984 Using sex differences to uncover novel microglia regulators of glioblastoma progression

Background

Glioblastoma Multiforme (GBM) is the most aggressive and treatment-resistant brain tumor. GBM progression is heavily influenced by non-malignant microglia, the brain resident macrophages, that get coerced into a symbiotic relationship with tumor cells causing them to adopt an immunosuppressive and tumor-protective state.^{1 2} GBM outcomes are also influenced by sex, with males having worse prognosis.³ Here, we investigate novel regulators of the GBM tumor-associated microglia (GBM-TAM) state and use male-biased gene expression to distinguish candidates most likely to drive GBM progression.

Methods

Human microglia were isolated from 18 GBM tumors, 10 intermediate/low-grade tumors, and 12 normal brain regions by FACS (CD11b⁺, CD45^mid, CX3CR1^mid, CD64⁺, and CCR2^low). Microglia were processed for bulk RNA-seq followed by differential gene expression and pathway analysis using DESeq2 and GSEA algorithms, respectively. CRISPRi experiments were conducted by integrating MPP1 and non-targeting control guide RNAs into dCas9-KRAB-expressing stem cells followed by differentiation into microglia. Differentiated microglia were treated with LPS for 3 hours to trigger inflammation then processed for RNA-seq.

Results

Increased abundance of immunosuppressive genes IL-10 and CD204, and decreased abundance of homeostatic genes P2RY12 and SALL1 was observed in GBM-TAMs compared to normal microglia, supporting the GBM-TAM state transition. Genes belonging to tumor-supportive pathways like cell proliferation, hypoxia, and glycolysis were also increased in GBM-TAMs. Since GBM is more aggressive in males, we distilled GBM-TAM genes to those most likely to promote tumor progression based on male-biased expression. Genes in inflammatory and cell proliferation pathways were male-biased, and none were female-biased. We used gene expression correlation analysis to find candidate regulators of the male-biased GBM-TAM pathways, prioritizing genes from the sex chromosomes given their role in establishing sex differences. X chromosome gene Membrane Palmitoylated Protein 1 (MPP1) was correlated with the GBM-TAM inflammatory pathway, and progressively increased in abundance with tumor grade. We tested the role of MPP1 in the inflammatory response using stem cell-derived microglia treated with LPS. MPP1 knockdown led to downregulation of genes involved in lipid metabolism, membrane composition, and the microtubule cytoskeleton, and upregulation of genes involved in inflammation, many belonging to the GBM-TAM inflammatory pathway.

Conclusions

We conclude that MPP1 is a novel regulator of the GBM-TAM state by mediating interactions and signaling between TAMs and tumor cells at the plasma membrane. Our results support an anti-tumor role for MPP1 by suppressing the TAM inflammatory program. Our ongoing work directly tests the function of MPP1 in tumor progression.

References

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