Stroma modulation of radiation response in head and neck squamous cell carcinoma: Insights from zebrafish larvae xenografts.

BACKGROUND: The tumour microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC) consists of different subtypes of cells that interact with the tumour or with each other. This study investigates the possibility of co-culturing HNSCC cells with different stroma cells in a zebrafish xenograft model, focusing on the effect of stroma cells on HNSCC growth and response to irradiation. MATERIAL AND METHOD: HNSCC metastatic cell line HSC-3 was used along with five types of stroma cells: normal gingival fibroblasts (NOF), cancer associated fibroblasts (CAF), macrophages, CD4+ T cells, and human umbilical vein endothelial cells (HUVEC). The mixture of HSC-3 cells and each-stroma cell type-was injected into 2-day post-fertilization zebrafish embryos, and the effect of stroma cells on tumour growth was tested. The study also aimed to mimic the HNSCC tumour by injecting a mixture of HSC-3 cells, CAFs, macrophages, and HUVECs into zebrafish embryos and testing the effect of these stroma cells on the cancer cells' response to irradiation compared to HSC-3-only tumours. RESULTS: CAFs had a significant inducement effect on tumour size, while HUVECs showed the opposite effect. The irradiated group of HSC-3-only tumour had a significantly smaller tumor cell area compared to the control, while the group with stroma cells and HSC-3 cells showed cancer cells being resistant to irradiation. CONCLUSION: This is the first report of co-culturing cancer cells with several types of stroma cells using a zebrafish xenograft model. This study also highlighted the role of stroma cells in turning the cancer cells from radioresponsive to radioresistant.

Occupation and tongue cancer in Nordic countries.

PURPOSE: Almost 200,000 tongue cancers were diagnosed worldwide in 2020. The aim of this study was to describe occupational risk variation in this malignancy. METHODS: The data are based on the Nordic Occupational Cancer (NOCCA) study containing 14.9 million people from the Nordic countries with 9020 tongue cancers diagnosed during 1961-2005. The standardized incidence ratio (SIR) of tongue cancer in each occupational category was calculated using national incidence rates as the reference. RESULTS: Among men, the incidence was statistically significantly elevated in waiters (SIR 4.36, 95% confidence interval (CI) 3.13--5.92), beverage workers (SIR 3.42, 95% CI 2.02-5.40), cooks and stewards (SIR 2.55, 95% CI 1.82-3.48), seamen (SIR 1.66, 95% CI 1.36-2.00), journalists (SIR 1.85, 95% CI 1.18-2.75), artistic workers (SIR 2.05, 95% CI 1.54-2.66), hairdressers (SIR 2.17, 95% CI 1.39-3.22), and economically inactive persons (SIR 1.57, 95% CI 1.42-1.73). Among women, the SIR was statistically significantly elevated only in waitresses (SIR 1.39, 95% CI 1.05-1.81). Statistically significant SIRs ≤ 0.63 were observed in male farmers, gardeners, forestry workers and teachers, and in female launderers. CONCLUSIONS: These findings may be related to consumption of alcohol and tobacco, but the effect of carcinogenic exposure from work cannot be excluded.

The effect of matrices on the gene expression profile of patient-derived head and neck carcinoma cells for in vitro therapy testing.

OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive tumor with a 5-year mortality rate of ~ 50%. New in vitro methods are needed for testing patients' cancer cell response to anti-cancer treatments. We aimed to investigate how the gene expression of fresh carcinoma tissue samples and freshly digested single cancer cells change after short-term cell culturing on plastic, Matrigel or Myogel. Additionally, we studied the effect of these changes on the cancer cells' response to anti-cancer treatments. MATERIALS/METHODS: Fresh tissue samples from HNSCC patients were obtained perioperatively and single cells were enzymatically isolated and cultured on either plastic, Matrigel or Myogel. We treated the cultured cells with cisplatin, cetuximab, and irradiation; and performed cell viability measurement. RNA was isolated from fresh tissue samples, freshly isolated single cells and cultured cells, and RNA sequencing transcriptome profiling and gene set enrichment analysis were performed. RESULTS: Cancer cells obtained from fresh tissue samples changed their gene expression regardless of the culturing conditions, which may be due to the enzymatic digestion of the tissue. Myogel was more effective than Matrigel at supporting the upregulation of pathways related to cancer cell proliferation and invasion. The impacts of anti-cancer treatments varied between culturing conditions. CONCLUSIONS: Our study showed the challenge of in vitro cancer drug testing using enzymatic cell digestion. The upregulation of many targeted pathways in the cultured cells may partially explain the common clinical failure of the targeted cancer drugs that pass the in vitro testing.

