Engineered Stem Cells Targeting Multiple Cell Surface Receptors in Tumors

Summary:

Multiple stem cell types exhibit inherent tropism for cancer, and engineered stem cells have been utilized as therapeutic agents to specifically target cancer cells. Recently, stem cells have been engineered to target multiple surface-receptors on tumor cells, as well as endothelial and immune cells in the tumor microenvironment. In this review, we discuss the rationales and strategies of developing multiple receptor targeted stem cells, their mechanisms of action, and the promises and challenges they hold as cancer therapeutics.

Introduction:

Since the identification of aberrant signal transduction through cell surface receptors as a common feature of many cancer types, the targeting of these pathways has proven to be a promising route in the development of cancer therapies. By tailoring therapeutic agents to selectively target surface receptors indicative of malignant cells, the cytotoxicity to neighboring cells can be significantly limited (1,2). However, resistance, both inherent and acquired, is a significant limitation on the efficacy monospecific antibodies and ligands targeting cell surface receptors due to the activation of alternative signaling pathways and receptors in cancer cells (3). Heterogeneity within and between tumors also limits the functionality of therapies targeting a single tumor biomarker (4,5,6). Due to its role in the tumor progression and resistance, the tumor microenvironment has also become a promising target for immune-based therapy (7). Therefore, targeting of multiple cell surface receptors on cancer cells and associated cells has the potential to target heterogeneous tumors, as well as impact the tumor microenvironment, and, therefore, has become an exciting new direction for targeted therapy in cancer (8) (Table 1).

Table 1:

Cell surface receptors expressed on tumor cells and within the tumor microenvironment that have been or have the potential to be* utilized in stem cell-delivered cell surface receptor targeting therapies and their respective targeting agents. Cell surface receptors with differential or unique expression on the surface of tumor cells or cells of the tumor microenvironment are attractive targets for cell surface receptor targeting therapies. *Stem cell (SC) delivered anti-tumor receptor targeting agents have not yet been explored for this receptor type. Abbreviations: TNFR – tumor necrosis factor receptor, DR4/5 – death receptor 4/5, EGFR – epidermal growth factor receptor, IFNAR - Interferon-α/β cell surface receptor complex, GPCR – G protein coupled receptor, BnR – Bombesin receptor, SSTR – somatostatin receptor, ET – endothelin receptors, FR – folate receptor, TfR – transferrin receptor, FGFR – fibroblast growth receptors, TRAIL - Tumor necrosis factor-related apoptosis inducing ligand, oHSV – oncolytic herpes simplex virus, TSP – thrombospondin, scFV- single chain variable fragment, BC – breast cancer, UC – uterine cancer, NB – neuroblastoma, GBM – glioblastoma, NK – natural killer AML – acute myeloid leukemia, ALL – acute lymphoblastic leukemia, LC – lung cancer, OC – ovarian cancer.

