Apoptotic cell overload drives multinucleated giant cell formation after neoadjuvant chemotherapy in colorectal cancer: molecular mechanisms and tumor-suppressive effects

Abstract Colorectal cancer is a common digestive malignancy. Neoadjuvant chemotherapy (NAC) can downstage tumors and increase R0 resection rates. In colorectal cancer specimens with favorable pathological regression after NAC, we observed CD68 + multinucleated giant cells (MGCs) preferentially localized in apoptotic cell-rich areas, whereas they were rarely detected in non-NAC specimens. However, the mechanisms driving MGC formation and their functional significance remain unclear. We established an in vitro apoptotic cell-macrophage co-culture system to assess how apoptotic cell burden and efferocytosis regulate macrophage fusion. The TREM2/TYROBP signaling axis was examined using molecular and pharmacological approaches. RNA sequencing, pathway enrichment, lysosome- and metabolism-related transcriptional analyses, and secretory profiling were performed to characterize efferocytosis-induced MGCs. Their effects on colorectal cancer cells and the tumor microenvironment were further evaluated in vitro and in tumor-bearing mouse models. Excessive apoptotic cell burden markedly promoted macrophage fusion and upregulated the fusion-related molecules DCSTAMP/Dcstamp and CDH1/Cdh1. Apoptotic cell overload shifted macrophages from a homeostatic/efferocytic state toward a fusion-prone MGC phenotype. TYROBP / Tyrobp knockdown or loss inhibited macrophage fusion. Mechanistically, apoptotic cell overload activated the TREM2/TYROBP-MAPK/c-FOS-DCSTAMP axis, thereby enhancing DCSTAMP transcription and macrophage fusion. Efferocytosis-induced MGCs showed reduced lysosome-related transcriptional programs and an altered secretory profile, including increased TNF-α expression. Functionally, MGCs inhibited colorectal cancer cell growth in vitro. In tumor-bearing mice, MGCs reduced CD206⁺ macrophage and regulatory T-cell abundance while increasing CD8 + T-cell infiltration, thereby contributing to tumor growth suppression. Together, these findings identify efferocytosis-induced MGCs as a distinct macrophage state with altered lysosomal and secretory programs, anti-tumor activity, and the potential to remodel the tumor microenvironment.