3087 - Multi-Omics Sequencing Unveils Tumor Microenvironment Remodeling Induced by Combined Radiotherapy and Immunotherapy in Microsatellite-Stable Locally Advanced Rectal Cancer

J. Wang^1,2, S. Wang^1,3, Y. Wang^1,2, L. Zhang^1,2, J. Wan^1,2, H. Zhang^1,2, L. Shen^1,2, F. Xia^1,2, F. X. Chen^1,3, and Z. Zhang^1,2; ¹Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, China, ²Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China, ³Institue of Biomedical Science, Fudan University, Shanghai, China

Purpose/Objective(s): The TORCH phase II clinical trial (NCT04518280) demonstrated notable improvements in tumor response in patients with microsatellite-stable (MSS) or proficient mismatch repair (pMMR) locally advanced rectal cancer (LARC) treated with radiotherapy (RT) and immune checkpoint inhibitors (ICIs). This study aims to investigate the mechanisms by which the combined approach enhances tumor response by remodeling the tumor microenvironment (TME) using multi-omics sequencing. Materials/

Methods: The study prospectively collected tumor samples from patients with MSS/pMMR LARC participating in the TORCH clinical trial. The samples were dissociated for single-cell RNA sequencing (scRNA-seq), and a subset underwent spatial transcriptomics sequencing (ST-seq) and multiplex immunohistochemistry (mIHC). Bioinformatic approaches were used to construct a TME atlas for MSS/pMMR LARC. Compositional analysis of cell populations was conducted to identify cell subtypes associated with therapeutic response. Functional phenotypes of cell subtypes were explored through differential gene expression and geneset enrichment analyses. Interactions within the TME were assessed using cell-cell communication analysis.
Results: A total of 45 tumor samples were collected from 36 patients with MSS/pMMR LARC, including 28 baseline samples and 17 samples after RT combined with ICIs. Totally, 254,231 single cells were obtained. The TME was significantly remodeled by the combination of RT and ICIs. Of the responders (Rs, N = 9), the combination therapy significantly increased the proportion of CD8⁺ Tem cells (p = 0.033), which were notably present around tertiary lymphoid structures (TLS) and exhibited progenitor exhausted T cell characteristics. Moreover, responders showed an increase in CD4⁺ Trm cell infiltration (p = 0.011), while the proportions of induced Tregs (iTregs, p = 0.006) and Th17 cells (p = 0.008) were significantly decreased. Radiotherapy combined with ICIs activated the myeloid innate immune response, resulting from a higher infiltration of classical monocytes and type II dendritic cells in Rs. This facilitated the activation of T-cell subsets. In contrast, the TME of non-responders (NRs, N = 8) after combination therapy was dominated by cells with immunosuppressive features, including iTregs, tumor-associated macrophages, and cancer-associated fibroblasts (CAFs). CAFs play a crucial role in orchestrating treatment resistance by recruiting immunosuppressive cells and maintaining epithelial cell stemness. These findings were validated by ST-seq.
Conclusion: Combining RT with ICIs significantly improves the treatment response for patients with MSS/pMMR LARC by activating immune effector cells and reducing of immunosuppressive components. Cancer-associated fibroblasts play a crucial role in mediating resistance to treatment, emphasizing the importance of targeting CAFs strategies in future research.