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.

Macrophages modulate migration and invasion of human tongue squamous cell carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) has a high mortality rate and the incidence is rising worldwide. Despite advances in treatment, the disease lacks specific prognostic markers and treatment modality. The spreading of OTSCC is dependent on the tumor microenvironment and involves tumor-associated macrophages (TAMs). Although the presence of TAMs is associated with poor prognosis in OTSCC, the specific mechanisms underlying this are still unknown. The aim here was to investigate the effect of macrophages (Mfs) on HSC-3 tongue carcinoma cells and NF-kappaB activity. We polarized THP-1 cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type Mfs. We then investigated the effect of Mfs on HSC-3 cell migration and NF-kappaB activity, cytokine production and invasion using several different in vitro migration models, a human 3D tissue invasion model, antibody arrays, confocal microscopy, immunohistochemistry and a mouse invasion model. We found that in co-culture studies all types of Mfs fused with HSC-3 cells, a process which was partially due to efferocytosis. HSC-3 cells induced expression of epidermal growth factor and transforming growth factor-beta in co-cultures with M2 Mfs. Direct cell-cell contact between M2 Mfs and HSC-3 cells induced migration and invasion of HSC-3 cells while M1 Mfs reduced HSC-3 cell invasion. M2 Mfs had an excess of NF-kappaB p50 subunit and a lack of p65 subunits both in the presence and absence of HSC-3 cells, indicating dysregulation and pro-tumorigenic NF-kappaB activation. TAM-like cells were abundantly present in close vicinity to carcinoma cells in OTSCC patient samples. We conclude that M2 Mfs/TAMs have an important role in OTSCC regulating adhesion, migration, invasion and cytokine production of carcinoma cells favouring tumor growth. These results demonstrate that OTSCC patients could benefit from therapies targeting TAMs, polarizing TAM-like M2 Mfs to inflammatory macrophages and modulating NF-kappaB activity.

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.

Histopathological spectrum of bone lesions associated with dental implant failure: osteomyelitis and beyond.

Early or late post-implant placement complications are usually localized infectious/inflammatory processes and treated accordingly. If the healing process does not take place within a reasonable timeframe, the possibility of a pathologic process beyond localized infection/inflammation should be suspected. We describe a radiological/histopathological spectrum of bony lesions ranging from inflammatory to malignant lesions surrounding failed dental implants. Five cases of mandibular dental implant failure that clinically, radiologically and histopathologically appeared to be inflammatory processes are presented. The failure of the dental implants was immediate in two cases and late in the remaining three. The radiological features were essentially similar for all five, and they included radiolucent or mixed radiolucent-radiopaque lesions with poorly defined borders. Three lesions were limited to the area of the failed implant, while the other two extended to a large part of the mandible. The histopathological findings ranged from acute osteomyelitis and chronic osteomyelitis with features of a fibro-osseous-like lesion and occasional rimming of atypical osteoblasts to osteogenic sarcoma that was admixed with a component of osteomyelitis (diagnosis of the latter was achieved only after a series of biopsies). In-depth investigative procedures are imperative in order to establish an accurate diagnosis whenever the histopathological diagnosis is inconsistent with persisting clinical signs and symptoms in bone lesions associated with failed dental implants.

The protective effect of p16(INK4a) in oral cavity carcinomas: p16(Ink4A) dampens tumor invasion-integrated analysis of expression and kinomics pathways.

