TMIC-107. Elucidating the Pro-Tumor Myeloid Response to Locally-Delivered Topotecan in Recurrent Glioblastoma

Abstract Glioblastoma (GBM) is the most common primary central nervous system tumor, with high associated mortality. In our recently completed phase 1b trial testing convection-enhanced delivery (CED) of topotecan for recurrent glioblastoma (rGBM), we observed a significant increase in myeloid cells post-treatment, which may contribute to unfavorable clinical outcomes by driving an anti-inflammatory, pro-tumor state. We hypothesized that the topotecan-associated myeloid cell response is driven by mesenchymal, pro-tumor macrophages, identifiable by the MARCO protein. We performed immunofluorescence staining on paired rGBM biopsies pre- and post-topotecan-CED treatment (n=5 patients) to quantify expression and spatial co-localization of myeloid markers Iba1, MSR1, and MARCO. Additionally, we performed deconvolution-based differential expression of bulk RNA-seq data from this cohort to extract a topotecan-induced myeloid signature. As validation, we analyzed single cell RNA sequencing of n=5 GBM biopsies treated ex-vivo with topotecan, and n=10 native rGBM biopsies from a literature cohort. Finally, we constructed a gene regulatory network (GRN) using PyScenic to identify upstream transcription factors. Immunofluorescence staining showed increased MARCO expression following CED topotecan, which colocalized with a heterogeneous range of myeloid populations, including Iba1+ MSR1+ (P=0.033), Iba1+ MSR1- (P=0.006), and double negative cells (P=0.004). Combined analysis of bulk RNA-seq patient samples and scRNA-seq slice cultures revealed a topotecan-induced transcriptomic macrophage signature, driven by MARCO. This signature was recapitulated as the primary macrophage MOFA factor in the untreated rGBM cohort (NES=1.43 by GSEA, P=0.001). GRN analysis identified CEBPB as a master regulator of the mesenchymal macrophage phenotype (NES=1.83 by GSEA, P=0.0001). CED topotecan is associated with robust expansion of a macrophage subpopulation that displays a MARCO+ mesenchymal signature. The transcription factor CEBPB is a key predicted master regulator of the MARCO-mesenchymal transcriptomic program. Our work therefore nominates CEBPB blockade as a therapeutic strategy for disrupting the pro-tumor macrophage population in the setting of CED of topotecan.