Inherent single-cell heterogeneity of the transcriptional response to hypoxia in cancer cells.

Hypoxia-inducible factor (HIF) is a master regulator of cancer cell adaptation to tumor hypoxia and is involved in cancer progression. Single-cell (sc) differences in the HIF response allow for tumor evolution and cause therapy resistance. These sc-differences are usually ascribed to tumor microenvironmental differences and/or clonal (epi)genetic variability. However, the sc-heterogeneity of the HIF response in otherwise identical clonal cells cultured under defined in vitro conditions has not yet been addressed. Therefore, we generated clonal HIF-1α- or HIF-2α-deficient cell lines and analyzed the sc-response to hypoxia. While HIF-1α and HIF-1 target mRNA sc-heterogeneity was slightly higher than global transcription or specific housekeeping mRNAs, HIF-2α and especially HIF-2 target mRNA sc-heterogeneity was extraordinary, and remained in independent subclones as well as following Isoform-specific HIFα ablation. Unexpectedly, neither HIF-2α mRNA nor nuclear protein levels correlated with target mRNA levels. Unsupervised but not supervised HIF target gene dimensionality reduction revealed the initial sample composition after scRNAseq, demonstrating that, owing to sc-heterogeneity, individual HIF target genes are not sufficient to unequivocally identify hypoxic cancer cells. In conclusion, the pronounced intrinsic sc-heterogeneity of the HIF response represents a hitherto unrecognized feature of cancer cells that impairs clinical HIF pathway-dependent cancer cell identification and targeting.