Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Background: Oral cavity squamous cell carcinoma (OCSCC) is an epithelial-derived cancer which is characterized by locoregional invasion and a high rate of recurrence. Importantly, OCSCC displays significant intratumoral heterogeneity, with tumor cells existing along a spectrum of epithelial-mesenchymal transition (EMT) phenotypes. EMT is a dynamic process by which epithelial cells assume characteristics of mesenchymal cells, thus increasing their capacity to invade and metastasize. Several studies have shown the impact of EMT on OCSCC prognosis, with “mesenchymal” tumors demonstrating worse disease-free and overall survival. Despite the characterization of EMT subpopulations within OCSCC, little is known about how intracellular communication impacts tumorigenesis, and how paracrine signaling factors might play a role in invasion.
Objective: The goal of this work is to better understand how paracrine crosstalk between EMT epithelial and mesenchymal subpopulations impacts cellular invasion in OCSCC.
Methods: Our lab has previously characterized a novel cell-based EMT model by sorting OCSCC cells based on E-Cadherin expression to create genetically distinct “epithelial-like” (SCC-9 E) and “mesenchymal-like” (SCC-9 M) subpopulations. Using this model, we performed transwell invasion assays in the presence or absence of SCC-9 EMT cell line conditioned media in the bottom chamber. We also performed ELISA-based genetically-informed analysis of secreted cytokines and chemokines in the conditioned media of each EMT cell line to identify paracrine signaling candidates. Protein expression data from the conditioned media was further correlated with mRNA sequencing information obtained from each OCSCC EMT cell line.
Results: “Epithelial-like” SCC-9 E cells exhibit essentially negligible transwell invasive potential at baseline, and addition of conditioned SCC-9 M media to the bottom chamber did not significantly change this. In contrast, “mesenchymal-like” SCC-9 M cells exhibit excellent baseline invasive capacity, with a mean of 96 migrated cells/20X field. Interestingly, upon addition of conditioned SCC-9 E media to the bottom transwell chamber, the invasive potential of SCC-9 M increased to 124 cells/20X field (30% increase), suggesting that a secreted factor(s) from SCC-9 E may augment SCC-9 M invasion. We next measured quantitative secreted factor expression in conditioned media to identify potential paracrine signaling candidates and found significantly increased expression of interleukin (IL)-6, IL-8, IL-11, IL-15, IP10, sFAS, and CXCL16 among others in SCC-9 E media compared to SCC-9 M, demonstrating a distinct “secretome” between the EMT subpopulations. In examining mRNA expression in the SCC-9 EMT lines both at the bulk mRNA level and single-cell RNA sequencing resolution, interleukin and cytokine signaling pathways were among the most differentially expressed, demonstrating additional opportunities for further paracrine signaling studies.
Conclusions: EMT is a critical program in cancer cell invasion and metastasis. We have identified OCSCC cell lines with distinct EMT profiles and significantly different invasive potentials. Interestingly, we have also identified potential secreted factors that might play a role in EMT crosstalk to promote OCSCC cancer cell invasion. Future studies will focus on precise identification of putative secreted pro-invasion factors, as well as mechanistic and in vivo studies.