Due to their poor immunogenicity, MSCs are introduced as appropriate candidates for clinical applications. Since MSCs have tumor tropism, they may be used as vehicles for tumor-specific delivery of anti-tumor molecules, such as immune related genes ⁽¹⁾. Application of adenoviral infected MSCs with IFN-β gene which could successfully inhibit the tumor growth and angiogenesis in melanoma, breast cancer, and glioma models have been reported previously ⁽¹⁾. Similarly, genes of in flammatory cytokines, such as IL-2 and IL-12, are known for activating anti-tumor immune responses when they were used with MSCs as gene carrires ⁽²⁾.

Other candidate genes for anti-tumor immunotherapy are anti-angiogenic chemokines among which, interferon-inducible protein-10 (IP-10 or CXCL10) has recently been introduced. IP-10 is secreted by several cell types such as monocytes, endothelial cells and fibroblasts in response to IFN-γ ⁽³⁾. This chemokine is a chemoattractant for monocytes, macrophages, T cells, NK cells and dendritic cells. Also, it has the ability to promote T cell adhesion to endothelial cells, antitumor activity, and to inhibit bone marrow colony formation and angiogenesis ^{(4, 5)}.

Melanoma tumor cells transfected with IP-10 gene showed outstanding reduction in tumor growth and promotion in comparison with vector-transfected tumor cells ⁽⁶⁾. It has recently been described that combination of anti-angiogenic therapy with chemotherapy has much better therapeautic effects ⁽⁷⁾. IP-10 expressing plasmid DNA and gemcitabine injection into tumor-bearing mice led to the inhibition of tumor growth through recruiting lymphocytes, induction of tumor cell apoptosis, and longer survival time in tumor-bearing mice ⁽⁷⁾. Yang et al have shown that IP-10 is involved in regulating the proliferation, survival, and activation of tumor-specific T cells other than mediating anti-angiogenic effects in tumor microenvironment ⁽⁸⁾. Thus, in vivo targeted expression of IP-10 is considered as a potentially useful approach for immunotherapy of cancer ⁽⁹⁾.

In contrast to IP-10, SDF-1 axis contributes to angiogenesis through induction of proangiogenic factors such as IL-8 and VEGF ⁽¹⁰⁾. SDF-1 has multiple physiological functions that include homing of hematopoietic stem cells to bone marrow during fetal life and marrow transplantation ⁽¹¹⁾, contribution to the homing of peripheral blood lymphocytes in peripheral lymphoid tissues such as lymph nodes and regulation of pre-B cell trafficking within bone marrow microenvironment as a chemoattractant factor ⁽¹²⁾. It has also pivotal roles in the development and function of nervous system ^{(13, 14)}.

About the role of SDF-1 in cancer promotion, it has been reported that myofibroblasts derived-SDF-1 increases the growth of breast cancer cells through angiogenesis and tumor cells proliferation ⁽¹⁵⁾. Therefore, antibodies against SDF-1 receptor, CXCR4, have the ability to inhibit SDF-1-CXCR4 interaction and prevent tumor growth and angiogenesis ⁽¹⁶⁾. Fernandis AZ et al reported that stimulation of MDA-MB-231 breast carcinoma cells by SDF-1 leads to release of MMP2 and MMP9 and to increase cellular motility and migration ⁽¹⁷⁾. In contrast, the epigenetic silencing of SDF-1 increases the metastasis of breast cancer cells and its forced expression decreases the number of metastases, in vivo ⁽¹⁸⁾.

In addition to the application of MSCs as novel immunological gene carriers, tumor promoting activity of these cells ^{(19, 20)} is a critical concern which can be modulated through transferring the immune related genes to these cells. For this purpose, present study aims to investigate the effects of IP-10 (as a potent anti-angiogenic factor) on the expression profile of tumor promoting molecules in adipose derived stem cells (ASCs). Results are compared to ASCs transfected with SDF-1 gene, as an angiogenic factor.

Among the different sources for isolating mesenchymal cells, bone marrow has been considered as the major source. However, MSCs has recently been found in other tissues such as adipose tissue ⁽²¹⁾. Although, MSCs isolated from both adipose tissue and bone marrow expanded easily in vitro, ASCs showed a higher proliferation rate and longer survival time in culture than bone marrow stem cells ⁽²²⁾. Adipose tissues are most safe and easy to harvest with low risk and are highly abundant in the human body ⁽²³⁾. Also, it is of particular interest due to the increased incidence of obesity and liposuction surgeries that are performed each year ⁽²¹⁾. Considering the advantages of ASCs, here we used the adipose tissue as a source for extracting MSCs.

