TMIC-110. Role of LRRC25 in local and systemic immunosuppression and its reversal

Abstract Immunosuppression in glioblastoma is a significant impediment to effectiveness in immunotherapy. We employed 3D-spatially mapped sampling and genome wide correlation analysis of RNA sequencing to identify an immunosuppressive signature comprising validated markers of multiple cell types, with LRRC25 as a key player. We confirmed the robustness of this signature in an independent internal cohort of 3D-spatially mapped GBM patients and TCGA. We observed strong co-variation of over one hundred genes, including markers for myeloid cells, neutrophils, and lymphoid cells (T and NK-cells) along with LRRC25. Published single-cell RNA-seq from glioma validated that LRRC25 is expressed by neutrophils and myeloid cells and absent from lymphoid-cells. The signature may therefore represent multiple interacting cell types. This is strongly supported by signature expression increasing when increasing the proportion of the three cell types bioinformatically by combining defined proportions of each via pseudobulking. LRRC25 expression varies spatially within tumors, as shown in single-cell data, the IVY-GAP laser capture microdissection database, and Visium spatial sequencing technologies. These data support and extend our 3D-spatially mapped samples showing upregulation in the tumor core compared to the periphery. We confirmed LRRC25 expression in myeloid cells in situ by RNAscope co-stained with anti-CD163, and in tumor cells by RNAscope and histopathology. HGG and LGG patients have LRRC25 upregulated locally (intratumoral) and systemically (circulating immune cells) in myeloid, T and NK cells relative to healthy donors. This suggests a potentially actionable role in local and systemic immunosuppression. CRISPRi-mediated LRRC25 knockdown in MACs-sorted patient peripheral myeloid cells enhances tumor-cell killing in T-cell co-culture assays. While significant efforts have focused on mechanisms of myeloid to T-cell immunosuppression, less focus is given to myeloid to NK cell, and neutrophil immunosuppression, despite the fact they exist at levels comparable in the tumor microenvironment. This work will be followed by further validation of functional immunosuppression.