Tetraspanin-driven metabolic plasticity and chemoresistance in disseminated breast cancer cells

Breast cancer (BC) is a major cause of cancer-related death in women, largely due to metastasis. Therapy-resistant Disseminated Cancer Cells (DCCs) can persist in a dormant state for long periods and serve as latent seeds of relapse. Their ability to evade detection and withstand treatment, such as chemotherapy, underscores the need to better understand DCC biology and to develop strategies to eliminate them before metastatic outgrowth. Across multiple orthotopic BC mouse models, including cell-line based or patient-derived xenografts, we observed extensive dissemination of tumor cells to multiple organs. DCCs persisted in a quiescent state for weeks yet retained metastatic capacity. Notably, whereas primary tumors and growing lung metastases responded to chemotherapy, DCCs remained resistant, indicating distinct survival mechanisms. To investigate the molecular properties of DCCs, we isolated the cells from secondary organs by flow cytometry and subjected them to transcriptomic profiling to uncover mechanisms underlying their biological function and resistance to chemotherapeutics. DCCs were analyzed across multiple secondary sites, including bone, kidney, and pancreas. This revealed specific differences between DCCs and mammary tumor cells, as well as remarkably conserved adaptive programs among DCCs from distinct organs, highlighting common survival strategies that may represent shared therapeutic targets. Gene signature analysis identified various cellular functions altered in DCCs. We observed reduced apoptotic responses, accompanied by enhanced DNA repair and striking metabolic rewiring as hallmarks of DCC persistence. Specifically, DCCs repressed glycolysis while inducing oxidative phosphorylation and fatty acid metabolism to establish a bioenergetic state that supports survival under therapeutic stress. Among the most highly upregulated genes in DCCs, we identified TSPAN8 and TSPAN1, which emerged as essential mediators of DCC metabolic adaptation and chemoresistance. These two members of the tetraspanin superfamily are transmembrane proteins that lack intrinsic enzymatic activity but organize membrane microdomains via interactions with partner receptors and signaling molecules. Their expression was strongly correlated in DCCs, suggesting a shared regulatory mechanism. Functional studies in vitro and in vivo demonstrated that ectopic TSPAN1/8 expression promotes metabolic adaptation and chemoresistance, whereas their knockdown disrupts the rewired metabolic network and sensitizes DCCs to chemotherapy. Collectively, our study establishes TSPAN1 and TSPAN8 as mediators of metabolic plasticity and chemoresistance in dormant DCCs. These findings provide a rationale to investigate combinatorial therapies integrating metabolic inhibitors with standard-of-care chemotherapy to eradicate dormant DCCs, the root drivers of metastatic recurrence.