mtDNA transfer dynamics in vivo can induce dose-dependent tumor cell dedifferentiation and heterogeneity

Tumor heterogeneity limits treatment efficacy and drives therapy resistance. Although mitochondrial genome (mtDNA) mutations occur in ~60% of solid tumors, their contribution to heterogeneity is unexplored. We deployed established rho0 B16 melanoma and 4T1 breast cancer mouse models and engineered isogenic tumor cells carrying unique homoplasmic single-nucleotide mtDNA variants used as tracking barcodes. Using single-cell ATAC-seq with mitochondrial genotyping (mtscATAC-seq / mgatk) and single-cell multiome (ATAC + gene expression) profiling, we mapped and quantified horizontal mtDNA exchange within tumors in vivo. Respiration-deficient mutant tumor cells selectively and stably acquired host mtDNA, and mtDNA transfer induced dose-dependent tumor cell dedifferentiation and heterogeneity.