Patient-derived organotypic tumor spheroids as a functional platform for predicting immunotherapeutic responses and guiding precision treatment for hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading cause of cancer mortality worldwide. While immune checkpoint blockade has revolutionized the treatment of many cancers, responses in HCC remain limited. Robust functional platforms capable of predicting individual responses to immunotherapy are urgently needed. In this study, we developed a patient-derived organotypic tumor spheroid (PDOTS) model that preserves the heterogeneity and immune microenvironment of HCC, enabling rapid and reliable assessment of targeted and immunotherapeutic responses. METHODS: Tumor tissues from 30 HCC patients were processed using a "Five-Point Clock" sampling method and cultured within a three-dimensional microfluidic chip supplemented with IL-2 and CD3/28 activator to maintain tumor-infiltrating lymphocyte activity. The genomic, immune, and histopathological fidelity of PDOTS relative to parental tumors was evaluated. Drug responses were assessed ex vivo and validated in matched patient-derived xenograft (PDX) models. Transcriptomic profiling was subsequently performed to identify gene signatures associated with treatment sensitivity and to construct a transcriptomic predictive model. RESULTS: =0.829, p<0.001) and predicted clinical outcomes in an external patient cohort treated with atezolizumab plus bevacizumab. CONCLUSIONS: These findings establish PDOTS as an immune-competent ex vivo platform for functional precision oncology and support the integration of functional testing with transcriptomic prediction to guide individualized immunotherapy in HCC.