An innovative patient-specific cholangiocarcinoma-on-chip as a platform for personalized therapy

Background and Aims Cholangiocarcinoma (CCA) is a deadly cancer with limited treatment options. The development of new therapies is urgently needed and Organ-On-Chip has emerged as a promising tool for studying diseases in a more reliable 3D environment. In this study, we evaluated the reliability of CCA-on-chip as a patient-specific platform by integrating the immune cells and assessing their migration based on patient characteristics. Materials and Methods Primary CCA cells were isolated from surgically resected patients at Humanitas Research Hospital. The CCA microenvironment was recapitulated in the device by co-culturing CCA cells and cancer-associated fibroblasts (CAFs) in the central channel, flanked by endothelial cells in one lateral channel. Results T-cells exhibited a high ability to migrate within the tumor niche, spreading throughout the central channel. Two culture conditions were compared to investigate the influence of crosstalk between CCA cells and CAFs on T-cell migration. In the monoculture, T-cells showed greater trafficking compared to the co-culture. Furthermore, T-cells formed aggregates surrounding tumor spheroids in the monoculture, while they appeared dispersed in the co-culture. Interestingly, the co-culture exhibited higher levels of immunosuppressive molecules, suggesting that CAFs could contribute to an immunosuppressive microenvironment. Furthermore, in immunohistochemistry, CCA patient-derived cells were divided into two groups according to CD3+ cells: high-infiltrating (HOT) patients showed increased T-cell migration compared to low-infiltrating (COLD) patients. T-cells exhibited higher migration in the monoculture in HOT and COLD patients compared to their respective co-culture, further corroborating the role of CAFs in influencing immune cell recruitment and immunosuppression. Indeed, the expression of chemoattractant or immunosuppressive molecules, such as CXCL9, CXCL10, IL6 and IL10, varied between HOT and COLD patients and between culture conditions, suggesting their roles in immune cell recruitment and immunosuppression. Conclusions Our CCA-on-chip platform recapitulates the heterogeneity of the tumor microenvironment, demonstrating differences in T-cell trafficking and the expression of immunomodulatory molecules between high-infiltrating and low-infiltrating patients. Cholangiocarcinoma (CCA) is a deadly cancer with limited treatment options. The development of new therapies is urgently needed and Organ-On-Chip has emerged as a promising tool for studying diseases in a more reliable 3D environment. In this study, we evaluated the reliability of CCA-on-chip as a patient-specific platform by integrating the immune cells and assessing their migration based on patient characteristics. Primary CCA cells were isolated from surgically resected patients at Humanitas Research Hospital. The CCA microenvironment was recapitulated in the device by co-culturing CCA cells and cancer-associated fibroblasts (CAFs) in the central channel, flanked by endothelial cells in one lateral channel. T-cells exhibited a high ability to migrate within the tumor niche, spreading throughout the central channel. Two culture conditions were compared to investigate the influence of crosstalk between CCA cells and CAFs on T-cell migration. In the monoculture, T-cells showed greater trafficking compared to the co-culture. Furthermore, T-cells formed aggregates surrounding tumor spheroids in the monoculture, while they appeared dispersed in the co-culture. Interestingly, the co-culture exhibited higher levels of immunosuppressive molecules, suggesting that CAFs could contribute to an immunosuppressive microenvironment. Furthermore, in immunohistochemistry, CCA patient-derived cells were divided into two groups according to CD3+ cells: high-infiltrating (HOT) patients showed increased T-cell migration compared to low-infiltrating (COLD) patients. T-cells exhibited higher migration in the monoculture in HOT and COLD patients compared to their respective co-culture, further corroborating the role of CAFs in influencing immune cell recruitment and immunosuppression. Indeed, the expression of chemoattractant or immunosuppressive molecules, such as CXCL9, CXCL10, IL6 and IL10, varied between HOT and COLD patients and between culture conditions, suggesting their roles in immune cell recruitment and immunosuppression. Our CCA-on-chip platform recapitulates the heterogeneity of the tumor microenvironment, demonstrating differences in T-cell trafficking and the expression of immunomodulatory molecules between high-infiltrating and low-infiltrating patients.