Understanding the cellular and molecular basis of carcinogenesis will be a primary step towards devising effective chemo preventive strategies. This proposal aimed to characterize CSCs during oral cancer progression and elucidate the effect of fibroblast interactions. The primary outcome of the project will be biomarkers that can be clinically applied in prediction and early detection and/or will provide possible candidates as novel chemoprevention targets.
The human cell lines HaCaT (non-neoplastic) and DOK (dysplastic) were used in the study. Assessment of self-renewal capacity was carried out in 3D cultures and under the effect of CAF-conditioned medium (CAF-CM) by spheroid formation. The spheroids generated were used for NanoString Analysis with Stem Cell Panel (199 genes) (outsourced to TheraCUES Innovations Pvt. Ltd., Bangalore). The analysis of the NanoString data was carried out using R programming. Correlation plots were used to compare the different batches of the samples. The analysis of NanoString data was carried out using nSolver 4.0 software. The p-value adjustment for the experiment was carried out using the Benjamin-Hochberg method. The genes identified will be validated in respective cell lines and patient samples.
Assessment of self-renewal capacity in 3D spheroids (day 21) indicated formation of a significantly larger number and size of spheroids in DOK cells (p<0.0001) as compared to HaCaT cells. A comparison of the CSC profile (NanoString) indicated that spheroids from DOK, when compared to HaCaT showed differential regulation of 13 genes, out of which major entities were from the WNT pathway (WNT5A, WNT4, WNT10A, WNT7A) in addition to other genes such as CCND1 and HDAC2. The effect on the CAF-induced CSC profile was also assessed in both the dysplastic and non-neoplastic cells; CAF-induced spheroids formed were significantly high in DOK (p<0.001). NanoString analysis identified 52 differentially expressed genes from CAF-induced spheroids in HaCaT and DOK. CDC42 and ALDH2 are uniquely up-regulated in HaCaT and DOK spheroids, while CD44, PLAU, KRT15 along with WNT/NOTCH pathway were commonly up-regulated in both CAF-induced spheroids. A comparison between the spheroid populations revealed that Collagenase and CTBP being uniquely up-regulated in DOK spheroids; further detailed analysis and validation is currently ongoing.
These results suggest that CSC-enriched spheroids with both 3D scaffolds and under the effect of CAF-conditioned medium show a differential profile in non-neoplastic and dysplastic cells. Further analysis is being carried out to identify markers that can differentiate non-neoplastic cells from dysplastic cells.