https://orcid.org/0000-0003-3011-2671
Figures
Abstract
Almost 380,000 new cases of oral cancer were reported worldwide in 2020. Oral squamous cell carcinoma (OSCC) accounts for 90% of all types of oral cancers. Emerging studies have shown association of Toll-like receptors (TLRs) in carcinogenesis. The present study aimed to investigate the expression levels and tissue localization of TRL1 to TRL10 and NF-κB between OSCC and healthy oral mucosa, as well as effect of Candida colonization in TRL expression in OSCC. Full thickness biopsies and microbial samples from 30 newly diagnosed primary OSCC patients and 26 health controls were collected. The expression of TLR1 to TLR10 and NF-κB was analyzed by immunohistochemistry. Microbial samples were collected from oral mucosa to detect Candida. OSCC epithelium showed lower staining intensity of TRL1, TRL2 TRL5, and TRL8 as compared to healthy controls. Similarly, staining intensity of TRL3, TRL4, TRL7, and TRL8 were significantly decreased in basement membrane (BM) zone. Likewise, OSCC endothelium showed lower staining intensity of TLR4, TLR7 and TLR8. Expression of NF-κB was significantly stronger in normal healthy tissue compared to OSCC sample. Positive correlation was found between the expression of NF-κB, TRL9 and TRL10 in basal layer of the infiltrative zone OSCC samples (P = 0.04 and P = 0.002, respectively). Significant increase in TRL4 was seen in BM zone of sample colonized with Candida (P = 0.01). According to the limited number of samples, our data indicates downregulation of TLRs and NF-κB in OSCC, and upregulation of TLR4 expression with presence of Candida.
Citation: Rusanen P, Marttila E, Amatya SB, Hagström J, Uittamo J, Reunanen J, et al. (2024) Expression of Toll-like receptors in oral squamous cell carcinoma. PLoS ONE 19(4): e0300437. https://doi.org/10.1371/journal.pone.0300437
Editor: Aditya K. Panda, Berhampur University, INDIA
Received: October 24, 2022; Accepted: February 28, 2024; Published: April 9, 2024
Copyright: © 2024 Rusanen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the Suomen Hammaslääkäriseura Apollonia (Finnish Dental Society Apollonia) and the Helsingin ja Uudenmaan Sairaanhoitopiiri (Helsinki University Central Hospital) Research Funds. JR was supported by Academy of Finland. RR-R was supported by NIHR Manchester Biomedical Research Centre. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Oral cavity cancer is ranked as one of the most common malignancies in the world with its prevalence rising yearly. Europe diagnoses more than 100,000 new cases each year. Over 90% of all oral malignancies are attributed to oral squamous cell carcinomas (OSSCs), with the mobile tongue being the most affected area [1]. Regardless of advances in treatment modalities, no significant improvement has been observed in the 5-year survival rate of OSCC in decades [2]. Smoking and drinking are deemed to be the key risk factors of OSCCs [3, 4]. Chronic candidiasis has been linked to the pathogenesis of oral carcinoma; however, the mechanism is unknown [5]. Several studies have confirmed that Candida species are able to produce mutagenic levels of carcinogenic acetaldehyde in vitro [6–8].
The role of chronic inflammation and the innate immune system in the development of cancer is widely recognized and a strong link between chronic inflammation and many types of cancers have been reported [9–12]. Anti-apoptotic effects of nuclear factor-kappaB (NF-kappaB), induction of tissue repair response and oxidative DNA damage are among the various mechanisms of inflammatory response that aid in promotion of carcinogenesis [9, 13, 14].
Toll like receptors (TLRs) are a family of trans-membranous pattern recognition receptors that recognize molecular patterns of microbial pathogens as well as those of endogenous origin. They have a key role in the activation of the innate immunity and provide a quick and highly efficient response to pathogens and host-derived molecules. TLRs are involved in maintenance of various aspects of tissue homeostasis via regulation of inflammatory and tissue repair responses [15]. Ten different types of TLRs are recognized in humans, TLR1-TLR10. Along with the cells of the immune system, these TLRs are also expressed by various non-immune cells such as basement membrane cells of oral mucosa and keratinocytes of skin [16, 17]. Additionally, body fluids such as saliva, plasma, breast milk, pleural fluid and amniotic fluid have been found to harbor soluble forms of TLRs [18–21]. Endothelial cells have been shown to express one or more TLRs when triggered by a stimulus such as infection by pathogens or by tissue damage signaling molecules [22]. The expression and signaling of TLRs in endothelial cells is a key factor to regulate angiogenesis. While angiogenic process is deemed crucial for tissue repair, endothelial dysfunction promoted by TLRs contributes to tissue damage in inflammatory and autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, and even cancer [22, 23]. Indeed, accumulating evidence points to a crucial role of TLRs in angiogenesis which is required for the growth of some tumors [23]. To date, the understanding of the ligand recognition, signaling and biological functions of TLR1-TLR9 is fairly clear. In contrast, the agonists, signaling, and function of TLR10 remains largely unknown [24].
