Single-Cell and spatial profiling of immune and tumor- Intrinsic mechanisms in response to neoadjuvant radiotherapy plus immunochemotherapy in microsatellite stable locally advanced rectal cancer

Background: Locally advanced rectal cancer (LARC) is recognized as a highly aggressive disease, characterized by frequent local recurrence and metastasis. Unlike microsatellite instability-high (MSI-H) tumors, microsatellite stable (MSS) LARC is immunologically cold and poorly responsive to immune checkpoint inhibitors (ICIs), necessitating multimodal treatment. The combination of chemoradiotherapy and ICIs in the neoadjuvant setting has shown promising clinical benefits in MSS LARC, but the underlying mechanisms of these therapeutic responses remain unclear. Understanding the role of the tumor microenvironment (TME) in therapy response could optimize patient stratification and guide the development of novel therapeutic strategies to improve outcomes in MSS LARC.

Methods: We performed a comprehensive analysis of longitudinal biopsies from 32 MSS LARC patients treated with short-course radiotherapy (SCRT) followed by CAPEOX and Envafolimab at pre-RT, post-RT, and post-immunochemotherapy (post-ICT) time points using single-cell RNA sequencing, single-cell immune repertoire sequencing, and spatial RNA sequencing. Patients were categorized into two groups based on pathologic response: pathological complete response (pCR; n=20) and non-pCR (NpCR; n=12).

Results: We constructed a comprehensive single-cell transcriptome atlas from 454,369 cells across 93 MSS LARC samples, which led to the identification of eight major cellular subsets, including cancer, immune, and stromal cells. At the cancer cell transcriptome level, therapyinduced transcriptional reprogramming was closely associated with clinical response. Following RT, tumor cells in pCR patients exhibited upregulated MHC-I and MHC-II pathways along with activation of type I and type III interferon (IFN) signaling cascades, suggesting a more immunogenic phenotype. In contrast, tumor cells from NpCR patients demonstrated increased expression of E2F targets, Myc targets, and oxidative phosphorylation (OXPHOS)- related genes, indicative of persistent tumor proliferation and metabolic reprogramming that may contribute to immune evasion. Within the immune landscape, a high level of pre-existing CXCL13? CD8? T cell infiltration in the TME, along with elevated tertiary lymphoid structures (TLS) levels, was associated with better responses to neoadjuvant therapy. Furthermore, RT enhanced the interaction between CXCL13? CD8? T cells and Bfoc/Bgc cells via the CXCL13- CXCR5 axis, facilitating the formation of TLS. Additionally, CXCL13? CD4? T cells also played a crucial role in TLS formation, particularly during the ICT phase, further supporting the importance of adaptive immune responses in treatment efficacy. However, in NpCR patients, RT induced a prominent upregulation of immunosuppressive molecules in myeloid cells, including LAMP3? dendritic cells (DCs), XCR1? DCs, CXCL11? monocytes, and IFIT1? neutrophils. The stromal components of the TME also contribute significantly to the immune landscape and therapeutic outcomes. CellChat analysis revealed a strengthened CXCL12- CXCR4 interaction between CXCL12? fibroblasts and CD8? T cells following ICT, indicating that ICT may enhance CXCL12? fibroblast-mediated CD8? T cell recruitment, thereby modulating the immune landscape and potentially improving anti-tumor immunity.

Conclusions: Together, these findings elucidate key immune and tumor-intrinsic mechanisms underlying the response and resistance to the combination of SCRT and immunochemotherapy in MSS LARC, which may inform novel therapeutic strategies to optimize patient stratification and enhance treatment efficacy.

Dr. Jia is an oncologist specializing in the identification of prognositic and therapeutic biomarkers in colorectal cancer (CRC). She holds a PhD in oncology from Zhejiang Universty. Her research focuses on elucidating key molecular and cellular mechanisms that influence CRC progression and treatment response, particularly in the context of immunotherapy and neoadjuvant strategies. Dr. Jia has contributed to multiple peer-reviewed publications and conference presentations and is actively involved in interdisciplinary collaborations.