The genomic alterations in glioblastoma influence the levels of CSF metabolites.

Cerebrospinal fluid (CSF) analysis is underutilized in patients with glioblastoma (GBM), partly due to a lack of studies demonstrating the clinical utility of CSF biomarkers. While some studies show the utility of CSF cell-free DNA analysis, studies analyzing CSF metabolites in patients with glioblastoma are limited. Diffuse gliomas have altered cellular metabolism. For example, mutations in isocitrate dehydrogenase enzymes (e.g., IDH1 and IDH2) are common in diffuse gliomas and lead to increased levels of D-2-hydroxyglutarate in CSF. However, there is a poor understanding of changes CSF metabolites in GBM patients. In this study, we performed targeted metabolomic analysis of CSF from n = 31 patients with GBM and n = 13 individuals with non-neoplastic conditions (controls), by mass spectrometry. Hierarchical clustering and sparse partial least square-discriminant analysis (sPLS-DA) revealed differences in CSF metabolites between GBM and control CSF, including metabolites associated with fatty acid oxidation and the gut microbiome (i.e., carnitine, 2-methylbutyrylcarnitine, shikimate, aminobutanal, uridine, N-acetylputrescine, and farnesyl diphosphate). In addition, we identified differences in CSF metabolites in GBM patients based on the presence/absence of TP53 or PTEN mutations, consistent with the idea that different mutations have different effects on tumor metabolism. In summary, our results increase the understanding of CSF metabolites in patients with diffuse gliomas and highlight several metabolites that could be informative biomarkers in patients with GBM.

Single-cell analysis of human glioma and immune cells identifies S100A4 as an immunotherapy target.

A major rate-limiting step in developing more effective immunotherapies for GBM is our inadequate understanding of the cellular complexity and the molecular heterogeneity of immune infiltrates in gliomas. Here, we report an integrated analysis of 201,986 human glioma, immune, and other stromal cells at the single cell level. In doing so, we discover extensive spatial and molecular heterogeneity in immune infiltrates. We identify molecular signatures for nine distinct myeloid cell subtypes, of which five are independent prognostic indicators of glioma patient survival. Furthermore, we identify S100A4 as a regulator of immune suppressive T and myeloid cells in GBM and demonstrate that deleting S100a4 in non-cancer cells is sufficient to reprogram the immune landscape and significantly improve survival. This study provides insights into spatial, molecular, and functional heterogeneity of glioma and glioma-associated immune cells and demonstrates the utility of this dataset for discovering therapeutic targets for this poorly immunogenic cancer.

HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion.

The FGFR3-TACC3 (F3-T3) fusion gene was discovered as an oncogenic molecule in glioblastoma and bladder cancers, and has subsequently been found in many cancer types. Notably, F3-T3 was found to be highly expressed in both untreated and matched recurrence glioblastoma under the concurrent radiotherapy and temozolomide (TMZ) treatment, suggesting that targeting F3-T3 is a valid strategy for treatment. Here, we show that the F3-T3 protein is a client of heat shock protein 90 (HSP90), forming a ternary complex with the cell division cycle 37 (CDC37). Deprivation of HSP90 or CDC37 disrupts the formation of the ternary complex, which destabilizes glycosylated F3-T3, and thereby suppresses F3-T3 oncogenic activity. Gliomas harboring F3-T3 are resistant to TMZ chemotherapy. HSP90 inhibitors sensitized F3-T3 glioma cells to TMZ via the inhibition of F3-T3 activation and potentiated TMZ-induced DNA damage. These results demonstrate that F3-T3 oncogenic function is dependent on the HSP90 chaperone system and suggests a new clinical option for targeting this genetic aberration in cancer.

Characterization of patient-derived bone marrow human mesenchymal stem cells as oncolytic virus carriers for the treatment of glioblastoma.