Receptor type	SC delivered anti-tumor targeting agent	Expression in tumor cells and cells of the tumor microenvironment	References
TNFRs
Death receptors 4 and 5 (DR4/5)	TRAIL	Expressed in non-malignant and malignant cells, suggested to be expressed at higher levels in malignant cells	120,121
CD137/4–1BB	CD137/4–1BB ligand (CD137L/4–1BBL)	Expressed on T cells	86
EGFRs
EGFRvIII	EGFR targeting antibodies or nanobodies (ENb)	Expressed only in glioma, novel variant	80
EGFR2/HER2	anti-HER2 antibodies (trastuzaumab)	Overexpressed in BC, gastric, cervical, UC, gallbladder and testicular cancers, bladder carcinomas, and extrahepatic cholangiocarcinomas	122
Nectin-1	oHSV	Expressed in neurons, as well as a variety of cancers including NB, GBM, and squamous cell carcinoma. Often predicative of oHSV susceptibility.	123,124
IFNAR	Type I interferons (IFNα, IFNβ)	Expressed on a variety of tumor cells (fibrosarcoma, cervical, colon, and lung carcinoma, etc.) and immune cells (T cells, NK cells, and dendritic cells)	64–66,125
CD36	TSP	Expressed on endothelial cells,present in highly vascularized tumors such as GBM	62
CD20	anti-CD20 antibodies (rituximab, scFv–CD20)	Exclusively expressed in B cell precursors, mature B cells, and B cell lymphoma (Hodgkin’s and non-Hodgkin lymphoma)	84
CD33	anti-CD33 antibodies (gemtuzumab ozogamicin (Mylotarg), anti- CD33–anti-CD3 bsAb)	Overexpressed on of the majority of AML cells	87
CD19	anti-CD19 antibodies (blinatumomab, anti-CD3–antiCD19 TandAb)	Expressed in B-precursor ALL and B cell lymphoma	87
CD3	anti-CD3 antibodies (anti-CD33–anti-CD3 bsAB, TandAb, blinatunmomab, anti-CD3scFv)	Expressed on T cells	86
GPCRs
BnR	*	Expressed in GBM, NB, non-small cell and small cell LC (novel receptor), intestinal, thymic, gastrointestinal, lung, and bronchial carcinoid tumors, OC, pancreatic, prostate, BC, head and neck, colon, UC, and renal cancers, Ewing sarcoma, pituitary tumors	126,127
SSTRs	*	Expressed in small cell LC, prostate, BC, gastric cancers, neuroendocrine tumors, colorectal and hepatocellular carcinoma	128
ET	*	Expressed in melanoma	129,130
Integrins
ανβn (ex ανβ3)	*	Expressed in activated endothelial cells and tumor cells (U87MG GBM cells)	131,132
α−3	*	Expressed in OC and BC, and melanoma	133
FR (α, β, γ)	*	Expressed in OCand BC, endometrial and renal cell carcinomas, lung andenocarcinomas and mesotheliomas	134
TfRs
TfR1 (CD71)	*	Overexpressed in glioma and GBM, expressed in BC and OC	135–137
FGFRs	*	Alterations in expression in BC, bladder, prostate, LC, and haematological malignancies	138

The multiple receptor targeting strategy is still limited by the unfavorable pharmacokinetics of potential therapeutics, due to rapid clearance from the tumor site and limited bio-transformation and availability. Additionally, the difficulty in maintaining sustained concentrations of biologically based therapeutics at the site of the tumor, particularly in the case of solid tumors where therapeutics are often unable reach the core of the tumor (9). The tumor-tropic nature of adult stem cells through solid tumors and to the sites of micro-metastases has made them attractive candidates for use as therapeutics in cancer. Stem cells can be engineered to stably express and then release biological therapeutics, thus becoming factories for the production of therapeutics, which can improve the half-lives of these agents and provide continuous exposure at the tumor-site (9,10). This circumvents the short half-lives of therapeutics such as antibodies and anti-cancer proteins, by permitting site-specific and long-term delivery of therapeutic agents. Stem cells delivered therapeutics are readily accessable at the site of tumors and can infiltrate solid tumors more readily than biological therapies delivered using traditional methods (9,10). The goal of this review is to discuss the recent advances the development of multiple cell surface receptor targeting therapies through the use engineered stem cells.

Stem cell types:

