Precision imaging of the spatial regulators of the hybrid epithelial/mesenchymal state in head and neck cancer - PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC), including oral cavity squamous cell carcinoma (OCSCC) is the sixth leading cause of cancer-related mortality, with the majority of deaths attributable to tumor metastasis and failures in treatment. Because most cases of OCSCC result from tobacco and alcohol exposure, these tumors are highly heterogeneous, greatly complicating diagnosis, treatment, and investigations into the biology of this disease. We recently performed dissociated single cell RNA-sequencing (scRNA-seq) in OCSCC and identified a hybrid epithelial/mesenchymal state (HEM) with some features of classical EMT, yet persistent expression of epithelial markers. Further investigation into HEM demonstrated its localization at the leading edge of tumors where it appears to drive invasion and metastasis as well as poor outcomes in OCSCC. How does HEM trigger worse outcomes in HNSCC patients? Based on recent multispectral, multiplexed imaging analysis, we have found that HEM leads to exclusion of tumor infiltrating lymphocytes including T-cells. Thus, understanding what underlying structural and biological features of a tumor dictate this phenotype of immune exclusion and induce HEM is of critical importance to advancing diagnostic biomarkers and targeted therapeutics for HNSCC. We hypothesize that HEM reflects the underlying tumor architecture and the complex interplay between cells in the OCSCC ecosystem. However, due to the heterogeneity of these tumors both biologically as well as spatially, bulk and/or dissociated transcriptomic and proteomic techniques forego this critical information. Our goal is to utilize advanced imaging approaches to mature a set of imaging-based biomarkers based on HEM and its interactions with the immune system, thereby creating opportunities to advance patient stratification and treatment of OCSCC. To test our hypothesis, we will: (1) Further define the effect of HEM on immune infiltration using MSI, especially as it relates to specific architectural features of tumor morphology. We will utilize MSI to analyze patient tumors treated with immune checkpoint inhibitors (ICI) to identify imaging-based metrics that predict response. Next, (2) we will combine proteomic, transcriptomic, and histopathologic approaches while retaining spatial information to obtain a more comprehensive understanding of the spectrum of HEM states and expression heterogeneity within the tumor environment that contributes to HEM. Finally, (3) we will establish pathways to both train and mentor the next generation of head and neck cancer investigators, creating both hands-on laboratory exchanges as well as portable education modules for use in community colleges. At the research level, testing these hypotheses will provide critical insights into the relationship between tumor- infiltrating immune cells and HEM, and the underlying expression states that direct HEM and ICI response more broadly. From a DEIA lens, our work will aim to increase the diversity of investigators in the HNSCC community, critical for the innovation needed to improve our ability to treat oral cancers, while also providing new ways to study oral biology and diseases.
