Isolation, characterization, and fibroblast uptake of bacterial extracellular vesicles from Porphyromonas gingivalis strains.

Periodontitis is an inflammatory condition caused by bacteria and represents a serious health problem worldwide as the inflammation damages the supporting tissues of the teeth and may predispose to systemic diseases. Porphyromonas gingivalis is considered a keystone periodontal pathogen that releases bacterial extracellular vesicles (bEVs) containing virulence factors, such as gingipains, that may contribute to the pathogenesis of periodontitis. This study aimed to isolate and characterize bEVs from three strains of P. gingivalis, investigate putative bEV uptake into human oral fibroblasts, and determine the gingipain activity of the bEVs. bEVs from three bacterial strains, ATCC 33277, A7A1-28, and W83, were isolated through ultrafiltration and size-exclusion chromatography. Vesicle size distribution was measured by nano-tracking analysis (NTA). Transmission electron microscopy was used for bEV visualization. Flow cytometry was used to detect bEVs and gingipain activity was measured with an enzyme assay using a substrate specific for arg-gingipain. The uptake of bEVs into oral fibroblasts was visualized using confocal microscopy. NTA showed bEV concentrations from 108 to 1011 particles/mL and bEV diameters from 42 to 356 nm. TEM pictures demonstrated vesicle-like structures. bEV-gingipains were detected both by flow cytometry and enzyme assay. Fibroblasts incubated with bEVs labeled with fluorescent dye displayed intracellular localization consistent with bEV internalization. In conclusion, bEVs from P. gingivalis were successfully isolated and characterized, and their uptake into human oral fibroblasts was documented. The bEVs displayed active gingipains demonstrating their origin from P. gingivalis and the potential role of bEVs in periodontitis.

Cytokine Levels in Saliva Are Associated with Salivary Gland Fibrosis and Hyposalivation in Mice after Fractionated Radiotherapy of the Head and Neck.

Cytokines are mediators of inflammation that could lead to fibrosis. The aim was to monitor cytokine levels in saliva and serum after locally fractionated radiotherapy of the head and neck in mice and investigate associations with salivary gland fibrosis and hyposalivation. C57BL/6 mice were randomized to sham or X-ray irradiation of 66 Gy in 10 fractions over 5 days. Blood and saliva were collected on days -7, 5, 35, 80, and 105 following cytokine analysis. The harvested submandibular salivary gland was assessed for the presence of fibrosis. Decision tree regression analysis was used to investigate whether cytokine levels could predict late endpoints in terms of hyposalivation or fibrosis. Significant formation of fibrosis in gland tissue and reduced saliva production was found after irradiation. The pro-inflammatory cytokines IL-1α, TNF, TIMP1, G-CSF, KC, and MIP-1α showed increased levels in saliva in irradiated mice and a strong correlation with late endpoints. The decision tree analysis largely separated controls from irradiated animals, with IL-1α being the strongest predictor. Pro-inflammatory cytokines in saliva, but not in serum, were associated with late endpoints. This indicates that cytokine expression in saliva is a good biomarker for local salivary gland damage with IL-1α as the strongest single predictor.

The spectrum and frequency of histopathological diagnosis of oral diseases in Oslo: Implications to oral pathology syllabus.

INTRODUCTION: To assure knowledge and skills in diagnostic work of oral diseases a continuously updated curriculum is essential. The first aim of the present study was to evaluate the spectrum and frequency of oral histopathological diagnoses signed out by oral pathologists at the Department of Pathology, Oslo University Hospital (OUS), Norway during a two-year period. The second aim was to compare the spectrum of histopathological diagnoses with the content of the current syllabus in oral pathology at the Faculty of Dentistry, University of Oslo (UiO). MATERIALS AND METHODS: In this retrospective cross-sectional study, all histological diagnosis signed out during 2015 and 2016 were included. All histopathological reports were analysed with regard to clinical information and histopathological diagnosis. The spectrum of histopathological diagnoses was compared to the diagnoses presented in lectures and courses for dental and dental hygienist students at UiO. RESULTS: Three thousand four hundred and two histopathological reports (47% males and 53% females) were included. The diagnoses were categorised into eight disease groups and the three most frequent disease groups were cysts, benign tumours/reactive lesions, and white, red, ulcerative and vesiculobullous lesions. The lateral periodontal cyst was more frequent than expected. CONCLUSIONS: We conclude that a minor revision of the syllabus is needed, although the most frequent oral conditions presented in this study are well covered in the oral pathology teaching in Oslo. A more clinical related teaching approach should be considered by categorising oral diseases according to, for example location and age groups.

