Studying Immunogenic Cell Death in Human Colorectal Cancer Organoids.

PURPOSE: Combination therapies of chemotherapeutic agents and immunotherapy have shown promising results in the treatment of cold tumors. Various chemotherapies trigger immunogenic cell death (ICD) and release of hallmarks immunogenic damage associated molecular patterns (DAMPs) that have been related with immunostimulatory activities, leading to better patient prognosis. We aim to optimize in vitro assays to detect DAMPs release in response to chemotherapeutic agents using a colorectal cancer (CRC) organoids model. METHODS: CRC patient-derived organoids (PDOs) were treated either with oxaliplatin (OXA) or with 5-fluorouracil (5FU) and viability was measured with CellTiter-Glo3D Cell viability assay. Calreticulin (CALR) and high mobility group box 1 protein (HMGB1) intracellular translocation was assessed in immunofluorescence microscopy and quantified via co-localization with wheat germ agglutinin (WGA) and DAPI. Extracellular release of adenosine triphosphate (ATP) was quantified via specific luminescence assays. RESULTS: The results showed that CRC PDOs release DAMPs in a patient-specific manner in response to OXA and 5FU treatments. CONCLUSION: This study successfully used immunofluorescence and luminescence methods to detect ICD-associated DAMPs release in CRC PDOs in response to chemotherapeutic treatments. This approach allows the recognition of patient-specific ICD activation and could help predict patient response to therapies.