Novel human lymph node-derived matrix supports the adhesion of metastatic oral carcinoma cells.

BACKGROUND: 3D culture is increasingly used in cancer research, as it allows the growth of cells in an environment that mimics in vivo conditions. Metastases are the primary cause of morbidity and mortality in cancer patients, and solid tumour metastases are mostly located in lymph nodes. Currently, there are no techniques that model the pre-metastatic lymph node microenvironment in vitro. In this study, we prepared a novel extracellular matrix, Lymphogel, which is derived from lymph nodes, mimicking the tumour microenvironment (TME) of metastatic carcinoma cells. We tested the suitability of the new matrix in various functional experiments and compared the results with those obtained using existing matrices. METHODS: We used both commercial and patient-derived primary and metastatic oral tongue squamous cell carcinoma (OTSCC) cell lines. We characterized the functional differences of these cells using three different matrices (human uterine leiomyoma-derived Myogel, human pre-metastatic neck lymph node-derived Lymphogel (h-LG), porcine normal neck lymph node-derived Lymphogel (p-LG) in proliferation, adhesion, migration and invasion assays. We also performed proteomic analyses to compare the different matrices in relation to their functional properties. RESULTS: OTSCC cells exhibited different adhesion and invasion patterns depending on the matrix. Metastatic cell lines showed improved ability to adhere to h-LG, but the effects of the matrices on cell invasion fluctuated non-significantly between the cell lines. Proteomic analyses showed that the protein composition between matrices was highly variable; Myogel contained 618, p-LG 1823 and h-LG 1520 different proteins. The comparison of all three matrices revealed only 120 common proteins. Analysis of cellular pathways and processes associated with proteomes of each matrix revealed similarities of Myogel with h-LG but less with p-LG. Similarly, p-LG contained the least adhesion-related proteins compared with Myogel and h-LG. The highest number of unique adhesion-related proteins was present in h-LG. CONCLUSIONS: We demonstrated that human pre-metastatic neck lymph node-derived matrix is suitable for studying metastatic OTSCC cells. As a whole-protein extract, h-LG provides new opportunities for in vitro carcinoma cell culture experiments.

Prognostic markers for oral cancer: An overview of the current status and directions for future research.

In the last decades, the search for biomarkers for response to therapy and prognosis, with potential of providing information about the likely course and outcome of disease, has been intense in oral squamous cell carcinomas. Although several biomarkers, ranging from genetic and molecular alterations to clinical and histopathological features, have been described, only a few of them are used in routine practice. The aim of this review is to outline the recent advances in oral squamous cell carcinomas biomarkers, exploring those related to clinical and histopathological features, cancer cells and components of the tumor microenvironment. Future directions, highlighting the main issues that limit the clinical application of many of the potential biomarkers, are discussed.

Variable roles of interleukin-17F in different cancers.

BACKGROUND: Interleukin (IL)-17 family is a group of six cytokines that plays a central role in inflammatory processes and participates in cancer progression. Interleukin-17A has been shown to have mainly a protumorigenic role, but the other members of the IL-17 family, including IL-17F, have received less attention. METHODS: We applied systematic review guidelines to study the role of IL-17F, protein and mRNA expression, polymorphisms, and functions, in cancer. We carried out a systematic search in PubMed, Ovid Medline, Scopus, and Cochrane libraries, yielding 79 articles that met the inclusion criteria. RESULTS: The findings indicated that IL-17F has both anti- and protumorigenic roles, which depend on cancer type and the molecular form and location of IL-17F. As an example, the presence of IL-17F protein in tumor tissue and patient serum has a protective role in oral and pancreatic cancers, whereas it is protumorigenic in prostate and bladder cancers. These effects are proposed to be based on multiple mechanisms, such as inhibition of angiogenesis, vasculogenic mimicry and cancer cell proliferation, migration and invasion, and aggravating the inflammatory process. No solid evidence emerged for the correlation between IL-17F polymorphisms and cancer incidence or patients' prognosis. CONCLUSION: IL-17F is a multifaceted cytokine. There is a clear demand for more well-designed studies of IL-17F to elucidate its molecular mechanisms in different types of cancer. The studies presented in this article examined a variety of different designs, study populations and primary/secondary outcomes, which unfortunately reduces the value of direct interstudy comparisons.