The prognostic role of combining Krüppel-like factor 4 score and grade of inflammation in a Norwegian cohort of oral tongue squamous cell carcinomas.

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor involved in inflammation, cancer development, and progression. However, the relationship between KLF4, inflammation, and prognosis in oral cancer is not fully understood. KLF4 expression levels were examined in a multicenter cohort of 128 oral squamous cell carcinoma (OSCC) specimens from the tongue (OTSCC) using immunohistochemistry. In two external KLF4 mRNA datasets (The Cancer Genome Atlas/The Genotype-Tissue Expression Portal), lower KLF4 mRNA expression was found in OSCC and head and neck squamous cell carcinomas (HNSCC) than in control oral epithelium. These data indicate that down-regulation of KLF4 mRNA is linked to OSCC/HNSCC progression. Using Cox-multivariate analysis, a significantly favorable 5-year disease-specific survival rate was observed for a subgroup of patients with a combination of high levels of KLF4 expression and inflammation. OSCC cell lines exposed to IFN-γ showed a significant upregulation of nuclear KLF4 expression, indicating a link between inflammation and KLF4 expression in OSCC. Overall, the current data suggest a functional link between KLF4 and inflammation. The combination of high KLF4 nuclear expression and marked/moderate stromal inflammation might be useful as a favorable prognostic marker for a subgroup of OTSCC patients.

Expression profile of SARS-CoV-2 cellular entry proteins in normal oral mucosa and oral squamous cell carcinoma.

OBJECTIVE: Besides angiotensin converting enzyme 2 (ACE2), an active involvement of proteases (FURIN and/or TMPRSS2) is important for cellular entry of SARS-CoV-2. Therefore, a simultaneous expression profiling of entry proteins in a tissue might provide a better risk assessment of SARS-CoV-2 infection as compared to individual proteins. In an attempt to understand the relative susceptibility of oral squamous cell carcinoma (OSCC) lesions as compared to the normal oral mucosa (NOM) for SARS-CoV-2 attachment/entry, this study examined the mRNA and protein expression profiles of ACE2, FURIN, and TMPRSS2 in the corresponding tissues using public transcriptomic and proteomics datasets. METHODS AND METHODS: Public transcriptomic and proteomics datasets (the Cancer Genome Atlas (TCGA)/the Genotype-Tissue Expression (GTEx), the Human Protein Atlas (HPA), and two independent microarray datasets) were used to examine the expression profiles of ACE2, TMPRSS2 and FURIN in NOM and OSCC. RESULTS: ACE2, TMPRSS2, and FURIN mRNAs were detected in NOM, however, at lower levels as compared to other body tissues. Except for moderate up-regulation of FURIN, expression levels of ACE2 and TMPRSS2 mRNA were unchanged/down-regulated in OSCC as compared to the NOM. CONCLUSIONS: These results indicate that NOM may serve as a possible site for SARS-CoV-2 attachment, however, to a lesser extent as compared to organs with higher expression levels of the SARS-CoV-2 entry proteins. However, the evidence is lacking to suggest that expression status of entry proteins predisposes OSCC lesions to additional risk for SARS-CoV-2 attachment/entry as compared to NOM.

High-risk human papilloma virus was not detected in a Norwegian cohort of oral squamous cell carcinoma of the mobile tongue.

OBJECTIVES: The presence of and the causative role of high-risk human papilloma virus (HPV) is a subject of controversy in oral squamous cell carcinoma (OSCC). The disagreement can be related to the misclassification of OSCC as oropharyngeal squamous cell carcinoma and/or lack of standard detection methods. This study aimed to examine the presence of transcriptionally active high-risk HPV in a homogenous Norwegian cohort of primary and second primary OSCC of the mobile tongue (oral tongue squamous cell carcinoma-OTSCC). METHODS: Tissue microarrays containing formalin-fixed and paraffin-embedded cores of 146 OTSCC from the anterior 2/3 of the tongue (n = 128 primary and n = 18 second primary) from a multicentric Norwegian cohort were examined for the presence of high-risk HPV by DNA- and RNA-in situ hybridization (ISH) assays and p16 immunohistochemistry. RESULTS: Transcriptionally active HPV (E6/E7 mRNA) was not identified in any of the OTSCC specimens. In parallel, no tumors were positive for HPV by DNA ISH. Although, 61 (42%) OTSCC demonstrated p16 positivity with varying staining intensity and subcellular localization, only two cases demonstrated strong and uniform p16-staining (both cytoplasmic and nuclear) in >70% of cancer cells. The absence of transcriptionally active high-risk HPV in this cohort of OTSCC indicates that high-risk HPV is an unlikely causative factor in the present material.

