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Inflammatory Mediators in Glioma Microenvironment Play a Dual Role in Gliomagenesis and Mesenchymal Stem Cell Homing: Implication for Cellular Therapy

      Abstract

      Glioblastoma is the most aggressive malignant primary brain tumor, with a dismal prognosis and a devastating overall survival. Despite aggressive surgical resection and adjuvant treatment, average survival remains approximately 14.6 months. The brain tumor microenvironment is heterogeneous, comprising multiple populations of tumor, stromal, and immune cells. Tumor cells evade the immune system by suppressing several immune functions to enable survival. Gliomas release immunosuppressive and tumor-supportive soluble factors into the microenvironment, leading to accelerated cancer proliferation, invasion, and immune escape. Mesenchymal stem cells (MSCs) isolated from bone marrow, adipose tissue, or umbilical cord are a promising tool for cell-based therapies. One crucial mechanism mediating the therapeutic outcomes often seen in MSC application is their tropism to sites of injury. Furthermore, MSCs interact with host immune cells to regulate the inflammatory response, and data points to the possibility of using MSCs to achieve immunomodulation in solid tumors. Interleukin 1β, interleukin 6, tumor necrosis factor α, transforming growth factor β, and stromal cell–derived factor 1 are notably up-regulated in glioblastoma and dually promote immune and MSC trafficking. Mesenchymal stem cells have widely been regarded as hypoimmunogenic, enabling this cell-based administration across major histocompatibility barriers. In this review, we will highlight (1) the bidirectional communication of glioma cells and tumor-associated immune cells, (2) the inflammatory mediators enabling leukocytes and transplantable MSC migration, and (3) review preclinical and human clinical trials using MSCs as delivery vehicles. Mesenchymal stem cells possess innate abilities to migrate great distances, cross the blood-brain barrier, and communicate with surrounding cells, all of which make them desirable “Trojan horses” for brain cancer therapy.

      Abbreviations and Acronyms:

      AMSC (adipose tissue–derived mesenchymal stem cell), BBB (blood-brain barrier), BMSC (bone marrow–derived mesenchymal stem cell), CED (convection-enhanced delivery), DC (dendritic cell), EGFRvIII (EGFR variant III), 5-FC (5-fluorocytosine), GBM (glioblastoma), GSC (glioma stem cell), IFN (interferon), IL (interleukin), MDSC (myeloid-derived suppressor cell), MHC (major histocompatibility complex), MSC (mesenchymal stem cell), NSC (neural stem cell), TAM (tumor-associated macrophage), TGF (transforming growth factor), TNF (tumor necrosis factor), UC-MSC (umbilical cord MSC)
      Article Highlights
      • Glioblastomas avoid elimination by the immune system through the secretion of immunomodulating factors.
      • Mechanisms for gliomagenesis involved in immunosuppression and tumor progression also mediate the recruitment of therapeutic and transplantable mesenchymal stem cells (MSCs) for cellular therapy.
      • Mesenchymal stem cells as cellular therapy may offer 2 advantages for glioma: site-specific targeting and immunomodulation via cargo delivery.
      • Mesenchymal stem cells (adipose tissue–derived MSCs, bone marrow MSCs, umbilical cord MSCs) display favorable results as therapeutic delivery vehicles and could be considered as a viable alternatives to neural stem cells because of their availability, ease of extraction, simple expansion protocols, and low immunogenicity permitting their transplant across major histocompatibility barriers.
      • Mesenchymal stem cells display promising potential as immunotherapy for glioblastoma via cargo delivery of immunostimulants.
      Glioblastoma (grade IV) is the most malignant form of primary brain cancer and makes up 15% of all primary brain tumors. Despite the most aggressive treatment strategies (radiation, chemotherapy, and surgery), patients have a median survival of approximately 15 months.
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      • et al.
      Cancer Genome Atlas Research Network
      Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.
      Tumors arising from glial cells account for 75% of malignant brain tumors and are classified by histopathologic and genetic findings. Grade IV gliomas are classified by IDH (for expansion of gene symbols, use search tool at www.genenames.org) alterations, p53, ATRX loss, and 1p/19q codeletions and stratified into 4 subtypes: classic, neural, mesenchymal, and proneural.
      • Verhaak R.G.
      • Hoadley K.A.
      • Purdom E.
      • et al.
      Cancer Genome Atlas Research Network
      Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.
      Genetic alterations and immunosuppression drive gliomagenesis, promoting tumor cell growth, proliferation, cellular invasion, and therapeutic resistance.
      • Charles N.A.
      • Holland E.C.
      • Gilbertson R.
      • Glass R.
      • Kettenmann H.
      The brain tumor microenvironment [published correction appears in Glia. 2012;60(3):502-514].
      Malignant tumors have been described as chronic injuries
      • Charles N.A.
      • Holland E.C.
      • Gilbertson R.
      • Glass R.
      • Kettenmann H.
      The brain tumor microenvironment [published correction appears in Glia. 2012;60(3):502-514].
      wherein inflammation plays a large role in advancing the proliferation, progression, and aggressiveness of tumor growth.
      • Dvorak H.F.
      Tumors: wounds that do not heal; similarities between tumor stroma generation and wound healing.
      One major problem encountered with the treatment of gliomas is the blood-brain barrier (BBB). This structural and biological barrier impedes accumulation of effective therapeutic concentrations into the tumor bulk. Administration of pharmacological agents are conservatively regimented due to the vulnerability of healthy cells and the risks of off-target effects ultimatley impeding effective pharmacological concentrations for therapeutic efficacy. This stringent balance of systemic toxicity vs tumor ablation has hindered the translation of therapies with strong tumoricidal effects that have otherwise shown robust efficacy, preclinically. Moreover, histopathologic and tumor composition studies have revealed considerable heterogeneity in the tumor bulk, rendering directed and targeted therapy even more complex.
      The tumor niche consists of stromal cells (endothelial, fibroblasts, pericytes), reactive astrocytes, tumor cells with varying lineage heterogeneity, and invading immune cells (microglia, macrophages, granulocytes, B cells, and T cells). However, the inability to stimulate an antitumor immune response is due to multiple soluble factors released by tumor cells that mediate immune reprogramming and allow the recruitment of immunosuppressive cells. Clinical data suggest extensive infiltration of peripheral monocytes that have assumed an immunosuppressive state; this infiltration and accumulation in the tumor bulk is directly correlated with glioma grade, with glioblastoma (grade IV) being the most infiltrated.
      • Pinton L.
      • Masetto E.
      • Vettore M.
      • et al.
      The immune suppressive microenvironment of human gliomas depends on the accumulation of bone marrow-derived macrophages in the center of the lesion.
      Mesenchymal stem cells (MSCs) from bone marrow (BMSCs), adipose tissue (AMSCs), or umbilical cord (UC-MSCs) have been preclinically investigated for the treatment of brain cancer by delivering various antiglioma cargo to modulate the tumor niche. An effective treatment strategy for glioma would preferentially target the tumor and enable the release of a therapeutic payload to transformed cells while sparing healthy cells in proximity. Mesenchymal stem cells have emerged as one potential cellular vehicle for the delivery of therapeutic cargo and may be an effective candidate as immune cargo delivery vehicles to brain cancer. The influence of inflammatory cytokines originating from the tumor niche enable MSCs to selectively migrate to tumor areas.
      • Li Q.
      • Wijesekera O.
      • Salas S.J.
      • et al.
      Mesenchymal stem cells from human fat engineered to secrete BMP4 are nononcogenic, suppress brain cancer, and prolong survival.
      ,
      • Zachar L.
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      • Rosocha J.
      Activation, homing, and role of the mesenchymal stem cells in the inflammatory environment.
      There is scarcity in the literature regarding the role of the immune system in glioma initiation, but strong evidence suggests that immune cells inhabiting the tumor niche are able to support gliomagenesis.
      • Galvão R.P.
      • Zong H.
      Inflammation and gliomagenesis: bi-directional communication at early and late stages of tumor progression.
      Such mechanisms include immunomodulation initiated by secretion of soluble factors,
      • Gomez G.G.
      • Kruse C.A.
      Mechanisms of malignant glioma immune resistance and sources of immunosuppression.
      induction of T-cell anergy,
      • Humphries W.
      • Wei J.
      • Sampson J.H.
      • Heimberger A.B.
      The role of Tregs in glioma-mediated immunosuppression: potential target for intervention.
      polarization of microglia and macrophages toward an immunosuppressive state,
      • Hussain S.F.
      • Yang D.
      • Suki D.
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      • Grimm E.
      • Heimberger A.B.
      The role of human glioma-infiltrating microglia/macrophages in mediating antitumor immune responses.
      extracellular matrix reconstruction to allow for tumor cell migration and invasion, and activation of the tumor stromal compartments for support and maintenance of cancer cell niche for survival. These aforementioned factors work together in synchrony to create a tumor microenvironment that favors tumor cells harboring a selective mutational advantage to evade immunosurveillance.
      Mesenchymal stem cells have widely been regarded as hypoimmunogenic, enabling MSC administration across major histocompatibility complex (MHC) barriers. While MSCs are not immunoprivileged, they are regarded as immunoevasive and largely go undetected by the immune system. Mesenchymal stem cells possess innate abilities to migrate great distances, cross the BBB, and communicate with surrounding immune cells, all of which make them desirable “Trojan horses” for brain cancer therapy. In this review, we will highlight (1) the bidirectional communication of glioma cells and tumor-associated immune cells, (2) the inflammatory mediators released by glioma that could be exploited for the recruitment and migration of therapeutic transplantable MSCs, and (3) future implications for utilizing MSCs as cargo delivery vehicles for glioblastoma (GBM) immunomodulation (Figure 1).
      Figure thumbnail gr1
      Figure 1Mode of mesenchymal stem cell (MSC) and immune cell migration and trafficking to glioblastoma (GBM). (1) Inflammatory cytokines released by GBM (2) promote extravasation of MSCs, lymphocytes, and monocytes and direct them toward tumor location. IL = interleukin; TGF = transforming growth factor; TNF = tumor necrosis factor.