A large body of evidence shows that p16(INK4a) overexpression predicts improved survival and increased radiosensitivity in HPV-mediated oropharyngeal squamous cell carcinomas.(OPSCC). Here we demonstrate that the presence of transcriptionally active HPV16 in oral cavity squamous cell carcinomas does not correlate with p16(INK4a) overexpression, enhanced local tumor immunity, or improved outcome. It is interesting that HPV-mediated oropharyngeal squamous cell carcinomas can be categorized as having a 'nonaggressive' invasion phenotype, whereas aggressive invasion phenotypes are more common in HPV-negative squamous cell carcinomas. We have developed primary cancer cell lines from resections with known pattern of invasion as determined by our validated risk model. Given that cell lines derived from HPV-mediated oropharyngeal squamous cell carcinomas are less invasive than their HPV-negative counterparts, we tested the hypothesis that viral oncoproteins E6, E7, and p16(INK4a) can affect tumor invasion. Here we demonstrate that p16(INK4a) overexpression in two cancer cell lines (UAB-3 and UAB-4), derived from oral cavity squamous cell carcinomas with the most aggressive invasive phenotype (worst pattern of invasion type 5 (WPOI-5)), dramatically decreases tumor invasiveness by altering expression of extracellular matrix remodeling genes. Pathway analysis integrating changes in RNA expression and kinase activities reveals different potential p16(INK4a)-sensitive pathways. Overexpressing p16(INK4a) in UAB-3 increases EGFR activity and increases MMP1 and MMP3 expression, possibly through STAT3 activation. Overexpressing p16(INK4a) in UAB-4 decreases PDGFR gene expression and reduces MMP1 and MMP3, possibly through STAT3 inactivation. Alternatively, ZAP70/Syk might increase MUC1 phosphorylation, leading to the observed decreased MMP1 expression.

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.

Key architectural changes in tumor-negative lymph nodes from metastatic-free oral cancer patients are valuable prognostic factors.

Regional lymph node (LN) metastasis in oral cancer patients is the most significant grave prognostic factor. We evaluated the relationship between clinical outcomes and different histopathological changes in tumor-negative LNs (LN0) selected from neck dissections without metastatic disease (pN0). A total of 435 LN0 selected from pN0 neck dissections (up to three nodes in each level) were scored for histopathological parameters of LN areas, capsule thickness, subcapsular and medullary sinus ectasia, lobular architecture and percent of cortical reactive follicles. These were compared to 328 LN0 selected from neck dissections with metastases (pN+) after exclusion of metastatic LNs. Data were presented by maximum scores of each parameter in I-III (close) and in IV-V (distant) levels. Limited data from level V and regression analyses inferred that the values in level IV represented the worst changes for most patients. Cox proportional hazard regression on each parameter in close and distant levels demonstrated that capsule thickness, number of lobules and percent of reactive follicles were significantly associated with time to death from disease. The higher the change in distant levels, the shorter the time to death, while the higher the change in close levels (given a stable change in distant levels), the longer the time to death. After adjustment for gender, age and location, only the effect of the percent of reactive follicles retained their significant effect. Logistic regression of metastases demonstrated that all parameters except for percent of reactive follicles were significantly associated with risk of metastases, with differences between close and distant levels similar to those found for time to death. After adjustment for gender, age and location, only the area and number of lobes retained their significance. The findings of this study suggested that selective histopathological changes in tumor-negative LNs in metastatic-free patients provide new valuable prognostic parameters.

Human bone marrow mesenchymal stem cells induce collagen production and tongue cancer invasion.

Tumor microenvironment (TME) is an active player in carcinogenesis and changes in its composition modify cancer growth. Carcinoma-associated fibroblasts, bone marrow-derived multipotent mesenchymal stem cells (BMMSCs), and inflammatory cells can all affect the composition of TME leading to changes in proliferation, invasion and metastasis formation of carcinoma cells. In this study, we confirmed an interaction between BMMSCs and oral tongue squamous cell carcinoma (OTSCC) cells by analyzing the invasion progression and gene expression pattern. In a 3-dimensional myoma organotypic invasion model the presence of BMMSCs inhibited the proliferation but increased the invasion of OTSCC cells. Furthermore, the signals originating from OTSCC cells up-regulated the expression of inflammatory chemokines by BMMSCs, whereas BMMSC products induced the expression of known invasion linked molecules by carcinoma cells. Particularly, after the cell-cell interactions, the chemokine CCL5 was abundantly secreted from BMMSCs and a function blocking antibody against CCL5 inhibited BMMSC enhanced cancer invasion area. However, CCL5 blocking antibody did not inhibit the depth of invasion. Additionally, after exposure to BMMSCs, the expression of type I collagen mRNA in OTSCC cells was markedly up-regulated. Interestingly, also high expression of type I collagen N-terminal propeptide (PINP) in vivo correlated with the cancer-specific mortality of OTSCC patients, whereas there was no association between cancer tissue CCL5 levels and the clinical parameters. In conclusion, our results suggest that the interaction between BMMSC and carcinoma cells induce cytokine and matrix molecule expression, of which high level of type I collagen production correlates with the prognosis of OTSCC patients.

