299 - Radiotherapy, the Tumor Immune Microenvironment through PD-L1+ Macrophages, and Immunotherapy Efficacy in pMMR Colorectal Cancer

02:25pm - 02:35pm ET

Room 160

Presenter(s)

Luoxi He, BS - Fudan University Shanghai Cancer Center, Shanghai, Shanghai

L. He¹, Y. Chen², R. Xu², Q. Zhou², H. Zhang², M. L. Zhou³, J. Wang², W. Yang², S. Chen², Y. Lin², Y. Lu³, L. Shen⁴, Y. Wang², Z. Zhang², Y. Wang⁵, S. Zhou², F. Xia², and Z. Zhang⁶; ¹Fudan University Shanghai Cancer Center, Shanghai, Shanghai, China, ²Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, China, ³Fudan University Shanghai Cancer Center, Shanghai, China, ⁴Department of Oncology, Shanghai Medical College of Fudan University, Shanghai, China, ⁵Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China, ⁶Department of Radiation Oncology, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University; Shanghai Clinical Research Center for Radiation Oncology; Shanghai Key Laboratory of Radiation Oncology, Shanghai, China

Purpose/Objective(s):

Radiotherapy profoundly reshapes the tumor immune microenvironment (TIME) and has emerged as a potential strategy to sensitize proficient mismatch repair (pMMR) colorectal cancer (CRC) to immunotherapy. Myeloid cells are central regulators of radiation–immune interactions, yet the macrophage populations driving post-radiation immune remodeling remain poorly defined. This study aims to identify radiotherapy-induced macrophage states and define their functional role in immune activation and immunotherapy sensitization.

Materials/Methods:

Single-cell RNA sequencing was performed on 6 paired pre- and post-radiotherapy biopsies from pMMR CRC patients receiving long-course radiotherapy (50 Gy/25 fractions). Key findings were validated by patients immunofluorescence, a BALB/c CT26 radiotherapy mouse model with flow cytometry, and macrophage–tumor cell co-culture assays with qPCR. TCGA datasets and clinical immunotherapy cohorts were analyzed to evaluate immune microenvironmental features and survival associations.

Results:

Single-cell analysis revealed a pronounced expansion of myeloid cells following radiotherapy, with subtype analysis identifying a dominant increase in a PD-L1⁺ macrophage population. This population was validated in patient samples by immunofluorescence and in the CT26 radiotherapy mouse model by flow cytometry. These macrophages exhibited a pro-inflammatory transcriptional program, characterized by high expression of IL1B and related cytokines, with NFKB1 identified as the core regulatory transcription factor. Metabolic flux analysis demonstrated a high-glycolysis and high-lactate metabolic phenotype, consistent with inflammatory activation. Tumors enriched for this macrophage population displayed enhanced T-cell cytotoxic functional signatures. Co-culture experiments showed that radiotherapy alone did not induce PD-L1 expression in macrophages; however, co-culture with irradiated tumor cells significantly upregulated PD-L1 and other pro-inflammatory cytokines, indicating tumor-macrophage signaling as the driver of this phenotype. TCGA analysis demonstrated that this macrophage signature was enriched in dMMR tumors, associated with a hot immune microenvironment. In immunotherapy-treated clinical cohorts, high expression of this signature, validated by mIHC and bulk RNA-seq, was associated with significantly improved survival outcomes.

Conclusion:

Our study defines a previously unrecognized radiotherapy-induced, metabolically reprogrammed PD-L1⁺ macrophage state that links inflammation, glycolysis, and adaptive immunity. This macrophage program represents a potential biomarker of immune-active tumors and a predictive signature for immunotherapy response, providing a conceptual framework for integrating radiotherapy with immunotherapy through targeted modulation of tumor-associated macrophage states.