Stem cells (SCs) are the source of all embryogenic tissue generation and any continued regeneration of tissue throughout adulthood. A variety of SC types have been explored as potential biologically based therapeutics, and from these studies adult stem cells seem to have the most promise for use in clinical studies (11,12,13). Embryonic SCs (ESCs) were the first SC type to be explored for possible sources of stem cell therapy but were abandoned primarily due to ethical concerns in the sourcing of these cells (14,15). Therefore, adult SCs, which can be successfully isolated from various sources within the body, have become the focus of stem cell-based therapies. The three primary types of adult SCs that have been explored for these purposes are mesenchymal SCs (MSC), neural SCs (NSCs), and induced pluripotent SCs (iPSCs). MSCs can be derived from bone marrow, adipose tissue, umbilical cord blood (UCB), and various other tissues (16). NSCs, also called neural precursor cells, do not refer to a single cell type but rather a heterogeneous population of self-renewing and multipotent cells present in both the developing and adult central nervous system (17). NSCs have shown particular promise in developing cell-based therapies for neurodegenerative disorders, but also hold potential for other conditions including certain cancers (12,17,18,19,20). iPSCs, which are generated from the reprogramming of somatic cells by expression of defined transcription factors, have emerged as a popular and effective SC type for therapy. These cells can be derived from a wide variety of starting cells, providing incredible accessibility compared to other SC types for the development of iPSC-based therapies and disease models (11,21). Engineered hematopoietic stem cells (HSCs) have also been explored for their potential to generate sustained anti-tumor effect by engraftment of engineered HSCs expressing either T-cell receptors (TCRs) or chimeric antigen receptors (CARs) (22). However, HSCs have not been explored for bifunctional tumor cell surface receptor targeting.

Stem cell homing and immunogenicity

SCs offer the opportunity to improve the efficacy of biological therapeutics by improving pharmacokinetics and biotransformation and producing continuous potent concentrations of therapeutics at the tumor site. Two properties, the abilities to evade the host immune system and to home to tumor loci, are necessary to establish an effective cellular therapy; however, if and to what extent these characteristics are present in SCs is still subject to debate. SCs have been documented to have immunosuppressive properties via the expression of cytokines and growth factors that can impact immune pathways within a host organism (23,24,25). The implantation of foreign SCs, both adult SCs and induced allogeneic donor SCs, has still been shown to elicit an immune response and lead to rejection of the cells in vivo (26,27,28,29,30). SCs have also been shown to have inherent immuno-modulatory effects. NSC implantation in the brain has been shown to induce an immunological response, indicated by infiltration of lymphocytes, and to induce of pro-inflammatory cytokines IL-1β and TNFα in the brain (25). Allogeneic MSCs have been shown to inhibit the activation of CD4+ T cells and to alter the humoural immune response both in vitro and in vivo (29). Furthermore in contrast to ESC derived cells, iPSCs have been shown to induce a T-cell dependent immune response in syngeneic recipients. This immunogenicity was attributed to differential cell surface marker expression and may in fact limit the efficacy of iPSC-based therapy (26). While allogeneic MSCs are less immunogenic than other allogeneic non-SC cell types, such as fibroblasts, they are not entirely immune privileged but rather they are able to escape host rejection transiently (29,31).

The second necessary characteristic for cellular delivery, migratory potential, was first demonstrated for neural SCs (NSC) and neural progenitor cells in xenograft mouse models (10). NSCs have been shown not only to integrate into primary tumors, but also to track to micro-metastases that are typical of brain tumors like glioblastoma (32). Tumor tropic characteristics have also been demonstrated in numerous SC types (33,34,35). Although the molecular mechanisms of tumor tropism are not yet completely understood, several chemokine-chemokine receptor pathways have been implicated in this characteristic. The most well studied of these is stromal cell-derived factor 1 (SDF1) and its receptor CXC-chemokine receptor 4 (CXCR4), which have been shown to have a significant role in the migration of NSCs, MSCs, ESCs, HSCs, and iPSCs (33,34,36–40). Other chemokine-chemokine receptor pairs such as SCF-c-Kit, HGF-c-Met, VEGF-VEGFR, PDGF-PDGFR, MCP-1-CCR2, and HMGB1-RAGE as well as cytokines that co-operate with G-protein coupled receptors and growth factor receptors have also been shown to be involved in stem cell recruitment and migration (35). The nature of the SC population such as heterogeneity, culture conditions, and expression of migratory factors can influence the migration of SCs to tumor sites (10). Additionally, the tumor microenvironment, particularly degree of hypoxia, extent of vascularization, and inflammation, can also play a role in the SC migration toward tumor loci (10). Tropism to tumor loci is a key feature of a variety of SC types that make them attractive for cell therapy in cancer, and the ability to use or enhance this feature may improve therapeutic potential of SCs. Better understanding the factors that influence SC tumor tropism and immune evasion can aid in developing SCs as tools for therapeutic delivery by capitalizing on the extent to which these characteristics are intrinsic and by potentially modifying the SCs or the tumor environment to promote these characteristics.

