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Oral tongue squamous cell carcinoma (OTSCC) is one of the most common cancers worldwide and is characterized by early metastasis and poor prognosis. Recently, we reported that extracellular interleukin‐17F (IL‐17F) correlates with better disease‐specific survival in OTSCC patients and has promising anticancer effects in vitro. Vasculogenic mimicry (VM) is the formation of an alternative vasculogenic system by aggressive tumor cells, which is implicated in treatment failure and poor survival of cancer patients. We sought to confirm the formation of VM in OTSCC and to investigate the effect of IL‐17F on VM formation. Here, we showed that highly invasive OTSCC cells (HSC‐3 and SAS) form tube‐like VM on Matrigel similar to those formed by human umbilical vein endothelial cells. Interestingly, the less invasive cells (SCC‐25) did not form any VM structures. Droplet‐digital PCR, FACS, and immunofluorescence staining revealed the presence of CD31 mRNA and protein in OTSCC cells. Additionally, in a mouse orthotopic model, HSC‐3 cells expressed VE‐cadherin (CD144) but lacked Von Willebrand Factor. We identified different patterns of VM structures in patient samples and in an orthotopic OTSCC mouse model. Similar to the effect produced by the antiangiogenic drug sorafenib, IL‐17F inhibited the formation of VM structures in vitro by HSC‐3 and reduced almost all VM‐related parameters. In conclusion, our findings indicate the presence of VM in OTSCC and the antitumorigenic effect of IL‐17F through its effect on the VM. Therefore, targeting IL‐17F or its regulatory pathways may lead to promising therapeutic strategies in patients with OTSCC., In this study, we first confirmed the presence of the vasculogenic mimicry (VM) phenomenon in oral cancer patients, in vitro and in vivo, which exhibited different molecular patterns and markers. Next, we reported a novel therapeutic effect of IL‐17F against VM formation in highly aggressive cancer cells. Overall, targeting IL‐17F or its regulatory pathways could lead to promising therapeutic strategies in patients with oral cancer and other solid tumors as well.

B7-H3 was the only molecule identified with prognostic potential from a recent systematic review of the prognostic value of immune checkpoints in oral cancer. We aimed to validate this finding in a multicenter international cohort. We retrospectively retrieved 323 oral tongue squamous cell carcinoma (OTSCC) samples from three different countries (Brazil, Finland, and Norway) for immunostaining and scoring for B7-H3. We evaluated tumor immunogenicity by analyzing the amount of tumor-infiltrating lymphocytes and divided the tumors into immune hot and cold. To increase the reliability of the results, both digital and manual visual scoring were used. Survival curves were constructed based on the Kaplan-Meier method, and the Cox proportional hazard model was utilized for univariate and multivariate survival analysis. B7-H3 expression was not significantly associated with overall or disease-specific survival in the whole OTSCC cohort. When divided into immune hot and cold tumors, high B7-H3 expression was significantly associated with poor disease-specific and overall survival in the immune hot group, depending on the scoring method and the country of the cohort. This was achieved only in the univariate analysis. In conclusion, B7-H3 was a negative prognosticator for OTSCC patient survival in the subgroup of immune hot tumors, and was not validated as a prognosticator in the full cohort. Our findings suggest that the immune activity of the tumor should be considered when testing immune checkpoints as biomarkers. Electronic supplementary material The online version of this article (doi:10.1007/s12105-020-01222-3) contains supplementary material, which is available to authorized users.

