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Next we wanted to understand the phenotypic changes associated with the in vitro transformed vimentin expressing cells. BP treated vimentin positive cells showed a significant increase in its in vitro invasive potential, although no significant difference was seen in the migration and proliferation potential of the two clones (Fig. 3A-E). Exogenously expressed vimentin levels remained unchanged till the end of BP treatment (Supplementary Fig. S4A and B). Surprisingly, neither BP treated vimentin expressing nor its vector control cells showed endogenous induction of vimentin expression, which suggests that perhaps it occurs very late in the process of transformation and further treatment with BP for several weeks, might be required to achieve this stage. The E-cadherin expression remained downregulated while expression of Oct-4, Sox2 and Nanog showed significant increase in BP treated vimentin positive cells (Fig. 3F, Supplementary Fig. S5A).  Additionally, BP treated vimentin positive cells showed no difference in the levels of p53, Ras, ?-catenin and EMT regulators (Snail, Slug, Twist), while a decline was seen in the protein level of involucrin (Supplementary Fig. S5A-C). Interestingly, tumorigenic potential of BP treated vimentin positive cells was significantly higher, as reflected by the increase in volume of subcutaneous tumors, in NOD-SCID mice. On the other hand control cells failed to form tumors and in one case they formed a cyst (Fig. 3G and Supplementary Table S5). Furthermore, IHC analysis confirmed the differential levels of vimentin between the vimentin positive and its control group (Fig. 3H).

3.4 Significant correlation was seen between high vimentin-low E-cadherin expression and lymph node metastasis, in OSCC patients

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Significant correlation was seen between expression levels of vimentin-E-cadherin and demographic/clinical parameters like age (p = 0.008), stage (p = 0.001), node status (p<0.001) and perineural invasion (p = 0.008) (Supplementary Table S6) in oral tumors. Further, a significant correlation was also seen between expression levels of vimentin-E-cadherin and histopathological grade of oral dysplasia (p = 0.002, n=25) (Supplementary Fig. S6 and Table S7). IHC analysis of the OSCC tissues (n=183) demonstrated a significant correlation between high vimentin and lymph node metastasis (p<0.001), between low E-cadherin and lymph node metastasis (p=0.005) and between high vimentin-low E-cadherin and lymph node metastasis (p<0.001) (Fig. 4A-D). Collectively, the IHC analysis suggested that high vimentin-low E-cadherin expression was strongly associated with the grade of the premalignant lesions and with lymph node metastasis in OSCC patients. Thus, high vimentin-low E-cadherin expression at an early stage might be useful to identify the high risk premalignant lesions which could progress into frank malignancy. However, further studies are required with statistically adequate cases of precancerous lesions and their long term, systematic clinical follow-up. Our study at this stage supports the potential of vimentin as an early diagnostic marker but additional rescue experiments involving knockdown of vimentin followed by treatment with BP and then chasing for an in vitro transformed state is required. The long term goal would be to follow-up the patients with premalignant lesions for longer time period and then assess whether, high-vimentin-low-E-cadherin expression correlates with the malignant transformation. Further, this in vitro carcinogenesis model may serve as a useful system to screen for vimentin associated molecules and anticancer drugs which specifically target vimentin expressing cancer cells eg. withaferin 24. In conclusion, our data suggests that forced expression of vimentin in the initial stage proves to be advantageous to the cell, in order to push itself towards transformation, in the presence of an additional carcinogenic trigger. 

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