Dan Dayan

Are the polarization colors of Picrosirius red-stained collagen determined only by the diameter of the fibers?

SummaryPolarization colors of various purified collagens were studied in fibers of similar thickness. Three different soluble collagens of type I, insoluble collagen type I, lathyritic collagen type I, two p-N-collagens type I, pepsin extract collagen type II, two soluble collagens type III, p-N-collagen type III, and soluble collagen type V were submitted to a routine histopathologic procedure of fixation, preparation of 5-μm-thick sections, staining with Picrosirius red and examination under crossed polars. Polarization colors were determined for thin fibers (0.8 μm or less) and thick fibers, (1.6–2.4 μm). Most thin fibers of collagens and p-N-collagens showed green to yellowish-green polarization collors with no marked differences between the various samples. Thick fibers of all p-N-collagens, lathyritic and normal 0.15 M NaCl-soluble collagens showed green to greenish-yellow polarization colors, while in all other collagens, polarization colors of longer wavelengths (from yellowish-orange to red) were observed. These data suggested that fiber thickness was not the only factor involved in determining the polarization colors of Picrosirius red-stained collagens. Tightly packed and presumably, better aligned collagen molecules showed polarization colors of longer wavelengths. Thus, packing of collagen molecules and not only fiber thickness plays a role in the pattern of polarization colors of Picrosirius red-stained collagens.

A preliminary study of activation of collagenase in carious human dentine matrix

Collagenolytic activity in human carious and non-carious dentine matrix was compared. Results confirmed the presence of latent collagenase in demineralized dentine and indicated a slow rate of degradation of collagen substrate. Collagenolytic activity was enhanced with the addition of trypsin-TPCK to the demineralized dentine. More activity was observed in the carious dentine, suggesting the presence of collagenase activators or partial enzymic destruction of the inhibitor in the collagen-collagenase-inhibitor complex. It seems that during dentine development collagenase-inhibitor complex is secreted and bound to collagen-dentine matrix, and the enzyme can be activated during the process of dental caries.

Cancer-associated fibroblasts and epithelial-mesenchymal transition in metastatic oral tongue squamous cell carcinoma

We examined cancer‐associated fibroblasts (CAFs) and a panel of immunohistochemical markers of epithelial‐mesenchymal transition (EMT) in 19 pair‐matched oral tongue squamous cell carcinoma (SCC) and metastatic tumors to regional lymph nodes (RLNs). α‐Smooth muscle actin (α‐SMA) was studied to identify CAFs. EMT was studied with syndecan‐1, Cadherin‐11, fibroblast‐specific protein (FSP)‐1, secreted protein acidic and rich in cysteine (SPARC) and Twist. Triple immunostaining in RLNs was used to highlight the carcinoma cells (E‐cadherin and Ki‐67) and their relationship to the CAFs (α‐SMA). We found that metastatic RLNs hosted CAFs similarly as in pair‐matched primary tumors. Expression of EMT markers is common in both primary and metastatic tumors. We demonstrate that metastatic carcinoma cells (Ki‐67 positive) downregulate E‐cadherin expression at the periphery of cancer islands, where they are in direct contact with CAFs. The supporting connective tissue microenvironment also commonly expresses syndecan‐1, Cadherin‐11, FSP‐1, and SPARC. In conclusion, CAFs are common to both primary and metastatic SCC. We hypothesize that CAFs not only promote tumor invasion but also facilitate metastases, either by cometastasizing and/or being recruited to lymph nodes. Evidence of EMT is common within primary tumors and metastatic SCC and may be further modulated by CAFs.

Tumor-host histopathologic variables, stromal myofibroblasts and risk score, are significantly associated with recurrent disease in tongue cancer