High mobility group box 1 (HMGB1) is an extremely conserved DNA-binding protein that stabilizes nucleosomes and facilitates gene transcription in mammalian cells. When released extracellularly, HMGB1 becomes an alarmin that can mediate systemic diseases. High mobility group box 1 signals via two main receptors: receptor for advanced glycation end-products (RAGE) and toll-like receptor-4 (TLR4). We hypothesized that HMGB1 expression is increased in patients with oral lichen planus (OLP) relative to healthy controls. Therefore, HMGB1 and its receptors were mapped in tissue biopsies from 25 patients with OLP and from 20 healthy controls by immunostaining and ImageJ analysis. High mobility group box 1 was induced in oral keratinocytes in all patients with OLP. The band-like cell infiltrate in patients with OLP revealed very strong staining for RAGE. Likewise, TLR4 was overexpressed throughout OLP mucosa which co-localized with HMGB1. In conclusion, we suggest that OLP could partly be an HMGB1-mediated condition by creating a proinflammatory loop cycle via RAGE- and TLR4-signalling axes, which may contribute to the chronicity of this disease.

Tongue squamous cell carcinoma (TSCC) has a poor prognosis due to its early metastasis through blood and lymphatic vessels. We undertook a systematic review to investigate the prognostic significance of blood microvessel density (MVD) and lymphatic vessel density (LVD) in TSCC patients. We carried out a systematic search in Ovid Medline, Scopus, and Cochrane libraries. All studies that evaluated the prognostic significance of MVD/LVD markers in TSCC were systematically retrieved. Our results showed that MVD/LVD markers, CD31, CD34, CD105, factor VIII, lymphatic vessel endothelial hyaluronan receptor‐1, and D2‐40 were evaluated in TSCC patients until 28 June 2018. Six out of 13 studies reported markers that were associated with poor prognosis in TSCC. Two out of three studies suggested that a high number of D2‐40+ vessels predicated low overall survival (OS); the third study reported that the ratio of D2‐40+ over factor VIII + vessels is associated with low OS. Most of the other markers had controversial results for prognostication. We found higher expression of MVD/LVD markers were commonly, but not always, associated with shorter survival in TSCC patients. It is therefore not currently possible to recommend implementation of these markers as reliable prognosticators in clinical practice. More studies (especially for D2‐40) with larger patient cohorts are needed.

We recently showed that extracellular interleukin-17F (IL-17F) correlates with better disease-specific survival in oral tongue squamous cell carcinoma (OTSCC) patients. However, the underlying mechanisms of such effect remain obscure. Here, we used qRT-PCR to assess the expression of IL-17F and its receptors (IL-17RA and IL-17RC) in two OTSCC cell lines (HSC-3 and SCC-25) and in normal human oral keratinocytes (HOKs). IL-17F effects on cancer cell proliferation, migration, and invasion were studied using a live-imaging IncuCyte system, and a Caspase-3/7 reagent was used for testing apoptosis. 3D tumor spheroids were utilized to assess the impact of IL-17F on invasion with or without cancer-associated fibroblasts (CAFs). Tube-formation assays were used to examine the effects of IL-17F on angiogenesis using human umbilical vein endothelial cells (HUVEC). OTSCC cells express low levels of IL-17F, IL-17RA, and IL-17RC mRNA compared with HOKs. IL-17F inhibited cell proliferation and random migration of highly invasive HSC-3 cells. CAFs promoted OTSCC invasion in tumor spheroids, whereas IL-17F eliminated such effect. IL-17F suppressed HUVEC tube formation in a dose-dependent manner. Collectively, we suggest that IL-17F counteracts the pro-tumorigenic activity in OTSCC. Due to its downregulation in tumor cells and inhibitory activity in in vitro cancer models, targeting IL-17F or its regulatory pathways could lead to promising immunotherapeutic strategies against OTSCC.

