Free-floating patient-derived organotypic tumor spheroids (PDOTS) from non-small cell lung cancer (NSCLC) tumors: a versatile tool for personalized testing of chemotherapeutic drugs.

Background and purpose: Patient-derived tumor 3D multicellular cultures are a novel non-small cell lung cancer (NSCLC) model for studying tumor biology and precision medicine, which recapitulates tumor morphology and gene expression profile. However, practical application is challenged by issues such as low establishment rates, long-term production difficulties, and the absence of immune microenvironment components. To address these issues, this study aimed to evaluate the efficacy of a novel method for generating free-floating patient-derived organotypic tumor spheroids (PDOTS) using a stimuli-responsive extracellular matrix (ECM)-mimicking gel. Experimental approach: Free-floating PDOTS were established from 18 NSCLC tumors and characterized by their morphology, marker expression, and extracellular matrix composition. Cell composition in PDOTS and their parental tumors was analyzed by flow cytometry, while RT-PCR was used to assess the expression of genes encoding signaling molecules. Finally, drug response and the expression of drug resistance genes were evaluated in the NSCLC PDOTS. Findings/Results: The PDOTS were successfully generated with a success rate exceeding 90%, forming spheroids within one week. These PDOTS preserved the parental tumor's morphology and included stromal and immune cells. Notably, 58% of the PDOTS maintained cytokine and growth factor expression profiles closely mimicked those of the original tumors. Furthermore, the PDOTS demonstrated varied responses to anticancer drugs, potentially influenced by differential expression of drug resistance-associated genes. Conclusion and implications: The high establishment rate and rapid production timeline of free-floating PDOTS using a stimuli-responsive ECM-mimicking gel make this approach a promising tool for advancing cancer biology research and evaluating therapeutic strategies with greater accuracy.