine receptor CC type 5 (CCR5) [84]. The migration of tumor cells to and from the metastatic site is mediated by SDF-1, a factor secreted by bone marrow MSCs, which interacts with the CXC receptor chemokine receptor type 4 (CXCR4) expressed in human tumor cell lines of the breast and prostate [33, 101, 102] (adapted from Sarah M. Ridge, Francis J. Sullivan and Sharon A. Glynn. Mesenchymal Stem Cells key players in cancer. Molecular Cancer, Feb. 2017 13:31 1-10. https://doi.org/10.1186/s12943-017-0597-8).

oncogenes and proto-oncogenes associated with tissue invasion, angiogenesis, and apoptosis (i.e., N-cadherin, vimentin, Twist, Snail, and E-cadherin) [48]. These molecular changes have been accompanied by morphological and growth alterations, which are characteristics of a more metastatic phenotype. It has been seen that 0.5 × 105 breast cancer cells co-injected subcutaneously with 1.3 × 106 MSCs derived from human bone marrow have significantly increased the rate of lung metastases in NOD/SCID mice. This effect was lost when the MSCs derived from bone marrow were injected separately from the tumor cells [83]. On the other hand, it has been shown that MSCs derived from bone marrow facilitate cancer cells [MCF-7, T47D low invasive cell lines, and factor 1 derived from stromal cells (SDF-1) null MDA-MB-231 highly aggressive] target to the bone marrow and modify the metastatic niche through the secretion of trophic factor (SDF-1 and CXCR4) and improved neovascularization in a xenogeneic mouse model (**Figure 3**) [96].

(**Figure 1**) (reviewed in [30]). In this regard, it is believed that MSCs suppress tumor growth by increasing the infiltration of inflammatory cells [97], inhibit angiogenesis [34], suppress Wnt and AKT signaling, and induce cell cycle arrest and apoptosis [32, 35, 36]. Recently, Ryu et al. reported that when the MSCs derived from adipose tissue were cultured at a high cell density, they synthesized IFN-beta, which then suppressed the growth of MCF-7 cells [98]. In addition, MSCs prepared with IFN-gamma or cultured with three-dimensional systems can express TRAIL, which induces specific apoptosis of tumor cells [97, 99]. In particular, it was demonstrated that in vitro culture of MSCs under hypoxic conditions increased cell proliferation. In addition, the expression of Rex-1 and Oct-4 was increased, leading to the conclusion that MSC scion was increased during hypoxia [100]. In addition, under hypoxic and starvation conditions, MSCs can survive through autophagy and release many antiapoptotic or pro-survival factors such as VEGF, FGF-2, PDGF, HGF, brain-derived neurotropic factor

articles/mesenchymal-stem-cells-exhibit-tgf-beta-dependent-tropism-gliomas-and-inhibit-angiogenesis).

**Figure 4.** Mesenchymal stem cells can perform homing to the tumor environment. Studies in murine models of gliomas have reported that they can be directed to the tumor site through TGF-beta signaling and, once there, they can suppress angiogenesis within the tumor microenvironment. The proposed mechanisms are the following in sequential order: (1) the glioma microenvironment contains high levels of the proangiogenic cytokine, IL-1 beta. (2) Signaling through the NF-kappa B axis increases the expression of Cathepsin B and activates extracellular matrix remodeling programs that promote angiogenesis. (3) The increase in beta IL-1 potentiates the signaling of PDGF-BB, which promotes the migration of endothelial progenitor cells. (4) Glioma stem cells within a tumor secrete TGF-beta and recruit MSCs through TGF-beta RII and the endoglin/CD105 co-receptor. (5) Within the glioma microenvironment, the presence of MSCs reduces the levels of beta IL-1, negatively regulating Cathepsin B and decreasing PDGF R-beta signaling. It is believed that the downregulation of these signaling cascades in the presence of MSCs inhibits angiogenesis, reduces the density of microvessels and suppresses glioma growth. (Adapted from: https://www.rndsystems.com/resources/

Multipotent Stromal Cells in a Tumor Microenvironment http://dx.doi.org/10.5772/intechopen.77345 71

#### **2.6. MSCs might inhibit tumor growth**

MSCs can not only secrete cell regenerative factors continuously but also secrete factors in response to other various stimuli [97]. Tumor progression is accompanied by hypoxia, starvation, and inflammation. Although many studies have shown that MSCs have tumor promoting properties, many other studies have shown that MSCs have tumor suppressor properties

