IDDF2024-ABS-0211 Modeling the modulation of microbiome metabolites on immunotherapy using a co-culture system of autologous colorectal cancer organoids and tumor-infiltrating lymphocytes

<h3>Background</h3> 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. <h3>Methods</h3> 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. <h3>Results</h3> 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. <h3>Conclusions</h3> 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.