Pathological changes in oral epithelium and the expression of SARS-CoV-2 entry receptors, ACE2 and furin.

BACKGROUND: Expression of angiotensin-converting enzyme (ACE)-2 and co-factors like furin, play key-roles in entry of SARS-CoV-2 into host cells. Furin is also involved in oral carcinogenesis. We investigated their expression in oral pre-malignant/malignant epithelial pathologies to evaluate whether ACE2 and furin expression might increase susceptibility of patients with these lesions for SARS-CoV-2 infection. METHODS: Study included normal oral mucosa (N = 14), epithelial hyperplasia-mild dysplasia (N = 27), moderate-to-severe dysplasia (N = 24), squamous cell carcinoma (SCC, N = 34) and oral lichen planus (N = 51). Evaluation of ACE2/furin membranous/membranous-cytoplasmic immunohistochemical expression was divided by epithelial thirds (basal/middle/upper), on a 5-tier scale (0, 1-weak, 1.5 -weak-to-moderate, 2-moderate, 3-strong). Total score per case was the sum of all epithelial thirds, and the mean staining score per group was calculated. Real time-polymerase chain reaction was performed for ACE2-RNA. Statistical differences were analyzed by One-way ANOVA, significance at p<0.05. RESULTS: All oral mucosa samples were negative for ACE2 immuno-expression and its transcripts. Overall, furin expression was weakly present with total mean expression being higher in moderate-to-severe dysplasia and hyperplasia-mild dysplasia than in normal epithelium (p = 0.01, each) and SCC (p = 0.008, p = 0.009, respectively). CONCLUSIONS: Oral mucosa, normal or with epithelial pathologies lacked ACE2 expression. Furin was weak and mainly expressed in dysplastic lesions. Thus, patients with epithelial pathologies do not seem to be at higher risk for SARS-CoV-2 infection. Overall, results show that oral mucosae do not seem to be a major site of SARS-CoV-2 entry and these were discussed vis-à-vis a comprehensive analysis of the literature.

Oral erythroplakia and oral erythroplakia-like oral squamous cell carcinoma - what's the difference?

BACKGROUND: Oral erythroplakia (OE) is a rare oral potentially malignant disorder, that has a high rate of malignant transformation. The definition of OE still lacks uniformity. In particular, lesions that look clinically like erythroplakias, but are histopathologically diagnosed as squamous cell carcinomas are still sometimes called erythroplakias. The purpose of this study is to present demographic and clinicopathologic features of a series of OEs and clinically oral erythroplakia -like squamous cell carcinomas (OELSCC), to study their differences and to discuss the definition of OE. METHODS: A multicenter retrospective case series of OEs and OELSCCs. Descriptive statistics were used to analyze the data. RESULTS: 11 cases of OEs and 9 cases of OELSCCs were identified. The mean age of the OE patients was 71 years and 72.7% were female, while the mean age of the OELSCC patients was 69 years, and all were female. 9% of the OE and 22% of the OELSCC patients had smoked or were current smokers. 72.7% of the OEs and 55.5% of OELSCCs were uniformly red lesions. 63.6% of the OE and 22% of the OELSCC patients had a previous diagnosis of oral lichenoid disease (OLD). The malignant transformation rate of OE was 9% in a mean of 73 months. CONCLUSIONS: OE and OELSCC may arise de novo or in association with OLD. Tobacco and alcohol use were not prevalent in the present cases. The clinical features of OEs and OELSCC are similar, but symptoms, uneven surface and ulceration may be more common in OELSCCs than in OEs. Clinical recognition of OE is important since it may mimic other, more innocuous red lesions of the oral mucosa. The diagnosis of OE requires biopsy and preferably an excision. Clarification of the definition of OE would aid in clinical diagnostics.

The profile of chronic hyperplastic candidiasis: a clinico-pathological study.