Creating cell surface receptor targeted stem cells

Unmodified SCs have been shown to have anti-tumor effects primarily attributed to factors secreted by the SCs as well as physical interactions between the SCs and tumor cells (41–48). Particularly, the antitumor effects of MSCs have been attributed in part to enhancing the host antitumor immune responses, demonstrating the benefits of the inherent immune-modulatory effects of SCs (43). However, quite promisingly SCs have been modified to target cancer cell surface receptors, greatly improving their anti-cancer efficacy. Commonly, this is achieved by viral transduction of SCs using lentivirus (LV) or safer alternatives such as retrovirus or adeno-associated virus (AAV) (10). Among the viral vectors, LVs have the unique ability to infect non-dividing cells, and therefore have been widely utilized in the creation of engineered stem cells. The anti-tumor agents that have been used to target cell surface receptors through engineered SCs include traditional biologics, such as antibodies and ligands, as well as oncolytic viruses (Figure 1).

Figure 1. Engineered receptor targeted stem cells promote tumor cell death:

SCs can be modified in a variety of ways to generate an anti-tumor response by targeting cell surface receptors. SC engineered to express therapeutic proteins or antibodies that function directly on tumor cells (TRAIL, EGF agonists, anti-CD20, CD33, CD19) or that act on the tumor microenvironment to elicit an immune response (IL-10, IL-18, IFNα/β) or on surround cells (stromal effectors or blood vessel effects (aaTSP-1). SCs loaded with oncolytic virus amplify within the SC eventually leading to its rupture and release of viral progeny. These viral particles will go on to infect tumor cells, killing them and inducing an immune response at the tumor site. Abbreviations: SC- stem cell, TRAIL- tumor necrosis factor-related apoptosis inducing ligand, IL- interleukin, IFN- interferon, EGF- epidermal growth factor, aaTSP-1- anti-angiogenic thrombospondin,

Targeting of Multiple Cell Surface Receptors

Single ligand targeting multiple receptors on tumor cells or receptors on tumor associated cell types:

While the use of SCs has been shown to improve the delivery of therapies targeting cell surface receptors, the targeting of multiple receptors with a single therapy has to potential to combat resistance and tumor heterogeneity (Figure 2). Pro-apoptotic protein tumor necrosis factor-related apoptosis inducing ligand (TRAIL), binds to death receptors 4 and 5 (DR4/5), which are expressed in a tumor specific manner and induce caspase mediated apoptosis upon TRAIL binding (49,50,51). In mouse models of highly malignant brain tumors, glioblastomas (GBM), both bone marrow-derived MSC and NSCs engineered to express a secretable (S) form of TRAIL (S-TRAIL) have shown anti-tumor effects (51,52,53,54). Additionally, MSC-TRAIL has recently been shown to be effective in preclinical models of pancreatic ductal adenocarcinoma, Ewing sarcoma, lung cancer, and melanoma (55,56,57,58). Photochemical internalization and nanoparticle-mediated transfections have also been used to engineer TRAIL secreting MSCs which also demonstrated an anti-tumor effect in preclinical models, indicating the potential for safer engineering of MSCs which may improve translation to the clinic (59,60). In addition to molecules that directly target multiple receptors on tumor cells, anti-angiogenic agents, such as three type 1 repeats (3TSR) of thrombospondin (TSP)-1, act as indirect effectors on tumor associated endothelial cells and blocking the ability of tumors to stimulate new blood vessel formation (61). Studies have shown that MSCs engineered to express anti-angiogenic (aa) 3TSR have anti-tumor efficacy by binding to CD36 receptors on tumor associated endothelial cells and preventing angiogenesis in a mouse model of GBM (61,62). aa3TSR also has the ability to bind to CD36 receptors on tumor cells and has been shown to upregulate expression of DR4/5 in tumor cells, priming these cells for treatment by S-TRAIL (62). Interferon (IFN)β belongs to type I interferons that bind to the interferon-α/β cell surface receptor complex (IFNAR) (63) and has direct anti-tumor activity (64,65,66). IFNβ also acts as an immunostimulatory molecule by indirectly provoking an antitumor response via modulation of the immune system (67,68,69). However, despite these bi-functional modes of action, the clinical translation of IFNβ treatments for cancer so far has been restricted by its short half-life and systemic toxicity (70,71,72). MSCs engineered to express mouse IFNβ have been shown to target multiple cell types in a resection model of mouse GBM. Encapsulated MSC-IFNβ placed in the tumor resection cavity, were able to simultaneously recruit CD4/CD8 T cells to the site of tumor resection and induce apoptosis in tumor cells, leading to increased survival in mice bearing GBMs (73).

