Evidence for etiologic field changes in tongue distant from tumor in patients with squamous cell carcinoma of the oral tongue.

Oral cancer is a paradigm of Slaughter's concept of field cancerization, where tumors are thought to originate within an area of cells containing genetic alterations that predispose to cancer development. The field size is unclear but may represent a large area of tissue, and the origin of mutations is also unclear. Here, we analyzed whole exome and transcriptome features in contralateral tumor-distal tongue (i.e. distant from the tumor, not tumor-adjacent) and corresponding tumor tissues of 15 patients with squamous cell carcinoma of the oral tongue. The number of point mutations ranged from 41 to 237 in tumors and from one to 78 in tumor-distal samples. Tumor-distal samples showed mainly clock-like (associated with aging) or tobacco smoking mutational signatures. Tumors additionally showed mutations that associate with cytidine deaminase AID/APOBEC enzyme activities or a UV-like signature. Importantly, no point mutations were shared between a tumor and the matched tumor-distal sample in any patient. TP53 was the most frequently mutated gene in tumors (67%), whereas a TP53 mutation was detected in only one tumor-distal sample, and this mutation was not shared with the matched tumor. Arm-level copy number variation (CNV) was found in 12 tumors, with loss of chromosome (Chr) 8p or gain of 8q being the most frequent events. Two tumor-distal samples showed a gain of Chr8, which was associated with increased expression of Chr8-located genes in these samples, although gene ontology did not show a role for these genes in oncogenic processes. In situ hybridization revealed a mixed pattern of Chr8 gain and neutral copy number in both tumor cells and adjacent nontumor epithelium in one patient. We conclude that distant field cancerization exists but does not present as tumor-related mutational events. The data are compatible with etiologic field effects, rather than classical monoclonal field cancerization theory. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Antigen peptide transporters are upregulated in squamous cell carcinoma of the oral tongue and show sex-specific associations with survival.

Transporter associated with antigen processing 1 (TAP1) and TAP2 serve pivotal roles in adaptive immunity. Tumor cells often show reduced antigen presentation on their surface as one mechanism to escape immune recognition. Whether downregulation of TAPs is a common mechanism of tumor immune evasion in squamous cell carcinoma of the oral tongue (SCCOT) is unclear. In the present study, samples from 78 patients with SCCOT and 17 patients with benign hyperplastic tongue lesions were analyzed for TAP1 and TAP2 expression by immunohistochemistry. The percentage of positive cells and staining intensity were scored. Associations with clinicopathological variables and survival outcome were also investigated. The results demonstrated that TAP1 and TAP2 levels were highly associated with each other in individual samples and were upregulated in SCCOT compared with benign lesions (P<0.001). The proportion of TAP1- or TAP2-positive tumor cells was >80% in all but two of the tumors, whereas 25.6 and 23.0% of the tumors showed weak intensity of TAP1 and TAP2, respectively. There were no significant associations with clinicopathological variables or survival outcomes between TAP-intermediate/strong and TAP-weak tumors. However, in patients <70 years old and with early stage SCCOT, male patients had better outcomes than female patients (log-rank P<0.05), and the best outcome was observed in male patients with intermediate/strong TAP expression. In conclusion, loss of TAP was not a frequent event in SCCOT and stronger TAP expression in male patients was associated with improved survival, providing further evidence for sex-specific immune modulation in cancer.

Cancer-Associated Fibroblasts Modulate Transcriptional Signatures Involved in Proliferation, Differentiation and Metastasis in Head and Neck Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAFs) are known to increase tumor growth and to stimulate invasion and metastasis. Increasing evidence suggests that CAFs mediate response to various treatments. HNSCC cell lines were co-cultured with their patient-matched CAFs in 2D and 3D in vitro models, and the tumor cell gene expression profiles were investigated by cDNA microarray and qRT-PCR. The mRNA expression of eight candidate genes was examined in tumor biopsies from 32 HNSCC patients and in five biopsies from normal oral tissue. Differences in overall survival (OS) were tested with Kaplan-Meier long-rank analysis. Thirteen protein coding genes were found to be differentially expressed in tumor cells co-cultured with CAFs in 2D and 81 in 3D when compared to tumor cells cultured without CAFs. Six of these genes were upregulated both in 2D and 3D (POSTN, GREM1, BGN, COL1A2, COL6A3, and COL1A1). Moreover, two genes upregulated in 3D, MMP9 and FMOD, were significantly associated with the OS. In conclusion, we demonstrated in vitro that CAF-derived signals alter the tumor cell expression of multiple genes, several of which are associated with differentiation, epithelial-to-mesenchymal transition (EMT) phenotype, and metastasis. Moreover, six of the most highly upregulated genes were found to be overexpressed in tumor tissue compared to normal tissue.

