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Radioresistance remains a major clinical challenge in glioblastoma (GB) therapy. However, the mechanism for the development of radioresistance in GB is unclear. Mounting evidence suggests that Galectin-1 (Gal1: a carbohydrate-binding protein galectin-1) contributes to the radioresistance of GB tumors. Recently, several Gal1-targeting compounds have emerged. OTX008 is a calixarene derivative designed to bind the Gal1 amphipathic β-sheet conformation. Our study aimed to reduce galectin-1 expression using OTX008 in a human GB pre-clinical model. For these studies an in vivo GL261 murine models of GB were utilized to assess efficacy of treatment with radiation plus OTX008. Our results demonstrated that inhibition of Gal-1 with OTX008 plus radiation showed significantly decrease in GB growth rate and improved survival in in vivo. OTX008 treatment plus radiation was associated with downregulation of Gal1 and Ki67 in treated tumors, as well as decreased microvessel density and VEGFR2 expression. Finally, combination studies showed OTX008 synergy with radiation therapies, principally when OTX008 was administered first. This study provides insights of Gal-1inhibitory effects of OTX008 and its enhancement of the efficacy of radiotherapy in mouse models of GB suggest that further studies are warranted.

Meningiomas are common intracranial brain tumors. Despite surgery and/or radiation therapy, recurrence rates occur in approximately 8–10 % of tumors. This may be due to the dysregulation of the cyclin D and cyclin-dependent kinase (CDK) pathway in malignant meningioma cells and radiation-resistant meningioma cells. The cyclin D-cyclin-dependent kinases 4 and 6 (CDK4/6)-retinoblastoma (Rb) pathway controls the cell cycle restriction point, and is commonly dysregulated in meningioma; making it a rational target for antimeningioma therapy. In this study, we interrogated the capacity of a CDK4/6 inhibitor, palbociclib, to activate RB function against Rb+ meningiomas cells in vitro, ex vivo and in vivo xenograft model. Meningioma tumors in SCID mice treated daily with intraperitoneal injections of palbociclib for 14 days dose dependly with X ray irradiation. Treatment effects were examined by immunoblot, cell viability, apoptosis, and cell cycle progression. Radiation resistance BENMEN1 cells were developed after exposure to repeated 320 kV X ray irradiation. Since CDK4 and CDK6 are proteins that are major part of a cell cycle regulatory pathway in meningioma that also includes p16, cyclin D, and the retinoblastoma (Rb) protein, we analyzed those parameters. Our both in vitro and in vivo data clearly demonstrate that pallbociclib (CDK 4/6 inhibitor) treatment reduced proliferation and has additional effects on cell cycling, including induction of an RB-associated G(1) arrest against Rb+ malignant meningioma cells and radiation-resistant meningioma cells. We did not see any significant effect of palbociclib on Rb – meningima cells. Our results also demonstrated that palbociclib treatment inhibits CDK4 and CDK6 expression and also decreases E2F target gene expression (CCNA2 and CCNE2). All together, our results provide strong evidence that palbociclib can be applied to Rb+ meningiomas as a therapeutic agent.

As chemokines are important mediators of cell–cell communication, we sought to identify commonly expressed chemokines in mouse and human medulloblastoma (MB) lines as surrogates to microglia/MB tumor cell interactions. We found CCL2 (chemokine (C-C motif) ligand 2) messenger RNA to be expressed by medulloblastoma lines. However, these lines did not express the CCL2 receptor, CCR2, which was found on microglia. The MP-1 medulloblastoma cell line is known to have low basal levels of CCL2. Hence, we overexpressed CCL2 plasmid with GFP tagging in the MP-1 medulloblastoma line to investigate the hypothesis that medulloblastoma-secreted CCL2 interacts with microglia to affect medulloblastoma growth and invasion. In medulloblastoma–microglia co-culture models, we observed an increase in the proliferation of CCL2-overexpressing clones. ELISA protein analyses revealed that interleukin-6 (IL-6) was consistently increased in the co-culture. Recombinant IL-6 enhanced the invasion of medulloblastoma cells when these were cultured alone, whereas a neutralizing antibody to IL-6 attenuated the microglia-stimulated invasion. This study has uncovered a mechanism by which medulloblastoma cells exploit microglia (M2-phenotype) for increased invasion. Specifically, medulloblastoma-derived CCL2 acts upon CCR2-bearing tumor-associated microglias (TAMs), which produce IL-6 to stimulate medulloblastoma cells, in a CCL2/CCR2/IL-6 loop. We targeted this loop by combining radiation therapy with 4-1BB (CD-137) monoclonal antibody (mAb), as CD-137 is involved in regulation of immune cell proliferation and survival. We re-educated microglia cells toward a cytotoxic M1-like phenotype and amplified effective anti-tumor immune responses in in vitro co-culture models. ELISA spot shows treatment with 50 ng of 4-1BB (CD137) + 1Gy X-ray radiation converted the microglia from M2 to M1 phenotype via a decrease in CCR2. This treatment induces the M1 phenotype microglia to induce MP-1 cell death. Based on our results, the use of 4-1BB mAb plus low-dose single fraction radiation may prove to be beneficial for the treatment of medulloblastoma.

