Background

CTCs predict an unfavourable prognosis and outcomes in cancers. Lowering of cytokeratin 18 expression is a hallmark of epithelial mesenchymal transition (EMT). Homogeneity and validation of CK18 expression in cancer cell lines and CTCs originating from distinct solid tumors is indeterminate and may contribute to non-specific counts. We hypothesize that the expression of CK18 in varied cell lines may differ quantitatively, and additionally may exhibit similar trends in CTCs enumerated from different tumor types.

Methods

CK18 variance in epithelial cell lines (e.g., A549+, MCF-7+, and MEF-) (n=192,269 cells) and CTCs (n=63) of different phenotypes was analyzed and compared. The fluorescence intensity was measured post-immunostaining, using motorized-automated, computer-assisted scanning, and through a customized ImageJ macro tool. The effect of anti-CK18 concentrations (0.06-6 μg/ml) and binding constants (Kb) was measured across all cell lines. CTCs were enumerated from head and neck squamous cell carcinoma (HNSCC) patients' blood samples (CTRI/2018/03/012905) and from clinical samples (e.g., breast, lung, colorectal (CRC), ovarian) using the clinically relevant OncoDiscover platform.

Results

CK18 mapping revealed diverse fluorescence intensities distribution in three cell lines, as well as in HNSCC, lung, breast, ovarian, and colorectal cancer CTCs (Table). In addition, the protein binding assay showed 8.65 x 10^3 Kb (M^-1) for MCF7 and 7.9 x 10^3 for A549 cells indicating concentration-dependent binding for CK18 expressing proteins on cells and may be varied in CTCs of different cancer types. Compared to the CK- cell line (MEF), the normalized CK18 intensity was higher by 290% and 310%, respectively, in MCF7 (breast) and A549 (lung) cells, demonstrating the variation in CK18 expression. On the other hand, CTCs showed significant diversity in CK18 expression with buccal mucosa revealing the lowest (67%), while CTCs of CRC origin demonstrated the highest expression (320%) (Table). CK18 intensity was represented across the cell lines and on CTCs enumerated from different cancer types.

Conclusions

Non-regulated CTC enumeration platforms pre-requisite critical validations to eliminate the non-specificity of CTC counts, which are highly imperative to clinical decisions in cancer management.

Clinical Trial Identification

CTRI/2018/03/012905.