[Tongue cancer: an ongoing problem which needs new therapeutic approaches].

Oral cancer together with oropharyngeal cancer constitutes the tenth most common malignancy in men worldwide. In spite of a general trend of mild decreases in the frequency of oral and pharyngeal cancers, there appears to be an increasing trend in the occurrence of oral tongue cancer (Localized in the anterior two-thirds of the tongue). Furthermore, in recent years, tongue cancer is on the rise in young patients (fifth decade and less). Tongue cancer is the most frequent type of malignancy in the oral cavity and is characterized by remarkable aggressive biological behavior. The 5-year survival rate in patients with tongue cancer has steadily remained lower than 50% during the last 40 years. This is in contrast to the considerable improvement in the survival rates that have been achieved in patients with solid tumors in other parts of the body, due to the introduction of advanced diagnostic means and new treatment modalities. Our recent studies have shown an important role that components of the tumor microenvironment (TME) play in the proliferation, invasion and local and distant spread of tongue cancer, which is not less than that of the cancer cells. The results of these studies suggest the development of new therapeutic modalities in tongue cancer, which should combine anti-cancer and anti-TME agents.

Nutraceuticals as new treatment approaches for oral cancer: II. Green tea extracts and resveratrol.

Nutraceuticals with anti-neoplastic potential are suitable candidates for extending the range of therapeutic options for several types of cancers. One of these malignancies is oral cancer of the squamous cell carcinoma type, for which current treatment approaches have not succeeded in improving long-term clinical outcome. We recently reviewed the beneficial effects of curcumin for the treatment of oral cancer. In the current review, we focused on the beneficial effects of other two nutraceuticals, green tea extracts [especially (-)-epigallocatechin-3-gallate (EGCG)] and resveratrol, in the treatment of oral cancer. In vivo and in vitro studies as well as clinical trials were reviewed, focusing on the beneficial effect of each of these plant-derived dietary agents, either alone or in combination with various pharmacological agents. We also presented the anti-cancer effects against cancer cells and against components of the tumor microenvironment. It emerged that the poor bioavailability of these nutraceuticals poses an obstacle to their exerting adequate anti-cancer potential. Ground-breaking studies employing new nanotechnology-based therapeutic approaches were presented.

Expression of the homeostasis-related markers, maspin, heat shock proteins 70 & 90, glutathione S-transferase, aquaporin 5 and NF-kB in young and old labial and palatal salivary glands.