Tumor budding score predicts lymph node status in oral tongue squamous cell carcinoma and should be included in the pathology report.

BACKGROUND: The majority of oral cavity cancers arise in the oral tongue. The aim of this study was to evaluate the prognostic value of tumor budding in oral tongue squamous cell carcinoma, both as a separate variable and in combination with depth of invasion. We also assessed the prognostic impact of the 8th edition of the American Joint Committee on Cancer's TNM classification (TNM8), where depth of invasion (DOI) supplements diameter in the tumor size (T) categorization. METHODS: Patients diagnosed with primary oral tongue squamous cell carcinoma were evaluated retrospectively. Spearman bivariate correlation analyses with bootstrapping were used to identify correlation between variables. Prognostic value of clinical and histopathological variables was assessed by Log rank and Cox regression analyses with bootstrapping using 5-year disease specific survival as outcome. The significance level for the hypothesis test was 0.05. RESULTS: One-hundred and fifty patients had available material for microscopic evaluation on Hematoxylin and Eosin-stained slides and were included in the analyses. Reclassification of tumors according to TNM8 caused a shift towards a higher T status compared to the previous classification. The tumor budding score was associated with lymph node metastases where 23% of the patients with low-budding tumors had lymph node metastases, compared with 43% of those with high-budding tumors. T-status, lymph node status, tumor budding, depth of invasion, and the combined tumor budding/depth of invasion score were all significantly associated with survival in univariate analyses. In multivariate analyses only N-status was an independent prognosticator of survival. CONCLUSION: Reclassification according to TNM8 shifted many tumors to a higher T-status, and also increased the prognostic value of the T-status. This supports the implementation of depth of invasion to the T-categorization in TNM8. Tumor budding correlated with lymph node metastases and survival. Therefore, information on tumor budding can aid clinicians in treatment planning and should be included in pathology reports of oral tongue squamous cell carcinomas.

Cancer cell line-specific protein profiles in extracellular vesicles identified by proteomics.

Extracellular vesicles (EVs), are important for intercellular communication in both physiological and pathological processes. To explore the potential of cancer derived EVs as disease biomarkers for diagnosis, monitoring, and treatment decision, it is necessary to thoroughly characterize their biomolecular content. The aim of the study was to characterize and compare the protein content of EVs derived from three different cancer cell lines in search of a specific molecular signature, with emphasis on proteins related to the carcinogenic process. Oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC) and melanoma brain metastasis cell lines were cultured in CELLine AD1000 flasks. EVs were isolated by ultrafiltration and size-exclusion chromatography and characterized. Next, the isolated EVs underwent liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Functional enrichment analysis was performed for a more general overview of the biological processes involved. More than 600 different proteins were identified in EVs from each particular cell line. Here, 14%, 10%, and 24% of the identified proteins were unique in OSCC, PDAC, and melanoma vesicles, respectively. A specific protein profile was discovered for each cell line, e.g., EGFR in OSCC, Muc5AC in PDAC, and FN1 in melanoma vesicles. Nevertheless, 25% of all the identified proteins were common to all cell lines. Functional enrichment analysis linked the proteins in each data set to biological processes such as "biological adhesion", "cell motility", and "cellular component biogenesis". EV proteomics discovered cancer-specific protein profiles, with proteins involved in processes promoting tumor progression. In addition, the biological processes associated to the melanoma-derived EVs were distinct from the ones linked to the EVs isolated from OSCC and PDAC. The malignancy specific biomolecular cues in EVs may have potential applications as diagnostic biomarkers and in therapy.

A combined histo-score based on tumor differentiation and lymphocytic infiltrate is a robust prognostic marker for mobile tongue cancer.

