Abstract 149: Heterogeneity in stromal-fibroblasts determines stemness-status in primary oral tumors

Cancer associated fibroblasts (CAFs) often demonstrate the characteristics of myofibroblast differentiation by producing ultrastructure of alpha-smooth muscle actin (αSMA). Clinical data have strongly linked high-αSMA expression in CAFs with poor-prognosis of oral cancer patients; however, it is unclear if the variable levels of αSMA in CAFs are the cause or the effect of aggressive cancer behaviour. Cancer stem cell-model provides a framework to understand the aggressive cancer behaviour; where small population of stem-like cancer cells (SLCCs) is associated with cancer initiation, progression, metastasis and relapse after therapy. Like normal stem cells, SLCCs may retain responsiveness to the microenvironmental signals for their self-renewal and differentiation. Therefore, in this study we tested if the CAFs with variable levels of αSMA in oral tumor-stroma may cause distinct effects on the SLCCs. Immunohistochemical staining of oral tumor tissues (n=46) distinctly demonstrated heterogeneity in levels of αSMA-positive CAFs. Interestingly, proliferation index as measured by percentage of Ki67 positive cells was significantly negatively correlated with αSMA score; whereas, the frequency of ALDH1A1 expressing oral-SLCCs was significantly positively correlated with αSMA score in primary tumor samples. Experiments with in vitro primary cultures of CAFs; established from oral tumors, suggested that the gene expression differences were indeed the basis of heterogeneity among CAFs. This further grouped these CAFs in two distinct clusters, which were termed as C1 and C2. Interestingly, the oral-CAFs belonged to C1 or C2 clusters showed low- or high- αSMA-score respectively. Similar to our results observed with tumor tissues, in vitro co-culture experiments comprehensibly demonstrated that the oral-CAF-subtype with lower score for αSMA (C1-type CAFs) were more supportive for cellular proliferation but suppressive for the self-renewal of oral-SLCCs. Further, data clearly exhibited for the first time the determining role of BMP4 secreted by the C1-type CAFs in restraining the self-renewal growth of oral-SLCCs. Taken together, the observed heterogeneity among CAFs and its role in regulation of self-renewal of oral-SLCCs could be of relevance for understanding the overall biology of oral tumors. By discovering the CAFs-mediated mechanisms which might restrain cancer cells to a less aggressive state, our work has provided an opportunity for prognostic classification of