Figure 2. Potentiating stem cell efficacy through bifunctional SCs:

The efficacy of engineered SCs can be improved by targeting multiple cell surface receptors with a single bifunctional SC, increasing specificity and overcoming resistance. Bifunctional SCs 1) release a single molecule, such as TRAIL, that simultaneously targets two cell surface receptors (DR4 and DR5); 2) release two different molecules that target distinct cell surface receptors, such as TRAIL and aaTSP-1, or release a bifunctional fusion protein to target two different cell surface receptors, such as ENb-TRAIL or scFv-CD20-TRAIL 3) release bifunctional fusion proteins to target and recruit immune cells from the tumor microenvironment such as T cells (anti-CD33-antiCD3 and anti-CD19-anti-CD3) bsAbs fusion proteins. Abbreviations: SC- stem cell, TRAIL-tumor necrosis factor-related apoptosis inducing ligand, 3TSP-1- anti-angiogenic thrombospondin, ENb- epidermal growth factor receptor nanobody, bsAb-bispecific antibody

SC engineered to express ligands targeting multiple receptors on cells in the tumor microenvironment:

Because tumor cells and the associated cells within the tumor microenvironment express a wide variety of cell surface receptors, one therapeutic strategy is to engineer SCs to simultaneously release multiple therapeutic proteins targeting multiple receptors on tumor and associated cells in the tumor microenvironment (74) (Figure 2). A growing number of studies are adopting this approach, utilizing bimodal SCs and SCs secreting bifunctional molecules and often taking advantage of synergistic biological relationships between two therapies. MSCs expressing both aa3TSR and S-TRAIL have been shown to target both DR4/5 on tumor cells and CD36 on tumor cells and tumor associated endothelial cells in TRAIL resistant GBMs, leading to extended survival times in mouse models (62). The dual expression of cytokines by SCs to target tumor cells and immune cells has also been shown to be effective. In a rat tumor model, SCs expressing both IL-18 and IFNβ significantly prolonged survival and inhibited tumor growth both by promoting tumor cell apoptosis and stimulating antitumor cytokine production and CD4/CD8 T cell infiltration of the intracranial gliomas. The robust antitumor response of MSC-IL18 and IFNβ indicated a synergistic effect of the two immunostimulatory cytokines when used in combination (75). Similarly, MSCs engineered to co-express the cytokines IL-10 and IFNγ reduced tumor growth in a mouse model of hepatocellular carcinoma by cell cycle arrest, demonstrated by increased expression of cell cycle inhibitors p21 and p27, as well as modulation of the MAPK pathway (76).

Stem cells expressing antibodies and ligands targeting multiple receptors:

Traditional antibodies have a highly conserved tetrameric structure consisting of a pair of heavy chains and a pair of light chains linked by disulphide bonds. Alternative antibody structures have been developed, including heavy chain-only antibodies (77): which do not have a CH1 domain, antigen-binding fragment or single-chain variable fragments (scFv): which contain only two variable domains, one from each of the heavy and light chains, and nanobodies: which consist of the only the heavy-chain variable fragment (V_HH) and are on the scale of nanometres (8). Antibody fragments have increased ability to penetrate tumor tissues, due to their small size, but their efficacy is limited because of their short half-life and rapid renal clearance in vivo (78).