Stimulation of various TLRs leads to the activation of different transcription factors, such as nuclear factor-kappaB (NF-κB). NF-κB regulates the genes that encode for expression of cytokines, chemokines, and co-stimulatory molecules such as TNF-α, IL-1β, and leukocyte and vascular adhesion molecules [25]. These immune and pro‐inflammatory mediators play essential roles in recruiting various inflammatory cells into the infection sites and activating the adaptive immune response later in infection. Some of these pro-inflammatory mediators can also activate NF-κB, and a type of positive regulatory loop may be formed that would aggravate and perpetuate the local inflammatory reactions. Since NF-κB plays a significant role in innate immunity, adaptive immunity, and cell proliferation processes, its activity is tightly regulated. Dysregulation of NF-κB activation pathway at any stage can lead to chronic inflammation, autoimmunity, and cancer [25, 26].
TLRs regulate a wide range of biological responses including immune and inflammatory responses during carcinogenesis [12]. TLRs may promote carcinogenesis through tumor promoting inflammatory signals, anti-apoptotic pathways, cell proliferative and fibroblast activation mechanisms, influencing either tumor cells or the tumor microenvironment [27]. Yet, the association of TLRs in OSCC is conflicting due to lack of adequate data and discrepancies between published studies [28]. However, several studies have demonstrated a role of TLR4 in the pathogenesis of cancer.
Downregulation of TLR4 has been reported to inhibit tumour growth and inflammation, cytokine secretion and to suppress metastasis of carcinoma in several cancers [29–32]. Moreover, according to the meta-analysis of Hao et al. 2018, the elevated expression of TLR4 in cancer patients is associated with poor overall survival and shorter disease-free survival [33]. In addition, a correlation between high TLR4 expression and worse survival rate in OSCC patients has been reported [34] and TLR-2, -4, and -9 seemed to predict invasive tumor growth [35]. Also, overexpression of TLR3 was associated with poorly differentiated OSCC and the polymorphism of TLR3 has been associated with the prognosis of transformation to malignant lesions in the mouth [36, 37].
The primary aim of this study was to investigate the expression levels and tissue localization of TLR1-TLR10 and NF-κB in mucosal biopsies from oral squamous cell carcinoma in comparison to that of healthy oral mucosa. In addition, we analyzed whether the presence of Candida colonization affects TLR expression in oral squamous cell carcinoma.
Material and methods
Study subjects
A total of 56 voluntary patients, 30 with newly diagnosed primary oral squamous cell carcinoma (OSCC) and 26 healthy controls (HC) treated at the Department of Oral and Maxillofacial Surgery, Helsinki University Hospital during 2007–2011 were enrolled in this study (Table 1). Patients who had received antimicrobial therapy (i.e. antibiotics, antifungals, or antiviral agents) within the past seven days and those diagnosed with HIV or hepatitis virus infection were excluded. All study patients signed an informed consent before inclusion. The study has been approved by the Ethics Committee of the Helsinki University Central Hospital (Ethical approval number 126/E6/07 25.4.2007). Written consent from all the subjects were taken according to the declaration of Helsinki. Of the 30 healthy controls (HC) enrolled in the study, 26 were included in the final analyses as four had inadequate amount of tissue for histopathological analyses.
Collection of histopathological samples
Full thickness biopsies including epithelial and stromal tissue were collected from OSCC patients from the site of active disease process according to normal clinical procedures. The biopsies from healthy control patients were collected from the buccal mucosa at the incision site immediately after surgical extraction of a retained wisdom tooth. The samples were fixed in 10% buffered formalin and embedded in paraffin.
Collection of microbial samples
Microbial samples for detection of Candida were collected using the filter paper sampling method as described in Rusanen et al., 2009 [38]. The samples were taken from the oral mucosa using a hydrophilic mixed cellulose ester MF-Millipore membrane filter (GSWP01300; Millipore Inc., Billerica, MA, USA, pore size 0.22 μm, diameter of 13 mm) [38]. The filter paper was placed gently on the oral mucosa for 30 s with the glossy side placed against the mucosa, after which it was placed into a sterile test tube containing 5 mL of sterile saline solution. All samples were cultured within 1 h of collection. Before culture, the samples were agitated for 30 s with five sterile Ø 3 mM glass beads. For the detection of yeasts, the samples were diluted 10-fold and 100 μl of the dilution, and the neat suspension were cultured on Sabouraud Dextrose Agar (SP; Saboraud Dextrose Agar [Lab M], Bacto Agar [Difco Laboratories, Basel, Switzerland] supplemented with penicillin [100,000 iu/mL] and streptomycin) and incubated for 48 hours at 37°C. After incubation, the numbers of yeasts were enumerated as colony forming units (CFU).