The aims of this study were investigation of clinical presentation, systemic factors, and long-term malignant transformation rate in chronic hyperplastic candidiasis versus leukoplakia. This is a retrospective case-controlled study of cases with chronic hyperplastic candidiasis and leukoplakia without dysplasia, diagnosed between 2000 and 2013. A database was created, and all additional biopsies from the same cases were searched up to 2022, for records of oral malignant transformation. Associations between microscopic diagnoses and clinical features of lesions and clinical outcomes of patients were performed. A study database included 116 patients, allocated to the group diagnosed with chronic hyperplastic candidiasis (CHC-group, 62) and to the group of leukoplakia without dysplasia (LKP-group, 54). Tongue and buccal mucosa were most frequently recorded in both groups. In CHC-group, significantly fewer cases presented as white lesions compared to LKP-group (P < 0.001); more were ulcerated or exophytic (P = 0.006 and P = 0.003, respectively). History of head and neck malignancy was significantly more frequent in CHC-group (P = 0.005), as were chemotherapy, (P = 0.019) radiotherapy (P = 0.0265), and immune-related conditions (P = 0.03). Within the follow-up period (2000-2022), in CHC-group, two cases (3.2%) had malignant transformation at the site of original biopsy, one was recurrence of previous carcinoma. In LKP-group, two cases (3.7%) had newly diagnosed carcinoma and one at the site of original biopsy; no significant differences were found between groups. In conclusion, medical background of immune-related conditions, head and neck malignancy, radiotherapy, and chemotherapy may play a role in predisposing for chronic hyperplastic candidiasis. Malignant transformation rate within CHC-group was low, and similar to that within LKP-group, representing a lower transformation rate than expected.

The effect of post-COVID-19 on gustatory and olfactory function: A preliminary case-controlled study.

AIMS: The aim of the study was to analyze objective and subjective olfactory/gustatory function in post-COVID-19 infection (PCI). MATERIALS AND METHODS: Patients with past PCR-confirmed COVID-19 infection and persistent olfactory/gustatory complaints were investigated. Olfactory threshold and identification, gustatory detection, identification, and magnitude scaling were tested. RESULTS: A total of 42 PCI subjects were compared to 41 age- and gender-matched controls with no COVID-19 history. All PCI tested had mild COVID-19 disease. Mean interval between COVID-19 confirmations to testing was 7.4 ± 3.1 months. PCI subjects complained of combined dysfunction in 85.7%, isolated olfactory or gustatory dysfunction in 7.1% each. Combined complaints were significantly higher in PCI (p < 0.001). Objective testing showed significantly higher prevalence of dysfunction in PCI versus controls for hyposmia (73.8%, 12.2%), anosmia (11.9%, 0%), odor identification (68.5%, 83.0%), hypogeusia (23% and 2.4%, respectively), and impaired magnitude scaling, (p < 0.05). All PCI subjects with hypogeusia had abnormal gustatory magnitude scaling. CONCLUSIONS: While most PCI subjects complained of combined gustatory and olfactory dysfunction, objective testing showed in the majority an isolated single sense dysfunction, with a low level of agreement between subjective and objective findings. Abnormal objective results for all olfactory and gustatory functions tested may suggest a central rather than peripheral mechanism, although concomitant mechanisms cannot be excluded.

Histopathological and histomorphometric analysis of glandular odontogenic cyst-A diagnostic aid.

