Background The majority of colorectal cancer (CRC) cases are classified as microsatellite stable (MSS) subtypes and typically show resistance to anti-PD-1 treatment. Here, we aim to develop a co-culture model of organoids and autologous tumor-infiltrating lymphocytes (TILs) derived from MSS CRC patients to identify gut microbiota metabolites with the potential for synergistic immunotherapy.

Methods CRC organoids and autologous TILs biobanks were simultaneously established from surgically resected tissues of CRC patients using optimized culture protocols. Caspase 3/7 probe-labeled flow cytometry and immunofluorescence staining were used to assess the efficacy of T cell-mediated killing against CRC organoids in a co-culture model. The behavioral characteristics of T cell attacks on CRC organoids were dynamically observed using the Live Cell workstation. The synergistic effect of butyrate on anti-PD-1 therapy was assessed using a CRC organoids and TILs co-culture model. The changes in the immune landscape were identified using single-cell RNA sequencing and flow cytometry.

Results A co-culture model of CRC organoid and autologous TILs was established to identify potential synergistic immunotherapy candidates. Butyrate combined with anti-PD-1 treatment significantly activated cytotoxic CD8⁺ T lymphocytes and promoted T cell-mediated killing of CRC organoids. In addition, tracing analysis revealed that T cells treated with butyrate and anti-PD-1 exhibited increased motility to infiltrate and kill CRC organoids. Mechanistically, the proportion of GNLY⁺CD8⁺ T cell subsets increased after treatment with butyrate combined with anti-PD-1 therapy. GNLY⁺ CD8⁺ T cells exhibited elevated levels of cytotoxic and chemokine genes compared to other CD8⁺ T cell subsets, indicating that they play a major subset in enhancing anti-tumor immunity driven by butyrate.

Conclusions We successfully established an autologous CRC organoids and TILs co-culture model, which can effectively identify microbiota metabolites as potential candidates to boost immunotherapy. Butyrate, identified in the co-culture model, could enhance the efficacy of anti-PD-1 through the GNLY⁺CD8⁺ cell subset and may serve as a promising adjuvant to augment anti-PD-1 therapy against CRC.