      Noxious Loop Between Tumor-Associated Immune Cells and Glioma

      Despite aggressive therapy, the progression of glioma in patients suggests a gross failure in host immune mechanisms. Various strategies in the clinic have employed immunostimulants to reactivate immune surveillence, such as interleukin (IL) 12 (ClinicalTrials.gov Identifier: NCT02079324), GM-CSF, tumor necrosis factor (TNF) α, and interferon (IFN) β (ClinicalTrials.gov Identifier: NCT02530047). Notwithstanding ongoing research and numerous strategies combating glioma, methods to reverse immunosuppression in patients have produced little success. This problem is generally due to cells gaining a mutational advantage over the course of the evolutionary process of gliomagenesis, rendering tumor cells virtually undetected by surveillance mechanisms. During tumor initiation and evolution, competent immune cells, such as microglia, natural killer cells, macrophages, and dendritic cells (DCs), attempt to destroy the tumor via the release of cytotoxic and proinflammatory factors such as TNF-α and IL-6, leading to the recruitment of helper CD4+ and cytotoxic CD8+ T cells from the periphery to the tumor bed.
      • Hambardzumyan D.
      • Gutmann D.H.
      • Kettenmann H.
      The role of microglia and macrophages in glioma maintenance and progression.
      This movment is aided by the release of the proinflammatory cytokine IL-1β, that contributes to the loss of BBB integrity via the expression of genes favoring vessel plasticity. Loss of BBB integrity permit infiltration of myeloid-derived suppressor cells (MDSCs) in the tumor bulk. Recruited macrophages up-regulate inducible nitric oxide synthase and secrete IFN-β, IL-12, and MCP1 in the growing tumor.
      • Colton C.A.
      Heterogeneity of microglial activation in the innate immune response in the brain.
      Tumor-infiltrating leukocytes, measured by the presence of CD45+ from human biopsy samples following resection, constitute 40.3% of grade IV gliomas; 85.6% of which are CD33+ MDSCs.
      • Pinton L.
      • Masetto E.
      • Vettore M.
      • et al.
      The immune suppressive microenvironment of human gliomas depends on the accumulation of bone marrow-derived macrophages in the center of the lesion.
      Composition of the tumor-associated macrophage (TAM) and polymorphonuclear cell subsets in MDSCs, defined by investigators as CD33+/HLA-DR+ antigen and CD33+/HLA-DR antigen constitute 64.7% and 15.8%, respectively, of the immune concentration found in tumor bed. TAMs and brain-resident microglia comprise the dominant nonneoplastic cell type in the tumor and largely contribute to the initial breakdown of immunosurveillance and eventual immunosuppression via the reciprocal release of multiple soluble factors: GM-CSF, SDF1, HGF/SF, CX3CL1, GDNF, ATP, MCP1, MCP3, (Figure 2).
      • Hambardzumyan D.
      • Gutmann D.H.
      • Kettenmann H.
      The role of microglia and macrophages in glioma maintenance and progression.
      In an effort to evaluate the prognostic potential of peripheral MDSCs on survival, investigators evaluated peripheral immune composition and concentration of MDSCs in 259 blood samples from patients with newly diagnosed and recurrent GBM. The study found that reduced MDSC concentrations in blood resulted in better outcomes
      • Alban T.J.
      • Alvarado A.G.
      • Sorensen M.D.
      • et al.
      Global immune fingerprinting in glioblastoma patient peripheral blood reveals immune-suppression signatures associated with prognosis.
      ; strategies to target MDSCs may offer a new avenue for GBM immunotherapy that would slow tumor growth at onset of disease.
      Figure thumbnail gr2
      Figure 2Immunosuppressive soluble factors. ATP = adenosine triphosphate; CSF-1 = colongy stimulating factor 1; CX3CL1 = chemokine ligand 1; GDNF = glial cell line-derived neurotrophic factor; GM-CSF = granulocyte-macrophage colony stimulating factor; HGF/SF = hepatocyte growth factor/scatter factor; MCP-1 = monocyte chemoattractant protein 1; MCP-3 = monocyte chemotactic protein; SDF-1 = stromal derived factor 1; TAM = tumor-associated macrophage.
      TAMs and MDSCs are associated with poor survival and contribute to innate immunosuppression via cross-talk with surrounding tumor cells.
      • Nishie A.
      • Ono M.
      • Shono T.
      • et al.
      Macrophage infiltration and heme oxygenase-1 expression correlate with angiogenesis in human gliomas.
      Results from one study suggest that the recruitment of MDSCs to the tumor bed is, in part, initiated by the secretion of macrophage migration inhibitory factor by tumor cells.
      • Otvos B.
      • Silver D.J.
      • Mulkearns-Hubert E.E.
      • et al.
      Cancer stem cell-secreted macrophage migration inhibitory factor stimulates myeloid derived suppressor cell function and facilitates glioblastoma immune evasion.
      Upon arrival, infiltrated microglia and macrophages transition from the proinflammatory “M1” state to an anti-inflammatory “M2” phenotype. This transition is facilitated by the uptake of tumor-derived factors such as MCP1,
      • Platten M.
      • Kretz A.
      • Naumann U.
      • et al.
      Monocyte chemoattractant protein-1 increases microglial infiltration and aggressiveness of gliomas.
      CSF1,
      • Alterman R.L.
      • Stanley E.R.
      Colony stimulating factor-1 expression in human glioma.
      and MCP3.
      • Okada M.
      • Saio M.
      • Kito Y.
      • et al.
      Tumor-associated macrophage/microglia infiltration in human gliomas is correlated with MCP-3, but not MCP-1.
      Over the course of tumor progression and through various tumor mechanisms, recruited macrophages lose their phagocytic ability, cytotoxic T-cell proliferation is inhibited, and there is an increase in infiltration of regulatory T cells, resulting in a chronic immunosuppressive state and tumor tolerance. With M2 or TAMs being the primary phenotype found in the tumor microenvironment, a more robust shift occurs, and an up-regulation of anti-inflammatory factors ensues.
      • Raes G.
      • Noël W.
      • Beschin A.
      • Brys L.
      • de Baetselier P.
      • Hassanzadeh G.H.
      FIZZ1 and Ym as tools to discriminate between differentially activated macrophages.
      These factors dampen tumor clearance and allow for a more permissive environment where cancer cells thrive.
      • Kong L.Y.
      • Wu A.S.
      • Doucette T.
      • et al.
      Intratumoral mediated immunosuppression is prognostic in genetically engineered murine models of glioma and correlates to immunotherapeutic responses.
      A toxic loop is now established within the tumor niche as more tumor-supportive immune cells, such as regulatory T cells and MDSCs, arrive.
      Soluble factors involved in the recruitment of MDSCs and leukocytes to the tumor have been implicated in the tropism and favored movement of neural stem cells (NSCs) and MSCs to glioma. These cells have been extensively inestigated as delivery vehicles for antiglioma cargo.

