OS11.6.A GEMISTOCYTIC TUMOR CELLS AND IMMUNE REACTIVE MICROGLIA PROGRAMMED FOR GLIAL SCARRING CHARACTERIZE T CELL CONTAINMENT IN IDH-MUTANT ASTROCYTOMA
Authors: van Hijfte L , Geurts M , de Heer I , Ghisai S , Balcioglu H , Hoogstrate Y , Vallentgoed W , Head R , Westerman B , Wesseling P , Joyce J , Debets R , French P 
Published: October 2024

Abstract

BACKGROUND
Isocitrate dehydrogenase 1/2 mutant (IDHmt) astrocytoma is considered a T cell-deprived tumor, yet little is known regarding the exact phenotype that relates to T cell exclusion. To aid better understanding of this mechanism, we systematically characterized the immune microenvironment in IDHmt astrocytoma.

MATERIALS AND METHODS
We performed multiplex immunofluorescence (IF) stainings for CD4+ and CD8+ T cells, B cells, tumor associated macrophages (TAMs) and tumor cells on 75 IDHmt astrocytoma samples from which we collected and analyzed 1295 images. We performed bulk RNA sequencing on 158 IDHmt astrocytoma samples, accompanied with single nucleus RNA sequencing and spatial RNA and protein profiling on samples from the same cohort. We integrated and analyzed all datasets to acquire a robust characterization of the microenvironment organization.

RESULTS
We demonstrated that when present, T cells were either distributed in the tumor stroma, or in the majority of cases, contained in the perivascular space (i.e. T cell cuffs). We found that the tumor stroma surrounding T cell cuffs showed significant local accumulation of gemistocytic tumor cells (GTCs), a tumor cell type that histologically shows a large eosinophilic cytoplasm and ectopically placed nucleus. GTC-high tumors (&gt;20% of all tumor cells) demonstrated enhanced expression of genes related to immune cell migration and activation that were reliably expressed by TAMs and T cells. Notably, we found that GTCs formed a unique transcriptional cell sub-cluster that was part of the ‘astrocyte-like’ tumor cell state. GTCs expressed markers such as CD44, TNC and CRYAB that are specific for glial scarring, a central nervous system specific mechanism for immune cell exclusion. Surrounding T cell cuffs, GTCs co-localized with TAMs that transcriptomically resembled reactive microglia and showed increased expression of the chemo-attractants CCL3 and CCL4. Together, GTCs and reactive microglia expressed receptor-ligand pairs that take part in reactive astrogliosis and glial scarring, such as Osteopontin (SPP1) - CD44 and IL-1β - IL1R1. We validated this spatial pattern on protein level using whole-slide multiplex IF stainings of GTC-low and GTC-high tumors and showed specific local accumulation of CD44 positive GTCs and TAMs around T cell cuffs in GTC high tumors.

CONCLUSION
Using different modalities of bulk, spatial and single cell profiling for RNA and protein, we uncovered a previously unknown spatial pattern of co-localizing GTCs and reactive microglia around T cell cuffs in IDHmt astrocytoma. Together, we show that IDHmt astrocytomas organize a cellular network of tumor cells and leukocytes that mimics glial scarring and can aid immune cell exclusion. Characterizations of these complex localized interactions between tumor- and immune cell subsets aids a more specific target selection for immune therapies.