Intraoral salivary glands undergo remarkable age-related morphologic changes. This study investigated the expression of a panel of molecular markers known for cellular homeostatic activity, dependent on age and location of the salivary glands. Samples taken from healthy subjects were classified according to age ("young" <45 years, n=51, and "old" ≥60 years, n=45) and location (lip, n=47 and palate, n=49). They were immunohistochemically stained for mammary serine protease inhibitor (maspin), heat shock protein (HSP)70, HSP90, glutathione S-transferase (GST), aquaporine5 (AQP5), and nuclear factor kappa-B (NF-κB) for assessment of their expression in acini and ducts, and in cytoplasmic and nuclear compartments. Results were expressed as the mean percentage of positively stained component per age group, gland location and type of cell and cellular compartment. Statistical analysis was performed by two-way ANOVA and crosstabs. The expression of maspin was lower in the old group in both the palatal and labial glands (acini and ducts, cytoplasm and nuclei) compared to the young group (p<0.05). In both age groups, when compared to labial glands, palatal glands exhibited higher expression of HSP70 (p<0.05) and lower expression of AQP5 (p<0.001) and NF-κB (p=0.018). Collectively, the low expression of factors capable of preserving cellular homeostasis (i.e., maspin and AQP5) vis-à-vis a high expression of factors that are also related to cell survival (i.e., HSPs) that was demonstrated in the old palatal glands may point to their high vulnerability to undergo selective phenotypic changes.

Nutraceuticals as new treatment approaches for oral cancer--I: Curcumin.

Oral squamous cell carcinoma (OSCC) is a growing global public health problem for which standard therapeutic strategies have failed to contribute significantly to improve the survival rates that have remained around 50% over the past three decades. Therefore, there is a pressing need for new therapeutic strategies. Curcumin is a natural dietary compound with known anti-neoplastic activities, hence its classification as a nutraceutical agent. This review presents the current in vitro and in vivo studies in which curcumin has been examined for its anti-cancer potential in treating OSCC. Its mechanisms of action are also beginning to become unveiled. The available studies have been focusing on the impact of curcumin on epithelial malignant cells, but overlooking the components of the tumor microenvironment. Curcumin has been emerging as a promising therapeutic agent in oral cancer, either alone or in combination with standard therapeutic agents, and will probably become of practical use once its route of administration has overcome its poor bioavailability.

Inflammatory cells of immunosuppressive phenotypes in oral lichen planus have a proinflammatory pattern of expression and are associated with clinical parameters.

OBJECTIVES: We sought to investigate the expression of cells with immunosuppressive/protumorigenic phenotypes in oral lichen planus (OLP), such as M2-tumor-associated macrophages (TAM2), myeloid-derived suppressive cells (MDSCs), and regulatory T cells (Tregs) in association with clinical parameters. MATERIALS AND METHODS: Cases of hyperkeratotic (HK)-OLP (n = 23) and erosive (E)-OLP (n = 26) were immunohistochemically stained to determine the percentages of CD163-TAM2, CD80-MDSCs, and FOXP3-Tregs of proinflammatory CD121a-Th17, CD4 and CD8 lymphocytes, and of cells positive for nuclear factor kappa B (NF-κB) and transforming growth factor beta. Clinical parameters included symptoms, treatment approach, treatment response, and others. RESULTS: The inflammatory infiltrate in HK-OLP and E-OLP contained immunosuppressive cells; however, their pattern of expression was compatible with a proinflammatory response [membranous CD163-TAM2 staining (not extracellular), CD80+ lymphocytes (not macrophages), and a few Tregs]. The presence of CD4+, CD8+, and CD121a+ T lymphocytes was extensive. TAM2 were more frequent in E-OLP than in HK-OLP (P = 0.017). A higher frequency of CD80+ lymphocytes was associated with partial to no response to treatment (P = 0.028). Nuclear expression of NF-κB in the inflammatory cells was absent. CONCLUSIONS: The pattern of expression of the immunosuppressive cells, together with numerous CD4+, CD8+, and Th17-CD121a+ lymphocytes, suggest an extensive proinflammatory response rather than an immunosuppressive/protumorigenic response. CLINICAL RELEVANCE: The frequency of selective types of inflammatory cells calls for individual profile analyses of inflammatory infiltrates and individually adjusted treatment.

Validation of the risk model: high-risk classification and tumor pattern of invasion predict outcome for patients with low-stage oral cavity squamous cell carcinoma.