OBJECTIVES: Compare recognized microscopic parameters, including variations in width, plaque-like thickenings, intra-epithelial microcysts, clefts, mucous, hob-nail, ciliated and clear cells, between glandular odontogenic cyst (GOC) and GOC-like cysts, investigate the extent of cyst circumference exhibiting these features, and inflammation. MATERIALS AND METHODS: Archival records of cysts with histological features of GOC evaluated between 2000 and2020 were retrieved. Slides were revised, and the expression of features throughout the cyst wall was analyzed. Cysts with at least 5 features were classified as GOC, cysts with 3-4 features as GOC-like. RESULTS: The study included 74 cysts, 47 males M, 25 females (2 unknown gender), aged 19-81 years, 62 (83.8%) GOC, 12 (16.2%) GOC-like. Mandible was involved in 44 (59.5%), maxilla in 30 (40.5%), 18 (25%) were associated with unerupted teeth. Cyst classified as GOC had significantly higher rates of all parameters investigated, (except ciliated and clear cells), than GOC-like cysts (p ≤ 0.05). 26 (40.6%) cases showed GOC features in >50% of cyst circumference, 21 (32.8%) involved 25-50%, 17 (26.6%) <25%. More than 50% circumference involvement was highly and independently predictive for a diagnosis of GOC, <25% was highly and independently predictive for GOC-like (p = 0.003). Hobnail cells (p = 0.008) and plaque-like thickenings (p = 0.038) were significantly more frequent in inflamed cysts. CONCLUSION: Besides the number and type of histological features, GOC can be characterized by their distribution within the cyst circumference (focal Vs diffuse), and it may serve as a new diagnostic aid. It is suggested that GOC and GOC-like may represent a single spectrum.

Unexpected Candidal Hyphae in Oral Mucosa Lesions-A Clinico-Pathological Study.

Background: Oral mucosal biopsies might harbor candidal hyphae (CH) in the absence of any clinical signs or symptoms. Aim: To assess oral mucosa biopsies for the frequency of unexpected CH and characterize their clinico-pathological features. Materials and Methods: All biopsy reports (2004−2019) were searched using CH/candida/candidiasis as key words. Cases with clinical diagnosis of oral candidiasis (OC) were excluded. Demographic data, health status, smoking habits, clinical features and diagnoses were collected. Statistical analysis included the chi-square test; significance was set at p < 0.05. Results: Of all the biopsies, 100 (1.05%) reported microscopical evidence of CH without typical clinical signs/symptoms of OC. Fifteen cases were from healthy, non-smoking patients. CH was common on buccal mucosa (38%) and lateral tongue (23%). The tip of tongue (OR = 54.5, 95% CI 9.02−329.4, p < 0.001) and lateral tongue (OR = 3.83, 95% CI 2.4−6.09, p < 0.001) were more likely to harbor CH-positive lesions. CH-positive lesions were diagnosed as epithelial hyperplasia (55%) and exophytic reactive lesions (30%). No correlation was found between CH and the grade of epithelial dysplasia. Conclusions: Microscopic evidence of CH embedded into oral epithelium without typical signs/symptoms of OC is rare, especially in healthy, non-smokers. Since CH was occasionally found in oral sites prone to local trauma and in association with reactive lesions, in absence of host co-morbidities, the contribution of local mechanical forces to CH embedment cannot be ruled out.

Minimizing MRONJ after Tooth Extraction in Cancer Patients Receiving Bone-Modifying Agents.

BACKGROUND: Medication-related osteonecrosis of the jaws (MRONJ) is a mucosal lesion of the maxillofacial region with necrotic bone exposure. MRONJ is believed to be multifactorial. Tooth extraction is debatably a risk factor for MRONJ. The targets of the present study were to examine MRONJ occurrence in patients using bone modifying agents (BMAs) for oncology indications and undergoing a dental extraction, and to assess whether suspected predisposing factors can predict MRONJ. MATERIALS AND METHODS: This retrospective, cohort study included all patients fitting the inclusion criteria and a large tertiary medical center. Data were obtained from the hospital's medical records using a structured questionnaire. RESULTS: We performed 103 extractions on 93 patients. Local inflammation/infection of the extraction site was most associated with a complication (p = 0.001) OR = 13.46, 95% CI = (1.71, 105.41), OR = 13.5. When the indication for extraction was periodontal disease, vertical root fracture, or periapical pathosis, the odds of developing MRONJ were 4.29 times higher than for all other indications (p = 0.1), OR = 4.29, 95% CI = (1.16, 15.85). A significant association was found between the time of onset of BMA treatment and time of extraction and the development of MRONJ, OR = 3.34, 95% CI = (1.01, 10.18). Other variables did not correlate with the development of MRONJ. CONCLUSION: Local inflammation/infection and onset of BMA treatment prior to extraction yield a 10.23 times higher chance of developing MRONJ following tooth extraction. Future protocols should use this information to minimize MRONJ incidence.

