Toward better immunotherapy for glioblastoma:Technical choices for preclinical studies and a novel treatment strategy

Glioblastoma (GBM) is a Grade 4 brain tumor with a poor prognosis of 15 months, despite aggressive standard-of-care. Recent advances in neuroimmunology and cancer immunotherapy have sparked interest in GBM immunotherapy; however, these therapies have been disappointing in the clinic. Preclinical glioma models are essential for exploring novel immunotherapies and characterizing the anti-glioma immune response. However, the character of the immune response against the immunocompetent murine glioma models, GL261 and CT2A, has not been well studied. In this work, we aimed to elucidate factors that influence the immune response to these models and investigate a novel GBM treatment strategy. Using the GL261 and CT2A models, we investigated the influence of immune response kinetics, tumor size, and expression of foreign proteins via flow cytometry and immunohistochemistry. We found that increasing the cell inoculum in ovalbumin-expressing GL261 boosted the T cell response in vivo. Both CT2A and GL261 models demonstrated a reduction in T cell function as the tumor progressed, with increased programmed cell death -1 (PD-1) expression in CT2A and reduced capacity to produce cytokines in GL261. Importantly, we found that luciferase expression in CT2A—not GL261—markedly increased the number of T cells in the brain and significantly altered CT2A’s responsiveness to anti-PD-1 antibody therapy. These results influenced our investigation into a novel treatment strategy for GBM. Based on clinical trials in which patients treated with neoadjuvant plus adjuvant anti-PD-1 antibody therapy had increased survival compared with treatment after surgery, we hypothesized that this survival advantage was due to the release of danger signals during surgery. Thus, we designed a treatment strategy with staged anti-PD-1 beginning before surgery and local, intratumoral injections of calreticulin, a potent phagocytosis inducer, to treat murine glioma. We found that our combination therapy increased the CD8:CD4 ratio and activation marker expression in microglia and macrophages in the brain. Notably, our combination strategy increased mouse survival in the CT2A model. Overall, our findings identify technical factors that can confound immunotherapeutic findings and present a novel treatment strategy for GBM with the initiation of anti-PD-1 antibody therapy followed by local injections of a danger signal.