Samrat Roy, Debabani Roy Chowdhury, Manoj Pandre, Sundarajan Kannan, Rajesh Kumar RK, Amit K. Sharma, John W. Ellingboe, Arnab Roy Chowdhury.

Dissecting tumorigenesis and metastatic properties of cell lines by phenotypic functional assays and plasticity ratio (PR), Mestastop Solutions Private Limited, Bangalore, India

Cancer cell plasticity plays a defining role in initial tumorigenesis and secondary metastasis. The epithelial to mesenchymal transition (EMT) is critical for the primary tumor cell population to promote metastasis, whereas the reverse mesenchymal to epithelial transition (MET) is important for tumorigenesis in secondary organs post seeding.

To understand the contribution of such plasticity in colorectal cancer, we have assessed the functional properties of four colorectal cancer cell lines (HT29, HCT 116, SW 480, Colo 205) in our comprehensive phenotypic metastasis assay platform and mapped  their respective plasticity ratios (PR, total vimentin / total E cadherin).  Next, we took cells with a low PR (HT 29) and engineered them (using plasmid vectors containing the gene of interest, in both constitutive and inducible strategies) to have a high PR, thus changing their functional behavior (e.g. extravasation, platelet binding) to more closely resemble wild type cells having a high PR (SW 480).

Interestingly, when we engineered SW 480 to increase its PR, we did not see a concomitant change in functional behavior. There was a decrease in extravasation and platelet binding suggesting a PR threshold and maxima exists for cellular behavior. Successful conversion of cells from low to high PR and vice versa helped us establish EMT and MET screening platforms in a 2D format; cells with high PR taking more time to go through MET. Variation in glutamine addiction, ROS formation, exosome secretion and stemness (CD44+ CD133) was also observed in cells with low and high PR.  No significant increase in either doubling time or chemoresistance was observed with increase in PR (20% only). Early in vivo experimental data with HT 29 engineered cells and a higher PR clone shows a marked difference in tumorigenesis; the former with lower PR showing high growth but the latter with higher PR forming only small tumors.

Our data suggests that irrespective of the genetic signatures of driver genes and mutational profile of cell lines, tumorigenesis and metastatic potential depends on the plasticity of the cell, which can be quantified by measuring the PR and subsequently mapped to the various cellular functions of the tumor cells. We are currently evaluating patient tumor samples and correlating the tumor PR with our functional assay platform to create a proprietary algorithm METSCAN™ that will help address both drug discovery needs and predictive assessment of primary tumor metastasis.

Poster 2 – AACR 2021