Margin status, a major prognostic parameter in oral cancer, was analyzed vis‐à‐vis the histopathologic parameters of risk scores and stromal myofibroblasts. Specimens of tongue carcinoma (n = 50) were submitted to a risk score assignment consisting of the worst pattern of invasion, lymphocytic infiltration, and perineural invasion. Frequency of stromal myofibroblasts (alpha‐smooth muscle actin stain) was assessed. A triple immunostaining assay with E‐cadherin, Ki‐67 and alpha‐smooth muscle actin was used to identify carcinoma cells undergoing epithelial–mesenchymal transition. Margins were considered ‘clean’ if the tumor was ≥5 mm away from them. Patients ≤60 years were considered as ‘young’. Kaplan–Meier survival analysis with univariate and Cox multivariate regression model with stepwise forward selection, and Fisher’s exact tests were used. Abundant myofibroblasts were found in 27 (54%) cases. Carcinoma cells devoid of E‐cadherin but amalgamated with the stromal myofibroblasts were identified in 18 (36%) cases. Local recurrence and overall survival were negatively influenced by abundance of stromal myofibroblasts (P = 0.004 and P = 0.008, respectively). High‐risk scores (P = 0.011), positive margins, and ‘young’ age (P = 0.027, each) had an unfavorable impact on recurrence. Multivariate analysis revealed that only abundance of stromal myofibroblasts had an independent adverse effect on local recurrence (hazard ratio [HR] 4.369; P = 0.014; 95% confidence interval [CI], 1.356–14.074). It seems that abundant stromal myofibroblasts (camouflaging some malignant cells) and high‐risk scores have an unfavorable impact on the risk of recurrence in particular in ‘young’ patients. Therefore, the treatment concept should be adjusted accordingly and target concomitantly the epithelial malignancy and its allied stroma. (Cancer Sci 2009)

Cancer-associated fibroblasts, a parameter of the tumor microenvironment, overcomes carcinoma-associated parameters in the prognosis of patients with mobile tongue cancer

Mobile tongue squamous cell carcinoma (MTSCC) is known for its strong propensity for regional metastasis and poor patient survival despite aggressive treatment, thus calling for new and reliable markers for predicting prognosis and guiding therapeutic management. Towards this end, three classes of markers were investigated: cancer-associated fibroblasts (CAFs; α-SMA positivity) as a representative of the tumor microenvironment, maspin (mammary serine protease inhibitor) as a tumor marker likely to be modulated by factors within the tumor microenvironment, and DNA content and Ki-67 labeling index as inbuilt tumor markers in 128 cases of MTSCC using immunohistochemistry and image cytometry. Of these markers, only CAF density was independently and relatively strongly associated with elevated mortality from MTSCC. The hazard ratio in the CAF-rich type of tumor microenvironment was 4.85 (95% CI 1.41-16.6, versus the CAF-poor) when adjusted by proportional hazards modeling for the center where the patient was managed, gender, tumor stage, presence of neck metastasis and age at diagnosis. CAF density was unrelated to non-MTSSC mortality. Given the strong association between increased CAF density and higher mortality in MTSCC, routine assessment of CAF density for disease course prognosis and inclusion as an integral part of treatment protocols are recommended.

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.

ANCIENT NEURILEMMOMA (SCHWANNOMA) OF THE ORAL CAVITY

Ancient neurilemmoma is a rare lesion of the oral cavity. The present article describes an additional case and discusses some difficulties in its histological diagnosis. The final diagnosis is based on both light and electron microscopy morphology. Immunohistochemical studies are, at present, only of supportive value.

Human Saliva-Derived Exosomes Comparing Methods of Isolation

ExoQuick-TCTM (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.

Age-related histomorphometric changes in labial salivary glands with special reference to the acinar component.

PURPOSE To evaluate histomorphometric age-related changes in labial salivary glands (LSG) of healthy subjects, with special reference to the mucous and seromucous acini. METHOD Investigation of age-related histomorphometric changes was conducted on 120 samples of LSG obtained from autopsy subjects free of salivary gland tumors/diseases. Samples were divided into young (< 30 years, n = 30), adult (30-60 years, n = 45) and old (> 60 years, n = 45) age groups. Measurements were performed on hematoxylin and eosin stained slides. Parenchymal components included acini and ducts. The acinar component was presently analyzed separately in respect to mucous acinar cells and seromucous acinar cells. Mucous acinar cells presented pale eosinophilic cytoplasm, while that of the seromucous was darker and basophilic. Stromal components included connective tissue, blood and lymphatic vessels, inflammatory infiltrate and adipose tissue. The mean volume fraction (V(v)) of all components in each age group was calculated. Statistical analysis was performed by one-way ANOVA and Tamhane tests. RESULTS The mean V(v) of the seromucous acinar cells decreased by 49.3% (p < 0.0001) and that of the mucous acinar cells decreased only by 28.5% (p = 0.0001). Changes in the ducts were statistically non-significant (p = 0.312). Of the stromal components, the mean V(v) of the adipose tissue showed the highest increase, 2768% (p < 0.0001) followed by the inflammatory infiltrate, 512% (p < 0.0001). CONCLUSIONS The marked decrease in the V(v) of seromucous acinar cells accompanied by a lower decrease in the V(v) of mucous acinar cells may explain age-related changes in LSG secretion. These findings could bear important implications regarding functional age-related changes of these glands.

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.