<p>High-grade serous tubo-ovarian cancer (HGSC) is marked by substantial inter- and intra-tumor heterogeneity. The tumor microenvironments (TME) of HGSC show pronounced variability in cellular make-up across metastatic sites, which is linked to poorer patient outcomes. The influence of cellular composition on therapy sensitivity, including chemotherapy and targeted treatments, has not been thoroughly investigated. In this study, we examined the premise that the variations in cellular composition can forecast drug efficacy. Using a high-throughput 3D in vitro tumoroid model, we assessed the drug responses of 23 distinct cellular configurations of tumoroids comprised of OVCAR3 HGSC cells, mesenchymal stem cells, HUVEC endothelial cells, and U937 monocytes to an assortment of five therapeutic agents, including carboplatin and paclitaxel. We identified that the overall pooled viability in response to these five drugs was highest among tumoroid compositions that contained a large number of myeloid cells, whereas the most sensitive tumoroids to these agents were comprised of only cancer cells. Additionally, we found that the “mesenchymal tumoroids” containing 400 or more mesenchymal stem cells were more sensitive to carboplatin than paclitaxel. By amalgamating our experimental findings with random forest machine learning algorithms, we assessed the influence of TME cellular composition on treatment reactions. Our findings reveal notable disparities in drug responses correlated with tumoroid composition, underscoring the significance of cellular diversity within the TME as a predictor of therapeutic outcomes. This research establishes a foundation for employing human tumoroids with varied cellular composition as a method to delve into the roles of stromal, immune, and other TME cell types in enhancing cancer cell susceptibility to various treatments.</p>