OBJECTIVE: Delta-24-RGD is an oncolytic adenovirus that is capable of replicating in and killing human glioma cells. Although intratumoral delivery of Delta-24-RGD can be effective, systemic delivery would improve its clinical application. Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) obtained from healthy donors have been investigated as virus carriers. However, it is unclear whether BM-hMSCs can be derived from glioma patients previously treated with marrow-toxic chemotherapy or whether such BM-hMSCs can deliver oncolytic viruses effectively. Herein, the authors undertook a prospective clinical trial to determine the feasibility of obtaining BM-hMSCs from patients with recurrent malignant glioma who were previously exposed to marrow-toxic chemotherapy. METHODS: The authors enrolled 5 consecutive patients who had been treated with radiation therapy and chemotherapy. BM aspirates were obtained from the iliac crest and were cultured to obtain BM-hMSCs. RESULTS: The patient-derived BM-hMSCs (PD-BM-hMSCs) had a morphology similar to that of healthy donor-derived BM-hMSCs (HD-BM-hMSCs). Flow cytometry revealed that all 5 cell lines expressed canonical MSC surface markers. Importantly, these cultures could be made to differentiate into osteocytes, adipocytes, and chondrocytes. In all cases, the PD-BM-hMSCs homed to intracranial glioma xenografts in mice after intracarotid delivery as effectively as HD-BM-hMSCs. The PD-BM-hMSCs loaded with Delta-24-RGD (PD-BM-MSC-D24) effectively eradicated human gliomas in vitro. In in vivo studies, intravascular administration of PD-BM-MSC-D24 increased the survival of mice harboring U87MG gliomas. CONCLUSIONS: The authors conclude that BM-hMSCs can be acquired from patients previously treated with marrow-toxic chemotherapy and that these PD-BM-hMSCs are effective carriers for oncolytic viruses.

An immune-competent, replication-permissive Syrian Hamster glioma model for evaluating Delta-24-RGD oncolytic adenovirus.

BACKGROUND: Oncolytic adenoviruses are promising new treatments against solid tumors, particularly for glioblastoma (GBM), and preclinical models are required to evaluate the mechanisms of efficacy. However, due to the species selectivity of adenovirus, there is currently no single animal model that supports viral replication, tumor oncolysis, and a virus-mediated immune response. To address this gap, we took advantage of the Syrian hamster to develop the first intracranial glioma model that is both adenovirus replication-permissive and immunocompetent. METHODS: We generated hamster glioma stem-like cells (hamGSCs) by transforming hamster neural stem cells with hTERT, simian virus 40 large T antigen, and h-RasV12. Using a guide-screw system, we generated an intracranial tumor model in the hamster. The efficacy of the oncolytic adenovirus Delta-24-RGD was assessed by survival studies, and tumor-infiltrating lymphocytes (TILs) were evaluated by flow cytometry. RESULTS: In vitro, hamGSCs supported viral replication and were susceptible to Delta-24-RGD mediated cell death. In vivo, hamGSCs consistently developed into highly proliferative tumors resembling high-grade glioma. Flow cytometric analysis of hamster gliomas revealed significantly increased T-cell infiltration in Delta-24-RGD infected tumors, indicative of immune activation. Treating tumor-bearing hamsters with Delta-24-RGD led to significantly increased survival compared to hamsters treated with phosphate buffered saline (PBS). CONCLUSIONS: This adenovirus-permissive, immunocompetent hamster glioma model overcomes the limitations of previous model systems and provides a novel platform to study the interactions between tumor cells, the host immune system, and oncolytic adenoviral therapy; understanding of which will be critical to implementing oncolytic adenovirus in the clinic.

Endovascular Selective Intra-Arterial Infusion of Mesenchymal Stem Cells Loaded With Delta-24 in a Canine Model.