      Stem Cell Applications for Glioma

      Stem cell applications have been studied extensively in the field of regenerative medicine and are currently making headway in solid cancers. The 2 most studied stem cell applications for brain cancer therapy are MSCs and NSCs. Their application relies on the tropic and homing capacity toward brain tumors and the therapeutic delivery of antiglioma cargo. Precise and targeted applications for brain tumors via tumor antigens and neoantigens are currently being explored to avoid off-target toxicity. However, MSCs and NSCs have been found to be effective vehicles that colocalize to tumor cells when administered locally and systemically. While preclinical applications are being explored for MSCs in GBM, NSCs are currently undergoing phase 1 and 2 clinical trials in human patients. The first human safety and feasibility phase 1 study with stem cell therapy employed genetically modified NSCs expressing cytosine deaminase, an enzyme that converts the prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic 5-fluorouracil. Engineered NSCs were administered intracerebrally during resection and patients were given a 7-day oral dose of 5-FC (NCT02015819/NCT01172964).
      • Mutukula N.
      • Elkabetz Y.
      Neural killer" cells: autologous cytotoxic neural stem cells for fighting glioma.
      Follow-up results documented safety, and autopsy results revealed NSC homing to distant microsatellite tumors in the brain. The concluded pilot study documented proof of concept regarding NSCs’ tropism to glioma. Furthermore, the study confirmed the diffusion of 5-FC out of NSCs into adjacent and highly proliferative brain tumor cells mediating cell killing by proxy.
      • Portnow J.
      • Synold T.W.
      • Badie B.
      • et al.
      Neural stem cell-based anticancer gene therapy: a first-in-human study in recurrent high-grade glioma patients.
      Current work is focused on dose escalation regimens to identify an optimal therapeutic dose for phase 2. Similarly, NSCs were engineered to express carboxylesterase enzyme that mediates the conversion of the prodrug CPT-11 (irinotecan) to SN-38, a topoisomerase 1 inhibitor potent at killing cancer cells. Application is currently under way in phase 1 clinical trials for the intracerebral injection of patients with recurrent glioblastoma (NCT02192359). Although the administration of a prodrug with NSC-enzyme therapy has been proven safe for the intracereberal injection in recurrent GBM, NSCs have also been engineered to deliver oncolytic adenoviral therapy with concomitant administration of chemotherapy and radiation in patients with primary GBM following a phase 1 clinical trial (NCT03072134). Preclinical data on human NSCs documented efficacy in patient-derived xenograft mouse models of human commercial U87 and U251 GBM lines.
      • Ahmed A.U.
      • Thaci B.
      • Tobias A.L.
      • et al.
      A preclinical evaluation of neural stem cell-based cell carrier for targeted antiglioma oncolytic virotherapy.
      ,
      • Tobias A.L.
      • Thaci B.
      • Auffinger B.
      • et al.
      The timing of neural stem cell-based virotherapy is critical for optimal therapeutic efficacy when applied with radiation and chemotherapy for the treatment of glioblastoma.
      Currently, NSC-mediated cargo delivery to GBM owes its therapeutic success to the “bystander effect.” Cancer cell death is mediated via the diffusion of drug or virus out of the exogenously delivered NSCs through cell junctions and into adjacent cancer cells. They have limited effects as naive NSCs compared with MSCs. Although NSCs are known to be hypoimmunogenic, most NSC-mediated delivery utilizes an immortalized commercial cell line (HB1.F3.CD21)
      • Portnow J.
      • Synold T.W.
      • Badie B.
      • et al.
      Neural stem cell-based anticancer gene therapy: a first-in-human study in recurrent high-grade glioma patients.
      ,
      • Metz M.Z.
      • Gutova M.
      • Lacey S.F.
      • et al.
      Neural stem cell-mediated delivery of irinotecan-activating carboxylesterases to glioma: implications for clinical use.
      that is well characterized and found to be stable over sequential passaging and culture propagation. However, oncogenic transformation and serial karyotyping during propagation of immortalized cell lines must be closely monitored. Autologous MSCs applications, such as those isolated from adipose tissue offer minimally invasive protocols for the procurement of stem cells. Nonimmortalized primary NSCs can be isolated from the periventricular zone of adult brains during surgery or from fetal brains; the amount required for therapeutic application (or multiple dosing) necessitates high procurement of NSCs to meet sufficient dose requirements for human trials. Consequently, consecutive culture passaging of primary NSCs increases the risk of potential lineage commitment and differentiation that could lead to loss of migratory ability toward gliomas. Current clinical applications of NSCs to brain tumors use allogeneic lines, with the assumption that NSCs are hypoimmunogenic and express low levels of MHC class I and II. A study investigated the immunogenicity of human NSCs with HLA-incompatible donors in a one-way mixed lymphocyte reaction found that NSCs stimulated lymphocyte in all nonmatched donors, suggesting that this sensitivity may be sufficient to enable immunorecognition of exogenously delivered and allogenically grafted NSCs. The investigators reported that although immunogenicity is low, it is not negligible.
      • Ubiali F.
      • Nava S.
      • Nessi V.
      • et al.
      Allorecognition of human neural stem cells by peripheral blood lymphocytes despite low expression of MHC molecules: role of TGF-β in modulating proliferation.
      Long-term risk would ultimately lead to activation of peripheral lymphocytes and eventual clearance of the therapeutic vehicle (NSCs) more rapidly.
      Although NSCs display a relatively good safety index for the treatment of gliomas, MSCs are becoming seriously considered for the purpose of glioma therapy. Mesenchymal stem cells are immunoevasive and hypoimmunogenic, isolation is minimally invasive, large expansion protocols for primary human clinical use can be achieved, and risks for cell rejection can be eliminated with the use of autologous MSCs, given ease of procurement. Thus, simplicity of extraction, little to no notable cell manipulation (ie, immortalization), intrinsic immunomodulatory ability, and their tropic capacity to areas of insult make MSCs optimal cellular therapeutics.