Immunohistochemical staining
Tissue sections, 4 μm in thickness, were prepared from the paraffin embedded samples and applied to glass slides. The sections were deparaffined in xylenes, followed by rehydration in graded ethanol, and washed in deonized H2O. To expose the antigenic determinants after formalin fixation and paraffin embedding, the sections were incubated in pepsin for 30 min at room temperature. Endogenous peroxidase activity was quenched in the sections by incubating in hydrogen peroxidase in methanol.
The optimal primary antibody immunoglobulin G (IgG) concentration for immunohistochemistry was selected for each TLR antibody in pilot experiments and according to our previous study [17, 39]. All tissue samples were stained using this antibody concentration to allow comparisons between staining intensity. The final IgG concentrations of the polyclonal anti-human antibodies used in this study are shown in the Table 2. The TLRs were visualized as specified in user manual (catalogue nos., PK-4001 and PK-4005; Vectastain ABC kit; Vector Laboratories, Peterborough, England).
For the immunohistochemical staining with NF-κB, the tissue sections were buffered in citrate, pH 6.0 and heated 10 minutes in microwave oven and incubated for one hour in room temperature with an optimally diluted NF-κB antibody according to our previous study [17] (Table 2). After the primary antibody incubation, the tissue sections were incubated separately with Dako REAL™ EnVision™ kit using Dako automated immunostaining instruments. The reactions were visualized by Dako REAL™ DAB+ Chromogen according to manufacturer instruction (catalogue number K5007, Dako Glostrup Denmark).
Tissue samples from chronic periodontitis were used as positive controls [39, 40]. Negative controls were obtained by omission of the primary antibodies. All the specimens were stained with periodic acid-Schiff (PAS) method to determine the presence or absence of secondary candidiasis.
Evaluation of immunostaining
The expressions of TLR1-TLR10 were analysed using a light microscope (Nikon Eclipse 80i). Results were scored semi-quantitatively and photographed using an attached camera (Nikon DS-Fi1). All samples and stainings were analysed and scored by two of the authors (PR and JH) blinded for clinical data. The staining intensity of the endothelium, basal cell layer, the basement membrane zone as well as the deep and superficial thirds of the epithelium were graded in a four-point scale as 0 = no staining, 1 = staining of approximately 1–33% of cells, 2 = staining of 34–66% and 3 = staining of 67–100%. In the OSCC samples the staining intensities were separately assessed in the infiltrative tumor tissue (infiltrative zone) and adjacent normal appearing squamous epithelium. The staining intensity of the endothelium were assessed throughout the OSCC samples.
Statistical analysis
Data was analyzed by using GraphPad Prism version 5.00 (GraphPad Inc. San Diego, California, USA). The two-tailed Mann Whitney test and Spearman’s rho (rS) were used for the analyses of correlations. The Wilcoxon signed-ranks test was used to compare the differences between the different layers of samples. P-values of less than 0.05 were considered statistically significant.
Result
Subjects
30 individuals, clinically and histopathologically diagnosed with oral squamous cell carcinoma (OSCC), were enrolled in the study. 12 of them were female (mean age 68.2 years, range 52–85 years) and 18 were male (mean age 61.4 years, range 31–80 years). The mean age of OSCC patients was 64.0 years (range 31–85). The anatomical distribution of the tumors were the tongue (n = 9), maxilla (n = 6), the mandibular gingiva (n = 6), the floor of mouth (n = 5), the alveolar ridge (n = 2), and the palate (n = 2).
30 healthy controls (HC) were enrolled in the study. Out of which 26 were included in the final analysis, as four of the collected tissues samples were deemed inadequate for histopathological analysis. Out of the selected healthy controls, 17 were female with the mean age of 32.2 years (range 19–56 years) and nine of them were male with the mean age of 26.1 years (range 18±36 years). The mean age of healthy controls was 30.8 years (range 18–56) (Table 1).
Expression of TLR1-TLR10
The staining intensities in the different layers are presented in Fig 1 and relevant findings are summarized in Table 3. When comparing the staining intensity of TLR1-TLR10 in the superficial and deep epithelial layers, basal layers, basement membrane (BM) zone, as well as in the endothelium, in general significantly stronger staining of several TLRs was observed in HC samples in comparison to normal appearing squamous epithelium in OSCC samples (Fig 2).
In general, the staining intensity of several TLRs was significantly stronger in healthy controls (HC; n = 26) compared to oral squamous cell carcinoma (OSCC; n = 30) samples. The expression was determined according to the staining intensity in a four-point scale as 0 = no staining, 1 = staining of approximately 1–33% of cells, 2 = staining of 34–66% of cells and 3 = staining of 67–100% of cells. The epithelium was divided into three different layers: the superficial (Superf), deep and basal epithelial layers as well as the basement membrane zone (BM) and endothelium (Endoth). In the OSCC samples the infiltrative zone (Inf) was assessed separately. The P-values in the differences in expression of TLR1-TLR10 in the two patient groups are presented (Mann-Whitney t-test).
