Development and clinical application of a primary cancer organoid-PBMC co-culture system for personalized immunotherapy

AIM: Many patients with non-small cell lung cancer (NSCLC) do not derive clinical benefit from immune checkpoint inhibitors (ICIs), and a reliable method for identifying potential responders is lacking. This study aims to establish an optimized patient-derived organoids (PDOs) culture system and develop a novel organoid-peripheral blood mononuclear cell (PBMC) co-culture platform to evaluate response to ICIs. METHODS: We optimized culture conditions for patient-derived NSCLC organoids by integrating conditional reprogramming techniques. ICIs response was assessed with an organoid-PBMC co-culture system. The accuracy of this ex vivo system was validated by comparing its results with efficacy of ICI therapy in both an in vivo humanized mouse xenograft model and the corresponding patient's clinical response. Moreover, the genomic stability of the organoids during serial passaging was monitored through gene sequencing. RESULTS: An optimal indirect co-culture system was established, culturing organoids with 25% Matrigel. This system consisted of 40 Gy-irradiated Swiss 3T3-J2 fibroblasts in an upper chamber and primary NSCLC cells in a lower chamber, using F-medium (DMEM/F-12 (3:1, v/v) + 10 µM Y-27632) supplemented with 0.5 µM A83-01. This method achieved an 85.59% (95/111) success rate. Using this platform, we evaluated PD-1 antibody efficacy with the organoid-PBMC co-culture system in 70 cases. For in vivo validation, a direct patient-derived organoid xenograft (PDOX) models were established in humanized mice reconstituted with the corresponding patient's PBMCs. Notably, the ex vivo results demonstrated a strong correlation with both the in vivo validation data and patients' actual clinical responses to ICIs, yielding a sensitivity of 100% and a specificity of 62.20%. CONCLUSION: We developed a novel, simple, rapid and reliable organoid-PBMC co-culture system for screening the response of NSCLC patients to ICI therapy.