Sequential Isolation and Characterization of Single CTCs and Large CTC Clusters in Metastatic Colorectal Cancer Patients

SIMPLE SUMMARY: The presence of cancer cells clusters is a frequent event capable of increasing their aptitude to survive in the bloodstream. Consistently, clusters ranging from 2–50 cancer cells are detected in about 50% of patients with metastatic cancers, including colorectal carcinoma. Although a deepened analysis of clusters might certainly offer new insights into the complexity of metastatic cascade, research in this field has come to a halt, since most circulating tumor cells isolation techniques are not compatible with large-sized clusters isolation. In the present study, we describe a sequential method to simultaneously isolate single and clustered circulating tumor cells from a single blood draw, opening new scenarios for an ever more precise characterization of colorectal cancer metastatic cascade. ABSTRACT: Circulating tumor cells (CTCs) detach from a primary tumor or its metastases and circulate in the bloodstream. The vast majority of CTCs are deemed to die into the bloodstream, with only few cells representing viable metastatic precursors. Particularly, single epithelial CTCs do not survive long in the circulation due to the loss of adhesion-dependent survival signals. In metastatic colorectal cancer, the generation of large CTC clusters is a very frequent occurrence, able to increase the aptitude of CTCs to survive in the bloodstream. Although a deepened analysis of large-sized CTC clusters might certainly offer new insights into the complexity of the metastatic cascade, most CTC isolation techniques are unfortunately not compatible with large-sized CTC clusters isolation. The inappropriateness of standard CTC isolation devices for large clusters isolation and the scarce availability of detection methods able to specifically isolate and characterize both single CTCs and CTC clusters finally prevented in-depth studies on the prognostic and predictive value of clusters in clinical practice, unlike that which has been described for single CTCs. In the present study, we validated a new sequential filtration method for the simultaneous isolation of large CTC clusters and single CTCs in patients with metastatic colorectal cancer at failure of first-line treatments. The new method might allow differential downstream analyses for single and clustered CTCs starting from a single blood draw, opening new scenarios for an ever more precise characterization of colorectal cancer metastatic cascade.