BACKGROUND: Delta-24-RGD, an oncolytic adenovirus, shows promise against glioblastoma. To enhance virus delivery, we recently demonstrated that human bone marrow-derived mesenchymal stem cells loaded with Delta-24-RGD (hMSC-D24) can eradicate glioblastomas in mouse models. There are no studies examining the safety of endovascular selective intra-arterial (ESIA) infusions of MSC-D24 in large animals simulating human clinical situations. OBJECTIVE: To perform canine preclinical studies testing the feasibility and safety of delivering increasing doses of hMSCs-D24 via ESIA infusions. METHODS: ESIA infusions of hMSC-D24 were performed in the cerebral circulation of 10 normal canines in the target vessels (internal carotid artery [ICA]/P1) via transfemoral approach using commercially available microcatheters. Increasing concentrations of hMSC-D24 or particles (as a positive control) were injected into 1 hemisphere; saline (negative control) was infused contralaterally. Toxicity (particularly embolic stroke) was assessed on postinfusion angiography, diffusion-weighted magnetic resonance imaging, clinical exam, and necropsy. RESULTS: ESIA injections were performed in the ICA (n = 7) or P1 (n = 3). In 2 animals injected with particles (positive control), strokes were detected by all assays. Of 6 canines injected with hMSC-D24 through the anterior circulation, escalating dose from 2 × 106 cells/20 mL to 1 × 108 cells/10 mL resulted in no strokes. Two animals had ischemic and hemorrhagic strokes after posterior cerebral artery catheterization. A survival experiment of 2 subjects resulted in no complications detected for 24-h before euthanization. CONCLUSION: This novel study simulating ESIA infusion demonstrates that MSCs-D24 can be infused safely at least up to doses of 1 × 108 cells/10 mL (107 cells/ml) in the canine anterior circulation using commercially available microcatheters. These findings support a clinical trial of ESIA infusion of hMSCs-D24.

RNAi technology targeting the FGFR3-TACC3 fusion breakpoint: an opportunity for precision medicine.

BACKGROUND: Fusion genes form as a result of abnormal chromosomal rearrangements linking previously separate genes into one transcript. The FGFR3-TACC3 fusion gene (F3-T3) has been shown to drive gliomagenesis in glioblastoma (GBM), a cancer that is notoriously resistant to therapy. However, successful targeting of F3-T3 via small molecular inhibitors has not revealed robust therapeutic responses, and specific targeting of F3-T3 has not been achieved heretofore. Here, we demonstrate that depleting F3-T3 using custom siRNA to the fusion breakpoint junction results in successful inhibition of F3-T3+ GBMs, and that exosomes can successfully deliver these siRNAs. METHODS: We engineered 10 unique siRNAs (iF3T3) that specifically spanned the most common F3-T3 breakpoint with varying degrees of overlap, and assayed depletion by qPCR and immunoblotting. Cell viability assays were performed. Mesenchymal stem cell-derived exosomes (UC-MSC) were electroporated with iF3T3, added to cells, and F3-T3 depletion measured by qPCR. RESULTS: We verified that depleting F3-T3 using shRNA to FGFR3 resulted in decreased cell viability and improved survival in glioma-bearing mice. We then demonstrated that 7/10 iF3T3 depleted F3-T3, and importantly, did not affect levels of wild-type (WT) FGFR3 or TACC3. iF3T3 decreased cell viability in both F3T3+ GBM and bladder cancer cell lines. UC-MSC exosomes successfully delivered iF3T3 in vitro, resulting in F3-T3 depletion. CONCLUSION: Targeting F3-T3 using siRNAs specific to the fusion breakpoint is capable of eradicating F3T3+ cancers without toxicity related to inhibition of WT FGFR3 or TACC3, and UC-MSC exosomes may be a plausible vehicle to deliver iF3T3.

Targeting the Tumor Microenvironment in Brain Metastasis.

Dynamic interplay between cancer cells and the surrounding microenvironment is a feature of the metastatic process. Successful metastatic brain colonization requires complex mechanisms that ultimately allow tumor cells to adapt to the unique microenvironment of the central nervous system, evade immune destruction, survive, and grow. Accumulating evidence suggests that components of the brain tumor microenvironment (TME) play a vital role in the metastatic cascade. In this review, the authors summarize the contribution of the TME to the development and progression of brain metastasis. They also highlight opportunities for TME-directed targeted therapy.

Microcatheter delivery of neurotherapeutics: compatibility with mesenchymal stem cells.

