Establishment of Tumor Organoids, Carcinoma-Associated Fibroblasts, and Counterpart Fibroblasts from the Same Esophageal Cancer Patient

In esophageal squamous cell carcinoma (ESCC), the acquisition of treatment resistance and tumor recurrence remain major clinical challenges. Carcinoma-associated fibroblasts (CAFs), key components of the tumor microenvironment, are known to influence ESCC progression and therapeutic response. However, many aspects of CAFs' roles in tumor malignancy remain unknown, presumably due to inter-patient variability in ESCC patients. Various human ESCC cell lines available for research are thought to partially mimic the nature of human carcinoma. They are also propagated in vitro for years and decades in a Petri dish, resulting in adaptation to the culture conditions. Moreover, human ESCC cell lines harbor various genetic and epigenetic alterations distinct among the original patients, resulting in inter-patient variability. Nevertheless, long-term cultured cancer cell lines have been employed in most previous studies for experimentation with patient-mismatched CAFs. Inter-patient variability within each cancer cell line and between cancer cells and CAFs might therefore cause reduced reproducibility and reliability of results. To eliminate inter-patient variability, three distinct cell types were isolated from the same patient: cancer organoids and CAFs from a surgically dissected ESCC region, and counterpart fibroblasts (CFs) from an adjacent non-tumorous region as patient-specific controls for CAFs. A detailed protocol is presented that enables the simultaneous isolation of cancer organoids, CAFs, and CFs. The cancer organoids grew well under three-dimensional conditions. CAFs and CFs stained positively for vimentin, one of the fibroblast markers, supporting the enrichment of these cells in fibroblastic lineages. The described protocol enables us to establish ESCC organoids and patient-matched fibroblasts, providing a valuable experimental platform that excludes inter-patient variability in studying tumor-stromal interactions.