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

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.

Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer

“…CTC clusters represent an intriguing CTC population, with profound recent interest due to their apparently increased metastatic potential; dissociation of CTC clusters decreases the rates of metastasis in mice, suggesting a central role for clusters in cancer spread [ 58 ]. We detected CTC clusters in 23% of our cohort, similarly to previous studies where clusters were detected in over 20% of CRC patients using membrane-based isolation [ 24 , 42 ]. These observations highlight the utility of membrane-based methods for capturing CTC clusters.…”

“…It would be interesting to apply a sequential filtration technique with membranes of different pore sizes (<8 µm) and investigate whether this improves the performance of membrane-based technologies. Capturing not only smaller CTC populations but also single CTCs and clusters from the same patient has been demonstrated in another membrane-based approach for CTC enrichment [ 24 ]. Such an approach enables comparative studies on paired single CTCs and CTC clusters to gain a better understanding on the role of these CTC subpopulations in CRC metastasis.…”

“…High-quality CTC enrichment technologies must enrich CTCs along the entirety of the EMT spectrum, which cannot be achieved by marker-based methods such as CellSearch, and reliance on such techniques limits our understanding of CTC biology and metastasis [ 21 ]. CTC clusters, often formed by CTCs in intermediate EMT, [ 22 , 23 ] have improved bloodstream survival and metastatic potential when compared to single CTCs, but most current marker-based methods, including CellSearch, fail to capture clusters effectively [ 24 , 25 , 26 ]. The decreased surface-area-to-volume ratio of cell clusters, in combination with mechanical disruption of clusters in devices with turbulent flow, contributes to the poor efficacy of CellSearch in cluster isolation [ 22 ].…”

“…Microfluidic and membrane-based techniques for CTC enrichment have demonstrated the ability to capture CTCs along the EMT axis, isolating a larger spectrum of CTC subpopulations relative to marker-based methods [ 23 ]. These advantages expand the repertoire of CTCs for subsequent analysis and provide greater success in the capture of CTC clusters, as demonstrated in particular with a modified ScreenCell method, although further validation with larger sample sizes and a range of cancer types is needed [ 24 ].…”

The Crosstalk Between Immune Infiltration, Circulating Tumor Cells, and Metastasis in Pancreatic Cancer: Identification of HMGB3 From a Multiple Omics Analysis

“…It seems to be promising that the treatment with HMGB3 as an anticancer target could inhibit the survival and metastasis of CTCs and the proliferation of primary tumor cells, which puts forward a new direction for the treatment of PDAC. Our research center has enriched and isolated CTCs by ScreenCell ® as previously described (a filtration method that allows the isolation of various CTCs by size, ensuring the diversity of CTCs) ( Francescangeli et al, 2021 ) and is currently conducting research on primary CTC culture. Once we manage to cultivate primary pancreatic CTCs, subsequent cytological experiments would be carried out to further verify the role of HMGB3 in CTCs.…”