OBJECTIVE: Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) have been used in clinical trials for the treatment of several neurological disorders. MSCs have been explored as a delivery modality for targeted viral therapeutic agents in the treatment of intracranial pathologies. Delta-24-RGD, a tumor-selective oncolytic adenovirus designed to target malignant glioma cells, has been shown to be effective in animal models and in a recent clinical trial. However, the most efficient strategy for delivering oncolytic therapies remains unclear. BM-hMSCs have been shown to home toward glioma xenografts after intracarotid delivery. The feasibility of selective intraarterial infusion of BM-hMSCs loaded with Delta-24-RGD (BM-hMSC-Delta-24) to deliver the virus to the tumor is being investigated. To evaluate the feasibility of endovascular intraarterial delivery, the authors tested in vitro the compatibility of BM-hMSC-Delta-24 with a variety of commercially available, clinically common microcatheters. METHODS: BM-hMSCs were cultured, transfected with Delta-24-RGD, and resuspended in 1% human serum albumin. The solution was then injected via 4 common neuroendovascular microcatheters of different inner diameters (Marathon, Echelon-14, Marksman, and SL-10). Cell count and viability after injection through the microcatheters were assessed, including tests of injection velocity and catheter configuration. Transwell assays were performed with the injected cells to test the efficacy of BM-hMSC-Delta-24 activity against U87 glioma cells. BM-hMSC-Delta-24 compatibility was also tested with common neuroendovascular medications: Omnipaque, verapamil, and heparin. RESULTS: The preinfusion BM-hMSC-Delta-24 cell count was 1.2 × 105 cells/ml, with 98.7% viability. There was no significant difference in postinfusion cell count or viability for any of the catheters. Increasing the injection velocity from 1.0 ml/min to 73.2 ml/min, or modifying the catheter shape from straight to tortuous, did not significantly reduce cell count or viability. Cell count and viability remained stable for up to 5 hours when the cell solution was stored on ice. Mixing BM-hMSC-Delta-24 with clinical concentrations of Omnipaque, verapamil, and heparin prior to infusion did not alter cell count or viability. Transwell experiments demonstrated that the antiglioma activity of BM-hMSC-Delta-24 was maintained after infusion. CONCLUSIONS: BM-hMSC-Delta-24 is compatible with a wide variety of microcatheters and medications commonly used in neuroendovascular therapy. Stem cell viability and viral agent activity do not appear to be affected by catheter configuration or injection velocity. Commercially available microcatheters can be used to deliver stem cell neurotherapeutics via intraarterial routes.

Stem Cell Therapy of Gliomas.

Stem cells (SC) are the seeds of tissue repair and regeneration that have been extensively investigated as tumor-tropic vectors for gene delivery to solid cancers. SC have an inherent glioma tropism that supports their use as reliable vehicles to deliver therapeutic gene products to brain neoplasms. Several types of adult SC (ASC) have been used to carry antiglioma agents, and neural SC (NSC) and mesenchymal SC (MSC) are the most studied. The therapeutic cargoes that have been tested include secreted proteins, converting enzyme/prodrug suicide combinations, oncolytic viruses, antibodies, and nanoparticles. Some of these preclinical studies have advanced to phase I clinical trials. Use of SC as carriers to deliver various antitumor agents could become a valuable therapeutic option for glioma patients in the future.

Ionizing radiation augments glioma tropism of mesenchymal stem cells.

OBJECTIVE Mesenchymal stem cells (MSCs) have been shown to localize to gliomas after intravascular delivery. Because these cells home to areas of tissue injury, the authors hypothesized that the administration of ionizing radiation (IR) to tumor would enhance the tropism of MSCs to gliomas. Additionally, they sought to identify which radiation-induced factors might attract MSCs. METHODS To assess the effect of IR on MSC migration in vitro, transwell assays using conditioned medium (CM) from an irradiated commercially available glioma cell line (U87) and from irradiated patient-derived glioma stem-like cells (GSCs; GSC7-2 and GSC11) were employed. For in vivo testing, green fluorescent protein (GFP)-labeled MSCs were injected into the carotid artery of nude mice harboring orthotopic U87, GSC7-2, or GSC17 xenografts that were treated with either 0 or 10 Gy of IR, and brain sections were quantitatively analyzed by immunofluorescence for GFP-positive cells. These GSCs were used because GSC7-2 is a weak attractor of MSCs at baseline, whereas GSC17 is a strong attractor. To determine the factors implicated in IR-induced tropism, CM from irradiated GSC7-2 and from GSC11 was assayed with a cytokine array and quantitative ELISA. RESULTS Transwell migration assays revealed statistically significant enhanced MSC migration to CM from irradiated U87, GSC7-2, and GSC11 compared with nonirradiated controls and in a dose-dependent manner. After their intravascular delivery into nude mice harboring orthotopic gliomas, MSCs engrafted more successfully in irradiated U87 (p = 0.036), compared with nonirradiated controls. IR also significantly increased the tropism of MSCs to GSC7-2 xenografts (p = 0.043), which are known to attract MSCs only poorly at baseline (weak-attractor GSCs). Ionizing radiation also increased the engraftment of MSCs in strong-attractor GSC17 xenografts, but these increases did not reach statistical significance. The chemokine CCL2 was released by GSC7-2 and GSC11 after irradiation in a dose-dependent manner and mediated in vitro transwell migration of MSCs. Immunohistochemistry revealed increased CCL2 in irradiated GSC7-2 gliomas near the site of MSC engraftment. CONCLUSIONS Administering IR to gliomas enhances MSC localization, particularly in GSCs that attract MSCs poorly at baseline. The chemokine CCL2 appears to play a crucial role in the IR-induced tropism of MSCs to gliomas.

