TMIC-59. GLIOBLASTOMA INDUCES THE DIFFERENTIATION AND RECRUITMENT OF NON-CANONICAL ANTI-TUMORAL NEUTROPHILS FROM SKULL BONE MARROW

Abstract

Abstract The effects of tumor-associated neutrophils (TANs) on glioblastoma biology remain poorly understood. Flow cytometric analysis of 5 newly diagnosed glioblastoma fresh tissue specimens surprisingly revealed a high fraction (21.0-34.1%) of TANs expressed MHCII, a marker of antigen-presenting cells not classically associated with neutrophils and not expressed in matched peripheral blood (PBNs). Transcriptomic profiling confirmed that patient TANs upregulated expression of MHCII subunits (HLA-DR), chaperones (HLA-DM), and costimulatory ligands (CD86/83). Ex vivo cocultures further demonstrated that TANs activated patient-matched naïve T cells in an MHCII-dependent manner, while PBNs did not (CD25 MFI fold-change: 1.1 vs 3.2, p< 0.001). The antitumoral relevance of this property was confirmed in a syngeneic mouse glioma model, wherein αLy6G-mediated neutrophil depletion in T-cell-competent mice (Balb/c, n= 13) yielded endpoint tumors that had reduced CD8+ T cell infiltration (p= 0.0024) and were 2.4-fold larger by BLI (p= 0.0383) than controls, but had no bearing on tumor burden in T-cell-deficient (athymic) mice. Given the absence of MHCII+ neutrophils in circulation, we interrogated the inducibility of this phenotype by tumor-conditioned media in murine marrow/blood, finding that only immature Ly6Glow bone marrow neutrophils had sufficient plasticity to express MHCII and process foreign antigen as measured by DQ-ovalbumin uptake/proteolysis. Because of the non-inducibility of PBNs, we investigated the hypothesis that MHCII+ TANs were recruited to the glioblastoma microenvironment from adjacent skull bone marrow by labeling this space with CFMDA in tumor-implanted mice; at 72h, skull marrow-derived neutrophils contributed disproportionately to MHCII+ TANs compared to all TANs (41.7% vs 9.3%). As confirmation, in the first-ever scRNA-seq of human TANs, we demonstrated via pseudotime analysis that MHCII+ neutrophils represent a developmental lineage seen in TANs but not matched PBNs. Given the immunostimulatory effects of this population, mechanisms to promote neutrophil egress and tumor infiltration from the skull marrow may have therapeutic value.