      MSC Applications for Glioma

      Mesenchymal stem cells are multipotent adult stem cells residing in various tissues and organs in the adult body. These cells have been widely used in the field of regenerative medicine for tissue regeneration, wound healing, vehicles for cargo delivery, and immunomodulation. It was originally believed that MSCs integrate into the site of injury and differentiate into surrounding stroma to enable tissue regeneration. This theory has since been challenged (due to poor MSC persistence and retention after implantation); it is believed that MSCs act in a paracrine fashion through site-specific modulation and can endogenously regulate tissue at the site of implantation, primarily through soluble factor secretion. In this fashion, MSCs act dynamically in response to an insult and regulate the inflammatory response in a context-dependent manner. Mesenchymal stem cells have been widely used in clinical trials as intrinsic modulators of chronic inflammation or autoimmune disorders.
      • Huang Y.Z.
      • Xie H.Q.
      • Silini A.
      • et al.
      Mesenchymal stem/progenitor cells derived from articular cartilage, synovial membrane and synovial fluid for cartilage regeneration: current status and future perspectives.
      Their multipotency, immune privilege, and immunomodulatory capabilities make them a viable source for autologous or allogenic applications. Allogenic MSCs have a wide portfolio of diverse applications for human clinical trials in solid tumors. Numerous preclinical and clinical trials are assessing their potential in several disease models such as Crohn disease, tissue repair, graft-vs-host, cancer, and neurodegenerative diseases.
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      • Gothelf Y.
      • Argov Z.
      • et al.
      Safety and clinical effects of mesenchymal stem cells secreting neurotrophic factor transplantation in patients with amyotrophic lateral sclerosis: results of phase 1/2 and 2a clinical trials.
      • Mazzini L.
      • Vescovi A.
      • Cantello R.
      • Gelati M.
      • Vercelli A.
      Stem cells therapy for ALS.
      • Kim T.I.
      Clinical trials with stem cells in digestive diseases and future perspectives [in Korean].
      Although MSCs are widely used in regenerative medicine, their applications in solid tumors are currently being explored in early-phase human clinical trials using ClinicalTrails.gov (search words: cancer, MSCs, GBM) (Table 1). In refractory ovarian carcinoma, administrations of allogenic MSCs from male donor(s) virally engineered to secret IFN-β were evaluated for their safety in a phase 1 single-center trial (NCT02530047). One study delivered MSCs engineered with an oncolytic measles virus encoding a sodium iodide symporter as treatment for recurrent ovarian cancer (NCT02068794). Another phase 1 clinical trial used allogenic MSCs to target prostate cancer cells, while another used adipose tissue–derived culture-expanded AMSCs against pancreatic carcinoma (NCT04087889). To determine the maximum tolerable dose and safety of genetically modified MSCs expressing GX-051 (IL-12), Other investigators have tested intratumoral injections of the modified cells in patients with head and neck cancer (NCT02079324). Similarly, another study evaluated the safety and tolerability of weekly infusions of autologous BMSCs infected with an oncolytic adenovirus (ICOVIR5) (NCT01983709).
      Table 1Human Clinical Trial Applications of MSCs in Solid Tumors
      Source of MSCDiagnosisTrial phaseRoute of administrationParameters to evaluateClinical trial IDCytokine, factors, or drug involved
      Bone marrowProstate cancer1IntravenousRatio of MSC genomic to prostate DNA in bodily fluids (blood, seminal vesicles) in resected prostateNCT01983709Toxins (not specified)
      Bone marrowOvarian carcinoma1IntraperitonealMaximum tolerated doseNCT02530047INF-β
      Adipose tissuePancreatic cancer1IntravenousClinical response and adverse effectsNCT04087889Not specified
      Adipose tissueRecurrent ovarian cancer1IntraperitonealSafety and tolerabilityNCT02068794Oncolytic measles virus encoding thyroidal sodium iodide symporter
      Response rate, progression-free survival, and overall survival
      Not specifiedHead and neck cancer1IntratumoralTo assess safety and tolerabilityNCT02079324GX-051 (IL-12)
      Bone marrowRefractory solid tumors1IntravenousTo assess safety and tolerabilityNCT01983709ICOVIR5
      Bone marrowGBM, gliosarcoma, anaplastic astrocytoma1Intra-arterialTo evaluate safety, toxicity, and immuno-mediated cytokine responsesNCT03896568Oncolytic adenovirus (DNX-2401)
      To evaluate progression-free survival
      GBM = glioblastoma; ID = identification number; IL = interleukin; INF = interferon; MSC = mesenchymal stem cell.
      In brain cancer, specifically for recurrent high-grade gliomas (including gliosarcoma, anaplastic astrocytoma, and GBM), an ongoing phase 1 trial is evaluating cytokine response, safety, toxicity, and progression-free survival in response to intra-arterial administration of allogeneic human BMSCs carrying oncolytic adenovirus (DNX-2401) (NCT03896568).
      Adipose tissue–derived MSCs have been a good source of stem cell therapy, not only for regenerative medicine but also as vehicles for antiglioma cargo to the central nervous system.
      • Kim N.
      • Cho S.G.
      Clinical applications of mesenchymal stem cells.
      ,
      • Rustad K.C.
      • Gurtner G.C.
      Mesenchymal stem cells home to sites of injury and inflammation.
      They have previously been reported to selectively migrate in vitro and in vivo to xenograft models of human gliomas and secrete antiglioma cargo such as BMP4,
      • Li Q.
      • Wijesekera O.
      • Salas S.J.
      • et al.
      Mesenchymal stem cells from human fat engineered to secrete BMP4 are nononcogenic, suppress brain cancer, and prolong survival.
      ,
      • Ganaha S.
      • Al-Kharboosh R.
      • Ruiz-Valls A.
      • Guerrero Cazares H.
      • Quinones-Hinojosa A.
      Human fat-derived mesenchymal stem cells bioengineered to secrete BMP4 are nononcogenic, suppress glioma, and prolong survival.
      resulting in tumor reduction and increased survival. As cells that are endogenously designed to maintain homeostasis, they have an intrinsic capacity to adhere to brain endothelium as well as migrate across the BBB, allowing them to travel systemically and home to areas of high chemotactic and growth factor secretion such as that of GBM,
      • Li Q.
      • Wijesekera O.
      • Salas S.J.
      • et al.
      Mesenchymal stem cells from human fat engineered to secrete BMP4 are nononcogenic, suppress brain cancer, and prolong survival.
      ,
      • Ganaha S.
      • Al-Kharboosh R.
      • Ruiz-Valls A.
      • Guerrero Cazares H.
      • Quinones-Hinojosa A.
      Human fat-derived mesenchymal stem cells bioengineered to secrete BMP4 are nononcogenic, suppress glioma, and prolong survival.
      making them desirable vehicles for cargo delivery. Studies have validated AMSC safety against human brain tumors without risk of oncogenic transformation.
      • Zuk P.A.
      • Zhu M.
      • Ashjian P.
      • et al.
      Human adipose tissue is a source of multipotent stem cells.
      ,
      • Kucerova L.
      • Matuskova M.
      • Hlubinova K.
      • Altanerova V.
      • Altaner C.
      Tumor cell behaviour modulation by mesenchymal stromal cells.
      Adipose tissue–derived MSCs are a convenient form of cell therapy and have displayed promising potential as primary cellular vehicles compared with NSCs (mainly due to limitations in supply and challenges in acquisition) or BMSCs (which require invasive aspirations and have a relatively low extraction yield, eventually declining in life span on implantation).
      • Mishra P.J.
      • Mishra P.J.
      • Glod J.W.
      • Banerjee D.
      Mesenchymal stem cells: flip side of the coin [published correction appears in Cancer Res. 2009;69(7):3240].
      Similarly, BMSCs can scavenge surrounding cells and modulate tissue via secretion of suppressive and/or supportive soluble factors mediating the polarization or enhanced activation of macrophages; BMSCs can act as innate immunomodulators in their own right without modification.
      • Vasandan A.B.
      • Jahnavi S.
      • Shashank C.
      • Prasad P.
      • Kumar A.
      • Prasanna S.J.
      Human mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a PGE2-dependent mechanism.
      Bone marrow–derived mesenchymal stem cells have been used in clinical trials for chronic inflammation and autoimmune disorders.
      • Huang Y.Z.
      • Xie H.Q.
      • Silini A.
      • et al.
      Mesenchymal stem/progenitor cells derived from articular cartilage, synovial membrane and synovial fluid for cartilage regeneration: current status and future perspectives.
      Their multipotency, immune privilege, and immunomodulatory capabilities make them a viable source for autologous or allogenic applications. Strategies using BMSCs to increase the infiltration of cytotoxic T cells and enhance natural killer cell surveillance are being investigated, with documented efficacy. Preclinically, AMCSs and BMSCs have been engineered to deliver immunoactivating factors such as TNF-related apoptosis-inducing ligand,
      • Sasportas L.S.
      • Kasmieh R.
      • Wakimoto H.
      • et al.
      Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy.
      IL-12,
      • Hong X.
      • Miller C.
      • Savant-Bhonsale S.
      • Kalkanis S.N.
      Antitumor treatment using interleukin-12-secreting marrow stromal cells in an invasive glioma model.
      and IL-18,
      • Xu G.
      • Jiang X.D.
      • Xu Y.
      • et al.
      Adenoviral-mediated interleukin-18 expression in mesenchymal stem cells effectively suppresses the growth of glioma in rats.
      resulting in the activation of natural killer cells and infiltration of tumor-specific CD4 and CD8 T cells. Furthermore, studies suggest that BMSCs can act as antigen-presenting cells in the presence of IFN-γ by up-regulating MHC class I and II.
      • Ströjby S.
      • Eberstål S.
      • Svensson A.
      • et al.
      Intratumorally implanted mesenchymal stromal cells potentiate peripheral immunotherapy against malignant rat gliomas.
      Primary regulators of MSC tropism to GBM are mediated by soluble factors reviewed in the following section (Table 2).
      Table 2Preclinical Applications of Mesenchymal Stem Cells in Glioblastoma
      Reference, yearCell line/speciesSource of MSC/speciesExperimental speciesIn vitro/in vivoResultsImplicated cytokine and factors involved in cross-talk/up-regulated in GBM
      Smith et al,
      • Smith C.L.
      • Chaichana K.L.
      • Lee Y.M.
      • et al.
      Pre-exposure of human adipose mesenchymal stem cells to soluble factors enhances their homing to brain cancer.
      2015
      Human U87Human/primary AMSCAthymic miceIn vitro/in vivoPreexposure to GBM in vitro enhanced its migratory potential in vivoTNF-α/yes
      Egea et al,
      • Ganaha S.
      • Al-Kharboosh R.
      • Ruiz-Valls A.
      • Guerrero Cazares H.
      • Quinones-Hinojosa A.
      Human fat-derived mesenchymal stem cells bioengineered to secrete BMP4 are nononcogenic, suppress glioma, and prolong survival.
      2011
      Human U87Human/primary BMSCAthymic miceIn vitro/in vivoPreincubation of BMSC with TNF enhanced its migratory potential to GBMTNF-α/yes
      Choi et al,
      • Choi S.A.
      • Lee J.Y.
      • Kwon S.E.
      • et al.
      Human adipose tissue-derived mesenchymal stem cells target brain tumor-initiating cells [published correction appears in PLoS One. 2015;10(7):e0132877].
      2015
      Human/primary GBMHuman/primary AMSCNAIn vitroMigratory ability of AMSCs toward BTICs is mediated by the cross-talk of brain cancer cells and MSCsCXCR4/SDF-1, IL-6R/IL-6, IL-8R/IL-8
      Shahrokhi et al,
      • Shahrokhi S.
      • Daneshmandi S.
      • Menaa F.
      Tumor necrosis factor-α/CD40 ligand-engineered mesenchymal stem cells greatly enhanced the antitumor immune response and lifespan in mice.
      2014
      Murine/4T1 –breast cancer lineMurine/primary isolated from BALB/c mice and modified with TNF/CD40BALB/c immuno-competentIn vitro/in vivoMSC (TNF/CD)– suppressed helper T cell type 2, and regulatory T cells. Down-regulation of IL-4, IL-10.T cell TNF-α/CD40
      Enhanced survival in murine models of subcutaneous breast cancer
      Carrero et al,
      • Carrero R.
      • Cerrada I.
      • Lledó E.
      • et al.
      IL1β induces mesenchymal stem cells migration and leucocyte chemotaxis through NF-κB.
      2012
      NAHuman/primary BMSCNAIn vitroBMSC increased the recruitment of leukocytes. Reciprocal recruitment observed on IL-1β stimulation of BMCSIL-1β
      Pacioni et al,
      • Pacioni S.
      • D'Alessandris Q.G.
      • Giannetti S.
      • et al.
      Human mesenchymal stromal cells inhibit tumor growth in orthotopic glioblastoma xenografts.
      2017
      Human/U87Human/primary AMSCAthymic ratsIn vivoSystemic administration of MSCs colocalized to GBM in vivoGBM-soluble factors (unidentified)
      Human/GSC1
      Pavon et al,
      • Pavon L.F.
      • Sibov T.T.
      • de Souza A.V.
      • et al.
      Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo.
      2018
      Primary human GBM lines sorted for CD133 (BTICs/ GSC)Human/primary UC-MSCAthymic miceIn vitro/in vivoUC-MSCs migrate specifically toward glioma stemlike cells (CD133) in vitro and in vivoMCP-1/CCL2
      SDF-1/CXCL12
      Lourenco et al,
      • Lourenco S.
      • Teixeira V.H.
      • Kalber T.
      • Jose R.J.
      • Floto R.A.
      • Janes S.M.
      Macrophage migration inhibitory factor-CXCR4 is the dominant chemotactic axis in human mesenchymal stem cell recruitment to tumors.
      2015
      Human/U87Human/primary BMSCNANABMSCs migrate to U87 and knockdown of CXCR4 in BMSC abrogated tumor tropismCXCR4
      Li et al,
      • Li Q.
      • Wijesekera O.
      • Salas S.J.
      • et al.
      Mesenchymal stem cells from human fat engineered to secrete BMP4 are nononcogenic, suppress brain cancer, and prolong survival.
      2014
      Human/ GBM276Human/commercialAthymic miceIn vitro/in vivoModified and unmodified AMSCs migrate to glioma in vitro and in vivo and substantially enhance tumor survivalCXCR4
      Human/GBM612AMSC (Invitrogen R7788-115) expressing human BMP4
      Li et al,
      • Li M.
      • Zeng L.
      • Liu S.
      • et al.
      Transforming growth factor-β promotes homing and therapeutic efficacy of human mesenchymal stem cells to glioblastoma.
      2019
      Primary human GBMHuman/primary AMSCAthymic miceIn vitro/in vivoPreconditioning AMSCs with TGF increased the homing ability in vitro and in vivo. Knockdown of TGF receptor abrogated AMSC homingTGF-β
      CXCR4
      AMSC = adipose tissue–derived mesenchymal stem cells; BMSC = bone marrow–derived mesenchymal stem cell; BTIC = brain tumor initiating cell; GBM= glioblastoma; GSC = glioma stem cell; IL = interleukin; MSC= mesenchymal stem cell; NA = not available; TGF-β = transforming growth factor β; TNF = tumor necrosis factor; UC-MSC = umbilical cord MSC. For expansion of gene symbols, use search tool at www.genenames.org.

