Characterization of GBM Immunophenotype in Murine Heterotopic and Orthotopic Models, Implications for Novel Treatment Modalities

Abstract

Immunosurveillance within the CNS is mediated by traditional cell types as well as microglia, which play dual roles as a CNS macrophage and antigen presenting cells. GBMs are comprised of a complex network of tumor cells, neurons and immune cells, yet the importance of these components remains to be characterized. We sought to describe the tumor microenvironment using hetero- and orthotopic modeling of murine GBM and characterize disparities in treatment outcomes. A GBM cell line generated from an adult astrocytic genetically engineered mouse model (GEMM) was used for all experiments. The baseline components of heterotopic and orthotopic GBM microenvironments were analyzed with FACS. Heterotopic flank GBM models were used to characterize the tumor microenvironment and assess response to hypofractionated radiation therapy. Mice were sacrificed or followed for tumor growth kinetics and overall survival. The sacrificed mice were assessed with FACS to evaluate changes in the microenvironment and draining lymphatics in response to the tumor (control) or the tumor and RT. A parallel experiment was done using an orthotopic GBM model. Tumors were had similar proportion of CD45+ myeloid cells (48±45%), of which about a third (30±4%) were CD11b+ cells. In addition, infiltrating CD8+ cells comprised 10% of the microenvironment. Similar findings were seen within the orthotopic model. Mice with establish flank tumors were treated with 6Gy x 3, a subset was monitored and a subset were sacrificed for analysis. Mice treated with RT alone were found to have profound reduction in tumor doubling time (367±9 days vs. 13±3 days, P=0.01). This translated to a 30-day OS of 100% vs. 57% (P=0.05) with no control mice alive by day 40 and 100% of treatment mice alive at day 90. Cured mice were subsequently re-challenged on the opposite flank, and they demonstrated complete immunity compared to naïve mice. FACS revealed increased proportion of M2 macrophage (CD11b+/F4/80+/CD206+) following RT and a near doubling of infiltrating CD8+ cells. However, in the orthotopic model, identical treatment with 6Gy x 3 had a modest survival benefit over observation (P=0.02), but survival is short-lived with all control mice requiring euthanasia by day 15-20 and all treatment mice by day 30-35. There are profound differences in tumor control following identical treatment with hypofractionated RT. These differences may be accounted for by the distinct microenvironmental found in the CNS and flank, respectively. Myeloid derived cells (CD45+) comprise nearly 50% of a GBM tumor, of which TAMS/MDSC make up the majority cell type, in both brain and flank models. However, a key difference between the two models is the presence of microglia, a component of CNS immunoregulation that requires additional investigation. Modulation of the GBM microenvironment may result in improved outcomes. Future work will investigate TAM modulation with CSF-1R inhibition in combination with hypofractionated RT.