The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here a team led by researchers at Memorial Sloan Kettering Cancer Center identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. They generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNAhi EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS.
Stromal-derived EVs harbor the mitochondrial genome
(A) qPCR of mu-mtDNA copy number (ND1 gene) in circulating EV DNA isolated from HTR and HTS tumor-bearing mice (n = 3 per group). (B) Electron microscopy (scale bar, 500 nm) and NanoSight analyses of EVs isolated from mCAFs. The concentration of particles is reported as mean ± SD. (C) Representative exosomal proteins identified by quantitative mass spectrometry of mCAF-derived EVs. (D) Ratio of mtDNA/nDNA level by qPCR (two unique primer sets were used for murine ND1, and one set each was used for human ND1, murine GAPDH, and human GAPDH) in EVs isolated from mCAFs and human bone marrow stromal cells (H-BMSCs, HS27a) ± DNase0 treatment to eliminate exogenous DNA contamination. (E) Schematic and representative PCR gel electrophoresis of mtDNA (ND1) and nDNA (GAPDH) from fractions following sucrose cushion purification of mCAF-derived EVs. Free DNA was eliminated by DNase0 digestion before extraction of EV-DNA. (F) mtDNA level as absolute copy number (qPCR, ND1) from HS27a EVs and cells. (G) Schematic and representative gel electrophoresis image of long-range PCR (three contiguous amplicons encoding the 16-kbp mtDNA genome from purified EVs ± DNase0. (H) Electropherogram of the DNA sequence of the ND1 gene showing a mutation conserved between HS27a cells and EVs.