      Mechanism of MSC Recruitment and Microenvironment Modulation

      Role of TNF-α

      Tumor necrosis factor α is a potent soluble cytokine involved in orchestrating response to systemic inflammation. This inflammatory cytokine is enigmatic in the tumor context, displaying tumoricidal effects against glioma cells as well as tumor-promoting abilities. Tumor necrosis factor α facilitates angiogenesis by up-regulating EGFR,
      • Kore R.A.
      • Abraham E.C.
      Inflammatory cytokines, interleukin-1 beta and tumor necrosis factor-alpha, upregulated in glioblastoma multiforme, raise the levels of CRYAB in exosomes secreted by U373 glioma cells.
      induces immune-cell suppression through NF-κβ and STAT3
      • Hoesel B.
      • Schmid J.A.
      The complexity of NF-κB signaling in inflammation and cancer.
      and down-regulates PTEN tumor suppressor gene in glioma.
      • Kim S.
      • Domon-Dell C.
      • Kang J.
      • Chung D.H.
      • Freund J.N.
      • Evers B.M.
      Down-regulation of the tumor suppressor PTEN by the tumor necrosis factor-α/nuclear factor-κB (NF-κB)-inducing kinase/NF-κB pathway is linked to a default IκB-α autoregulatory loop.
      Paradoxically, TNF-α knockout resulted in larger tumors and reduced overall survival in immunocompetent animals bearing GL261 glioma. Study results suggest TNF is involved in reduced macrophage infiltration in histolopathological examinations, suggesting TNF plays a tumor-suppressive role along with its tumor supportive capabilities.
      • Teo G.S.
      • Ankrum J.A.
      • Martinelli R.
      • et al.
      Mesenchymal stem cells transmigrate between and directly through tumor necrosis factor-α-activated endothelial cells via both leukocyte-like and novel mechanisms.
      Despite its biological versatility in cancer, TNF-α is enriched in patient-derived human GBM–conditioned media, and preincubation of human AMSCs with TNF-α enhanced AMSCs migration toward glioma in vitro.
      • Smith C.L.
      • Chaichana K.L.
      • Lee Y.M.
      • et al.
      Pre-exposure of human adipose mesenchymal stem cells to soluble factors enhances their homing to brain cancer.
      Macrophages and AMSC migration is recruited, in part, by the secretion of TNF-α from tumor microenvironments.
      • Hoesel B.
      • Schmid J.A.
      The complexity of NF-κB signaling in inflammation and cancer.
      ,
      • Baek S.J.
      • Kang S.K.
      • Ra J.C.
      In vitro migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors.
      Similarly, systemically injected human BMSCs, preconditioned with TNF (50 ng/mL), migrated substantially in vivo in murine models bearing human U87 GBM, measured 72 hours postinjection.
      • Egea V.
      • von Baumgarten L.
      • Schichor C.
      • et al.
      TNF-α respecifies human mesenchymal stem cells to a neural fate and promotes migration toward experimental glioma.
      Similarly, preincubation of human glioma stem cells (GSCs) with AMSC-conditioned media resulted in the decreased expression of inflammatory IL-6 and IL-8 factors in tumor cells post-incubation. These inflammatory cytokines are implicated in immunosuppression and function as angiogenic and mitogenic factors, their overexpression confers a worse prognosis in patients with GBM. Analogously, a study documented a marked reduction of IL-8 and IL-6 protein expression in indirect coculture assays of human GBM and AMSCs, revealing that soluble factors released by AMSCs, and the response of GBM to these factors in direct or indirect coculture similarly resulted in a decreased expression of IL-6 and IL-8.
      • Choi S.A.
      • Lee J.Y.
      • Kwon S.E.
      • et al.
      Human adipose tissue-derived mesenchymal stem cells target brain tumor-initiating cells [published correction appears in PLoS One. 2015;10(7):e0132877].
      Although glioma-secreted factors have been found to recruit AMSCs and macrophages, studies suggest that MSCs display endogenous therapeutic effects against glioma cells, outside the context of cargo delivery.
      Similar to AMSCs, BMSCs directly influence dendritic cell activation. In a 4T1 subcutaneous murine breast cancer model, virally engineered BALB/c BMSCs with TNF-α/CD40L exhibited enhanced dendritic cell maturation markers (CD86, CD40, and MHC class II) indicative of antigen presentation.
      • Shahrokhi S.
      • Daneshmandi S.
      • Menaa F.
      Tumor necrosis factor-α/CD40 ligand-engineered mesenchymal stem cells greatly enhanced the antitumor immune response and lifespan in mice.
      Furthermore, BMSCs cocultured with DCs increased expression of proinflammation cytokines (IL-12, IFN) and decreased anti-inflammatory expression, indicated by the reduction of IL-4, IL-10, and transforming growth factor (TGF).
      • Shahrokhi S.
      • Daneshmandi S.
      • Menaa F.
      Tumor necrosis factor-α/CD40 ligand-engineered mesenchymal stem cells greatly enhanced the antitumor immune response and lifespan in mice.
      These studies illustrate the ability of BMSCs to engage in mechanisms that facilitate antitumor immunity by potentially enhancing DC activation.