The risk model is a validated outcome predictor for patients with head and neck squamous cell carcinoma (Brandwein-Gensler et al. in Am j surg pathol 20:167-178, 2005; Am J Surg Pathol 34:676-688, 2010). This model may potentially shift treatment paradigms for patients with low-stage cancers, as current protocols dictate that they might receive only primary surgery. Here we test the hypothesis that the Risk Model has added prognostic value for low-stage oral cavity squamous cell carcinoma (OCSCC) patients. 299 patients with Stage I/II OCSCC were characterized according to the risk model (Brandwein-Gensler et al. in Am J Surg Pathol 20:167-178, 2005; Am J Surg Pathol 34:676-688, 2010). Cumulative incidence and competing risk analysis were performed for locoregional recurrence (LRR) and disease-specific survival (DSS). Receiver operating characteristic analyses were performed for worst pattern of invasion (WPOI) and the risk categories. 292 patients were analyzed; 30 T1N0 patients (17%) and 26 T2N0 patients (23%) developed LRR. Disease-specific mortality occurred in 9 T1N0 patients (6%) and 9 T2N0 patients (10%). On multivariable analysis, the risk model was significantly predictive of LRR (p = 0.0012, HR 2.41, 95% CI 1.42, 4.11) and DSS (p = 0.0005, HR 9.16, 95% CI 2.65, 31.66) adjusted for potential confounders. WPOI alone was also significantly predictive for LRR adjusted for potential confounders with a cut-point of either WPOI-4 (p = 0.0029, HR 3.63, 95% CI 1.56, 8.47) or WPOI-5 (p = 0.0008, HR 2.55, 95% CI 1.48, 4.41) and for DSS (cut point WPOI-5, p = 0.0001, HR 6.34, 95% CI 2.50, 16.09). Given a WPOI-5, the probability of developing locoregional recurrence is 42%. Given a high-risk classification for a combination of features other than WPOI-5, the probability of developing locoregional recurrence is 32%. The Risk Model is the first validated model that is significantly predictive for the important niche group of low-stage OCSCC patients.

The hypoxic tumor microenvironment regulates invasion of aggressive oral carcinoma cells.

Invasion is an important hallmark of cancer involving interactions between the tumor microenvironment and the cancer cells. Hypoxia, low oxygen level, is related to increased invasion and metastasis in many cancers. The aim was to elucidate the effect of hypoxia on invasion of oral squamous cell carcinoma cells (OSCCs), and the applicability of a novel 3-dimentional myoma organotypic invasion model in hypoxia experiments. OSCC cell lines (primary oral carcinoma derived cells UT-SCC-43A, recurrent oral carcinoma cells UT-SCC-43B and aggressive tongue carcinoma cells HSC-3) were studied for their migration and invasion capabilities under normoxia, hypoxia, and in the presence a hypoxia-mimicker cobalt chloride. As expected, the recurrent UT-SCC-43B cells were significantly more aggressive than the primary tumor derived cells. In contrast to tongue carcinoma HSC-3 cells, they only mildly responded to hypoxia in the migration or invasion assays, indicating a cell line specific response of hypoxia on the invasive potential. The modification of the organotypic human tissue-derived matrix via the removal of various yet unidentified soluble factors by rinsing the tissue resulting in stripped matrix substantially changed the invasion pattern of HSC-3 cells and the outcomes of hypoxic treatments. Only in the stripped tissue hypoxia significantly increased invasion, whereas in native intact tissue the induced invasion was not observed. This demonstrates the importance of the soluble factors to the invasion pattern and to the hypoxia response. A metastasis and poor prognosis marker, hypoxia-regulated lysyl oxidase (LOX), was present in the myoma tissue, but could be removed by rinsing. The inhibition of LOX resulted in a decrease in invasion area, but only very mildly in invasion depth. Thus, it may have a role in the modulation of the invasion pattern. Another hypoxia-related poor prognosis marker carbonic anhydrase 9 (CAIX) was induced in HSC-3 cells both by the hypoxic exposure and interestingly in invading HSC-3 cells inside the tissue even in normoxic conditions. In conclusion, this suggests that the intact myoma organotypic model offers optimally hypoxic surroundings, thus being an excellent human tumor microenvironment mimicker.