We wanted to evaluate the prognostic value of common histopathological variables in a large cohort of patients with cancer in the mobile tongue as such information can be important for treatment stratification of the individual patient, especially for patients with low-stage disease. In addition, we wanted to investigate whether an alternative scoring model with fewer options would compromise the prognostic value. One hundred fifty patients with oral tongue squamous cell carcinomas that were treated in curative intent and with available HE-stained tumor sections were included. We reclassified all tumors and performed univariate and multivariate survival analyses of histopathological and clinical variables. For the complete cohort, lymph node status, grade of differentiation, perineural infiltration, and lymphocytic infiltration were independent prognosticators. In the low-stage disease group, independent prognostic factors were tumor size, grade of differentiation, and lymphocytic infiltrate. For patients with low-stage disease, a histo-score combining the scores for tumor differentiation and lymphocytic infiltrate identified a group of patients with particularly low survival, as patients with moderately or poorly differentiated tumors and little lymphocytic infiltrate had a less favorable 5-year survival outcome than patients in the high-stage disease group. This study shows that a histo-score combining tumor differentiation and lymphocytic infiltration should be given special consideration in treatment planning. Our results also illustrate that many variables can be scored with fewer options than previously suggested to increase their reproducibility, and still maintain their prognostic value.

Grading of oral squamous cell carcinomas - Intra and interrater agreeability: Simpler is better?

BACKGROUND: Numerous studies have been presented on histological grading of oral squamous cell carcinomas (OSCC) for predicting survival, but uncertainty of their usefulness rises due to discordances of results. A scoring system should be robust and well validated, and intra- and interrater agreement can be used as a tool to visualize the strength of reproducibility. METHODS: Here, we present an intra- and inter-observer study on evaluation of OSCC using some of the most common histopathological parameters. The observers were from different Norwegian university hospitals, and calibration to ensure accuracy was first performed. Percentage of the agreement was calculated for the score made by the individual observer at different times, as well as between pairs of observers. RESULTS: The evaluation made by the same observer at two different time points (intrarater) correlated better than observations made by different participants (interrater). In an attempt to increase the rate of agreement, many of the parameters were either dichotomized into simply low- and high grade, or to a three-tier system when more than three options in the original design. This increased the concurrence with 15.4% for the intrarater and with 23% for the interrater comparisons. CONCLUSION: High agreement for histopathological parameters can be difficult to obtain on hematoxylin and eosin staining in scoring systems with many options. A simpler system might be more advantageous to achieve higher degree of reproducibility.

Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography.

Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60-140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.

Prognostic molecular markers in cancer - quo vadis?

Despite the tremendous number of studies of prognostic molecular markers in cancer, only a few such markers have entered clinical practise. The lack of clinical prognostic markers clearly reflects limitations in or an inappropriate approach to prognostic studies. This situation should be of great concern for the research community, clinicians and patients. In the present review, we evaluate immunohistochemical prognostic marker studies in oral squamous cell carcinomas (OSCC) from 2006 to 2012. We comment upon general issues such as study design, assay methods and statistical methods, applicable to prognostic marker studies irrespective of cancer type. The three most frequently studied markers in OSCC are reviewed. Our analysis revealed that most new molecular markers are reported only once. To draw conclusions of clinical relevance based on the few markers that appeared in more than one study was problematic due to between-study heterogeneity. Currently, much valuable tissue material, time and money are wasted on irrelevant studies.

Nuclear and cytoplasmic expression of Met in oral squamous cell carcinoma and in an organotypic oral cancer model.

Met, the hepatocyte growth factor receptor, is important in transducing signals for tumour growth and metastasis. The aim of this study was to examine the pattern of Met expression and its value as a prognostic factor in oral squamous cell carcinomas (OSCCs). The material consisted of 53 OSCCs and five healthy controls from normal oral mucosa supplied with cell lines, 10 organotypic models supplied with oral cancer cells, and three organotypic models supplied with normal keratinocytes. Met protein expression was assessed by immunohistochemistry and western blotting. Met expression was scarce and limited to the basal layer in normal oral mucosa, but was more extensive in the tumours. Cytoplasmic expression of Met was found in the majority of the tumours, and nuclear expression was found in 72%, including a high fraction of the cells located at the invasive front. Organotypic models with normal or malignant oral cells yielded principally similar results as in the mucosa and the cancers, respectively. A smaller amount of Met immunoreactivity was detected, by western blotting, in the nuclear fraction of cultured oral cancer cells. In conclusion, Met was upregulated in OSCCs and was also found in the nucleus. However, Met was not a marker for prognosis in this study.

Expression of the T-cell-specific adapter protein in oral epithelium.