**Figure 4.** Mesenchymal stem cells can perform homing to the tumor environment. Studies in murine models of gliomas have reported that they can be directed to the tumor site through TGF-beta signaling and, once there, they can suppress angiogenesis within the tumor microenvironment. The proposed mechanisms are the following in sequential order: (1) the glioma microenvironment contains high levels of the proangiogenic cytokine, IL-1 beta. (2) Signaling through the NF-kappa B axis increases the expression of Cathepsin B and activates extracellular matrix remodeling programs that promote angiogenesis. (3) The increase in beta IL-1 potentiates the signaling of PDGF-BB, which promotes the migration of endothelial progenitor cells. (4) Glioma stem cells within a tumor secrete TGF-beta and recruit MSCs through TGF-beta RII and the endoglin/CD105 co-receptor. (5) Within the glioma microenvironment, the presence of MSCs reduces the levels of beta IL-1, negatively regulating Cathepsin B and decreasing PDGF R-beta signaling. It is believed that the downregulation of these signaling cascades in the presence of MSCs inhibits angiogenesis, reduces the density of microvessels and suppresses glioma growth. (Adapted from: https://www.rndsystems.com/resources/ articles/mesenchymal-stem-cells-exhibit-tgf-beta-dependent-tropism-gliomas-and-inhibit-angiogenesis).

oncogenes and proto-oncogenes associated with tissue invasion, angiogenesis, and apoptosis (i.e., N-cadherin, vimentin, Twist, Snail, and E-cadherin) [48]. These molecular changes have been accompanied by morphological and growth alterations, which are characteristics of a more

**Figure 3.** Interaction of tumor cells with MSCs during cancer progression. MSCs can interact with tumor cells at the primary site of the tumor and during metastasis by promoting cancer progression and invasion. One of the mechanisms involved in these processes is that MSCs induce EMT in tumor cells through close cell-cell contact, which could be due in part to the secretion of TGF-β [38, 82]. Studies have shown that secretion of osteopontin (OPN) by tumor cells, induces MSCs to secrete chemokine (motif CC) ligand 5 (CCL5) by stimulating the metastasis of the cancer cell through interaction with its specific chemokine receptor CC type 5 (CCR5) [84]. The migration of tumor cells to and from the metastatic site is mediated by SDF-1, a factor secreted by bone marrow MSCs, which interacts with the CXC receptor chemokine receptor type 4 (CXCR4) expressed in human tumor cell lines of the breast and prostate [33, 101, 102] (adapted from Sarah M. Ridge, Francis J. Sullivan and Sharon A. Glynn. Mesenchymal Stem Cells key players in cancer. Molecular

rate of lung metastases in NOD/SCID mice. This effect was lost when the MSCs derived from bone marrow were injected separately from the tumor cells [83]. On the other hand, it has been shown that MSCs derived from bone marrow facilitate cancer cells [MCF-7, T47D low invasive cell lines, and factor 1 derived from stromal cells (SDF-1) null MDA-MB-231 highly aggressive] target to the bone marrow and modify the metastatic niche through the secretion of trophic factor (SDF-1 and CXCR4) and improved neovascularization in a xenogeneic mouse model

MSCs can not only secrete cell regenerative factors continuously but also secrete factors in response to other various stimuli [97]. Tumor progression is accompanied by hypoxia, starvation, and inflammation. Although many studies have shown that MSCs have tumor promoting properties, many other studies have shown that MSCs have tumor suppressor properties

MSCs derived from human bone marrow have significantly increased the

breast cancer cells co-injected subcutane-

metastatic phenotype. It has been seen that 0.5 × 105

Cancer, Feb. 2017 13:31 1-10. https://doi.org/10.1186/s12943-017-0597-8).

70 Stromal Cells - Structure, Function, and Therapeutic Implications

**2.6. MSCs might inhibit tumor growth**

ously with 1.3 × 106

(**Figure 3**) [96].

