Mitochondrial transcript abundance in breast cancer circulating tumour cells may reflect both blood-derived transcripts and biological heterogeneity

Circulating tumour cells (CTCs) are rare cells frequently profiled on a single cell level. A key indicator of single-cell transcriptome quality is the percentage of reads encoded by genes in the mitochondrial genome (MT-reads). No systematic data exists on MT-reads content in CTCs, making it unclear whether this parameter can reliably indicate CTCs’ transcriptome quality. We collected RNA-Seq profiles of 349 single breast cancer (BC) CTCs and 129 white blood cells (WBCs) enriched with four methods to assess the level of MT-reads and the expression of markers of the most abundant blood components—red blood cells (RBCs) and platelets (PLTs). The median percentage of PLTs and RBCs transcripts in CTCs was 2% (0–38%) and 0.8% (0.0006-84%), respectively, with significant differences between enrichment methods ( p < 2.2 −16 ). As a result, 44% of CTCs showed MT-reads level exceeding 10%, commonly considered a threshold indicating low-quality cells. Level of MT-reads in CTCs was frequently positively correlated with PLTs and RBCs markers; though in selected datasets increased MT-reads correlated with higher expression of oxidative phosphorylation and mitochondria biogenesis genes. Isolated CTCs themselves showed varied degree of cancer cells characteristics depending on the applied criterion (BC and WBC marker levels, aneuploidy score), underlining the challenges in CTCs identification. To conclude, the proportion of MT-reads in single CTCs is not a reliable standalone indicator of CTCs’ transcriptome. Elevated MT-read fraction in CTCs may arise from multiple factors, including technical aspects of blood sample processing, potential contributions of platelet- and erythroid-derived transcripts or intrinsic biological processes occurring in CTCs.