      Role of IL-1β

      Glioblastomas produce large quantities of IL-1β, which plays a crucial role in glioma aggressiveness and survival.
      • Tarassishin L.
      • Casper D.
      • Lee S.C.
      Aberrant expression of interleukin-1β and inflammasome activation in human malignant gliomas.
      ,
      • Yeung Y.T.
      • McDonald K.L.
      • Grewal T.
      • Munoz L.
      Interleukins in glioblastoma pathophysiology: implications for therapy.
      To evaluate IL-1β activation–induced changes in glioma, human GBM U251 cell line, corresponding to an aggressive “mesenchymal” subtype, was stimulated with recombinant IL-1β in vitro. Proteomic analysis of the secretome revealed 2 biological nodes enriched in U251 on stimulation: (1) extracellular remodeling mechanisms and (2) cellular communication. Specifically, IL-8 and CCL2, two principal components of monocyte recruitment, were substantially up-regulated.
      • Tarassishin L.
      • Lim J.
      • Weatherly D.B.
      • Angeletti R.H.
      • Lee S.C.
      Interleukin-1-induced changes in the glioblastoma secretome suggest its role in tumor progression.
      The tumor microenvironment has an inflammatory signature that may favor the movement of MSCs via the up-regulation of surface receptors whose common ligands are expressed by the tumor, such as the receptor for IL-8. A study analyzing the response of human BMSCs on IL-1β stimulation revealed notable up-regulation of cellular mechanisms related to migration, cellular adhesion, host defense, and immunoregulation through NF-κβ.
      • Carrero R.
      • Cerrada I.
      • Lledó E.
      • et al.
      IL1β induces mesenchymal stem cells migration and leucocyte chemotaxis through NF-κB.
      Furthermore, IL-1β treatment enhanced BMSC migration and improved the recruitment of neutrophiles and monocytes in vitro. Consequently, blockade of transcription factor NF-κβ in BMSCs reduced BMSC migration and recruitment of leukocytes. This finding suggests that the influences of immune cell recruitment by BMCSs is mediated via NF-κβ and enhanced by the presence of IL-1β.
      • Carrero R.
      • Cerrada I.
      • Lledó E.
      • et al.
      IL1β induces mesenchymal stem cells migration and leucocyte chemotaxis through NF-κB.
      As mentioned earlier, CCL2 is a monocyte recruitment factor implicated in peripheral immunoresponse, and its up-regulation is enhanced in the presence of IL-1β. When neutralizing antibodies were used to block CXCR2, BMSC migration decreased by 45%, implicating the IL-8/CXCR2 axis in the chemotactic regulation of BMSC migration.
      • Ringe J.
      • Strassburg S.
      • Neumann K.
      • et al.
      Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2.
      Adipose tissue–derived MSCs have also displayed the same tropic behavior toward brain cancer stem cells using an orthotopic human GBM model in athymic rats. Systemically administered AMSCs injected through the common carotid artery and femoral vein were able to extravasate through the disrupted BBB and localize in the brain tumor.
      • Pacioni S.
      • D'Alessandris Q.G.
      • Giannetti S.
      • et al.
      Human mesenchymal stromal cells inhibit tumor growth in orthotopic glioblastoma xenografts.
      Umbilical cord MSCs also display enhanced migration toward glioma. Gliomas secrete high levels of CCL2; UC-MSCs up-regulated chemokine receptor type 2 and CCL2 receptors and displayed directed migratory ability to a specific subset of stemlike brain tumor–initiating cells expressing CD133+. It is suggested that chemokine receptor type 2 expressed by UC-MSCs enabled the directed migration toward brain tumor cells secreting CCL2. This finding was corroborated using dose-dependent administration of CCL2 in transwell migrations experiments.
      • Pavon L.F.
      • Sibov T.T.
      • de Souza A.V.
      • et al.
      Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo.
      Investigators further found iron nanoparticle–labeled UC-MSCs migrate toward brain tumor cells tracked by magnetic resonance imaging. Although UC-MSCs migrated toward the tumor, there was no decrease in tumor size in an immunosuppressed rat model of human GBM. This result may be due to the source of MSCs and isolation protocols, thus highlighting the need for protocol standardization for pre-clinical studies using MSC administration. Despite different sources of MSCs (bone marrow, adipose tissue, umbilical cord), migration preference toward glioma is maintained presumably through the expression of receptors on MSCs and associate ligands secreted by the brain tumors.

      Role of SDF-1/CXCR4

      One mechanism of MSC tropism to tumor is through the CXCR4/SDF-1α axis. This receptor-ligand complex plays a vital role in cell migration and inflammation. CXCR4 is up-regulated in GSCs by 25- to 89-fold compared with noninvasive tumor cells and increases with tumor grade.
      • Ehtesham M.
      • Winston J.A.
      • Kabos P.
      • Thompson R.C.
      CXCR4 expression mediates glioma cell invasiveness.
      CXCR4 colocalizes with SDF-1α and is frequently found in regions of angiogenesis, necrosis and degenerative environments.
      • Maestroni G.J.
      • Hertens E.
      • Galli P.
      Factor(s) from nonmacrophage bone marrow stromal cells inhibit Lewis lung carcinoma and B16 melanoma growth in mice.
      ,
      • Richardson P.J.
      CXCR4 and glioblastoma.
      CXCR4 is a G protein–coupled receptor expressed on glioma cells, microglia, neurons, and astrocytes. SDF-1α (CXCL12), the ligand for CXCR4, is also found on endothelial cells lining the BBB vessels and is suggested to mediate the adhesion and transcytosis of immune and stem cells into the tissue; it is proposed that the administration of cellular therapies, such as MSCs, cross the endothelium of the BBB via the CXCR4/SDF-1α pathway.
      • Schmidt N.O.
      • Przylecki W.
      • Yang W.
      • et al.
      Brain tumor tropism of transplanted human neural stem cells is induced by vascular endothelial growth factor.
      Binding of SDF-1 to CXCR4 plays an essential role in MSC trafficking. Substantial reduction of BMSC migration toward the U87 GBM cell line was observed with the use of a CXCR4 receptor antagonist in a 3-dimensional invasion assay and similarly displayed reduced tropism in vivo in a pulmonary metastasis models. Studies suggest that BMSC homing is due, in part, by CXCR4.
      • Lourenco S.
      • Teixeira V.H.
      • Kalber T.
      • Jose R.J.
      • Floto R.A.
      • Janes S.M.
      Macrophage migration inhibitory factor-CXCR4 is the dominant chemotactic axis in human mesenchymal stem cell recruitment to tumors.
      This axis plays a crucial role in cell recruitment during central nervous system injury and may be an important player in recruiting reparative cells during tissue insult.
      • Lee B.C.
      • Lee T.H.
      • Avraham S.
      • Avraham H.K.
      Involvement of the chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1alpha in breast cancer cell migration through human brain microvascular endothelial cells.
      SDF-1α/CXCR4 destabilizes the integrity of the BBB vasculature, resulting in enhanced recruitment of cells from the circulation.
      • Lee B.C.
      • Lee T.H.
      • Avraham S.
      • Avraham H.K.
      Involvement of the chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1alpha in breast cancer cell migration through human brain microvascular endothelial cells.
      This signaling pathway mediates actin polymerization and pseudopodia formation, allowing for immune and MSC transcytosis across the vasculature to areas where a high concentration of SDF-1 is present. This axis may be one of the cardinal regulators of cell homing to brain tumor microenvironments. A cytokine array profile was conducted to investigate the cross-talk of AMSCs in coculture with GBM. The CXCR4–SDF-1 axis was identified as a major regulator in MSC trafficking toward gliomas.
      • Choi S.A.
      • Lee J.Y.
      • Kwon S.E.
      • et al.
      Human adipose tissue-derived mesenchymal stem cells target brain tumor-initiating cells [published correction appears in PLoS One. 2015;10(7):e0132877].
      Similar to BMSCs, AMSCs significantly respond to SDF-1 released by glioma cells and have been engineered to overexpress CXCR4 to enhance homing to tumors.