(**Figure 1**) (reviewed in [30]). In this regard, it is believed that MSCs suppress tumor growth by increasing the infiltration of inflammatory cells [97], inhibit angiogenesis [34], suppress Wnt and AKT signaling, and induce cell cycle arrest and apoptosis [32, 35, 36]. Recently, Ryu et al. reported that when the MSCs derived from adipose tissue were cultured at a high cell density, they synthesized IFN-beta, which then suppressed the growth of MCF-7 cells [98]. In addition, MSCs prepared with IFN-gamma or cultured with three-dimensional systems can express TRAIL, which induces specific apoptosis of tumor cells [97, 99]. In particular, it was demonstrated that in vitro culture of MSCs under hypoxic conditions increased cell proliferation. In addition, the expression of Rex-1 and Oct-4 was increased, leading to the conclusion that MSC scion was increased during hypoxia [100]. In addition, under hypoxic and starvation conditions, MSCs can survive through autophagy and release many antiapoptotic or pro-survival factors such as VEGF, FGF-2, PDGF, HGF, brain-derived neurotropic factor (BDNF), SDF-1, IGF-1 and IGF-2, transforming growth factor-beta (TGF-b), and IGF-2 binding protein (IGFBP-2) [101, 102]. These factors inhibit the apoptosis of tumor cells and promote tumor proliferation, whereas normal MSCs do not acquire these properties. In addition to the mitogenic properties of growth factors secreted by MSCs, VEGF and FGF-2 can mediate Bcl-2 expression, delaying apoptosis [103], while indirect angiogenic factors can induce VEGF expression and FGF-2 [104]. In addition, SDF-1 was reported to prevent drug-induced apoptosis of chronic lymphocytic leukemia (CLL) cells [105]. In addition, VEGF, FGF-2, HGF, and IGF-1 expressed by MSCs have been reported to stimulate angiogenic and antiapoptotic effects after hypoxic conditioning [101, 106]. Although little is known about how MSCs under hypoxic conditions exert support effects on tumor cells directly, growth factors stimulated by MSCs, stimulated by hypoxia, can provide tumor support effects in the tumor microenvironment through angiogenic and antiapoptotic effects (**Figure 4**).

apoptosis. Finally, when ADSC was introduced in pancreatic adenocarcinoma, the tumor did not grow [107]. Similarly, tumor cells that were cultured with MSC in vitro were also stopped in the G1 phase [111]. However, when the nonobese diabetic-severe combined immunodeficient mice were injected with MSCs and tumor cells, their growth was more increased compared to the injection of tumor cells alone. Although it has been reported that MSCs can induce arrest of the cell cycle of tumor cells in vitro, little is known about the exact mechanisms. In our experiment, the delay or arrest of the cell cycle can be induced in certain types of tumor cells and under certain co-culture conditions (type of medium, cell concentration, or co-culture time). While we cannot explain the exact mechanism (s), several studies performed by different groups, including hours, have shown that the arrest of the tumor cell cycle occurs. It has been shown that MSCs derived from human bone marrow interfere in vitro with small cell lung cancer (A549), esophageal cancer (Eca-109), Kaposi's sarcoma, and proliferative kinetics

of the leukemic cell line [112]. The above was not only observed when 0.5 × 105

exposed to medium conditioned by MSC; the cells were stopped during the G1 phase of the cell cycle in both cases by the negative regulation of Cyclin D2 and the induction of apoptosis [111]. MSCs from other sources, including MSCs derived from human fetal skin and MSCs derived from adipose tissue, have also inhibited the growth of human liver cancer cell lines [32], breast cancer (MCF-7) [111], and primary leukemic cells by reducing their proliferation,

derived from human bone marrow in nude mice carrying Kaposi's sarcoma has inhibited the growth of tumor cells [32]. A similar effect has been observed in an animal model of hepatocellular carcinoma and pancreatic tumors, since the alteration of cell cycle progression has led to the decrease of cell proliferation [30, 31]; the same has happened with melanoma due to increased apoptosis of capillaries [34], and the growth of colon carcinoma in rats has been inhibited when rat EMFs (cell line MPC1cE) were co-mapped with tumor cells in a relationship 1:1 or 1:10 [33]. MSCs derived from human fetal skin (Z3 cell line) also delayed liver tumor growth and decreased tumor size when injected with the same number of cells from the H7402 cell line in SCID mice [36]. Injection of MSCs derived from human adipose tissue (1 × 103

) into established pancreatic cancer xenografts has led to apoptosis and the abrogation of tumor growth in nude (nude) Swiss mice [31]. The role of MSCs in cancer remains paradoxical. Evidence to date has suggested that they are pro as well as antitumorigenic [113–115] and such discrepancy seems to depend on the isolation and expansion conditions, the source and dose

The main signaling pathway involved in the control of cell survival is the pathway of phosphoinositide 3-kinase (PI3K)/AKT and WNT/beta-catenin. The activation of this pathway induces proliferation, growth, and migration, and increases cellular metabolism [116–118]. In the biology of a tumor cell, numerous studies have reported the requirement for the activation of the AKT-signaling cascade for the migration, invasion, and survival of tumor cells. Additionally, the WNT pathway has also been associated with the development of various types of carcinomas, including breast, liver, colon, skin, stomach, and ovary [119]. In a murine

of the cell, the route of administration, and the model tumor used.

**2.9. MSCs and regulation of cellular signaling**

colony formation, and oncogene expression [30, 32]. Intravenous injection of 4 × 106

were cultured together with 0.5 × 105

mm3

tumor cells

73

MSCs

cells/

MSCs derived from human bone marrow but also when

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