Conceptual changes in ameloblastoma: Suggested re-classification of a "veteran" tumor.

OBJECTIVES: The merging of ameloblastoma (AM) with mural unicystic ameloblastoma (UAM-M) was suggested by the 2017 WHO based on similar treatment needs. In an international multicenter study, we investigated the characteristics of their merged product (merged-AM) and raised the possibility of unifying AM and UAM (total-AM). MATERIALS AND METHODS: AM and UAM (luminal/intraluminal/mural), separate and combined, were analyzed for demographic/clinical/radiological features. ANOVA and chi-square tests were followed by univariate and multivariate analyses, and significance was set at p < .05. RESULTS: The patients' mean age was 39.6 ± 20.3 years in merged-AM (147 AM, 76 UAM-M), 45.1 ± 19.4 years in AM (p = .009). Merged-AM comprised 51.3% multilocular/48.7% unilocular tumors, AM comprised 72.5%/27.5%, respectively (p < .001). Merged-AM was associated with impacted teeth in 30.8%, AM in 18% (p = .023). The probability of merged-AM for multilocularity increased by 2.4% per year of age (95%CI 0.6-4.2, p = .009). Association with impacted teeth decreased by 7.9% per year of age (95%CI 1.9-14.39, p = .009). Merged-AM did not differ from total-AM (p > .05). CONCLUSIONS: Merged-AM partially differed from AM, but differences appeared to diminish in an age/time-wise manner. Merged-AM and total-AM were nearly indistinguishable. Therefore, AM and UAM may be considered a continuous spectrum of one type of tumor, further necessitating revision of the treatment approaches.

Expression of stem cell markers in stroma of odontogenic cysts and tumors.

BACKGROUND: The stroma of odontogenic cysts/tumors may confer them differential biological behavior. We aimed to investigate the immunoexpression of stem cell markers (Nanog, SOX2, Oct4, and CD34) in the stroma of odontogenic cysts and tumors. CD34 was investigated exclusively as a marker for stromal fibroblast/fibrocyte cells (CD34 + SFCs). CD34 + SFCs were also investigated ultrastructurally. METHODS: Ten cases each of primary odontogenic keratocyst (OKC), recurrent OKC, dentigerous cyst, ameloblastoma, unicystic ameloblastoma, odontogenic myxoma, and 7 syndromic OKC were included. Results were represented as the mean score (%) of positive cells/field for each marker for each study group. For CD34 + SFCs, results are presented as the mean number of cells/field for each type of lesion. Kruskal-Wallis and Spearman's correlation statistical tests were used; significance was set at P < .05. RESULTS: All markers except Oct4 were expressed by stromal cells in all lesions. Expression of SOX2 was significantly higher in tumors than in cysts (P < .05). CD34 + SFCs were more frequent in cysts than in tumors. Ultrastructurally, CD34 + SFCs were identified for the first time in odontogenic lesions and showed characteristic bipolar/dendritic morphology. CONCLUSION: Among examined stromal stem cell markers, only SOX2 distinguished tumors from cysts. CD34 + SFCs may also contribute to the biological behavior of odontogenic lesions.

Palatal Erythema with Histological Psoriasiform Pattern: An Enigmatic Oral Finding Shared by a Range of Conditions.

