Recent single cell expression profiling of head and neck squamous cell carcinoma (HNSCC) identified a partial epithelial-to-mesenchymal transition (p-EMT) subpopulation of cancer cells. We have previously demonstrated an association of p-EMT subpopulations with adverse clinical and pathologic features, in both TCGA expression data and an independent cohort of single institutional patients. Here, we aim to understand cellular co-expression of EMT markers and spatial relationships with immune infiltrates in oral cavity HNSCC. Paraffin blocks of 10 oral cavity HNSCC patients whose tumors had previously undergone single cell expression profiling were characterized by a 13-marker panel of tumor cell, p-EMT, epithelial differentiation, and immune markers. Multiplexed immunofluorescence was used to perform simultaneous staining within two serial tissue sections. Multispectral imaging was performed using wide field microscopy (Vectra 3), and algorithms were used for spectral unmixing, tissue and cell segmentation, and cell phenotyping. Following user training, tissue and cell segmentation and cell phenotyping were successfully performed in an automated fashion. Whole slides containing entire tissue sections were analyzed, enabling high throughput analysis of hundreds of thousands of individual cells per tumor. p-EMT quantification by tumor, as measured by simultaneous signal for p-EMT markers, showed consistent co-localization, in accordance with prior single cell expression profiling data. We found that individual cells defined as p-EMT positive, based on co-expression of two or more p-EMT markers, co-localized to the leading edge of tumor nests, in close apposition to the stroma. Co-localization of LAMB3 and LAMC2 was the most robust marker combination defining this subpopulation. In addition, helper and cytotoxic T cells were identified across tumors, including activated and exhausted T-cell subsets. The relationships of these cellular subpopulations to other immune cells, which may drive immune cell exhaustion, were explored by radial measurements associated with cell densities and intercellular distances. Tumor and immune cell profiling using 13 markers across serial sections enabled high throughput characterization of individual cells with spatial information in a manner not previously possible. Distinct spatial relationships among p-EMT and epithelial tumor cells were identified, including a potential correlation with immune cells. This technology may have clinical utility in HNSCC, which includes predicting the need for therapy and response to immunotherapy.

Citation Format: Joao Paulo Oliveira-Costa, Anuraag S. Parikh, Linda T. Neiman, Derin Sevenler, Doyeon Koo, Chenyue Lu, William C. Faquin, Itay Tirosh, Jeremy D. Richmon, Kevin S. Emerick, Daniel G. Deschler, Mark A. Varvares, Derrick T. Lin, Sidarth V. Puram, Bradley E. Bernstein, Shannon L. Stott. Multiplexed immunofluorescence and multispectral imaging-based quantification of tumor and immune cell populations reveals spatial relationships in oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1132.