Abstract Background: Glioblastoma (GBM) is characterized by inevitable recurrence and profound therapy resistance, particularly to immunotherapy, and effective options for recurrent disease remain extremely limited. An immunosuppressive tumor microenvironment dominated by tumor-associated macrophages (TAMs) plays a central role in attenuating antitumor immunity and sustaining therapy resistance. However, the contact-dependent signaling mechanisms linking GBM cells and TAMs to tumor recurrence and treatment failure remain poorly defined. Methods: We integrated publicly available scRNA-seq datasets from primary and recurrent GBM, as well as from immunotherapy responders and non-responders to map tumor-immune cell communication. Bulk transcriptomic data from TCGA-GBM and two CGGA cohorts (CGGA-693 and CGGA-325) were analyzed to determine the association of CD99 expression with patient survival, activation of signaling pathways, and oncogenic hallmark gene signatures. Spatial transcriptomics were used to assess the spatial proximity of CD99-high tumor cells and macrophages and the local signaling activity. For experimental validation, we performed in vitro and in vivo studies to assess whether CD99 blockade enhances the efficacy of anti-tumor immunotherapy. Results: Single-cell communication analysis revealed that macrophage populations were enriched and transcriptionally activated in recurrent GBM and immunotherapy non-responders, with CD99 homophilic-mediated interactions between macrophages and tumor cells specifically enriched in these settings. Across TCGA and CGGA cohorts, high CD99 expression was associated with worse overall survival (TCGA-GBM, p = 0.017; CGGA-693, p = 0.047; CGGA-325, p = 0.0039), enhanced JAK/STAT signaling, higher EMT pathway scores, and elevated expression of TGF-β, IL-10, and M2-related gene signatures. Spatial transcriptomics confirmed that CD99-high tumor cells were preferentially neighbored by macrophages, and these niches displayed elevated JAK2/STAT3 activity. Experimentally, under immunotherapy-mimicking conditions, CD99 blockade reduced JAK2/STAT3 signaling and EMT marker expression in GBM cells, lowered TGF-β/IL-10 secretion, and diminished M2 polarization of macrophages in co-culture. In mouse models, combining CD99 inhibition with anti-PD-L1 further suppressed tumor growth and attenuated the tumor-promoting effect of co-injected macrophages. Conclusions: These findings suggest CD99 homophilic engagement between GBM cells and macrophages as a critical amplifier of a JAK2/STAT3-EMT-M2 polarization loop that drives therapy resistance. Targeting CD99-mediated tumor-macrophage crosstalk may represent a promising strategy to overcome resistance and improve outcomes in GBM. Citation Format: Tongchao Jiang, Zilu Huang, Nan Li, Haishuang Sun, Tongcui Jiang. CD99 homophilic signaling between tumor-associated macrophages and glioblastoma cells regulates response to immunotherapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3307.
Jiang et al. (Fri,) studied this question.