Glioblastoma (GB) is the most common and highly aggressive adult brain cancer. Dysfunction of receptor tyrosine kinases, such as ALK, c-Met and VEGF have all been implicated in the progression of this malignancy and resistance to therapy. Traditional therapeutic strategies targeting ALK/c-Met or VEGF have not yet given satisfactory outcomes. Myxoma virus (MYXV) is a novel oncolytic virus that has been shown to replicate in GB models. Here, we investigated the efficacy of MYXV as monotherapy, or in combination with 1 Gy radiation plus c-Met/ALK inhibitor crizotinib and VEGF inhibitor bevacizumab in different cell lines (GL261, T9, and U118) surgically obtained from GB patients. We investigated the molecular mechanisms of apoptotic death by Western blots. Colorimetric assay demonstrated that treatment with MYXV plus radiation with crizotinib or bevacizumab caused apoptosis due to an increase in activation of caspase-3 and decrease in expression of Bcl-2 proteins in GB cells and tissues. Most importantly, MYXV plus radiation significantly enhanced the anti-cancer effect of crizotinib, bevacizumab and temozolomide, in GB cell lines as well as in fresh tumor biopsies from GB patients. Our studies also suggest that induction of apoptosis by MYXV does not depend on the functional status of the GB cell lines or in the GB tissues. Further studies in different animal models of GB are warranted in the future to determine whether MYXV in combination with radiation, crizotinib or bevacizumab may be useful as anti-cancer agent for the management of human GBs in vivo.

Medulloblastoma cells in situ are surrounded by microglia, suggesting medulloblastoma–microglia interactions to produce various outcomes. As chemokines are important mediators of cell–cell communication, we sought to identify common chemokines in mouse and human medulloblastoma lines. We found CCL2 (chemokine (C-C motif) ligand 2) messenger RNA was expressed by Group 3 and 4 medulloblastoma lines. However, these lines did not express the CCL2 receptor, CCR2, which was found on microglia. The MP-1 medulloblastoma cell line expresses low basal levels of CCL2. Hence, we overexpressed CCL2 plasmid with GFP tagging in MP-1 medulloblastoma cells to investigate the hypothesis that medulloblastoma-secreted CCL2 interacts with microglia to affect medulloblastoma growth and invasion. In medulloblastoma–microglia co-culture models, we observed increased proliferation of CCL2-overexpressing clones. ELISA protein analyses revealed that interleukin-6 (IL-6) was consistently increased in the co-culture. Recombinant IL-6 enhanced the invasion of medulloblastoma cells when these were cultured alone, whereas a neutralizing antibody to IL-6 attenuated the microglia-stimulated medulloblastoma invasion. This study reveals a mechanism by which medulloblastoma cells exploit microglia (M2-phenotype) for increased invasion. Specifically, medulloblastoma-derived CCL2 acts upon CCR2-bearing tumor associated microglias (TAMs), which produce IL-6 to stimulate medulloblastoma cells, in a CCL2/CCR2/IL-6 loop. We targeted this loop by combining radiation therapy with a 4-1BB (CD-137) monoclonal antibody (mAb), as CD-137 is involved in regulation of immune cell proliferation and survival. We re-educated microglia cells toward a cytotoxic M1-like phenotype and amplified effective anti-tumor immune responses in co-culture models. ELISA spot shows treatment with 50 ng of 4-1BB + 1Gy X-ray radiation converted the microglia from M2 to M1 phenotype via a decrease in CCR2. This treatment induces the M1 phenotype microglia to induce MP-1 cell death. Based on our results, the use of 4-1BB mAb plus radiation may prove to be beneficial for the treatment of medulloblastoma by targeting CCL2/CCR2/IL-6 loop.

Medulloblastoma (MB) is the most common malignant brain tumor in children, and has been historically classified based on clinical markers (patient age, presence of metastases at diagnosis, extent of resection) and histopathological characteristics (classic, desmoplastic/nodular, and large cell/anaplastic). However, the recent gene expression profiling of a large number of tumors across multiple studies has provided evidence of four distinct medulloblastoma molecular subgroups (Wnt, Shh, Group 3, and Group 4), associated with remarkably different prognoses. While current therapies for medulloblastoma are often toxic, and not always effective, immunotherapy, designed to target these subgroups, may offer therapeutic improvement. Although monoclonal antibody (mAb) therapy has not yet been shown to be effective for the treatment of brain tumors, recent data indicate that radiation therapy can induce immunogenic modulation characterized by cell-surface phenotypic changes leading to augmented tumour cell/cytotoxic T-cell interaction. We examined the ability of radiation to upregulate mAb therapy targets. Our data showed that low dose radiation significantly increased cell-surface and total protein expression of mAb targets CD137 (a member of the TNF receptor superfamily), FGF, CD20, HER2, Major Histocompatability Complex (MHC) class I and intercellular adhesion molecule-1 (ICAM-I) in 3 out of 3 medulloblastoma cell lines. These findings highlight a mechanism for combining radiation with immunotherapy, to potentially expand the effect of targeted therapy in brain tumor treatment.