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 cells cultured under defined in vitro conditions has not yet been addressed. Therefore, we analyzed the sc-response to hypoxia in nonclonal cell lines and multiple clonal derivatives, including HIF-1α or HIF-2α knockouts. While HIF-1α and HIF-1 target mRNA sc-heterogeneity was slightly higher than global transcription or specific housekeeping messenger RNAs (mRNAs), HIF-2α and especially HIF-2 target mRNA sc-heterogeneity was extraordinary, and remained in independent clones following HIFα knockouts. 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 scRNA-seq, 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.