SCs can be modified by viral or non-viral methods to express either full antibodies or antibody fragments (79). The epidermal growth factor receptor (EGFR), has also been a target of SC delivered antibody therapy. SCs engineered to express EGFR and its glioma associated variant (EGFRvIII) targeting nanobodies (ENb), which consist solely of the antigen-specific domain (V_HH), have been shown to reduce EGFR signaling in vitro and inhibit tumor growth in a mouse model of gliomas (80). In an effort to take advantage of the efficient cell-mediated delivery of antibodies for ligand-based therapies, researchers recently engineered NSCs containing phosphatase and tensin homolog (PTEN) with the leader sequence from human light-chain immunoglobulin G (IgG) to target inactivated PTEN signaling in GBM. This antibody-ligand fusion molecule showed increased secretion from NSCs and potential to transfer to neighboring cells, demonstrating an increased potential for NSC delivered PTEN therapy (81).

Although the area of SC delivered bifunctional proteins is still in its infancy, several studies have explored the delivery of single bi-functional molecules targeting two different cell surface receptors (9) (Figure 2). Due to the low expression levels of DR 4/5 and EGFR in adult SCs, SCs can be engineered to express a nanobody against EGFR (ENb) fused to TRAIL (ENb-TRAIL) without significant auto-toxicity. SC-ENb-TRAIL was shown to outperform SC-ENb alone in mouse tumor models by inhibiting EGFR and inducing caspase-mediate cell death (80). Recent studies have shown that ENb-TRAIL blocks EGFR signaling via the binding of ENb to EGFR which in turn induces DR5 clustering at the plasma membrane and thereby primes tumor cells to caspase-mediated apoptosis (82). MSCs expressing a dimeric EGFR-specific diabody single-chain TRAIL (Db-scTRAIL) were also shown to have antitumor activity against colorectal cancer in vitro and in vivo, particularly in combination with bortezomib, a proteasome inhibitor (83). MSCs loaded with a bifunctional protein consisting of scFv-CD20 fused with S-TRAIL was shown to inhibit cell proliferation and induce apoptosis in CD20 positive lymphoma cells. Additionally, in a xenograft model of B-cell lymphoma, i.v. injection of MSC-scFvCD20-TRAIL significantly inhibited tumor growth when compared to MSC-TRAIL without any noticeable toxicity (84).

T-cell engaging bispecific antibodies (bsAbs) are a promising tool for cancer treatment as they establish a transient synapse between T cells and a tumor cell by surface antigen binding on tumor cell with one arm and simultaneously recruiting T cells via the signal transmitting CD3 domain of the T-cell receptor complex (85) (Figure 2). Previous studies have explored MSCs for delivering a fully humanized anti-CD33-anti-CD3 bsAb, which is capable of redirecting human T cells against CD33-expressing leukemic cells. MSC expressing anti-CD33-anti-CD3 bsAb were shown to redirect T cells efficiently against CD33 presenting target cells and eliminated autologous acute myeloid leukemia (AML) cells over time. The immune response against AML cells was further enhanced further by providing T cells an additional co-stimulus via the CD137-CD137 ligand axis through MSCs expressing CD137L (86). More recent studies have shown that MSCs secreting TandAb, a tetravalent bispecific tandem diabody for CD3 and CD19, were also effective in a mouse model of B-cell lymphoma by inhibiting tumor growth (87).