      Role of IL-6

      Interleukin 6 is a soluble constituent involved in the malignant progression of glioma.
      • Shan Y.
      • He X.
      • Song W.
      • Han D.
      • Niu J.
      • Wang J.
      Role of IL-6 in the invasiveness and prognosis of glioma.
      Interleukin 6 promotes renewal, invasion, and angiogenesis. In glioma, elevated ligand and receptor expression is associated with poor survival.
      • Tchirkov A.
      • Khalil T.
      • Chautard E.
      • et al.
      Interleukin-6 gene amplification and shortened survival in glioblastoma patients.
      The survival-promoting actions of IL-6 include suppression of immunosurveillance via the recruitment (and stimulation) of MDSCs and tumor-associated neutrophils. This induction cripples the response of surrounding helper T cell type 1 and cytolytic T cells, ultimately leading to T-cell dysfunction and an inhibition of tumor clearance. Interleukin 6 is notably implicated in GBM, and stimulation of brain tumor cells with IL-6 promotes the top 3 signal transduction pathways involved in gliomagenesis: (1) p42/p44-MAPK (mitogen-activated protein kinase); this pathway is deregulated in approximately one-third of all cancers and is highly involved in the sensing and processing of stress signals,
      • Dhillon A.S.
      • Hagan S.
      • Rath O.
      • Kolch W.
      MAP kinase signalling pathways in cancer.
      (2) PI3K/AKT, a signaling pathway implicated in enhancing angiogenesis, activating epithelial to mesenchymal transition for increased invasion, and the promotion of metastasis,
      • Crespo S.
      • Kind M.
      • Arcaro A.
      The role of the PI3K/AKT/mTOR pathway in brain tumor metastasis.
      and lastly, (3) JAK-STAT3 pathway, which blocks tumor recognition by immune cells and promotes cell cycling progression and inhibition of apoptosis.
      • Nicolas C.S.
      • Amici M.
      • Bortolotto Z.A.
      • et al.
      The role of JAK-STAT signaling within the CNS.
      Invasion and migration were enhanced in human GBM lines (T98G and U251), with increased exposure to soluble IL-6 documented by scratch assays and in vivo studies.
      • Liu Q.
      • Li G.
      • Li R.
      • et al.
      IL-6 promotion of glioblastoma cell invasion and angiogenesis in U251 and T98G cell lines.
      In other cancers, such as breast, a cytokine screen from conditioned breast cancer media identified IL-6 as a regulator of BMSC migration. Enhanced migration was dose dependent, confirmed by the addition of increasing amounts of IL-6 in the lower chamber of transwell assays with dose-dependent response.
      • Anton K.
      • Banerjee D.
      • Glod J.
      Macrophage-associated mesenchymal stem cells assume an activated, migratory, pro-inflammatory phenotype with increased IL-6 and CXCL10 secretion.
      Glioma environments are under chronic inflammation, and IL-6 is one of the cytokines highly implicated in the chronic inflammatory phenotype often associated with GBM. Tumor-associated macrophages represent a large bulk of the nonneoplastic cells in the tumor and are large producers of IL-6. Mesenchymal stem cell migration toward IL-6 production could be exploited for targeted therapy to MDSCs using MSCs as delivery vehicles to transport immune-related cargo to the tumor niche for MDSC suppression or TAM polarization.

      Role of TGF-β

      The TGF-β family has been extensively linked to several diseases and highly implicated in malignant brain tumors for playing a dual role in regulating brain tumor stem cell activity. This “dual” role comes from its regulatory function in maintaining tissue homeostasis. Pretreatment of AMSCs with TGF-β enhanced the expression of pancreatic cancer stem marker CD144 and NANOG,
      • Kabashima-Niibe A.
      • Higuchi H.
      • Takaishi H.
      • et al.
      Mesenchymal stem cells regulate epithelial-mesenchymal transition and tumor progression of pancreatic cancer cells.
      while other members of the TGF-β family, such as BMP4, can attenuate brain tumor stem cell progression. In a human glioma xenograft model, undifferentiated human brain tumor cells with high CD133+, alluding to a more stem phenotype, responded to BMP4 by activating their related receptors and triggering the SMAD signaling pathway to induce differentiation and subsequently decrease tumor burden in vitro and in vivo.
      • Li Q.
      • Wijesekera O.
      • Salas S.J.
      • et al.
      Mesenchymal stem cells from human fat engineered to secrete BMP4 are nononcogenic, suppress brain cancer, and prolong survival.
      ,
      • Piccirillo S.G.
      • Reynolds B.A.
      • Zanetti N.
      • et al.
      Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.
      In order to be classified as an MSC, one inclusion criteria is the presence of the transmembrane TGF-β coreceptor CD105 on the cell surface. The receptor is responsible for mediating the directed homing of AMSCs toward microenvironments with high TGF-β expressions, such as that of gliomas. Preexposure of human AMSCs to TGF-β for 48 hours resulted in increased migration in vitro through a transwell assay and displayed morphological changes in lamellipodia formation in scratch assays. In vivo, locally implanted AMSCs preexposed to TGF-β displayed enhanced migration toward primary human glioma cells. Adipose tissue-derived MSC migration was reduced on TGF-β receptor AMSC knockdown.
      • Li M.
      • Zeng L.
      • Liu S.
      • et al.
      Transforming growth factor-β promotes homing and therapeutic efficacy of human mesenchymal stem cells to glioblastoma.

