Irradiation-Modulated Murine Brain Microenvironment Enhances GL261-Tumor Growth and Inhibits Anti-PD-L1 Immunotherapy

Abstract

PurposeClinical evidence suggests radiation induces changes in the brain microenvironment that affect subsequent response to treatment. This study investigates the effect of previous radiation, delivered six weeks prior to orthotopic tumor implantation, on subsequent tumor growth and therapeutic response to anti-PD-L1 therapy in an intracranial mouse model, termed the Radiation Induced Immunosuppressive Microenvironment (RI2M) model.Method and MaterialsC57Bl/6 mice received focal (hemispheric) single-fraction, 30-Gy radiation using the Leksell GammaKnife® Perfexion™, a dose that does not produce frank/gross radiation necrosis. Non-irradiated GL261 glioblastoma tumor cells were implanted six weeks later into the irradiated hemisphere. Lesion volume was measured longitudinally by in vivo MRI. In a separate experiment, tumors were implanted into either previously irradiated (30 Gy) or non-irradiated mouse brain, mice were treated with anti-PD-L1 antibody, and Kaplan-Meier survival curves were constructed. Mouse brains were assessed by conventional hematoxylin and eosin (H&E) staining, IBA-1 staining, which detects activated microglia and macrophages, and fluorescence-activated cell sorting (FACS) analysis.ResultsTumors in previously irradiated brain display aggressive, invasive growth, characterized by viable tumor and large regions of hemorrhage and necrosis. Mice challenged intracranially with GL261 six weeks after prior intracranial irradiation are unresponsive to anti-PD-L1 therapy. K-M curves demonstrate a statistically significant difference in survival for tumor-bearing mice treated with anti-PD-L1 antibody between RI2M vs. non-irradiated mice. The most prominent immunologic change in the post-irradiated brain parenchyma is an increased frequency of activated microglia.ConclusionsThe RI2M model focuses on the persisting (weeks-to-months) impact of radiation applied to normal, control-state brain on the growth characteristics and immunotherapy response of subsequently implanted tumor. GL261 tumors growing in the RI2M grew markedly more aggressively, with tumor cells admixed with regions of hemorrhage and necrosis, and showed a dramatic loss of response to anti-PD-L1 therapy compared to tumors in non-irradiated brain. IHC and FACS analyses demonstrate increased frequency of activated microglia, which correlates with loss of sensitivity to checkpoint immunotherapy. Given that standard-of-care for primary brain tumor following resection includes concurrent radiation and chemotherapy, these striking observations strongly motivate detailed assessment of the late effects of the RI2M on tumor growth and therapeutic efficacy.

An untapped window of opportunity for glioma: targeting therapy-induced senescence prior to recurrence

“…In contrast, irradiated p21-/- mice failed to exhibit radiation-induced senescence and did not promote tumor aggressiveness demonstrating that the senescent microenvironment was necessary to increase glioma aggressiveness. Although these and similar studies have been performed in multiple cell lines within immunocompetent models (GL261 13 , 73 , CT-2A 13 , NS2262 13 , and DBT 74 ), we have observed a similar phenotype in six of ten glioblastoma patient-derived xenograft lines after 15 Gy of pre-irradiation 72 . Interestingly, pre-radiation extended survival in the two lines with the shortest time to moribund in vivo (GBM10 and 12), while survival in two other highly aggressive lines (GBM39 and 123) was not impacted by pre-radiation.…”

Distinguishing Tumor Admixed in a Radiation Necrosis (RN) Background: 1H and 2H MR With a Novel Mouse Brain-Tumor/RN Model

“…The morphology of lesions in this post-irradiation implantation model is strikingly distinct from lesions resulting from gliomas implanted into non-irradiated brains. These findings are robust and have been observed in two syngeneic orthotopic murine malignant glioma models in two different mouse strains: i) DBT cells implanted in Balb/c mice ( 11 ) and ii) GL261 cells implanted in C57BL/6 mice ( 12 ).…”

“…For many years, we have studied the late effects of radiation on normal brain tissue, having developed and characterized extensively a mouse model of late time-to-onset RN ( 9 – 18 ). Recently, we have described the late persisting impact of previous radiation on subsequent tumor growth ( 11 ) and on the responsiveness of tumors to immunotherapy ( 12 ). Of late, we have employed this radiation-biology platform to develop a novel mouse model that recapitulates many of the clinical features of recurrent malignant gliomas.…”

Radiotherapy‐Induced Astrocyte Senescence Promotes an Immunosuppressive Microenvironment in Glioblastoma to Facilitate Tumor Regrowth

“…[ 17 ] In addition, delayed effects of IR on the TME might lead to aggressive tumor regrowth and immunotherapy resistance. [ 29 ] Though senescent glial cells, including astrocytes, have been discovered in neurodegenerative disease for a long period of time, radiation‐induced astrocyte senescence has recently been identified by co‐immunostaining of P16 INK4A and GFAP in irradiated GBM patient tissues. [ 13 ] Based on these results, Eliot et al have also verified that IR triggered widespread senescence in mouse brains by co‐immunostaining of GFAP and senescence markers.…”