Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, with the latter displaying an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the intravasation of CTC clusters are largely unknown. Here, we compare gene expression of hypoxic and normoxic CTC clusters to identify genes that are not only upregulated as a consequence to hypoxia, but that may also play a pivotal role in promoting CTC clusters generation and metastasis. We isolated a total of 31 live CTCs from a breast cancer patient (BR61) and two breast cancer xenografts (BR16 and LM2) and labeled them with HypoxiaRed, a cell-permeable dye that tags hypoxic cells based on their nitroreductase activity, allowing to compare the gene expression profile of hypoxic versus normoxic CTC clusters. Following previously published protocol for parallel DNA and RNA sequencing from individual cells, transcriptomes of lysed cells were separated and amplified according to the Smart-Seq2. Subsequently, libraries were prepared with Nextera XT (Illumina) and sequenced on NextSeq75, single read for RNA. Final hypoxic condition was defined as HypoxiaRed-positivity as well as expression of HIF1α and VEGFA.