      Current Immunotherapy for Glioma

      Immunotherapy is designed to stimulate the immune system or inhibit mechanisms that promote immunosuppression. Glioblastoma is a “cold tumor” in that it harbors a low mutational load, making it difficult to mount an effective immune response. However, the GBM immune environment primarily consists of MDSCs and regulatory T cells involved in actively suppressing the immune response through PDL1 and CTLA4. Antigen-presenting cells present tumor antigens to the adaptive immune system through CD80/CD86 ligand interacting with CD28 costimulatory receptor on T cells; T-cell stimulation is controlled via CTLA4 an antistimulatory and inhibitory signal that is often overexpressed in GBM and inversely correlated with survival. Competitive binding of CD80/CD86 to CTLA4 (due to its overexpression) dampens T-cell activation and ultimately leads to immunosuppression. Ipilimumab, a CTLA4 inhibitor, has undergone clinical trials with unremarkable results for GBM. Similarly, PDL1 is overexpressed on tumor cells; binding of PDL1 to programmed cell death 1 on leukocytes leads to T-cell suppression and self-tolerance by dampening the inflammatory response. Although programmed cell death 1 checkpoint inhibitors (atezolizumab, nivolumab, pembrolizumab) resulted in augmented T-cell diversity, immunoinfiltration, and increased cytokine production in GBM, a study reported associated molecular alterations and clinical response correlated to tumor evolution, suggesting that timing of administration is crucial for advantageous prognosis.
      Despite the limited immunotherapies for patients with GBM, several immunotherapeutic vaccines were able to reach phase 3 clinical trials.
      • Lim M.
      • Xia Y.
      • Bettegowda C.
      • Weller M.
      Current state of immunotherapy for glioblastoma.
      Rindopepimut,
      • Weller M.
      • Kaulich K.
      • Hentschel B.
      • et al.
      German Glioma Network
      Assessment and prognostic significance of the epidermal growth factor receptor vIII mutation in glioblastoma patients treated with concurrent and adjuvant temozolomide radiochemotherapy [published correction appears in Int J Cancer. 2016;139(4):E9].
      ICT-107,
      • Phuphanich S.
      • Wheeler C.J.
      • Rudnick J.D.
      • et al.
      Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma.
      and DCVax-L
      • Liau L.M.
      • Black K.L.
      • Prins R.M.
      • et al.
      Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens.
      ,
      • Liau L.M.
      • Prins R.M.
      • Kiertscher S.M.
      • et al.
      Dendritic cell vaccination in glioblastoma patients induces systemic and intracranial T-cell responses modulated by the local central nervous system tumor microenvironment.
      are at the top of this list for the following reasons. Rindopepimut, also known as CDX-110 or PEPvIII, is a peptide that mimics and targets EGFR variant III (EGFRvIII). EGFR variant III is an active mutant form of EGFR that is exclusively expressed in 25% to 30% of patients with GBM.
      • Calinescu A.A.
      • Kamran N.
      • Baker G.
      • Mineharu Y.
      • Lowenstein P.R.
      • Castro M.G.
      Overview of current immunotherapeutic strategies for glioma.
      Rindopepimut is a vaccine that depends on a single immunogenic peptide, which exclusively targets the EFGRvIII neoantigen expression on the tumor cells and limits the risk of toxicities.
      • Lim M.
      • Xia Y.
      • Bettegowda C.
      • Weller M.
      Current state of immunotherapy for glioblastoma.
      One of the disadvantages is the heterogeneity of the EGFRvIII expression on the GBM cells, which leads to outgrowth of tumor cells lacking this antigen.
      • Lim M.
      • Xia Y.
      • Bettegowda C.
      • Weller M.
      Current state of immunotherapy for glioblastoma.
      ,
      • Calinescu A.A.
      • Kamran N.
      • Baker G.
      • Mineharu Y.
      • Lowenstein P.R.
      • Castro M.G.
      Overview of current immunotherapeutic strategies for glioma.
      ICT-107 is a multipeptide vaccine that consists of patient-derived DCs incubated ex vivo with tumor peptides.
      • Phuphanich S.
      • Wheeler C.J.
      • Rudnick J.D.
      • et al.
      Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma.
      The proof of concept of this vaccine was challenging because similar peptides might not share the immunogenicity between humans and mice, and the gene overexpression might not be the same in mouse glioma models and human tumors.
      • Lim M.
      • Xia Y.
      • Bettegowda C.
      • Weller M.
      Current state of immunotherapy for glioblastoma.
      ,
      • Phuphanich S.
      • Wheeler C.J.
      • Rudnick J.D.
      • et al.
      Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma.
      Last, but not least, DCVax-L, the longest-acting vaccine in history for GBM,
      • Liau L.M.
      • Black K.L.
      • Prins R.M.
      • et al.
      Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens.
      acts by utilizing whole tumor lysate from patient GBM tissue to generate autologous DCs.
      • Polyzoidis S.
      • Ashkan K.
      DCVax®-L—developed by Northwest Biotherapeutics [published correction appears in Hum Vaccin Immunother. 2015;11(7):1881].
      However, this vaccine is considered challenging because it requires the collection of the patient’s tumor tissue and then processing the tissue to activate the autologous DCs.
      • Liau L.M.
      • Black K.L.
      • Prins R.M.
      • et al.
      Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens.
      ,
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      Dendritic cell vaccination in glioblastoma patients induces systemic and intracranial T-cell responses modulated by the local central nervous system tumor microenvironment.
      Furthermore, given the impermeable nature of the BBB, the delivery of the targeted agents to the brain is considered challenging. Therefore, several approaches have been considered for transiently breaching the BBB. First, the traditional approach consists of the injection of a hyperosmotic solution before the administration of the therapeutics that induce endothelial cell shrinkage and increased vascular leakage in the brain parenchyma.
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      ,
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      Additional increased effects of mannitol-temozolomide combined treatment on blood-brain barrier permeability.
      Second, focused ultrasound allows for a substantial increase in BBB permeability via the application of fine-tuned acoustic pressure to the brain that could be utilized for therapeutic delivery. Focused ultrasound produced more desirable results in breaching the BBB when used in combination with microbubbles.
      • Parodi A.
      • Rudzińska M.
      • Deviatkin A.A.
      • Soond S.M.
      • Baldin A.V.
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      ,
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      Clinical trial of blood-brain barrier disruption by pulsed ultrasound.
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      Additionally, photodynamic therapy is an approach that utilizes light irradiation via the use of photosensitive molecules. This application is beneficial in delivering large molecules and nanoparticles, but it is limited to a small area of the brain.
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      Lastly, the convection-enhanced delivery approach (CED) allows for intratumoral local drug injection via a catheter. Despite the invasive nature of the approach, CED has documented efficacy and safety with several therapeutic agents. Furthermore, in vitro and in vivo studies found that CED might increase the invasiveness of the glioma cell by activating the CXCR4-CXCL12 signaling pathway. This adverse effect can be avoided by coadministration of the CXCR4 antagonist AMD3100.
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      Current status of intratumoral therapy for glioblastoma.
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      Although BBB permeability remains a challenge, therapeutic approaches that exploit endogenous mechanisms to mediate extravasation and migration across the BBB into tissue may prove more effective at targeted delivery to GBM.

      Limitations in MSC Applications

      For years, MSC transplant has been regarded as safe with a wide array of therapeutic applications. However, low survival on engraftment and short-term retention have hampered MSC therapeutic efficacy. Cell-cell communication and adhesion play an essential role in the viability and proliferative capacity of stem and stromal cells, such as MSCs. During isolation and engraftment, anchorage-dependent mechanisms are disrupted, and cells undergo a form of programmed cell death referred to as anoikis. Poor engraftment is due to loss of extracellular matrix anchoraging at the site of implantation.
      • Lee S.
      • Choi E.
      • Cha M.J.
      • Hwang K.C.
      Cell adhesion and long-term survival of transplanted mesenchymal stem cells: a prerequisite for cell therapy.
      Strategies to improve cell retention may benefit from strategies that inhibit anoikis (eg, encapsulation of stem cells in biodegradable biomaterial). Although transplant is essential for regenerative medicine, therapeutic strategies utilizing MSCs for cargo delivery or immunoactivation would benefit from the transient effects of delivery, reducing potential risks of stable or chronic persistence of exogenously delivered therapeutic cells in tissue. Additionally, a prevailing theory in MSC biology suggests that MSCs act in a “hit-and-run” fashion and exert their therapeutic function on resident cells for stable tissue repair on arrival. Clinically, isolation methods, ex vivo expansion, route of administration, concentration, and synthetic modifications must be considered for each approach exclusively to harness the full potential of MSC therapy.
      Although the dynamics of MSC homing to the site of injury have yet to be fully understood,
      • Rustad K.C.
      • Gurtner G.C.
      Mesenchymal stem cells home to sites of injury and inflammation.
      MSCs display considerable potential as cellular vehicles because they are recruited by cytokines substantially up-regulated and secreted by gliomas. Studies have suggested that the mobilization strategies used by MSCs may be similar to the strategies used by leukocytes to home to areas of insult,
      • Teo G.S.
      • Ankrum J.A.
      • Martinelli R.
      • et al.
      Mesenchymal stem cells transmigrate between and directly through tumor necrosis factor-α-activated endothelial cells via both leukocyte-like and novel mechanisms.
      and MSCs can travel concomitantly during injury with immune cells to mediate repair. The aforementioned homing process of mesenchymal migration toward inflammation and immune-infiltrated loci has been observed in response to various inflammatory diseases.
      • Vasandan A.B.
      • Jahnavi S.
      • Shashank C.
      • Prasad P.
      • Kumar A.
      • Prasanna S.J.
      Human mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a PGE2-dependent mechanism.

      Future Implications

      Glioblastoma is a highly complex tumor that exploits several mechanisms to evade clinically approved therapeutics. The clinical experience of immunotherapy in current GBM treatment paradigms has revealed modest success, partially due to the cytokine secretion profile that blocks intratumoral cytotoxic T-cell migration and activation, resulting in an immunosuppressive state. Reeducating components of the immune system rather than ablating them may be a more effective approach for targeting the GBM tumor microenvironment. Delivery of cargo that induces immune activation (eg, IL-12, IFN, IL-2,) via MSCs may be a logical therapeutic approach in reactivating tumor surveillance mechanisms. Studies of GBM immunotherapy in immunocompetent animal models or humanized mouse models that accurately predict GBM response or escape to new treatment strategies are needed and may enable new discoveries in elucidating the mechanisms mediating the immunosuppression often associated with GBM.

      Conclusion

      Factors secreted in the tumor microenvironment can have multiple effects such as tumorigenesis, immune cell recruitment, and recruitment of exogenous cellular vehicles. Many of the regulatory mechanisms mentioned in this review skew immune infiltrating cells such as TAM (of the innate immune response) T cells (of the adaptive immune response) toward a type 2 phenotype that is often tumor promoting. These mechanisms can all take place simultaneously, dynamically or bidirectionally within the tumor niche, eventually resulting in immunosuppression. Potential interplay between the immune system and exogenous stem cells must be further examined, and a strategic avenue could explore the possibility of immune reeducation. Soluble factors released by tumors are implicated in MSC recruitment to GBM and can mediate site-specific delivery of a therapeutic cargo. We suggest that this characteristic be harnessed for immunomodulation in GBM by MSC via the delivery of an immune-activating or immune-enhancing cargo.

      Acknowledgments

      The views expressed in this article are those of the authors and do not necessarily represent those of the National Institutes of Health.
      Dr Lara-Velazquez thanks Consejo Nacional de Ciencia y Tecnología (CONACYT) and Plan of Combined Studies in Medicine (MD/PhD), National Autonomous University of Mexico (PECEM UNAM).

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