The staining intensity of TLR1, TLR2, TLR3, TLR4, TLR5, TLR7 and TLR8 in the normal appearing adjacent tissue in OSCC samples showed significantly lower staining intensity compared to the samples of healthy controls.
Epithelium.
In the superficial layers of the epithelium the staining intensities of TLR2 (P = 0.0039), TLR5 (P = 0.025), and TLR8 (P<0.0001) were significantly stronger in HC samples compared to superficial OSCC. In the deep layers of the epithelium the staining intensities of TLR2 (P = 0.0037) and TLR8 (P<0.0001) were significantly stronger in HC samples compared to the same of adjacent normal appearing squamous epithelium in OSCC samples. In the basal layer, the staining intensity of TLR1 was significantly stronger in HC samples compared to adjacent normal appearing squamous epithelium in OSCC samples (P = 0.03) (Fig 1).
Basement membrane zone.
In the basement membrane (BM) zone, the staining intensity of TLR3 (P = 0.029), TLR4 (P<0.0001), TLR7 (P<0.0001), and TLR8 (P<0.0001) were clearly / substantially stronger in HC samples than in the normal appearing adjacent tissue in OSCC samples (Figs 1 and 3).
Strong staining intensity of TLR 4 in HC samples in A) BM zone and B) endothelium (red arrow, x40). C) TLR4 positive staining in the BM zone in the healthy appearing tissue in OSCC sample (red arrow, x20). D) TLR 4 positive staining in OSCC infiltrative zone (red arrow, x20). HC: healthy control, OSCC: oral squamous cell carcinoma, BM: basement membrane.
Endothelium.
In the endothelium, the staining intensity of TLR4 (P = 0.0005), TLR7 (P = 0.001), and TLR8 (P = 0.0076) were significantly stronger in HC samples than in the normal appearing adjacent tissue in OSCC samples (Figs 1 and 3).
Comparison between infiltrative zone and adjacent normal appearing epithelium in OSCC samples.
In general, in the adjacent normal appearing epithelium the staining intensity of all TLRs decreased from the superficial layers towards the deeper parts of the epithelium. In the infiltrative zone several TLRs showed an increased staining intensity compared to different layers of the adjacent normal appearing epithelium: the difference was significant in TLR1 (basal cell layer; P = 0.046), TLR2 (basal cell layer; P = 0.015), TLR4 (deep layer and basal cell layer; P = 0.016 and 0.0036, respectively), TLR8 (basal cell layer; P = 0.0021) and TLR9 (basal cell layer; P = 0.035).
In superficial layer of adjacent normal appearing epithelium in OSCC samples the staining intensity of TLR2 (P = 0.018) and TLR7 (P = 0.016) were significantly stronger compared to the infiltrative zone. The staining intensities in the different layers in OSCC are presented in Fig 4.
In the infiltration zone TLR1, TLR2, TLR4, TLR8 and TLR9 showed significantly increased staining intensity compared to the basal cell layer of adjacent normal appearing epithelium. Similarly, the staining intensity of TLR4 was significantly higher in the infiltration zone compared to the deep epithelial layer of adjacent normal appearing epithelium. In opposite, TLR2 and TLR7 showed an increased staining intensity in the superficial layer compared to the infiltration zone. The expression was determined according to the staining intensity in a four-point scale as 0 = no staining, 1 = staining of approximately 1–33% of cells, 2 = staining of 34–66% of cells and 3 = staining of 67–100% of cells. The epithelium was divided into three different layers: the superficial (Superf), deep and basal epithelial layers. The P-values in the differences in expression of TLR1-TLR10 are presented (Mann-Whitney t-test): * = P < 0.05; ** = P < 0.005.
Association of Candida colonization and the expression of TLR1-10
Candida colonization was seen in 23% of the OSCC patients and 4% of the healthy controls. In the normal appearing adjacent tissue in OSCC samples, there was a significant positive correlation between Candida colonization and TLR4 expression in the BM zone (P = 0.012).
Expression of NF-κB
The expression of NF-κB was significantly stronger in HC samples compared to the normal appearing adjacent OSCC samples in the superficial and basal layer of the epithelium (P = 0.02 and 0.0064, respectively). The expression of NF-κB was significantly stronger in the infiltrative zone compared to the adjacent normal appearing mucosa in OSCC in all the epithelial layers (superficial, P = 0.008; deep, P = 0.003; basal, P = 0.01) (Table 3).
Associations between expressions of TLR1-10 and NF-κB
There was a positive correlation between the expression of NF-κB and TLR9 as well as TLR10 in the basal layer of the infiltrative zone in OSCC samples (P = 0.04 and P = 0.002, respectively).
However, no significant correlations were seen in the staining intensities of TLR1-10 and NF-κB in any of the epithelial layers of the HC samples or the adjacent normal appearing mucosa of OSCC samples (Table 3).