CAFs affect the proliferation and treatment response of head and neck cancer spheroids during co-culturing in a unique in vitro model.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors for which the overall survival rate worldwide is around 60%. The tumor microenvironment, including cancer-associated fibroblasts (CAFs), is believed to affect the treatment response and migration of HNSCC. The aim of this study was to create a biologically relevant HNSCC in vitro model consisting of both tumor cells and CAFs cultured in 3D to establish predictive biomarkers for treatment response, as well as to investigate the impact of CAFs on phenotype, proliferation and treatment response in HNSCC cells. METHODS: Three different HNSCC patient-derived tumor cell lines were cultured with and without CAFs in a 3D model. Immunohistochemistry of the proliferation marker Ki67, epidermal growth factor receptor (EGFR) and fibronectin and a TUNEL-assay were performed to analyze the effect of CAFs on both tumor cell proliferation and response to cisplatin and cetuximab treatment in tumor spheroids (3D). mRNA expression of epithelial-mesenchymal transition (EMT) and cancer stem cells markers were analyzed using qRT-PCR. RESULTS: The results demonstrated increased cell proliferation within the tumor spheroids in the presence of CAFs, correlating with increased expression of EGFR. In spheroids with increased expression of EGFR, a potentiated response to cetuximab treatment was observed. Surprisingly, an increase in Ki67 expressing tumor cells were observed in spheroids treated with cisplatin for 3 days, correlating with increased expression of EGFR. Furthermore, tumor cells co-cultured with CAFs presented an increased EMT phenotype compared to tumor cells cultured alone in 3D. CONCLUSION: Taken together, our results reveal increased cell proliferation and elevated expression of EGFR in HNSCC tumor spheroids in the presence of CAFs. These results, together with the altered EMT phenotype, may influence the response to cetuximab or cisplatin treatment.

The effect of 2D and 3D cell cultures on treatment response, EMT profile and stem cell features in head and neck cancer.

BACKGROUND: Head and Neck Squamous Cell Carcinoma (HNSCC) tumors are often resistant to therapies. Therefore searching for predictive markers and new targets for treatment in clinically relevant in vitro tumor models is essential. Five HNSCC-derived cell lines were used to assess the effect of 3D culturing compared to 2D monolayers in terms of cell proliferation, response to anti-cancer therapy as well as expression of EMT and CSC genes. METHODS: The viability and proliferation capacity of HNSCC cells as well as induction of apoptosis in tumor spheroids cells after treatment was assessed by MTT assay, crystal violet- and TUNEL assay respectively. Expression of EMT and CSC markers was analyzed on mRNA (RT-qPCR) and protein (Western blot) level. RESULTS: We showed that HNSCC cells from different tumors formed spheroids that differed in size and density in regard to EMT-associated protein expression and culturing time. In all spheroids, an up regulation of CDH1, NANOG and SOX2 was observed in comparison to 2D but changes in the expression of EGFR and EMT markers varied among the cell lines. Moreover, most HNSCC cells grown in 3D showed decreased sensitivity to cisplatin and cetuximab (anti-EGFR) treatment. CONCLUSIONS: Taken together, our study points at notable differences between these two cellular systems in terms of EMT-associated gene expression profile and drug response. As the 3D cell cultures imitate the in vivo behaviour of neoplastic cells within the tumor, our study suggest that 3D culture model is superior to 2D monolayers in the search for new therapeutic targets.