Circulating tumor cells (CTC) in the bloodstream not only provide us a sampling of the tumor population but are also keys to the metastatic process. Most CTC enrichment devices are coupled to downstream marker analysis by immunocytochemistry that define CTC as CD45-negative and epithelial marker (e.g. EPCAM)-positive cells. Our group recently reported a new class of tumor-derived cells in circulation, the circulating tumor-endothelial cluster, in colorectal cancer. This demonstrated that tumor-associated cells in circulation hitherto considered malignant may instead and unexpectedly provide key insights to the tumor microenvironment. To effectively classify the circulating cell milieu in lung cancer, we used a single-cell technique RNA and mutation analysis (scrm-PCR) we have described previously to characterize DNA and RNA from the same cell.
We enriched for CTC from whole blood of non-small cell lung cancer (NSCLC) patients using silicon microsieves with uniformly spaced 8 to 10uM circular pores. We manually selected captured single cells or cell clusters for characterization. 50 single cell or cell clusters from 5 NSCLC patients and 2 healthy subjects were probed for a curated panel of 27 RNA markers and hotspot DNA mutations in EGFR, TP53 and KRAS by scrm-PCR.
Classic epithelial markers such as EPCAM are largely unexpressed in CD45-negative cells in NSCLC. DNA mutations were undetected in all isolated cells except for 1 cell. CD45-negative cell clusters in NSCLC exhibit a different phenotypic profile as compared to our report of tumor-endothelial in colorectal cancer, failing to express classic endothelial markers such as VWF, MCAM and CDH5. We identified important morphological features correlating with biological phenotypic classification of these cells, allowing us to establish a map of the circulating cell milieu of NSCLC.
This is the first systematic same-cell combined mutation and expression analysis of circulating cells in NSCLC patients. With this novel approach, we report the presence of predominantly tumor-associated cells rather than malignant cells in circulation, and highlight the potential of these cells as biomarkers of NSCLC and its tumor microenvironment.
Clinical trial identification
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Institute of Bioengineering and Nanotechnology
Agency for Science, Technology and Research (A*STAR)
All authors have declared no conflicts of interest.