Discussion
The present study analyzed the staining intensity and immunolocalization of TLR1-10 and NF-κB in the different epithelial layers, basement membrane (BM) zone, endothelium, and infiltrative zone of oral squamous cell carcinoma (OSCC) tissue sections and results were compared with the staining intensity in healthy oral mucosa from control patients. In addition, it was analyzed whether the presence of Candida colonization affects TLR expression in oral squamous cell carcinoma.
In general, the healthy appearing tissue in OSCC samples showed lower staining intensity of several TLRs compared to the healthy controls (HC). The difference was statistically significant in the staining intensity of TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, and TLR8.
The BM zone showed the most variation as the staining intensity of TLR 3, 4, 7, and 8 were significantly decreased in normal appearing adjacent tissue in OSCC samples compared to those of healthy control samples. Different studies have demonstrated the presence of soluble forms of toll-like receptors in saliva, plasma, breast milk, amniotic fluid, and other body fluids [18, 20]. Therefore, it is possible to speculate that BM zone also harbor soluble forms of TLR fragments produced by the basal epithelial cells and were stained during the analysis.
In the epithelium, we did not find any statistical differences in TLR4 staining intensities between HC and OSCC samples. This is in contrast with the study of Yang et al. 2016, who demonstrated that the expression of cytoplasmic TLR4 is increased in tissue samples of OSCC compared to healthy oral mucosa [41]. The differences might be attributed to the different sampling areas of interest: in the study of Yang et al., the TLR4 expression was scored only from the epithelium. In our study, the TLR4 expression was scored also from the BM-zone, endothelium, and infiltrative zone, separately.
In healthy appearing tissue samples from OSCC patients with candidal colonization at the tumor site, the staining intensity of TLR4 was significantly increased at the BM zone when compared to OSCC samples with no Candida (P = 0.01). This is in line with the study where epithelial cells of the oral mucosa have been shown to upregulate TLR4 upon stimulation of Candida albicans [42]. This might be due to the higher levels of the mutagenic acetaldehyde: We have previously demonstrated that a significantly more quantity of mutagenic acetaldehyde is produced by the Candida samples collected from OSCC and oral lichenoid lesions sites of the patients than those from the patients with no candida colonization [6].
When comparing infiltration zone with adjacent normal appearing epithelium in OSCC tissue, several TLRs showed an increased staining intensity in the infiltration zone compared to the basal cell layer of adjacent normal appearing epithelium. The difference was statistically significant in TLR1, TLR2, TLR4, TLR8 and TLR9. In addition, the staining intensity of TLR4 was significantly higher in the infiltration zone compared to the deep epithelial layer of adjacent normal appearing epithelium. These findings are in line with the earlier studies which compared the expression of TLRs between tumoral tissue and adjacent normal epithelial tissue in OSCC samples [43–45].
The staining for NF-κB in the superficial epithelium and basal cell layer was significantly lower in the normal appearing adjacent tissue in OSCC samples compared to the control samples (P = 0.02, P = 0.0064, respectively). This might be attributed to the lower staining for several TLRs as seen in our study. Nevertheless, the expression of NF-κB was significantly stronger in the infiltrative zone compared to the healthy appearing adjacent tissue in OSCC samples in all the epithelial layers. This may indicate that NF-kB contributes to tumor growth and hematologic and lymphatic metastasis [46]. In fact, as seen in our study, the staining intensity of most TLRs in the infiltrative zone was more intense compared to the basal layer of the healthy appearing tissue in OSCC samples. Significant association between NF-κB staining and TLR9 and TLR10 was seen in basal layer in the samples from patients with OSCC. However, no such association was seen in control samples.
When comparing our results to a very limited number of previous studies that used immunohistochemical methods to define the differences in TLR staining intensity and immunolocalization between OSCC and healthy control tissue, we find points that need to be studied in more detail. Several studies have used the tissue adjacent to the tumor as healthy tissue controls [45, 47, 48]. Carcinogenesis is a multifactorial cascade which is affected by numerous intrinsic and extrinsic factors. These factors lead to the loss of control of cell cycle and finally to tumor progression. Many of these factors also affect other parts of the oral mucosa leading to the fact that the normal appearing mucosa adjacent to the tumor is also altered. The results of our previous study supports this notion: staining intensity of tumor suppressor protein p53 was found to be noticeably elevated in both the infiltrative zone and healthy appearing adjacent mucosa of OSCC samples [49]. Tumor suppression protein p53 is a transcription factor involved in apoptosis and control of cell cycle and plays a vital role in the preservation of genetic stability and thus prevention of progression of cancer [50].
We acknowledge some limitations in our study, such as the difference in age between the OSCC patients and healthy controls. This could partly be attributed to the fact that OSCC is more common in the adult population, whereas the control samples taken during the surgical removal of third molars, is mainly performed in young adults. However, it is unlikely that the age gap would have a major impact on the findings because, in contrast to adaptive immune responses, the innate responses are not generally significantly affected by aging [51]. In fact, in this study, we did not find any age-related correlations with the immunostaining of any TLRs. A limitation of this study is that in this study only immunohistochemical staining was used to compare the level of expression of TLR and NF-kB between the OSCC and HC samples. The expression of TLR and NF-kB should ideally also be analyzed using additional techniques, such as measuring mRNA levels by qPCR.
