The tumor microenvironment is complex and cannot be adequately recapitulated using conventional two-dimensional in vitro assays. Three-dimensional multicellular tumor spheroids (MCTS) offer a versatile platform to study heterotypic cell interactions, extracellular matrix (ECM) deposition, and drug screening in a controlled setting. Although technical advances have been made, there is still a lack of standardization among the different MCTS-forming methodologies. In fibroblast-containing MCTS, it is unclear how the initial cancer cell-fibroblast ratio affects MCTS architecture, functionality, and resemblance to in vivo tumors. Here, we systematically investigated how varying stromal content shapes MCTS architectural, molecular, and functional characteristics. Four cancer cell lines with distinct in vivo stromal signatures were co-cultured with fibroblasts at defined ratios to generate spheroids with increasing stromal content. At defined time points, spheroids were analyzed via histology, live fluorescence microscopy, immunofluorescence, flow cytometry, and gene expression assays to quantify growth kinetics, cell organization, proliferation, ECM deposition, and phenotypic states. We demonstrated that cancer cell identity and fibroblast proportion determine spheroid compactness, internal architecture, desmoplastic activity, and proliferation. Notably, fibroblast-rich spheroids displayed an increased ECM deposition and upregulation of genes related to fibroblast activation and ECM remodeling. These trends observed in MCTS were in line with patterns identified in vivo mouse xenograft and patient-derived samples. Finally, a drug testing proof-of-concept validation revealed that increasing stromal content reduces sensitivity to chemotherapeutics, with cancer cell-fibroblast MCTS recapitulating treatment responses more accurately than cancer cell homospheroids. Taken together, our study enables the standardization of parameters and identification of variables that influence the desmoplastic reaction within MCTS. This knowledge may contribute to the manufacturing of MCTS with desired morphological and functional features, aiming to support their integration in bioreactor-based advanced in vitro models for tackling complex biological questions. STATEMENT OF SIGNIFICANCE: We established a reproducible strategy to engineer fibroblast-containing multicellular tumor spheroids (MCTS) with tunable stromal content and desmoplastic activity. By systematically varying the cancer cell-fibroblast ratio, we demonstrated a proportional and controllable increase in extracellular matrix deposition. Furthermore, fibroblast inclusion induced coordinated transcriptional, secretory, and functional changes that scale with stromal abundance and recapitulate key tumor-type-specific phenotypic states observed in murine xenografts and human tumor specimens. Together, these findings provide a standardized and scalable framework for generating MCTS with defined stromal properties, enhancing the relevance and reproducibility of 3D in vitro tumor models. This platform enables controlled interrogation of tumor-stroma interactions and offers a practical foundation for studying stromal modulation of therapy response.
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