The multifunctional T-cell-specific adapter protein (TSAd) was originally described in T cells but is also expressed in epithelial cells from the respiratory tract and in endothelium. In this study, we found expression of TSAd messenger RNA (mRNA) and protein in both human and murine oral mucosal epithelium as well as in human primary oral keratinocyte cell cultures. In TSAd(-/-) mice, the mucosa and skin appeared macroscopically normal, but severe disturbances were observed in the fine structures of the basal membrane and intercellular epithelial spaces upon analysis using transmission electron microscopy. Oral epithelial cells from TSAd(-/-) mice displayed decreased migration compared with cells from wild-type mice, whereas overexpression of TSAd in a human epithelial cell line resulted in impaired proliferation. This study is the first to show that TSAd is expressed in normal oral mucosa, that it is important for the normal ultrastructural morphology of the epithelium and the basal membrane, and that it is involved in the migration and proliferation of oral keratinocytes.

Induction of invasion in an organotypic oral cancer model by CoCl2, a hypoxia mimetic.

Invasion is a hallmark of malignancy. The aim of this study was to develop an in vitro model that can be used for experimental studies of cancer cell invasion. The organotypic oral cancer model was constructed by growing oral squamous cell carcinoma (OSCC) cells on a collagen matrix in which normal human fibroblasts were incorporated. Immunohistochemical staining of the model showed that the expression of invasion-related molecules such as phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), cyclooxygenase-2 (COX-2), p75(NTR), and hepatocyte growth factor receptor (Met) was similar to that seen in OSCC. Treatment of the model with cobalt chloride (CoCl(2)) to mimic hypoxic conditions increased cancer cell invasion, defined as the appearance of cancer cell islands protruding into the matrix. Models treated with CoCl(2) showed increased expression of p75(NTR) and laminin-5 in the cancer cells, and a more pronounced fragmentation of collagen IV in the basal membrane area, in contrast to models that were left untreated. The results indicate that the present model is well suited for studies on cancer cell invasion in the matrix and that the addition of CoCl(2) on day 3 of the experiment is indicated because it markedly increases the invasion and improves the model.

A study of phosphorylated ERK1/2 and COX-2 in early stage (T1-T2) oral squamous cell carcinomas.

BACKGROUND: Histomorphological grading at the invasive front of oral squamous cell carcinomas (OSCCs) may provide useful prognostic information. In the present study, we investigated the presence and prognostic value of activated phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2) and cyclo-oxygenase-2 (COX-2) both at the invasive front and in central/superficial parts of OSCCs. METHODS: Using immunohistochemistry, we assessed the presence of p-ERK1/2 and COX-2 in 53 early stage OSCCs. Clinical data were recorded prospectively. The end point was disease-free survival. RESULTS: p-ERK1/2 staining was present in almost all tumours. The staining was mostly nuclear in the cells of the invasive front and either nuclear or nuclear/cytoplasmic in central/superficial tumour parts. COX-2 was observed in almost all tumours (98%) and the staining was often restricted to focal areas. Most tumours were COX-2 negative at the invasive front. The lowest P-value in survival analyses was P = 0.06 for p-ERK1/2 at the invasive front. COX-2, the histomorphological grading systems and TNM stage were of no prognostic value. CONCLUSION: p-ERK1/2 was present in almost all tumours and p-ERK1/2 may be a prognostic marker at the invasive front of OSCCs. In early stage OSCCs, most tumours did not express COX-2 at the invasive front.

Molecular based treatment of oral cancer.

Given the increase in the age distribution of the population, an increase in cancer incidence rates are to be expected. Oral cancer is a disfiguring disease that continues to increase in incidence, particularly in the young, and to an extent that cannot be fully explained by increased exposure to known risk factors. Despite extensive research on treatment modalities towards oral cancer, the 5-year survival rate of this disease has not been improved over the last 4-5 decades. These facts strongly favour chemoprevention-systemic medication to revert, stop, or delay the carcinogenic process-as an approach to treating oral cancer. A chemopreventive approach to oral cancer most likely should encompass a combination of drugs targeting metabolic pathways relevant to oral carcinogenesis. Candidate drugs are retinoids and selective inhibitors of cyclooxygenase-2, epidermal growth factor receptor (EGFR), and peroxisome proliferator activated receptors (PPARs). Chemopreventive trials so far have used surrogate intermediate biomarkers as measurement of treatment effect. However, the efficiency of any drug for chemopreventive use should be assessed through a prospective randomized trial and evaluated by the only definitive end point for prevention of cancer, the incidence rates of new carcinomas.