OSCC is a multifactorial disease linked with various risk factors and no single factor has been recognized as having independent influence on the prognosis of OSCC. During the progression of oncogenesis from a normal healthy cell to a pre-malignant or a potentially malignant cell, mutations in several DNA molecules occur leading to the loss of control of the growth of cell which eventually lead to uncontrolled proliferation of the cells leading to the cancer. To determine the genetic mechanisms involved in OSCC, the mutations should ideally also be analyzed using genetic techniques such as qPCR in order to measure mRNA levels in the tissue sample. In this study only immunohistochemical staining was used to compare the level of expression of TLR and NF-kB between the OSCC and HC samples and is one of the limitations of this study. However, qPCR techniques do not provide information about the specific site within the tissue sample from where the signal is originated. While, in this study we have analyzed immunolocalization and the staining intensity of TLR1-10 and NF-κB in the different epithelial layers, basement membrane (BM) zone, endothelium, and infiltrative zone of tissue sections. In addition, a limitation of our study is the low number of sample size due to the limited amount of tissue available.
In conclusion, all the TLRs and NF-κB, and their co-localization in the epithelium, basement membrane zone and endothelium were mapped for the first time. The differences in the expression of several TLRs and NF-κB between the infiltrative zone and healthy appearing adjacent tissue in OSCC samples forms a significant finding. Additional studies are required to determine the role of soluble forms of TLRs observed in BM zone.
References
- 1. Kalogirou EM, Tosios KI, Christopoulos PF. The Role of Macrophages in Oral Squamous Cell Carcinoma. Frontiers in Oncology. Frontiers Media S.A.; 2021. pmid:33816242
- 2. Cao R, Zhang J, Jiang L, Wang Y, Ren X, Cheng B, et al. Comprehensive Analysis of Prognostic Alternative Splicing Signatures in Oral Squamous Cell Carcinoma. Front Oncol. 2020;10. pmid:32984057
- 3. Homann N, Tillonen J, Meurman JH. Increased salivary acetaldehyde levels in heavy drinkers and smokers: a microbiological approach to oral cavity cancer. Carcinogenesis. Oxford Academic; 2000 Apr. pmid:10753201
- 4. Scully C, Bagan J V. Oral squamous cell carcinoma: Overview of current understanding of aetiopathogenesis and clinical implications. Oral Diseases. Oral Dis; 2009. pp. 388–399. pmid:19371401
- 5. Scully C. Oral cancer aetiopathogenesis; past, present and future aspects. Med Oral Patol Oral Cir Bucal. 2011;16: 306–311. pmid:21441876
- 6. Marttila E, Uittamo J, Rusanen P, Lindqvist C, Salaspuro M, Rautemaa R. Acetaldehyde production and microbial colonization in oral squamous cell carcinoma and oral lichenoid disease. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;116: 61–68. pmid:23619349
- 7. Uittamo J, Siikala E, Kaihovaara P, Salaspuro M, Rautemaa R. Chronic candidosis and oral cancer in APECED-patients: Production of carcinogenic acetaldehyde from glucose and ethanol by Candida albicans. International Journal of Cancer. 2009. pp. 754–756. pmid:18975379
- 8. Marttila E, Bowyer P, Sanglard D, Uittamo J, Kaihovaara P, Salaspuro M, et al. Fermentative 2-carbon metabolism produces carcinogenic levels of acetaldehyde in Candida albicans. Mol Oral Microbiol. 2013;28: 281–291. pmid:23445445
- 9. Coussens LM, Werb Z. Inflammation and cancer. Nature. Nature; 2002. pp. 860–867. pmid:12490959
- 10. Moss SF, Blaser MJ. Mechanisms of disease: Inflammation and the origins of cancer. Nature Clinical Practice Oncology. Nat Clin Pract Oncol; 2005. pp. 90–97. pmid:16264881
- 11. Landskron G, De La Fuente M, Thuwajit P, Thuwajit C, Hermoso MA. Chronic inflammation and cytokines in the tumor microenvironment. Journal of Immunology Research. Hindawi Publishing Corporation; 2014. pmid:24901008
- 12. Rich AM, Hussaini HM, Parachuru VPB, Seymour GJ. Toll-like receptors and cancer, particularly oral squamous cell carcinoma. Frontiers in Immunology. Frontiers Media S.A.; 2014. pmid:25309546
