Introduction: CNS innate immune cells, microglia and macrophages, are the largest component of the inflammatory infiltrate in glioblastoma (GBM)[1]. They initially participate in tumor surveillance, but are subverted by GBM[2]. Immunotherapies have proven incredibly successful in cancers such as melanoma[3], but not in GBM partly because GBM-associated microglia and macrophages (GAMMs) are not well understood. Not only are GAMMs phenotypically complex, but there is a lack of studies in human tissue, especially of newly diagnosed IDH-mutant GBMs. We hypothesize IDH-wildtype and –mutant GAMMs display divergent inflammatory phenotypes that contribute to differing prognoses. Additionally, we suspect the content and inflammatory phenotype of GAMMs varies between patients. Understanding GAMM heterogeneity will allow for informed immunotherapy development and precision application.

Methods: Newly diagnosed, untreated human IDH-wildtype and -mutant GAMMs were isolated using flow cytometry and cultured for collection of conditioned media and analysis of secretory products. Automated segmentation with a widefield high-content analysis system was used to quantitate GAMM content and inflammatory phenotype in frozen sections. GAMM profiles in publicly available single-cell RNA-sequencing databases between human IDH-wildtype and -mutant GBMs were compared using new bioinformatics techniques. A summary of methods is shown in Fig. 1.

Results: Interestingly, while IDH-mutant GAMMs had more markers of activation than their wildtype counterparts, there was less GAMM content overall (Fig. 2 and 3). Surprisingly marked variation in GAMM infiltration exists between GBMs ranging from ~0-70% of specimen cells. A mixture of pro- and anti-inflammatory microglia and macrophages were found in each GBM (Fig. 4).

Conclusions: Taken together, microglia and macrophage activation status and content differs between IDH-mutant and -wild type GBMs. The highly variable GAMM profile suggests the success of immunotherapies hinges on taking a precision medicine approach. For example, microglia and macrophage-devoid GBMs would not benefit from therapies that target GAMMs.

Patient Care: By demonstrating the considerably differences in microglia and macrophage content and inflammatory phenotype between different human GBMs, we show that immunotherapies targeting these innate immune cells should be applied in microglia/macrophage-rich GBMs, and would likely not be effective in microglia/macrophage-poor GBMs. Furthermore, the differences between IDH-mutant and -wildtype GBM-associated microglia and macrophages have implications on why IDH-mutant GBM patients have better prognoses, informing future therapeutic tactics.

Learning Objectives: By the conclusion of this session, participants should be able to: 1) Describe the main differences between microglia and macrophages in IDH-mutant and -wildtype GBMs 2) Discuss, in small groups,what implications this variable innate immunophenotype has on current immunotherapy development and selection, 3) Identify future strategies for assessing microglia and macrophage response that encompass quantitation of microglia and macrophage content and inflammatory phenotype within GBM.

References: 1. Darmanis, S. et al. Single-Cell RNA-Seq Analysis of Infiltrating Neoplastic Cells at the Migrating Front of Human Glioblastoma. Cell Rep 21, 1399-1410 (2017). 2. Poon, C.C., Sarkar, S., Yong, V.W. & Kelly, J.J.P. Glioblastoma-associated microglia and macrophages: targets for therapies to improve prognosis. Brain 140, 1548-1560 (2017). 3. Luke, J.J., Flaherty, K.T., Ribas, A. & Long, G.V. Targeted agents and immunotherapies: optimizing outcomes in melanoma. Nat Rev Clin Oncol 14, 463-482 (2017).