Glioblastoma, the most aggressive form of glioma, has a 5-year survival rate of <5%. While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. This study quantifies the temporal and spatial localization of immune cell populations and mediators during glioma development. Eightweek old male C57Bl/6 mice were orthotopically inoculated with 1x10 6 Gl261 cells and tumor morphology, local and systemic immune cell populations, and plasma cytokines/chemokines assessed at day 0, 1, 3, 7, 14, and 21 post-inoculation by magnetic resonance imaging, chromogenic immunohistochemistry, multiplex immunofluorescent immunohistochemistry, flow cytometry and multiplex immunoassay respectively. From day 3 tumors were distinguishable with >30% Ki67 and increased tissue vascularization (p<0.05). Increasing tumor proliferation/malignancy and vascularization were associated with significant temporal changes in immune cell populations within the tumor (p<0.05) and systemic compartments (p = 0.02 to p<0.0001). Of note, at day 14 16/24 plasma cytokine/chemokines levels decreased coinciding with an increase in tumor cytotoxic T cells, natural killer and natural killer/T cells. Data derived provide baseline characterization of the local and systemic immune response during glioma development. They reveal that type II macrophages and myeloid-derived suppressor cells are more prevalent in tumors than regulatory T cells, highlighting these cell types for further therapeutic exploration.

Brain, lung, and colon tissue experience deleterious immune-related adverse events when immune-oncological agents or radiation are administered. However, there is a paucity of information regarding whether the addition of radiation to immuno-oncological regimens exacerbates the tissue inflammatory response. We used a murine model to evaluate sub-acute tissue damage and the systemic immune response in C57Bl/6 mice when administered systemic anti-programmed cell death protein 1 (αPD-1) immunotherapy alone or in combination with stereotactic fractionated 10 gray/5 X-ray radiation to normal brain, lung or colon tissue. The model indicated that combinatorial αPD-1 immunotherapy and radiation may alter normal colon cell proliferation and cerebral blood vasculature, and induce systemic thrombocytopenia, lymphopenia, immune suppression, and altered immune repertoire (including interleukin-1β). Therein our data supports close monitoring of hematological and immune-related adverse events in patients receiving combination therapy.

Mesothelioma is an aggressive cancer with limited treatment options and a poor prognosis. Phytocannabinoids possess anti-tumour and palliative properties in multiple cancers, however their effects in mesothelioma are unknown. We investigated the anti-cancer effects and potential mechanisms of action for several phytocannabinoids in mesothelioma cell lines. A panel of 13 phytocannabinoids inhibited growth of human (MSTO and H2452) and rat (II-45) mesothelioma cells in vitro, and cannabidiol (CBD) and cannabigerol (CBG) were the most potent compounds. Treatment with CBD or CBG resulted in G0/G1 arrest, delayed entry into S phase and induced apoptosis. CBD and CBG also significantly reduced mesothelioma cell migration and invasion. These effects were supported by changes in the expression of genes associated with the cell cycle, proliferation, and cell movement following CBD or CBG treatment. Gene expression levels of CNR1, GPR55, and 5HT1A also increased with CBD or CBG treatment. However, treatment with CBD or CBG in a syngeneic orthotopic rat mesothelioma model was unable to increase survival. Our data show that cannabinoids have anti-cancer effects on mesothelioma cells in vitro and alternatives of drug delivery may be needed to enhance their effects in vivo.

INTRODUCTION Glioma is a debilitating and early fatal cancer arising in the glial cells of brain. Glioblastoma, the most aggressive form of gliomas, has a 5-year survival rate of 5% with treatment options limited to surgery, radiotherapy or chemotherapy with temozolomide. OBJECTIVE While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. In this study we quantified the temporal and spatial localisation of immune cell populations and mediators during glioma development. METHODS Mice were inoculated with 1x106 Gl261 cells at AP0.1mm, ML1.0mm, DV2.6mm Bregma. Tumour morphology, local and systemic immune cell populations were assessed at Day-0, 1, 3, 7, 14, and 21 post-inoculation by MRI, immunohistochemistry for Ki67+ proliferation and CD31+ vessel density; local immune infiltrate by multiplex immunofluorescence (VECTRA®;PerkinElmer); systemic immunity in spleen, bone marrow and peripheral blood by 16-parameter flow cytometry (LSRFortessa™;BD Biosciences); and multiplex immunoassay (Bio-Plex®;Bio-Rad) for plasma cytokines/chemokines. RESULTS From Day-3 tumours were distinguishable with high Ki67+ (> 30%) and increased tissue vascularisation (26.38±1.45 vessels/field; p< 0.05). Ki67 remained high until Day-21 with visible necrotic regions, increased vessel density and lumen area within tumour region. Increasing tumour proliferation/malignancy and vascularisation were associated with significant temporal changes in immune cell populations within the tumour (p< 0.05) and systemic compartments (p=0.02- p< 0.0001). Of note, at Day-14 NK, M1, PMN-MDSCs and M-MDSCs in the tumour infiltrate declined, coinciding with a decrease in 16/24 plasma cytokine/chemokines levels. Tumour infiltrating immune cell populations did not correlate with peripheral blood populations. However, a decrease in plasma cytokine/chemokine levels may indicate ‘immune exhaustion’. CONCLUSIONS The data derived provide baseline characteristics to study changes associated with multi-modal treatment strategies and to sequence therapies to maintain immune modulation. This information will contribute to the identification of novel combination therapies.