It has been evidenced that MSCs inhibit the proliferation and responses of both primary and activated T cells through Th2 type cytokine production ⁽²⁶⁾. They also contribute to induction of tumor infiltrating T lymphocytes toward the regulatory T cells ^{(24, 27)}. MSCs’ derived soluble factors, such as indolamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2),Transforming Growth Factor-β1 (TGFβ1), Hepatocyte Growth Factor (HGF) ^(28–30), Leukemia Inhibitory Factor (LIF) ⁽³¹⁾, and HLA-G5 ⁽³²⁾ are considered as known immuno-modulatory mechanisms of these cells. MSCs have the ability to transdifferentiate into Cancer Associated Fibroblasts (CAFs) and express the main molecular markers of CAFs, including SDF-1, under the influence of TGF-β ^{(33, 34)}. Interestingly, MSCs are one of the main sources of TGF-β in the tumor microenvironment which is named as the master regulator of metastasis through EMT mechanism ⁽²⁰⁾.

In contrast to the tumor promoting effects of MSCs, they are known as one of the best gene carriers for the delivery of immunological genes to the tumor microenvironment ⁽³⁵⁾ because of their ability to access the inflammatory sites after i.v. administration ⁽³⁶⁾. Based on these reports, we hypothesized that transfection of ASCs with an angistatic gene, such as IP-10, may modulate the functional properties of ASCs and present them as better gene carriers for cancer gene therapy. Thus, we firstly aimed to increase the efficiency of our transfection methods, including electroporation and lipofectamin. The best result of transfection with IP-10 or SDF-1 plasmids was obtained by electroporation because both genes were expressed in large amounts in our transfected ASCs by this method. Then we assessed the effect of IP-10 and SDF-1 overexpression on the expression of tumor promoting cytokines. As mentioned in result section, transfection of ASCs by IP-10 led to reduced amounts of Bcl-2 and MMP2 transcripts in ASCs.

It has been demonstrated that IP-10 may act as similar as IL-12, regarding its antitumor effects ⁽³⁷⁾. Luster et al genetically engineered tumor cells to secrete high levels of murine IP-10 and demonstrated that IP-10 has a strong antitumor effect in vivo that is mediated through the recruitment of lymphocytes, neutrophils, and monocytes ⁽³⁸⁾. It has also been shown that the overexpression of MIG, IP-10, and CXCL11 in the local tumor inhibits tumor growth and metastasis through the angiostatic effect or activation of T lymphocytes or NK cells ⁽³⁹⁾.

These findings and our results which showed downregulation of MMP2 and Bcl-2 in IP-10 transfected ASCs explain the importance of IP-10 in tumor immunotherapy. Additionally, combination therapy with IP-10 and other antitumor agents, such as IL-12, may have more significant tumor regressive effects compared to IP-10 or IL-12 alone ⁽³⁷⁾. However, upregulation of VEGF after using IP-10 in ASCs is not consistent with other investigations which showed the inhibiting effects of IP-10 on the endothelial cells ^{(40, 41)}. Thus, the present results may impose us to do further studies for better elucidation.

Controversial results have been obtained after transfecting different types of cells by SDF-1 gene. Evidences showed that saturation of CXCR4 through autocrine SDF-1 production reduces chemotaxis to the target organs which were rich in SDF-1 ⁽¹⁸⁾. Interestingly, adenoviral gene delivering of SDF-1 to tumors might inhibit the growth of preexisting tumors through attraction and accumulation of dendritic cells to the tumor site ⁽⁴²⁾. SDF-1 has been introduced as an effective adjuvant to augment anti-tumor responses through recruitment of CXCR4 expressing T cells ⁽⁴³⁾. In contrast, there are several reports showing the tumor promoting effects of SDF-1 for instance in angiogenesis of the tumor ^{(15, 16)}. Treatment of human colon epithelial cells with SDF-1 can induce the production of IL-8 as well as GROα ⁽⁴⁴⁾. Stimulation with SDF-1 led to secretion of a variety of MMPs and to cellular motility ^{(17, 45)}. Our results confirmed the tumor promoting effects of SDF-1 because CXCR4, Bcl-2, MMP2, VEGF, IL-10, and IGF-1 had more expressions after transfecting with SDF-1 gene. Thus, we conclude that the production of SDF-1 in tumor microenvironment may contribute to the tumor growth and metastasis.

Overall, it is concluded that adipose tissue is a suitable source for extracting stem cells and applying them as immunological gene carriers to the tumor site. In addition, among the candidate genes IP-10 may be introduced as an anti-tumor therapeutic agent to suppress tumor progression and metastasis. However, more investigations in experimental models are required.