The effect of interleukin-17F on vasculogenic mimicry in oral tongue squamous cell carcinoma.

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.

The critical effects of matrices on cultured carcinoma cells: Human tumor-derived matrix promotes cell invasive properties.

The interaction between squamous cell carcinoma (SCC) cells and the tumor microenvironment (TME) plays a major role in cancer progression. Therefore, understanding the TME is essential for the development of cancer therapies. We used four (primary and metastatic) head and neck (HN) SCC cell lines and cultured them on top of or within 5 matrices (mouse sarcoma-derived Matrigel®, rat collagen, human leiomyoma-derived Myogel, human fibronectin and human fibrin). We performed several assays to study the effects of these matrices on the HNSCC behavior, such as proliferation, migration, and invasion, as well as cell morphology, and molecular gene profile. Carcinoma cells exhibited different growth patterns depending on the matrix. While fibrin enhanced the proliferation of all the cell lines, collagen did not. The effects of the matrices on cancer cell migration were cell line dependent. Carcinoma cells in Myogel-collagen invaded faster in scratch wound invasion assay. On the other hand, in the spheroid invasion assay, three out of four cell lines invaded faster in Myogel-fibrin. These matrices significantly affected hundreds of genes and a number of pathways, but the effects were cell line dependent. The matrix type played a major role in HNSCC cell phenotype. The effects of the ECMs were either constant, or cell line dependent. Based on these results, we suggest to select the most suitable matrix, which provides the closest condition to the in vivo TME, in order to get reliable results in in vitro experiments.

The effect of fascin 1 inhibition on head and neck squamous cell carcinoma cells.

Fascin 1 plays important pro-metastatic roles in head and neck carcinoma (HNSCC) migration, invasion, and metastasis. However, limited advancement in targeting metastasis remains a major obstacle in improving HNSCC patients' survival. Therefore, we assessed the therapeutic potential of fascin 1 targeted inhibition and its potential prognostic value in HNSCC patients. Using in vitro and in vivo approaches, we investigated the effect of compound G2, a novel fascin 1 inhibitor, on HNSCC cells migration, invasion, and metastasis. High-throughput screening (HTS) was used to assess cytotoxic activity of compound G2 alone or combined with irradiation. We also evaluated the prognostic potential of fascin 1 in HNSCC patients. Interestingly, compound G2 reduced carcinoma cells migration and invasion in vitro and inhibited metastasis in vivo. Moreover, HTS revealed a modest cytotoxic activity of the compound G2 on HNSCC cell lines. Irradiation did not synergistically enhance the compound G2-mediated cytotoxic activity. Survival analyses showed that high fascin 1 immunoexpression, at the tumor invasive front, was associated with cancer-specific mortality in the advanced stages of HNSCC. Collectively, our findings suggest that fascin 1 represents a promising anti-metastatic therapeutic target and a useful prognostic marker in patients with HNSCC. Novel anti-metastatic agents could provide a valuable addition to cancer therapy.

Human myoma tissue-based extracellular matrix models for testing the effects of irradiation on the HPV positive cells.

BACKGROUND: This study was designed to investigate the invasion of human papillomavirus (HPV) positive human cervical carcinoma cell lines in human leiomyoma-based extracellular matrices in vitro, and to test the suitability of the model for studying the irradiation effects on the cancer cell invasion. METHODS: HPV positive cervical carcinoma cell lines SiHa and CaSki, and HPV negative squamous cell carcinoma cell line HSC-3 were used. CaSki cells contain around 600 copies of HPV 16 virus in the genome, whereas SiHa have only 1-2 copies per cell. Cells were analyzed using two different human tumor derived extracellular matrix methods (3D myoma disc model, and Myogel Transwell invasion assay). Cultures were irradiated with 4 Gy. Myoma invasion area and the depth of invasion were measured with ImageJ 1.51j8 software. Statistical analyses were performed with SPSS Statistics (IBM SPSS® Statistics 25). RESULTS: All cells invaded through Myogel coated Transwell membranes and within myoma discs. In myoma discs, a difference in the invasion depth (p = 0.0001) but not in invasion area (p = 0.310) between the HPV positive cell lines was seen, since SiHa (less HPV) invaded slightly better than CaSki (more HPV). HSC-3 cells (HPV negative) invaded deepest (p = 0.048) than either of the HPV positive cell line cells. No difference was detected in the invasion area (p = 0.892) between HPV positive and HPV negative cells. The ionized radiation significantly reduced the invasion depth of HSC-3 (p = 0.008), SiHa (p = 0.0001) and CaSki (p = 0.005). No significant effect on the invasion area was detected in any of the cell lines. However, a significant difference was observed between SiHa and CaSki in the reduction of the invasion depth after radiation (p = 0.013) as the reduction was greater with SiHa than CaSki. CONCLUSIONS: Both solid and gelatinous human leiomyoma-based extracellular matrix models were suitable platforms to study the invasion of HPV positive cervical carcinoma cells in vitro. SiHa cells with less HPV copy number cells invaded slightly better and were slightly more sensitive to irradiation than CaSki cells with high HPV copy number. However, there was no drastic differences between the invasion properties of these carcinoma cells.