Immunohistochemical features of 3,3',4,4'-tetrachloroazobenzene-induced rat gingival lesions.

Gingival lesions of squamous hyperplasia, cystic keratinizing hyperplasia (CKH), and squamous cell carcinoma (SCC) can be induced in rats treated by chronic gavage with 10-100 mg/kg 3,3',4,4'-tetrachloroazobenzene. We evaluated gingival squamous hyperplasia (GSH), CKH, and SCC for the immunohistochemical pattern of expression of carcinogenesis-associated markers. The 3 types of lesions and controls were stained with proliferation markers (proliferating cell nuclear antigen [PCNA] and cyclin-D1), tumor-suppressor markers (ß-catenin and mammary serine protease inhibitor [maspin]) and stroma-related markers (α-smooth muscle actin [SMA] and osteonectin/SPARC). The lesions had common immunohistochemical characteristics that differed in their expression patterns among the various diagnoses. PCNA and cyclin-D1 expression was higher in GSH, CKH, and SCC than in controls. The normal membranous expression of ß-catenin was lower in GSH, and almost absent in CKH and SCC. Maspin expression was similar in GSH and controls, whereas both CKH and SCC showed decreased expression. SMA and/or osteonectin/SPARC were seen in stromal cells in CKH and SCC. Collectively, there appears to be a progression from hyperplastic and cystic lesions toward malignancy based on the morphological changes, supported by the expression of carcinogenesis-associated proteins. The exact sequence of events leading to SCC remains to be defined in a time-dependent manner.

Epithelial salivary gland tumors in two distant geographical locations, Finland (Helsinki and Oulu) and Israel (Tel Aviv): a 10-year retrospective comparative study of 2,218 cases.

Salivary gland tumors (SGTs) of epithelial origin are relatively rare, and worldwide reports show considerable variations in their epidemiology. The aim of this study was to examine, for the first time, the records of SGTs from two very distant geographical locations, Finland (two medical centers) and Israel (one medical center) between 1999 and 2008, based exclusively on the 2005 WHO classification of head and neck tumors, and to compare those data to the other available (single-center) studies that used the same classification. A total of 2,218 benign and malignant tumors diagnosed in the three centers were analyzed. Differences in classification of the tumors were found between the two geographical locations as well as between the two centers from Finland. There was a higher ratio of benign-to-malignant SGTs in the Finnish centers (5.4:1 and 7:1) compared to the Israeli center (2:1), a higher frequency of tumors of minor salivary glands in the Israeli center (34%) than in the Finnish centers (4 and 11%), and a higher frequency of malignant SGTs in the minor salivary glands in Israel (64.5%) than in Finland (10.9 and 27%). The diversity of these multicenter data are compatible with reports from different parts of the world. We conclude that conducting epidemiologic surveys based on the latest WHO classification provides clinicopathologic correlations on SGTs that seem to be characteristic even in small geographical regions.

E-cadherin in oral SCC: an analysis of the confusing literature and new insights related to its immunohistochemical expression.

E-cadherin plays a crucial structural role in cell-cell contacts in epithelial tissues, and a functional role in signaling pathways that regulate cell proliferation, differentiation, and survival. Reduced immunoexpression of E-cadherin adhesions is largely considered as being equivalent to defective functionality and malignancy, and has been used as a prognostic parameter. A critical analysis of studies on E-cadherin immunoexpression in oral carcinomas revealed a wide range of both technical and interpretational aspects. This paper highlights biological characteristics of E-cadherin with respect to its expression in normal and neoplastic epithelial cells and to its interrelations with the tumor microenvironment that can have an impact on immunohistochemical results and their application in the clinical setting.