Targeting TREM2+ and TIMD4+ Macrophages Reprograms the Tumor Microenvironment and Enhances PD-1 Blockade Efficacy in Intrahepatic Cholangiocarcinoma

Abstract

Background: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. Macrophages are crucial components of the tumor microenvironment (TME). Triggering receptor expressed on myeloid cells 2-positive (TREM2+) macrophages have recently emerged as potent pro-tumor progress players in several cancers. However, the heterogeneity and tumor-promoting mechanisms of macrophages especially TREM2+ macrophages in ICC remain systematically unexplored. Therefore, this study aimed to systematically elucidate the heterogeneity of macrophages in ICC, with a specific focus on the pro-tumor role of TREM2+ macrophages. Methods: We collected tumor tissues, adjacent nontumor tissues, and tumor-adjacent border tissues from 16 ICC patients. These samples were subjected to single-cell RNA sequencing and spatial transcriptomic sequencing, followed by comprehensive analysis. The findings were further validated through flow cytometry, immunohistochemistry, multiplex immunofluorescence, and in vitro cellular assays. Furthermore, we established an ICC model using genetically engineered (Trem2−/−) mice as a validation cohort to corroborate the discoveries from human samples. Results: Macrophages are important prognostic factors in ICC, with functional heterogeneity. TREM2+ macrophages in tumor tissues promote immune suppression by exhausting CD8+ T cells and inducing the generation of regulatory T cells to produce. At the tumor border region, TREM2+ macrophages enhance ICC invasion via the urokinase-type plasminogen activator–urokinase-type plasminogen activator receptor axis and induce hepatic stellate cell conversion to myofibroblastic cancer-associated fibroblast, forming a physical immune barrier that impedes CD8+ T cell infiltration in tumor tissues. TIMD4+ macrophages in adjacent tissues also suppress CD8+ T cell function. Combining anti-Trem2 and anti-Timd4 with anti-Pd-1 antibodies significantly improved treatment outcomes in the ICC mice. Conclusions: TREM2+ and TIMD4+ macrophages are key regulators of the immunosuppressive TME in ICC. Targeting Trem2 and Timd4 enhances the effectiveness of anti-Pd-1 therapy in ICC mouse models.