Mesenchymal stromal cells for the delivery of oncolytic viruses in gliomas.

Mesenchymal stromal cells (MSCs) are a type of adult stem cell that has been exploited for the treatment of a variety of diseases, including cancer. In particular, MSCs have been studied extensively for their ability to treat glioblastoma (GBM), the most common and deadly form of brain cancer in adults. MSCs are attractive therapeutics because they can be obtained relatively easily from patients, are capable of being expanded numerically in vitro, can be easily engineered and are inherently capable of homing to tumors, making them ideal vehicles for delivering biological antitumoral agents. Oncolytic viruses are promising biological therapeutic agents that have been used in the treatment of GBMs, and MSCs are currently being explored as a means of delivering these viruses. Here we review the role of MSCs in the treatment of GBMs, focusing on the intersection of MSCs and oncolytic viruses.

MiR-506 suppresses proliferation and induces senescence by directly targeting the CDK4/6-FOXM1 axis in ovarian cancer.

Ovarian carcinoma is the most lethal gynaecological malignancy. Better understanding of the molecular pathogenesis of this disease and effective targeted therapies are needed to improve patient outcomes. MicroRNAs play important roles in cancer progression and have the potential for use as either therapeutic agents or targets. Studies in other cancers have suggested that miR-506 has anti-tumour activity, but its function has yet to be elucidated. We found that deregulation of miR-506 in ovarian carcinoma promotes an aggressive phenotype. Ectopic over-expression of miR-506 in ovarian cancer cells was sufficient to inhibit proliferation and to promote senescence. We also demonstrated that CDK4 and CDK6 are direct targets of miR-506, and that miR-506 can inhibit CDK4/6-FOXM1 signalling, which is activated in the majority of serous ovarian carcinomas. This newly recognized miR-506-CDK4/6-FOXM1 axis provides further insight into the pathogenesis of ovarian carcinoma and identifies a potential novel therapeutic agent.

Post-transcriptional regulatory network of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions.

Epithelial-to-mesenchymal transition (EMT) and its reverse process, mesenchymal-to-epithelial transition (MET), play important roles in embryogenesis, stem cell biology, and cancer progression. EMT can be regulated by many signaling pathways and regulatory transcriptional networks. Furthermore, post-transcriptional regulatory networks regulate EMT; these networks include the long non-coding RNA (lncRNA) and microRNA (miRNA) families. Specifically, the miR-200 family, miR-101, miR-506, and several lncRNAs have been found to regulate EMT. Recent studies have illustrated that several lncRNAs are overexpressed in various cancers and that they can promote tumor metastasis by inducing EMT. MiRNA controls EMT by regulating EMT transcription factors or other EMT regulators, suggesting that lncRNAs and miRNA are novel therapeutic targets for the treatment of cancer. Further efforts have shown that non-coding-mediated EMT regulation is closely associated with epigenetic regulation through promoter methylation (e.g., miR-200 or miR-506) and protein regulation (e.g., SET8 via miR-502). The formation of gene fusions has also been found to promote EMT in prostate cancer. In this review, we discuss the post-transcriptional regulatory network that is involved in EMT and MET and how targeting EMT and MET may provide effective therapeutics for human disease.