Long standing, asymptomatic, well-demarcated erythema of the hard palate with a histopathological psoriasiform pattern comprises a challenging diagnosis. We present a series of patients with such clinical and histological findings and discuss the possible diagnoses. We collected all patients with palatal erythematous lesions that had well-documented clinical examination. Excluded were patients with definitive diagnosis of oral infections (e.g. candidiasis), neoplastic/pre-neoplastic lesions, auto-immune diseases, reactive lesions, blood disorders and vascular malformations. Thirteen patients (six females, seven males, age range 11-56 years) were included. Histopathologically, a psoriasiform pattern was observed in all biopsied lesions. One patient was diagnosed with hereditary mucoepithelial dysplasia (HMD) and four with cutaneous psoriasis. The remaining eight patients were otherwise healthy. A combination of persistent, asymptomatic palatal erythematous lesion with psoriasis-like histopathology may represent an oral manifestation of HMD or psoriasis, concomitant to extra-oral features. In lack of any known medical background, the term "oral psoriasiform mucositis" is suggested.

Oral cancer-associated fibroblasts predict poor survival: Systematic review and meta-analysis.

OBJECTIVES: To perform systematic review and meta-analysis on correlations between cancer-associated fibroblasts (CAFs) and the risk of death for patients with oral squamous cell carcinoma. SUBJECTS AND METHODS: English literature (1966-2018) was systematically analyzed for studies that immunohistochemically assessed CAF density by alpha-smooth muscle actin and presented 5 year survival rates by Kaplan-Meier plots. Mean age of patients, proportion of male/female patients, and male/female majority (>50% male/female patients) per study were also collected. Significance level for statistical models was p < 0.05. RESULTS: Meta-analysis comprised 11 studies/1,040 patients. Univariate Cox regressions showed that high CAF density was a negative prognostic factor in studies with female and male majority [OR 5.329 (95% CI 3.223-8.811), p < 0.001, and OR 2.208 (95% CI 1.717-2.839), p < 0.001, respectively]. High CAF density with male majority was associated with a more favorable prognosis [OR 0.996 (95% CI 0.979-1.013), p < 0.001]. Multivariate Cox regressions showed that death risk was significantly higher among patients with high CAF density compared to low CAF [OR 2.741 (95% CI 2.220-3.384) p < 0.001]. High mean age and male proportion were significantly protective [OR 0.940 (95% CI 0.925-9.955), p < 0.001, OR 0.125 (95% CI 0.018-0.867), p = 0.035), respectively]. CONCLUSIONS: CAFs increased death risk, male majority, and higher mean age were protective. A clinically validated cutoff for CAF density could serve as a reliable prognostic tool.

Markers of the pre-metastatic niche "knock on the door" of metastasis-free cervical lymph nodes in patients with oral cancer.

AIM: To assess expression of some markers of the pre-metastatic niche (PMN) in lymph nodes (LNs) of oral cancer patients. MATERIALS: LNs from metastatic-free neck dissections (LN0/N0, N = 43) and metastatic-free LNs in the vicinity of metastasis-containing LNs (LN0/N+, N = 30) were immuno-histochemically stained for lysyl oxidase (LOX), fibronectin (FN), vascular-endothelial growth factor receptor (VEGFR)-1 and matrix metalloproteinase (MMP)-9. Staining was assessed as 0 (no or weak staining), 1 (strong stain in 25% cells or extracellular area), 2 (same as 1 but in up to 50%) and 3 (same as 1 but in > than 50% of cells/area). Assessment was performed in the lymph node capsule (CAP), sub-capsular sinus (SCS) and medullary sinus (MS). In addition, sections were stained with picrosirius red and examined with polarized microscopy for assessing the distribution of polarization colors of the collagen fibers in the LN capsular area. RESULTS: All examined LNs were positive for markers of the PMN. In general, the distribution and intensity of the immunoreactivity was similar between the LN0/N0 and LN0/N+, with only a few differences regarding expression of LOX in the capsule (p = 0.002) and VEGFR1 and MMP9 in the SCS (p = 0.023 and p < 0.001, respectively). Picrosirius red stain and polarized microscopy revealed a disrupted arrangement and distribution of the collagen fibers in both LN0/N0 and LN0/N + . CONCLUSION: Markers for PMN were shown for the first time to be expressed in cervical LN0/N0 from patients with oral cancer, suggesting the increased permissive pathway remotely paved by the primary oral tumor for the incoming metastatic cells.

