189 - Single-Cell RNA Sequencing to Reveal Cellular Mechanisms Driving Differential Responses to Radiotherapy Combined with Systemic Therapy and Immunotherapy in MSS Locally Advanced Rectal Cancer with vs. without Liver Metastases

Purpose/Objective(s): The efficacy of cancer treatment is closely related to the tumor stage. The TORCH study enrolled patients with microsatellite stable (MSS) locally advanced rectal cancer (LARC), while the MIRACLE-1 and MIRACLE-2 studies (briefly referred to as MIRACLE) enrolled MSS LARC patients with metastases. Both studies employed similar combination treatment of radiotherapy, systemic therapy and PD-1 inhibitor. Notably, over 50% of patients in the TORCH study achieved complete response (CR), whereas almost no patient in the MIRACLE study achieved CR. This study aims to elucidate the cellular mechanisms underlying the distinct treatment responses.

Materials/Methods: We collected 48 primary tumor samples (29 from TORCH patients and 19 from MIRACLE patients with liver metastases) at baseline. Single-cell RNA sequencing (scRNA-seq) was performed to characterize differences in the tumor microenvironment of MSS LARC with versus without liver metastases.

Results: TORCH tumor samples exhibited higher proportions of CXCR4⁺ CD8⁺ effector memory T cells (T_EM), CD4⁺ follicular helper T cells (T_FH), and CD4⁺ tissue-resident memory T cells (T_RM), alongside lower proportions of regulatory T cells (T_reg), compared to MIRACLE tumor samples. Cell-cell interaction identification revealed that SPP1⁺ macrophages could regulate T cell function. Compared to samples without SPP1+ macrophages, those with SPP1⁺ macrophages showed lower proportions of CXCR4⁺ CD8⁺ T_EM and CD4⁺ T_RM but a higher proportion of T_reg, suggesting an immunosuppressive role for SPP1⁺ macrophages. The higher proportion of SPP1⁺ macrophages and enhanced SPP1 signaling interactions between SPP1⁺ macrophages and T cells in MIRACLE tumor samples may partly explain their poorer response to immunotherapy-based neoadjuvant therapy. Additionally, tumor cells and cancer-associated fibroblasts (CAFs) may suppress CD8⁺ T cell function via the IGF pathway, with a higher communication probability observed in MIRACLE compared to TORCH.

Conclusion: MSS LARC with and without liver metastases at baseline treated with immunotherapy-based neoadjuvant therapy exhibited distinct treatment responses, potentially associated with specific cell interactions such as SPP1⁺ macrophages regulating T cells. These findings provide insights into the cellular mechanisms of treatment resistance and highlight potential targets for improving efficacy in LARC with metastases. (NCT04518280) (NCT05359393) (NCT05359406)