In vitro humanized 3D microfluidic chip for testing personalized immunotherapeutics for head and neck cancer patients.

OBJECTIVES: Immunotherapy and personalized medicine therapeutics are emerging as promising approaches in the management of head and neck squamous cell carcinoma (HNSCC). In spite of that, there is yet no assay that could predict individual response to immunotherapy. METHODS: We manufactured an in vitro 3D microfluidic chip to test the efficacy of immunotherapy. The assay was first tested using a tongue cancer cell line (HSC-3) embedded in a human tumour-derived matrix "Myogel/fibrin" and immune cells from three healthy donors. Next, the chips were used with freshly isolated cancer cells, patients' serum and immune cells. Chips were loaded with different immune checkpoint inhibitors, PD-L1 antibody and IDO 1 inhibitor. Migration of immune cells towards cancer cells and the cancer cell proliferation rate were evaluated. RESULTS: Immune cell migration towards HSC-3 cells was cancer cell density dependent. IDO 1 inhibitor induced immune cells to migrate towards cancer cells both in HSC-3 and in two HNSCC patient samples. Efficacy of PD-L1 antibody and IDO 1 inhibitor was patient dependent. CONCLUSION: We introduced the first humanized in vitro microfluidic chip assay to test immunotherapeutic drugs against HNSCC patient samples. This assay could be used to predict the efficacy of immunotherapeutic drugs for individual patients.

Adipose-Derived Mesenchymal Stem Cells do not Affect the Invasion and Migration Potential of Oral Squamous Carcinoma Cells.

Mesenchymal stem cells (MSCs) are commonly isolated from bone marrow and adipose tissue. Depending on the tissue of origin, MSCs have different characteristics and physiological effects. In various cancer studies, MSCs have been found to have either tumor-promoting or tumor-inhibiting action. This study investigated the effect of adipose tissue-MSCs (AT-MSCs) and bone marrow-MSCs (BM-MSCs) on global long interspersed nuclear element-1 (LINE-1) methylation, the expression level of microenvironment remodeling genes and cell proliferation, migration and invasion of oral tongue squamous cell carcinoma (OTSCC). Additionally, we studied the effect of human tongue squamous carcinoma (HSC-3)-conditioned media on LINE-1 methylation and the expression of microenvironment remodeling genes in AT-MSCs and BM-MSCs. Conditioned media from HSC-3 or MSCs did not affect LINE-1 methylation level in either cancer cells or MSCs, respectively. In HSC-3 cells, no effect of MSCs-conditioned media was detected on the expression of ICAM1, ITGA3 or MMP1. On the other hand, HSC-3-conditioned media upregulated ICAM1 and MMP1 expression in both types of MSCs. Co-cultures of AT-MSCs with HSC-3 did not induce proliferation, migration or invasion of the cancer cells. In conclusion, AT-MSCs, unlike BM-MSCs, seem not to participate in oral cancer progression.

Prognostic value of blood and lymphatic vessel markers in tongue cancer: A systematic review.

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.

PCR-based zebrafish model for personalised medicine in head and neck cancer.

