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Myeloid cells comprise the majority of immune cells in tumors, where their content and composition is determined by tumor type and driver mutation. While these cells are essential for shaping the tumor microenvironment, promoting tumor growth, and contributing to therapeutic resistance, targeting tumor-associated myeloid cells, including bone-marrow-derived monocytes and neutrophils, has not been successful in the clinics. Monocyte chemoattractant protein (MCP) family, comprising of Ccl2, Ccl7, Ccl8, Ccl12, are essential for monocytes trafficking to the tumor sites. To eliminate monocyte recruitment, we leveraged CRISPR/Cas-9 based gene editing tool to generate a mouse strain that is devoid of all MCP genes, which we termed quadruple MCP knockout (qMCP-/-). Using these mice in combination with genetically engineered mouse models (GEMM) of glioblastoma (GBM), we abolished tumor monocyte infiltration. Due to the functional redundancy of MCP family members, we show that targeting individual MCP genes leads to compensation by other MCPs. In contrast, when all MCPs are genetically deleted and monocyte recruitment is abolished, neutrophil infiltration ensues. Single-cell RNA sequencing revealed that intratumoral neutrophils promoted proneural-to-mesenchymal transition in GBM, and supported tumor aggression by facilitating hypoxia response via TNF production. Remarkably, pharmacologic or genetic interventions that suppress both monocytes and neutrophil infiltration improve the survival of GBM-bearing mice. Taken together, our findings establish that specific subsets of myeloid cells can influence the dynamism of tumor microenvironment, and they emphasize the importance of targeting both monocytes and neutrophils simultaneously for effective GBM immunotherapy.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The only intervention that has improved the survival rate of GBM patients over the past several decades has been combining temozolomide (TMZ) with radiotherapy (RT), which increased median survival by only ~2.5 months to where it currently stands at ~15 months. Unfortunately, all GBM patients eventually die due to tumor recurrence. Intrinsic or acquired resistance to TMZ is a significant contributing factor to tumor progression, and various mechanisms have been suggested, including deficiencies in DNA mismatch repair (MMR) genes such as MSH6. To test the biological significance of Msh6 in GBM growth and resistance to TMZ and immunotherapy, we have generated immunocompetent genetically engineered mouse models (GEMMs) of GBM with germline loss of Msh6. To generate tumors with reduced or abrogated Msh6, we utilized GEMMs based on the RCAS/tv-a gene transfer system in combination with mice that exhibited heterozygous and homozygous loss of Msh6. When PDGFB was overexpressed in combination with the silencing of Tp53 to induce tumors, tumor-bearing mice with reduced or deficient Msh6 demonstrated increased tumor growth and shorter survival time compared to wild-type tumor-bearing mice. Our data demonstrate that two weeks of TMZ treatment at a clinically relevant dose of 25 mg/kg provides a significant survival advantage in WT-tumor-bearing mice, while no efficacy was observed in tumor-bearing mice with either heterozygous or homozygous loss of Msh6. We are currently evaluating the mechanism by which of Msh6 confers TMZ-resistance.