Cancer-associated fibroblasts in the tumor microenvironment of tongue carcinoma is a heterogeneous cell population.

OBJECTIVES: To examine different immunophenotypes of cancer-associated fibroblasts (CAFs) in tongue squamous cell carcinoma (TSCC) and to investigate how they related to clinical outcomes. METHODS: Serial sections from 54 cases of TSCC were immunohistochemically stained with α-smooth muscle actin (αSMA, CAF marker) to determine CAF density, and double-immunostained with αSMA combined with CD80 and CD86 (myeloid/monocytic-derived cell markers), Nanog (mesenchymal stem cell marker) and CD133 (hematopoietic/endothelial stem cell marker). Density of cells co-expressing these marker combinations was semi-quantitatively assessed in 5 randomly selected high power fields within the tumor area and scored as 1 - one-to-five stained cells in each field, 2 - more than 5 stained cells in each field; any finding less than score 1, was allocated a score of 0. RESULTS: There were 26 CAF-poor, 16 CAF-rich and 12 CAF-intermediated cases. CD86+αSMA+ cells were the most frequent (80.4%) followed by CD80+αSMA+ (72%) and Nanog+αSMA+ cells (56%). The CD133+αSMA+ phenotype was found only in association with blood vessels. High density of αSMA+ CAFs was associated with disease recurrence and poor survival (p < 0.05). Increased density of CD86+αSMA+ cells was significantly associated with CAF-rich tumors and with poor survival (p < 0.05). CONCLUSION: In TSCC, CAFs demonstrate heterogeneous and overlapping phenotypes with the myeloid/monocytic type being the most frequent and having an impact on the clinical outcomes. Further studies are needed in order to further characterize CAF phenotypes in carcinomas of various oral sites, as this may open new frontiers for personalized medicine.

Atherosclerotic carotid plaque on panoramic radiographs: neural network detection.

AIM: Atherosclerotic carotid plaques (ACPs) constitute the main etiological factor in about 15% of strokes. ACPs can be detected on routine dental panoramic radiographs. As these are one of the most commonly performed dental images, they can be used as a source of available data for computerized methods of automatic detection of ACPs in order to significantly increase their timely diagnosis. The aim of this study was to present the potential of applying deep learning methodology to detect ACPs on routine panoramic radiographs with the ultimate goal of preventing strokes. METHODS: The Faster Region-based Convolutional Neural Network (Faster R-CNN) for deep learning was used. The operation of the algorithm was assessed on a small dataset of 65 panoramic images. As the available training data was limited, data augmentation was performed by changing the brightness and randomly flipping and rotating cropped regions of interest in multiple angles. Receiver operating characteristic (ROC) analysis was performed to calculate the accuracy of detection. RESULTS: ACPs were detected with a sensitivity of 75%, a specificity of 80%, and an accuracy of 83%. The ROC analysis showed a significant area under curve (AUC), different from 0.5. CONCLUSIONS: The novelty of the study lies in showing the efficiency of a deep learning method for the detection of ACPs on routine panoramic images based on a small dataset. Further improvement is needed as regards the application of the algorithm to the level of introducing this methodology in routine dental practice for stroke prevention.

FTIR-based spectrum of salivary exosomes coupled with computational-aided discriminating analysis in the diagnosis of oral cancer.

