Transcriptomic and Immunophenotypic Characterization of Tumor Immune Microenvironment in Squamous Cell Carcinoma of the Oral Tongue

Abstract

The tumor immune microenvironment of oral tongue squamous cell carcinoma may be accountable for differences in clinical behavior, particularly between different age groups. We performed RNA expression profiling and evaluated tumor infiltrating lymphocytes (TILs) and their T-cell subsets in order to assess the functional status of oral tongue squamous cell carcinoma tumor microenvironment and detect potentially clinically useful associations. Archival surgical pathology material from sixteen oral tongue squamous cell carcinoma patients was microscopically evaluated for TIL densities. RNA was extracted from macrodissected whole tumor sections and normal controls and RNA expression profiling was performed by the NanoString PanCancer IO 360 Gene Expression Panel. Immunostains for CD4, CD8 and FOXP3 were evaluated manually and by digital image analysis. Oral tongue squamous cell carcinomas had increased TIL densities, numerically dominated by CD4 + T cells, followed by CD8 + and FOXP3 + T cells. RNA expression profiling of tumors versus normal controls showed tumor signature upregulation in inhibitory immune signaling (CTLA4, TIGIT and PD-L2), followed by inhibitory tumor mechanisms (IDO1, TGF-β, B7-H3 and PD-L1). Patients older than 44 years showed a tumor microenvironment with increased Tregs and CTLA4 expression. Immunohistochemically assessed CD8% correlated well with molecular signatures related to CD8 + cytotoxic T-cell functions. FOXP3% correlated significantly with CTLA4 upregulation. CTLA4 molecular signature could be predicted by FOXP3% assessed by immunohistochemistry (R² = 0.619, p = 0.026). Oral tongue squamous cell carcinoma hosts a complex inhibitory immune microenvironment, partially reflected in immunohistochemically quantified CD8 + and FOXP3 + T-cell subsets. Immunohistochemistry can be a useful screening tool for detecting tumors with upregulated expression of the targetable molecule CTLA4.

Keywords: CD4-positive T lymphocytes; CD8-positive T lymphocytes; Genetic transcription; Immunohistochemistry; Oral cavity; Regulatory T lymphocytes; Squamous cell carcinoma; Tongue; Tumor infiltrating lymphocytes.

Fig. 1

Tumor infiltrating lymphocytes and PD-L1 expression in oral tongue squamous cell carcinoma. a Brisk tumor infiltrating lymphocytes (score 75%, H&E × 200); b Low density of tumor infiltrating lymphocytes (score 10%, H&E × 200); c High expression of PD-L1 (combined positive score: 126, × 100); d Case negative for PD-L1 (combined positive score: 0.75, × 100)

Fig. 2

Immunohistochemical assessment of CD4 + , CD8 + and FOXP3 + T-cell subsets in oral tongue squamous cell carcinoma. High densities of CD4 + (27.97%) (a), CD8 + (14.15%) (b) and FOXP3 + (7.22%) (c) T-cells. Contrast with low densities of CD4 + (10.9%) (d), CD8 + (0.11%) (e) and FOXP3 + (2.77%) (f) T-cells

Fig. 3

Transcriptomic profiling of oral tongue squamous cell carcinoma. a Gene expression heatmap illustrating unsupervised hierarchical clustering accounting for the differential expression of several molecular signatures between tumors and their normal controls; b Forest plot illustrating upregulated, unchanged and downregulated gene expression signatures between tumors and their normal controls