- 13. Balkwill F, Mantovani A. Inflammation and cancer: Back to Virchow? Lancet. Elsevier Limited; 2001. pp. 539–545. pmid:11229684
- 14. Karin M, Greten FR. NF-κB: Linking inflammation and immunity to cancer development and progression. Nature Reviews Immunology. Nat Rev Immunol; 2005. pp. 749–759. pmid:16175180
- 15. Rakoff-Nahoum S, Medzhitov R. Toll-like receptors and cancer. Nature Reviews Cancer. Nat Rev Cancer; 2009. pp. 57–63. pmid:19052556
- 16. Kumar H, Kawai T, Akira S. Toll-like receptors and innate immunity. Biochemical and Biophysical Research Communications. Biochem Biophys Res Commun; 2009. pp. 621–625. pmid:19686699
- 17. Rusanen P, Marttila E, Uittamo J, Hagström J, Salo T, Rautemaa-Richardson R. TLR1-10, NF-κB and p53 expression is increased in oral lichenoid disease. PLoS One. 2017;12: e0181361. pmid:28715461
- 18. ten Oever J, Kox M, van de Veerdonk FL, Mothapo KM, Slavcovici A, Jansen TL, et al. The discriminative capacity of soluble Toll-like receptor (sTLR)2 and sTLR4 in inflammatory diseases. BMC Immunol. 2014;15: 1–10. pmid:25406630
- 19. Srinivasan M, Kodumudi KN, Zunt SL. Soluble CD14 and toll-like receptor-2 are potential salivary biomarkers for oral lichen planus and burning mouth syndrome. Clinical Immunology. 2008;126: 31–37. pmid:17916440
- 20. Zunt SL, Burton L V., Goldblatt LI, Dobbins EE, Srinivasan M. Soluble forms of Toll-like receptor 4 are present in human saliva and modulate tumour necrosis factor-α secretion by macrophage-like cells. Clin Exp Immunol. 2009;156: 285–293. pmid:19292767
- 21. LeBouder E, Rey-Nores JE, Rushmere NK, Grigorov M, Lawn SD, Affolter M, et al. Soluble Forms of Toll-Like Receptor (TLR)2 Capable of Modulating TLR2 Signaling Are Present in Human Plasma and Breast Milk. The Journal of Immunology. 2003;171: 6680–6689. pmid:14662871
- 22. Salvador B, Arranz A, Francisco S, Córdoba L, Punzón C, Llamas MÁ, et al. Modulation of endothelial function by Toll like receptors. Pharmacological Research. Academic Press; 2016. pp. 46–56. pmid:27073018
- 23. Grote K, Schütt H, Schieffer B. Toll-like receptors in angiogenesis. The Scientific World Journal. Scientific World Journal; 2011. pp. 981–991. pmid:21516292
- 24. Su SB, Tao L, Deng ZP, Chen W, Qin SY, Jiang HX. TLR10: Insights, controversies and potential utility as a therapeutic target. Scand J Immunol. 2021;93: e12988. pmid:33047375
- 25. Karin M, Lawrence T, Nizet V. Innate immunity gone awry: Linking microbial infections to chronic inflammation and cancer. Cell. Elsevier B.V.; 2006. pp. 823–835. pmid:16497591
- 26. Pal S, Bhattacharjee A, Ali A, Mandal NC, Mandal SC, Pal M. Chronic inflammation and cancer: Potential chemoprevention through nuclear factor kappa B and p53 mutual antagonism. Journal of Inflammation (United Kingdom). BioMed Central Ltd.; 2014. pmid:25152696
- 27. Pradere JP, Dapito DH, Schwabe RF. The Yin and Yang of Toll-like receptors in cancer. Oncogene. Nature Publishing Group; 2014. pp. 3485–3495. pmid:23934186
- 28. Fang SL, Kong XB, Zhang ZQ. Association of toll-like receptors with the risk of oral squamous cell carcinoma. J Cancer Res Ther. 2018;14: S180–S183. pmid:29578170
- 29. Li D, Jin Y, Sun Y, Lei J, Liu C. Knockdown of toll-like receptor 4 inhibits human NSCLC cancer cell growth and inflammatory cytokine secretion in vitro and in vivo. Int J Oncol. 2014;45: 813–821. pmid:24889928
- 30. Wang Y, Cai J, Zeng X, Chen Y, Yan W, Ouyang Y, et al. Downregulation of toll-like receptor 4 induces suppressive effects on hepatitis B virus-related hepatocellular carcinoma via ERK1/2 signaling. BMC Cancer. 2015;15. pmid:26514586
- 31. H Y, H Z, P F, X Z, H W, X X, et al. Reduced expression of Toll-like receptor 4 inhibits human breast cancer cells proliferation and inflammatory cytokines secretion. J Exp Clin Cancer Res. 2010;29. pmid:20618976
- 32. Ye K, Wu Y, Sun Y, Lin J, Xu J. TLR4 siRNA inhibits proliferation and invasion in colorectal cancer cells by downregulating ACAT1 expression. Life Sci. 2016;155: 133–139. pmid:27177773