BACKGROUND: Currently, in vivo model for personalised cancer drug testing is challenging. A zebrafish larvae xenograft model has been applied in recent years to cancer research, particularly for drug testing purposes, showing promising results in drug testing against patient-derived tumour xenografts. Currently, these xenograft models apply imaging techniques to measure drug efficacy. However, this method carries several limitations, including timely imaging, thereby reducing the available number of tested fish and drugs. Here, we propose a PCR-based fast assay to evaluate drug efficacy in a zebrafish larvae xenograft model. METHODS: We tested two primary and corresponding metastatic head and neck squamous cell carcinoma (HNSCC) cell lines and patient-derived tongue cancer sample applying zebrafish larvae xenograft model. Cisplatin efficacy was tested using imaging technique and compared the results with PCR-based methods. Drug screening of eight compounds was applied on both cell lines and patient sample using PCR. RESULTS: In a head-to-head comparison, all the three techniques (imaging, quantitative PCR, and droplet digital PCR) showed similar reduction of the cancer cells growth after cisplatin treatment. Using the quantitative PCR assay, we demonstrated a dose-dependent response of HNSCC cells to cisplatin. Drug screening results of four HNSCC cell lines and patient sample revealed different drug efficacy between tested cancer cells. CONCLUSION: We introduce a novel, easy, fast and cost-effective PCR-based in vivo zebrafish larvae assay to test the response of cell lines and clinical tumour samples to anti-cancer drugs. This method goes hand-by-hand with the commonly used imaging assay.

MiR-455-3p, miR-150 and miR-375 are aberrantly expressed in salivary gland adenoid cystic carcinoma and polymorphous adenocarcinoma.

BACKGROUND: Adenoid cystic carcinoma (AdCC) and polymorphous adenocarcinoma (PAC) are included among the most common salivary gland cancers. They share clinical and histological characteristics, making their diagnosis challenging in specific cases. MicroRNAs (miRNA) are short, non-coding RNA sequences of 19-25 nucleotides in length that are involved in post-transcriptional protein expression. They have been shown to play important roles in neoplastic and non-neoplastic processes and have been suggested as diagnostic and prognostic markers. METHODS: This study, using quantitative RT-PCR, investigated miR-150, miR-455-3p and miR-375 expression, in order to identify a possible molecular distinction between AdCC and PAC. RESULTS: miRNA-150 and miRNA-375 expression was significantly decreased in AdCC and PAC compared with salivary gland tissue controls, whilst miRNA-455-3p showed significantly increased expression in AdCC when compared to PAC, (P < 0.05). miR-150, miR-357 and miR-455-3p expression in AdCC, PAC and control was not associated with age, gender nor with anatomic site (major and minor salivary glands) (P > 0.05). CONCLUSION: MiR-455-3p could be used as a complimentary tool in the diagnosis of challenging AdCC cases.

The prognostic value of immune checkpoints in oral squamous cell carcinoma.

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.

Immune checkpoints indoleamine 2,3-dioxygenase 1 and programmed death-ligand 1 in oral mucosal dysplasia.

BACKGROUND: Oral mucosal dysplasia is a histologic feature of potentially malignant disorders that is associated with the risk of transformation to carcinoma. Dysplastic cells use many strategies during their transformation to cancer, including escape from the immune mediated destruction. We hypothesized that adaptive immunity is inhibited by activation of distinct immune checkpoint molecules, such as indoleamine 2,3-dioxygenase 1 (IDO1) and programmed death-ligand 1 (PD-L1). METHODS: We collected 63 oral dysplasia samples from 47 patients. Nine biopsies from alveolar mucosa were taken during wisdom teeth extractions were used as healthy controls. Tissue samples were stained and scored for IDO1 and PD-L1. Additionally, dysplasia grades and inflammatory cell infiltration were evaluated. Eight patients were followed up to 36 months to evaluate dysplasia progression, inflammation, and immune checkpoint molecules expression. RESULTS: Dysplastic epithelium had significantly lower IDO1 expression than that of healthy controls. PD-L1 positive cells in the lamina propria were mainly in dysplastic samples and seldom in healthy controls. Dysplasia grade was associated negatively with epithelium IDO1 and positively with IDO1 and PD-L1 expression in the lamina propria. There was a positive association between dysplasia grade and level of inflammatory cell infiltration. During follow-up, dysplasia grade, inflammatory cell infiltration, and the immune checkpoint expression fluctuated over time. CONCLUSIONS: Immune checkpoint molecules IDO1 and PD-L1 are modulated during oral epithelial dysplastic changes, and their expression is associated with inflammatory cell infiltration in the lamina propria. As immune checkpoint molecules expression fluctuates over time, these molecules are not useful as biomarkers for oral mucosal dysplasia progression.

