Figure 1. Flowchart of head and neck squamous cell carcinoma (HNSCC) cohort used in this study. (A) 47 HNSCC formalin-fixed paraffin-embedded (FFPE) specimens were stained using a seven-color multiplex immunohistochemistry (IHC) panel to distinguish CD44v6+tumor cells, CD163+macrophages, CD19+B cells, CD8+T cells, CD3+CD8− FoxP3− T cells (CD4+T helper cells) and FoxP3+regulatory T cells. 29 out of those 47 were HPV-negative surgical resection specimens. These resection specimens were used for assigning immunotypes (figure 2), descriptive analyses (figure 4), and immune cellular neighborhoods (figure 5). For 11 out of the 29 tumors, secretome data was available of overnight cultures from matched fresh single-cell suspensions (figure 3). All 29 resection specimens were used to compare the TIME from different anatomical sites: OCSCC (n=12), hypopharynx SCC (HSCC, n=9) and larynx SCC (LSCC, n=8, figure 7). Lastly, 18 out of 47 HNSCC FFPE specimens were oropharynx SCC (OPSCC) biopsies for the comparison between the TIME of HPV-positive (n=6) and HPV-negative (n=12) OPSCC (figure 6). (B) 12 HNSCC FFPE specimens were stained using a five-color multiplex IHC panel to distinguish pan-cytokeratin (pan-CK)+tumor cells, CD103+tumor-resident, CD103− recruited, Ki67+proliferating and Ki67− non-proliferating CD8+T cells. All 12 resections were used for the characterization of infiltrating CD8+T cells (online supplemental figure 2). 6 out of those 12 specimens overlapped between the seven-color and five-color multiplex IHC panels, explaining the total number of 53 unique specimens. HHPV, human papillomavirus; OCSCC, oral cavity SCC; TIME, tumor immune microenvironment.