- 33. B H, Z C, B B, M Y, S Y, Y F, et al. Role of TLR4 as a prognostic factor for survival in various cancers: a meta-analysis. Oncotarget. 2018;9. pmid:29560134
- 34. Ren WH, Zhang LM, Liu HQ, Gao L, Chen C, Qiang C, et al. Protein overexpression of CIRP and TLR4 in oral squamous cell carcinoma: An immunohistochemical and clinical correlation analysis. Medical Oncology. 2014;31: 1–9. pmid:25027624
- 35. Mäkinen LK, Atula T, Häyry V, Jouhi L, Datta N, Lehtonen S, et al. Predictive role of toll-like receptors 2, 4, and 9 in oral tongue squamous cell carcinoma. Oral Oncol. 2015;51: 96–102. pmid:25264223
- 36. K Z, G S, N J, R K, M B-M, M O, et al. Association of TLR2, TLR3, TLR4 and CD14 genes polymorphisms with oral cancer risk and survival. Oral Dis. 2014;20. pmid:23796347
- 37. Chuang HC, Huang CC, Chien CY, Chuang JH. Toll-like receptor 3-mediated tumor invasion in head and neck cancer. Oral Oncol. 2012;48: 226–232. pmid:22070917
- 38. Rusanen P, Siikala E, Uittamo J, Richardson M, Rautemaa R. A novel method for sampling the microbiota from the oral mucosa. Clin Oral Investig. 2009;13: 243–246. pmid:18797939
- 39. Beklen A, Hukkanen M, Richardson R, Konttinen YT. Immunohistochemical localization of Toll-like receptors 1–10 in periodontitis. Oral Microbiol Immunol. 2008;23: 425–431. pmid:18793367
- 40. Ali A, Natah S, Konttinen Y. Differential expression of Toll-like receptors in chronic hyperplastic candidosis. Oral Microbiol Immunol. 2008;23: 299–307. pmid:18582329
- 41. Yang J, Liu D, Khatri KS, Wang J, Zhang G, Meng C, et al. Prognostic value of toll-like receptor 4 and nuclear factor-κBp65 in oral squamous cell carcinoma patients. Oral Surg Oral Med Oral Pathol Oral Radiol. 2016;122: 753–764.e1. pmid:27727111
- 42. Weindl G, Naglik JR, Kaesler S, Biedermann T, Hube B, Korting HC, et al. Human epithelial cells establish direct antifungal defense through TLR4-mediated signaling. Journal of Clinical Investigation. 2007;117: 3664–3672. pmid:17992260
- 43. Li L, Zhou Z, Mai K, Li P, Wang Z, Wang Y, et al. Protein overexpression of toll-like receptor 4 and myeloid differentiation factor 88 in oral squamous cell carcinoma and clinical significance. Oncol Lett. 2021;22. pmid:34594427
- 44. Visioli F, Nunes JS, Pedicillo MC, Leonardi R, Santoro A, Zannoni GF, et al. TLR4 Expression in Ex-Lichenoid Lesions—Oral Squamous Cell Carcinomas and Its Surrounding Epithelium: The Role of Tumor Inflammatory Microenvironment. Biomolecules. 2022;12. pmid:35327577
- 45. Kauppila JH, Korvala J, Siirilä K, Manni M, Mäkinen LK, Hagström J, et al. Toll-like receptor 9 mediates invasion and predicts prognosis in squamous cell carcinoma of the mobile tongue. 2015;44: 571–577. Available: https://onlinelibrary.wiley.com/doi/full/10.1111/jop.12272
- 46. Lakshminarayana S, Augustine D, Rao RS, Patil S, Awan KH, Venkatesiah SS, et al. Molecular pathways of oral cancer that predict prognosis and survival: A systematic review. J Carcinog. 2018;17: 7. pmid:30766450
- 47. Kauppila JH, Mattila AE, Karttunen TJ, Salo T. Toll-like receptor 5 (TLR5) expression is a novel predictive marker for recurrence and survival in squamous cell carcinoma of the tongue. Br J Cancer. 2013;108. Available: /pmc/articles/PMC3593548/ pmid:23287987
- 48. Sun Z, Luo Q, Ye D, Chen W, Chen F. Role of toll-like receptor 4 on the immune escape of human oral squamous cell carcinoma and resistance of cisplatin-induced apoptosis. Mol Cancer. 2012;11: 33. pmid:22583829
- 49. Marttila E, Rusanen P, Uittamo J, Salaspuro M, Rautemaa-Richardson R, Salo T. Expression of p53 is associated with microbial acetaldehyde production in oralsquamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol. 2021;131: 527–533. pmid:33858805
- 50. Massano J, Regateiro FS, Januário G, Ferreira A. Oral squamous cell carcinoma: review of prognostic and predictive factors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102: 67–76. pmid:16831675
- 51. Olivieri F, Rippo MR, Prattichizzo F, Babini L, Graciotti L, Recchioni R, et al. Toll like receptor signaling in “inflammaging”: microRNA as new players. Immun Ageing. 2013;10: 11. pmid:23506673