MDK+ cancer-associated fibroblasts promote immunotherapy resistance through mitochondrial reprogramming in breast cancer

Abstract

Immunotherapy has improved outcomes for patients with breast cancer, yet therapeutic resistance remains a major clinical challenge. Therefore, we integrated single-cell and spatial transcriptomic data from breast cancer cohorts treated with immune checkpoint inhibitors to identify resistance-associated cellular populations and pathways, with validation across five independent bulk RNA-seq immunotherapy cohorts. Cancer-associated fibroblasts (CAFs) were isolated from anti-PD-1-resistant breast tumors for functional studies, including in vitro co-culture and in vivo models. Mitochondrial function and metabolic alterations were assessed by flow cytometry, transmission electron microscopy, ATP and adenosine quantification, and JC-1 staining. Tumor mitochondrial metabolism was markedly upregulated in anti-PD-1 non-responders and consistently associated with poor therapeutic response across validation cohorts. MDK-expressing CAFs were enriched in resistant tumors, exhibited strong interactions with tumor cells, and were negatively associated with intratumoral CD8⁺ T cell infiltration. Single-cell source contribution and depletion analyses identified CAF-derived MDK as the major paracrine mediator linking stromal signaling to tumor metabolic adaptation and immune suppression. Mechanistically, MDK activated SDC1-dependent PI3K/AKT/mTOR signaling in cancer cells, promoting mitochondrial metabolic reprogramming, ATP production, and CD39/CD73-dependent extracellular adenosine accumulation, thereby suppressing CD8⁺ T cell cytotoxicity. Receptor-depletion experiments further identified SDC1 as the principal functional receptor mediating these effects. Importantly, CAF-specific MDK depletion or pharmacological MDK inhibition enhanced anti-PD-1 efficacy and restored CD8⁺ T cell effector activity in vivo. Collectively, MDK⁺ CAFs facilitate anti-PD-1 resistance via mitochondrial rewiring and adenosine-mediated immunosuppression, supporting the MDK-SDC1 axis as a potential therapeutic target for overcoming resistance and improving immunotherapy efficacy in breast cancer.