Stem cells loaded with bi-functional Oncolytic Viruses targeting multiple receptors

Oncolytic viruses (OVs) are viruses that either naturally or by genetic modification have the ability to selectively infect and replicate in tumor cells, while sparing surrounding healthy tissue (88,89). Oncolytic viruses make use of cell surface receptors to gain entry into cells (89). However, clearance of the virus by the host immune system and off-target infection of noncancerous cells makes systemic administration of OVs ineffective, while insufficient viral spread in intratumoral injection also limits this route of delivery (90). SCs have been explored as a possible means of overcoming issues in OV delivery by shielding the virus from anti-viral immunity and directing viral particles more directly toward the tumor. A variety of SC types, including MSCs, have been explored for the delivery of oncolytic adenovirus either systemically or intratumorally (91,92,93,94,95). However, only limited number of OVs have been engineered that offer the utilization of at least two different cell surface receptors, one for the viral entry and other for the ligand/antibody expressed by the SC loaded with OVs. Recently, MSCs loaded with adenovirus were used to modify hepatocellular carcinoma (HCC) cells in vivo to present anti-CD3scfv on their surface. Once presented, this surface marker activated lymphocytes leading to cancer cell specific lysis and inhibited tumor growth in mouse models (96). Furthermore, OVs have been genetically engineered to express various agents targeting tumor cell receptors and/or immune cell receptors with the intention of improving their specificity and efficacy. oHSV engineered with TRAIL has been shown to inhibit tumor progression and prolong survival in mice bearing resistant intracerebral tumors (97). While MSC delivered oncolytic adenovirus expression TRAIL has been shown to be effective in preclinical models of pancreatic cancer, demonstrating the potential for SC delivery of OVs with additional anti-tumor agents (98).

Stem cell released Extracellular Vesicles

A variety of cells types release extracellular vesicles (EVs), which can contained a variety of biological molecules including mRNA, miRNA, proteins and lipids (99). EVs may be taken up by target cells by fusion with the plasma membrane, binding to receptors on the cell surface, or by endocytosis by phagocytosis (100). Two of the most well described ligand-receptor complexes involved in exosome endocytosis are low-density lipoprotein (LDL) and its receptor (LDLR), and transferrin (Tf) and the transferrin receptor (TfR). Other protein-protein interactions linked to EV intake include lectins, adhesion molecules, such as cadherins and integrins, heparin sulfate proteoglycans, and T-cell immunoglobulins and mucins, including EGF/EGFR (101). EVs, which include both exosomes and microvesicles, may be responsible for the paracrine influences of MSCs on adjacent cells by transfer of biological material to enhance communication between cell types (102,103). MSC delivered EV-miRNA mimics (miR-124 and miR-125) to GBM cells, which are deficient in expression of these miRNAs has been shown to have antitumor effects in a mouse model of GBM (104). In addition to miRs, MSCs have also been used to delivery anti-miRs, which block miR function. MSC derived exosomes were used to deliver anti-miR-9 to GBM cells sensitizing these cells to temozolomide, and thus reverse chemoresistance (100,105) Exosomes from MSCs engineered to express an siRNA against GRP78, which is overexpressed in Sorafenib resistant cancer cells, were also shown to effective in combination with Sorafenib against hepatocellular carcinoma cells in vitro and in vivo, further demonstrating the ability SC derived exosomes to reverse chemoresistance (106). SC derived exosomes have also been used to target the tumor microenvironment. Exosomes derived form mESCs engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF) were shown to inhibit the growth mouse models of lung cancer by activating CD8⁺ T effector responses and acting as a prophylactic cancer vaccine (107). Menstrual MSC derived exosomes have been identified as inhibitors of angiogenesis in prostate, breast cancer, and oral squamous cell carcinomas by a reduction of VEGF secretion by endothelial cells leading to anti-tumor effects (108,109).

Perspectives

The use of engineered SCs to target multiple cell surface receptors has the potential to overcome some of the most difficult challenges in developing effective cancer treatments: resistance, tumor heterogeneity, and poor pharmacokinetics. The success of anti-cancer SCs in preclinical studies has been exciting for researchers in the field; however, as these therapies begin to be translated to the clinic, significant caution must be taken to ensure safety and efficacy of these therapies. Improvements in the homing of SCs, developing effective methods of non-viral engineering, and a strong understanding of the mechanisms underlying the efficacy of anti-tumor SCs are all essential for the continued pursuit of these therapies.