PURPOSE: To determine the Fourier-transform infrared (FTIR) spectra of salivary exosomes from oral cancer (OC) patients and healthy individuals (HI) and to assess its diagnostic potential using computational-aided models. METHODS: Whole saliva samples were collected from 21 OC patients and 13 HI. Exosomes were pelleted using differential centrifugation (12,000g, 120,000g). The mid-infrared (IR) absorbance spectra (900-5000 cm- 1 range) were measured using MIR8025 Oriel Fourier-transform IR equipped with a PIKE MIRacle ZnSe attenuated total reflectance attachment. Machine learning techniques, utilized to build discrimination models for the absorbance data of OC and HI, included the principal component analysis-linear discriminant analysis (PCA-LDA) and support vector machine (SVM) classification. Sensitivity, specificity and the area under the receiver operating characteristic curve were calculated. RESULTS: IR spectra of OC were consistently different from HI at 1072 cm- 1 (nucleic acids), 2924 cm- 1 and 2854 cm- 1 (membranous lipids), and 1543 cm- 1 (transmembrane proteins). The PCA-LDA discrimination model correctly classified the samples with a sensitivity of 100%, specificity of 89% and accuracy of 95%, and the SVM showed a training accuracy of 100% and a cross-validation accuracy of 89%. CONCLUSION: We showed the specific IR spectral signature for OC salivary exosomes, which was accurately differentiated from HI exosomes based on detecting subtle changes in the conformations of proteins, lipids and nucleic acids using optimized artificial neural networks with small data sets. This non-invasive method should be further investigated for diagnosis of oral cancer at its very early stages or in oral lesions with potential for malignant transformation.

Fermented Lingonberry Juice Inhibits Oral Tongue Squamous Cell Carcinoma Invasion In Vitro Similarly to Curcumin.

BACKGROUND: Oral tongue squamous cell carcinoma (OTSCC) cells are highly proliferative and invasive. Lingonberry contains several polyphenolic compounds similar to curcumin. We hypothesize that fermented lingonberry juice (FLJ) has an anti-invasive and anti-proliferative effect on OTSCC cells similarly to curcumin, which is known to be anti-carcinogenic. MATERIALS AND METHODS: FLJ, curcumin dissolved in ethanol, or curcumin loaded in Candida extracellular vesicles (EVs) were added to more (HSC-3) and less aggressive (SCC-25) OTSCC cells. Cell proliferation was measured with a 5-bromo-2'-deoxyuridine kit and invasion in the three-dimensional Myogel spheroid assay. Statistical analyses were completed with one-way ANOVA and Bonferroni post-hoc testing. RESULTS: Both FLJ and curcumin significantly reduced the proliferation and invasion of HSC-3 and SCC-25 cells. The effects of curcumin were not improved when cells were treated with curcumin loaded within EVs. CONCLUSION: Our results suggest that FLJ, like curcumin, has an anti-carcinogenic effect on aggressive OTSCC cells in vitro.

Morphological and molecular features of oral fluid-derived exosomes: oral cancer patients versus healthy individuals.

PURPOSE: Oral cancer (OC) patients are at high risk to develop recurrent disease or secondary primary cancers with no available biomarkers to detect these events until a visible lesion is readily present and diagnosed by biopsy. Exosomes secreted by cancer cells are involved in tumor growth, invasion and metastasis. We aimed to determine morphological and molecular differences between oral fluid (OF)-derived exosomes of OC patients and those isolated from healthy individuals (HI). METHODS: OF from OC patients (n = 36) and HI (n = 25) was initially assessed by nanoparticle tracking analysis (NTA). Following ultracentrifugation, exosomal pellets of OC patients and HI were morphologically examined by transmission electron microscopy and atomic force microscopy (AFM). Enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) were used to analyze the expression of exosomal markers--CD9, CD81 and CD63. RESULTS: NTA showed that OC samples of OF had a significantly higher concentration of nanoparticles/ml (p = 0.01) and modal nanoparticle size (p = 0.002) compared to HI. The difference in size was structurally highlighted by AFM three-dimensional images applied on exosomal pellets. ELISA and WB showed differential expression of exosomal markers in OC exosomes compared to HI: lower expression of CD81 and CD9 in contrast to a higher expression of CD63 (~53 kDa). CONCLUSIONS: OF-derived exosomes from OC patients differ both morphologically and molecularly from exosomes present in HI. This study is a baseline that provides a starting point for finding exosomal biomarkers for early detection of malignant changes in high-risk patients without overt clinical signs/lesions.