Altered Expression of Toll-like Receptors in Human Oral Epithelium in Oral Lichenoid Reactions.

Oral lichenoid reactions (OLRs) are chronic inflammatory lesions induced by contact with allergens. Toll-like receptors (TLRs) are members of pattern-recognition receptor superfamily. Once activated, TLRs induce production of cytokines and chemokines, thus leading to inflammatory reaction in host tissue. In the present study, we aimed to investigate the potential role of TLRs in the initiation and perpetuation of OLRs, in which TLRs induce innate immune responses mounted against allergens. TLRs, 1 through 10, were mapped in tissue samples obtained from healthy donors and OLR patients using real-time quantitative reverse transcription polymerase chain reaction, immunostaining, and image analyses. We found that the immunoreactivity for all TLRs was increased in OLRs, except for TLR5, which was noticeably reduced. Gene analysis revealed that TLR1, TLR2, TLR4, TLR7, TLR8, and TLR9 transcripts were upregulated in OLRs compared with controls. In contrast, expression of TLR3, TLR5, and TLR6 genes were negatively regulated in OLRs. TLR10 remained unchanged in both groups. In conclusion, TLRs expression is deranged in OLRs in which TLRs could be sensitized by allergens and haptens derived from dental restorations. TLR reactivity is further enhanced by recruitment of T lymphocytes forming a diffuse lymphocytic infiltrate and thus creating a proinflammatory loop cycle. These findings suggest that TLRs are involved in OLRs and pave the way for alternative cost-effective therapeutic intervention.

Distinctive expression pattern of interleukin-17 cytokine family members in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers in both genders. Even though interleukin (IL)-17A was shown to play an important role in intestinal tumourigenesis and CRC, other IL-17 family members were not studied well. We therefore studied the expression of IL-17 cytokine family members in CRC. Ten healthy colons and ten CRC mucosa were immunostained for IL-17B, IL-17C, IL-17E, and IL-17F, and their receptors IL-17RA, IL-17RB, and IL-17RC. Double immunofluorescence staining of the CRC mucosa was done for IL-17B with markers of neutrophils, endothelial cells, macrophages, T cells, mast cells, or fibroblasts. While IL-17B was increased in CRC with a strong presence both in the epithelial and stromal compartments, IL-17C showed different expression depending on the grade of differentiation and IL-17E remained unchanged. In contrast, IL-17F was decreased in CRC compared to healthy control. Colon epithelial cells stained positive for IL-17RA, IL-17RB, and IL-17RC in both healthy control and CRC. Neutrophils were the main source of IL-17B in the stroma. IL-17 family members demonstrated distinct expression patterns in CRC, suggesting a differential role exerted by each member in colon carcinogenesis.

Cathepsin K expression is increased in oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is an idiopathic T-cell-mediated mucosal inflammatory disease. Cathepsin K (Cat K) is one of the lysosomal cysteine proteases. It is involved in many pathological conditions, including osteoporosis and cancer. The expression and role of Cat K in OLP are unknown. METHODS: Twenty-five oral mucosal specimens diagnosed histopathologically as OLP and fourteen healthy controls (HC) were used to study the immunohistochemical (IHC) expression of Cat K. Colocalization of Cat K with CD1a, Melan-A, CD68, CD45, mast cell tryptase (MCT), and Toll-like receptors (TLRs) 4 and 9 were studied using double IHC and/or immunofluorescence (IF) staining. Expression of Cat K was also evaluated in OLP tissue samples before and after topical tacrolimus treatment. RESULTS: Cat K was expressed in a higher percentage of cells in the epithelial zone, and the staining intensity was stronger in the stroma in OLP compared to controls (P < 0.001). In OLP, Cat K was present mostly in melanocytes and macrophages and sporadically in basal keratinocytes, endothelial cells, and extracellularly. Cat K was found also in some fibroblasts in HC and OLP samples. Coexpression of Cat K and TLRs 4 and 9 was seen in some dendritic cells (presumably melanocytes) and macrophages. In OLP, tacrolimus treatment reduced the expression of Cat K in the epithelium but increased it in the stroma. CONCLUSIONS: These results suggest that Cat K is involved in the pathogenesis of OLP. Cat K possibly takes part in the modulation of matrix molecules and cellular receptors.