A simple liquid 3D cell culture paradigm models oxidative mitochondrial metabolism of epithelial breast cancer cells with relevance for lung metastases.

Three-dimensional (3D) cell culture systems have emerged as powerful tools for modeling tumor biology in ex vivo settings. However, the diverse array of available 3D culture methods presents challenges in selecting the most appropriate model for specific research questions. This study provides a comparative analysis of breast cancer cells (SUM149, IBC-3, and MDA-MB-468) in the mammosphere culture (SphC) model or an "emboli" culture (EmC) model, which enrich for cancer stem cells and epithelial features, respectively. The EmC model, designed originally for inflammatory breast cancer, is characterized by media viscosity and mechanical rocking of the culture vessel. Notably, cells in EmC showed a distinct and durable reduction in cell proliferation ex vivo while demonstrating increased capacity to establish experimental lung metastases in vivo. Ultrastructural quantitative analysis of electron microscopy images suggested that cells in EmC acquire nuclear and mitochondrial features that resemble those of tumor tissue. Proteomics, single-cell transcriptomics, and metabolic flux analyses showed that cells in EmC and SphC favor mitochondrial oxidative metabolism (OXPHOS) and glycolysis, respectively. EmC rendered cells hypersensitive to OXPHOS inhibition, but more resistant to oxidative stress. Several genes associated with lung metastasis, including ID1, were specifically enriched in EmC. Given the emerging role of OXPHOS in cancer cell survival during dissemination and as established metastases, we propose that the EmC paradigm is a suitable ex vivo model to study signaling pathways relevant for tumor tissue and to assess drug sensitivities and resistance mechanisms of metastatic breast cancer cells ex vivo. SIGNIFICANCEThis study provides an in-depth characterization of a resource-efficient yet powerful 3D culture paradigm to improve the physiological relevance of ex vivo approaches. Applicable to epithelial cancers, this model offers a platform to accelerate the discovery of physiologically relevant signaling pathways and specific cancer cell vulnerabilities.