Human &beta, defensin 2 (hBD-2) is a potent antimicrobial peptide that participates in defense against invading bacteria. We recently showed that bacterial components and histamine, through histamine H4 receptor (H4R), are involved in the pathogenesis of the potentially malignant lesion, oral lichen planus (OLP). However, the underlying mechanisms remain unknown. We, therefore, investigated the role of hBD2&ndash, histamine crosstalk signaling in promoting OLP pathology. Biopsies from OLP and oral tongue squamous cell carcinoma (OTSCC) patients, and healthy controls were used. Two OTSCC cell lines and normal human oral keratinocytes (HOKs) were used. HBD-2 and other targets were mapped by immunostaining and analyzed by ImageJ2 software. The highly sensitive droplet-digital PCR technology and qRT-PCR were utilized to study the clinically derived and in vitro samples, respectively. H4R was challenged with the specific agonist HST-10 and inverse agonist ST-1007. HBD-2 was highly induced in OLP lesions. In contrast, hBD2 expression was attenuated in OTSCC tissues, while very low levels of hBD-2 messenger RNA (mRNA) were observed in OTSCC cells. Together with tumor necrosis factor-&alpha, (TNF-&alpha, ), histamine upregulated hBD-2 mRNA expression in HOKs. Activation of H4R seems to modulate the expression of epithelial hBD-2. These findings suggest the involvement of hBD-2 in the pathogenesis of OLP and may, thus, be harnessed for therapeutic interventions in OLP.

Background Despite the importance of immune checkpoints in immunotherapy, the prognostic value of these molecules remains controversial in oral squamous cell carcinoma (OSCC). We performed a systematic review to investigate the prognostic significance of the immune checkpoints in OSCC. Materials A systematic search was conducted in Ovid Medline, Scopus and Cochrane libraries, and all studies that evaluated the prognostic significance of immune checkpoints in OSCC were systematically retrieved. Results Twelve immune checkpoints/modulators were studied for their prognostic values in OSCC patients between 1985 and 2017. Seven immune checkpoints (FKBP51, B7-H4, B7-H6, ALHD1, PD-L1, B7-H3 and IDO1) were reported to be associated with poor patients' survival in at least one study, and five (CTLA-4, TLT-2, VISTA, PD-L2 and PD-1) did not have a significant prognostic value. PD-L1 results were controversial as it was reported to be associated with both better and worse patients' survival. Conclusions Even though immune checkpoint markers had high expectation for OSCC prognostication, our systematic review revealed that the majority of them had been studied only once. The other molecules, which had been studied more than once, had controversial findings, except B7-H3.

Purpose: Recent reports indicate that histamine and its novel, high-affinity histamine H4 receptor (H4R) play a role in carcinogenesis, and thus H4R signalling has become a focus of increasing interest in the pathogenesis of many cancers. The roles of H4R in oral epithelial dysplasia (OED), and oral tongue squamous cell carcinomas (OTSCC) are unknown. The purpose of this study was to assess H4R expression in OTSCC patients and in cancer cell lines. Methods: Biopsies taken from OED, OTSCC, and healthy oral mucosa were studied by immunostaining. Primary human oral keratinocytes (HOKs) and two cancer cell lines (HSC-3 and SCC-25) were used for the in vitro studies. Quantitative real-time polymerase chain reaction was used to measure oncogene expression in the stimulated HOKs. Results: H4R-immunoreactivity was significantly reduced in OED and OTSCC samples, especially in the samples that had higher histopathological grades, which were also associated with noticeably increased mast cell counts. The presence of H4R in HSC-3 cells had clearly waned, in contrast to the HOKs. Gene expression data indicated that histamine-relevant inflammatory and environmental elements may participate in the regulation of some studied oncogenes. Conclusions: The results suggest a possible association between H4R and oral carcinogenesis. Furthermore, our findings raise a potential implication of histamine-mediated factors in the regulation of oncogenes, possibly via mast cells, as crucial components of tumor microenvironment. The identification of new elements that govern oral cancer development is highly important for the development of novel therapeutic approaches in OTSCC.

Recent reports indicate that histamine and its novel, high-affinity histamine HBiopsies taken from OED, OTSCC and healthy oral mucosa were studied by immunostaining. Primary human oral keratinocytes (HOKs) and two OTSCC-derived cell lines (HSC-3 and SCC-25) were used for the in vitro studies. Quantitative real-time PCR was used to measure oncogene expression in the stimulated HOKs.We found that HOur results suggest an association between H