Recent studies have aimed to address clinical limitations on the use of engineered SCs, such as the use of viral transduction in the generation of therapeutic SC lines, which has safety concerns due to immunogenicity and oncogenic effects of viruses (59). The use of non-viral polylysine-modified polyethylenimine (PEI-PLL) copolymer-based transfection to generate engineered MSCs (110) indicate that such methods may hold to key for safe and expedient translation of modified SCs to clinical settings. A further limitation in the clinical application of SC delivered cancer therapies is the potential for immune rejection after implantation. Therefore, the generation of immune evasive SCs may hold the potential for advancing these therapies. Recently the studies have demonstrated methods of generating hypoimmunogenic or immunosuppressive SCs either by inactivation of the major histocompatibility complex (MHC) class I and II genes and activation of CD47 or by ex vivo induction of apoptosis (111,112). Both of these strategies could improve the safety of SC based therapies by reducing the risk of adverse immune response to transplantation.

The continued development of more clinically relevant tumor models will improve the potential for therapeutic success of treatments developed in the lab. Profiling of patient-derived tumors can be used to establish new targets for therapies, aid in understanding the mechanisms of existing therapies, and determine what tumors will respond to what therapies based on their specific cancer profile (113). Engineered SCs offer the opportunity to develop more personalized medicine, where the therapeutic SCs can be developed to have optimal efficacy against a specific tumor and its profile. Furthermore, establishing more true-to-life animal models would aid in the screening of SC based therapies. As of now, most animal models rely on the surgical introduction of cancer cells from cancer cell lines into immunocompromised host animals. This model poorly reflects the true development and subsequent environment of tumors and have limited utility in screening therapies, especially when there is an immunomodulatory component. Genetically engineered mouse model that develop tumors without introduction of outside cells or incorporating patient-derived tumor samples into animal models may hold more utility in testing therapies that depend significantly on the tumor microenvironment (10).

Clinical trials testing gene engineered SC targeting single cell surface receptors have been completed or are currently active. Genexine, Inc. sponsored trial, to establish the safety and efficacy of MSCs expressing IL-12 (GX-051) in patients with head and neck cancers in 2014. trial was conducted at MD Anderson Cancer Center in 2015 to establish tolerable doses of MSCs expressing IFNβ in patients with ovarian cancer. Furthermore, trial is currently recruiting patients for MSCs expressing TRAIL (TACTICAL) for non-small cell lung cancer. MSCs loaded with oncolytic adenovirus (ICOVIR-5) were also explored for their safety and efficacy in metastatic neuroblastoma and melanoma (114,115). Finally, clinical trials for NSCs expressing cytosine deaminase in combination with 5-Fluorocytosine () or 5-Fluorocytosine and Leucovorin () have either been completed or are active respectively. While these single receptor targeting SC delivered therapies are being explored in the clinic, multiple cell surface receptor targeting SC based therapies have not yet reached clinical trial stage.

SC based therapies, particularly those targeting multiple aberrant signaling pathways, offer the potential to significantly improve the efficacy of biologically based therapies that have previously shown limited efficacy due to unfavorable pharmacokinetics, resistance, or tumor heterogeneity. Engineered SC-based therapies can also be combined with other non-cell delivered immune-based therapies or traditional chemotherapies (116,117) to potentially further enhance efficacy by targeting multiple pathways simultaneously. Furthermore, engineered SC-based therapies have an enormous potential to be used in combination with other cell based cancer therapies, such as CAR-T cells. However, as with all newly developing therapies, caution must be taken to prioritize safety and preserve the integrity of field moving forward. As clinical trials have begun, the safety of these therapies will continue to be tested rigorously while preclinical trials will continue to explore the potential of these therapies. SCs engineered to express receptor-targeted ligands have also been frequently combined with suicide therapy like HSV-TK to provide synergistic therapeutic effect and improve the safety of therapeutic SCs by ensuring their eradication post-treatment (110,118). Recently, the novel suicide gene iCasp9, which binds to the bioinert small molecule (AP20187) leading to cell death, has been explored in combination with MSC-TRAIL with promising results demonstrating coexistence between the suicide and anti-cancer mechanisms (119).