Caveolin-1 accumulation in the tongue cancer tumor microenvironment is significantly associated with poor prognosis: an in-vivo and in-vitro study.

BACKGROUND: Caveolin-1 (CAV1) may be upregulated by hypoxia and acts in a tumor-dependent manner. We investigated CAV1 in tongue squamous cell carcinoma (TSCC) and its association with clinical outcomes, and studied in vitro possible ways for CAV1 accumulation in the tumor microenvironment (TME). METHODS: TSCC cases (N = 64) were immunohistochemically stained for CAV1. Scores were separately assessed in the tumor and TME and plotted for association with recurrence and survival (univariate analysis with log-rank test). In vitro studies were performed on a 3D myoma organotypic model, a mimicker of TME. Prior to monoculturing HSC-3 tongue cancer cells, the model underwent modifications in oxygenation level (1%O2 hypoxia to upregulate CAV1) and/or in the amount of natural soluble factors [deleted by 14-day rinsing (rinsed myoma, RM), to allow only HSC-3-derived factors to act]. Controls included normoxia (21%O2) and naturally occurring soluble factors (intact myoma, IM). HSC-3 cells were also co-cultured with CaDEC12 cells (fibroblasts exposed to human tongue cancer). CAV1 expression and cellular distribution were examined in different cellular components in hypoxic and rinsed myoma assays. Twist served as a marker for the process of epithelial-mesenchymal transition (EMT). Exosomes isolated from HSC-3 media were investigated for containing CAV1. RESULTS: Expression of CAV1 in TSCC had a higher score in TME than in the tumor cells and a negative impact on recurrence (p = 0.01) and survival (p = 0.003). Monocultures of HSC-3 revealed expression of CAV1 mainly in the TME-like myoma assay, similar to TSCC. CAV1+, alpha-smooth muscle actin (αSMA) + and Twist + CAF-like cells were observed surrounding the invading HSC-3, possibly reflecting EMT. RM findings were similar to IM, inferring action of HSC-3 derived factors, and no differences were seen when hypoxia was induced. HSC-3-CaDEC12 co-cultures revealed CAV1+, αSMA+ and cytokeratin-negative CAF-like cells, raising the possibility of CaDEC12 cells gaining a CAF phenotype. HSC-3-derived exosomes were loaded with CAV1. CONCLUSIONS: Accumulation of CAV1-TME in TSCC had a negative prognostic value. In vitro studies showed the presence of CAV1 in cancer cells undergoing EMT and in fibroblasts undergoing trans-differentiation to CAFs. CAV1 delivery to the TME involved cancer cell-derived exosomes.

Human saliva-derived exosomes: comparing methods of isolation.

ExoQuick-TC(TM) (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.

Insights into the role of components of the tumor microenvironment in oral carcinoma call for new therapeutic approaches.

The research on oral cancer has focused mainly on the cancer cells, their genetic changes and consequent phenotypic modifications. However, it is increasingly clear that the tumor microenvironment (TME) has been shown to be in a dynamic state of inter-relations with the cancer cells. The TME contains a variety of components including the non-cancerous cells (i.e., immune cells, resident fibroblasts and angiogenic vascular cells) and the ECM milieu [including fibers (mainly collagen and fibronectin) and soluble factors (i.e., enzymes, growth factors, cytokines and chemokines)]. Thus, it is currently assumed that TME is considered a part of the cancerous tissue and the functionality of its key components constitutes the setting on which the hallmarks of the cancer cells can evolve. Therefore, in terms of controlling a malignancy, one should control the growth, invasion and spread of the cancer cells through modifications in the TME components. This mini review focuses on the TME as a diagnostic approach and reports the recent insights into the role of different TME key components [such as carcinoma-associated fibroblasts (CAFs) and inflammation (CAI) cells, angiogenesis, stromal matrix molecules and proteases] in the molecular biology of oral carcinoma. Furthermore, the impact of TME components on clinical outcomes and the concomitant need for development of new therapeutic approaches will be discussed.