A novel gain-of-function mutation of TGF-ß receptor II promotes cancer progression via delayed receptor internalization in oral squamous cell carcinoma.

We present a novel gain-of-function mutation of TGF-ß receptor II (TßRII) found in human oral squamous cell carcinoma (OSCC). Expression of E221V/N238I mutant TßRII enhanced TGF-ß signaling. Mutation of TßRII conferred cells higher migratory and invasive capabilities and MMP-2 activity. In mouse tumor model, mutant tumors exhibited poor differentiation and E-cadherin relocalization to the cytosol. Lipid-raft-dependent endocytosis of TßRII was attenuated in mutant TßRII, suggesting that enhancement of TGF-ß signaling by this mutation is due to delayed TßRII internalization. Taken together, our results show a novel gain-of-function TßRII mutation, which enhances TGF-ß signaling leading to more invasive phenotypic changes in human OSCC.

A triazine compound S06 inhibits proinvasive crosstalk between carcinoma cells and stromal fibroblasts via binding to heat shock protein 90.

Carcinoma-associated fibroblasts (CAFs) promote tumor invasion by secreting soluble factors. A tagged triazine library was screened in our novel transwell coculture model of CAF and oral squamous cell carcinoma (OSCC). We discovered compound S06, which reduced OSCC invasion by inhibiting secretion of CAF-derived proinvasive chemokines. The N-terminus of Hsp90 was found to be the cellular target of S06. Importantly, S06 did not induce hepatic toxicity, a side effect associated with well-known Hsp90 inhibitors. Moreover, S06 inhibited tumor cell migration in a zebrafish xenograft model. Our results demonstrate that Hsp90 is a novel target for stromal-based therapy to modulate proinvasive molecular crosstalk within the tumor microenvironment. Furthermore, S06 represents a new class of Hsp90 inhibitor and is an attractive candidate for anticancer drug development.

Evaluation of vitrification for cryopreservation of teeth.

PURPOSE: The aim of this study was to investigate whether vitrification in the cryopreservation of periodontal ligament (PDL) cells could be useful for tooth banking. METHODS: In step 1, primary cultured human PDL cells were cryopreserved in 100% conventional cryopreservation media and 100% vitrification media (ESF40 media) in different temperatures for 2 weeks. In step 2, a series of modified vitrification formulae named T1 (75% vitrification media + 25% F-media), T2 (50% vitrification media + 50% F-media) and T3 (25% vitrification media + 75% F-media) were used to store PDL cells for 2 weeks and 4 weeks in liquid nitrogen. MTT assay was performed to examine the viability of PDL cells. RESULTS: Maximum cell viability was achieved in cells stored in 100% conventional cryopreservation media at -196 (positive control group) in step 1. Compared to the positive control group, viability of the cells stored in 100% vitrification media was very low as 10% in all test conditions. In step 2, as the percentage of vitrification media decreased, the cell viability increased in cells stored for 2 weeks. In 4-week storage of cells in step 2, higher cell viability was observed in the T2 group than the other vitrification formulae while the positive control group had the highest viability. There was no statistically significant difference in the cell viability of 2-week and 4-week stored cells in the T2 group. CONCLUSIONS: These observations indicate 100% vitrification media is not successful in PDL cell cryopreservation. Conventional cryopreservation media is currently the most appropriate media type for this purpose while T2 media would be interesting to test for long-term storage of PDL cells.

Tumor-stromal crosstalk in invasion of oral squamous cell carcinoma: a pivotal role of CCL7.

Recent studies have shown that stromal fibroblasts have a more profound influence on the initiation and progression of carcinoma than was previously appreciated. This study aimed at investigating the reciprocal relationship between cancer cells and their associated fibroblasts at both the molecular and cellular level in oral squamous cell carcinoma (OSCC). To identify key molecular regulators expressed by carcinoma-associated fibroblasts (CAF) that promote cancer cell invasion, microarrays were performed by comparing cocultured OSCC cells and CAF with monoculture controls. Microarray and real-time PCR analysis identified marked upregulation of the chemokine (C-C motif) ligand 7 (CCL7) in cocultured CAF. ELISA showed an elevated level of CCL7 secretion from CAF stimulated by coculture with OSCC cells. CCL7 promoted the invasion and migration of OSCC cells, and the invasiveness was inhibited by treatment with CCL7 neutralizing antibody. OSCC cells were shown to express CCR1, CCR2 and CCR3, receptors for CCL7, by RT-PCR. In addition, treatment with anti-CCR1 or anti-CCR3 antibody inhibited CCL7-induced OSCC cell migration, implicating that CCL7 promotes cancer cell migration through CCR1 and CCR3 on OSCC cells. Cytokine antibody array analysis of the supernatant from OSCC cell culture revealed that interleukin-1alpha was an inducer of CCL7 secretion by CAF. This study confirms the reciprocal relationship of the molecular crosstalk regulating the invasion of OSCC and describes new potential targets for future therapy.

Morphogenesis and biological significance of spindle cell transformation in a spindle cell carcinoma.

Our study investigated the histogenesis and biological significance of spindle cell carcinoma (SpCC) by comparing with moderately-well-differentiated squamous cell carcinoma (SCC) cell lines. Snail mRNA expression was readily detectable in the SpCC cell line, while E-cadherin was undetectable. SpCC cells showed lower proliferative and invasive activities than two other SCC cell lines. Culturing under air-liquid interface conditions promoted squamous cell differentiation, whereas fibroblastic differentiation after submerged culture with collagen, suggesting that the microenvironment may be a regulating factor of spindle cell differentiation as well as Snail expression and spindle cell change may not always entail the invasive behavior.

Collagen-based co-culture for invasive study on cancer cells-fibroblasts interaction.

The roles of tumor stroma in carcinogenesis are still unclear. This study was aimed at designing an in vitro model for investigating the effects of stromal fibroblasts in the invasive growth of squamous cell carcinoma. Using two cancer cell lines, we performed three-dimensional co-culture with dermal equivalents to evaluate the effects of fibroblasts in cancer invasion. In vitro models for cellular interaction study were designed as follows: a collagen gel-based direct co-culture model (C-Dr) and a collagen gel-based indirect co-culture model (C-In). The invasive growth was found only in the dermal equivalents with fibroblasts. MMP-2 activity could be induced by direct contact between cancer cells and stromal fibroblasts. Cathepsin D was also highly expressed when co-cultured with cancer cells and fibroblasts. The present study demonstrated that the presence of fibroblasts is essential in cancer invasion and that collagen gel-based co-culture models might be useful for invasive study.

Cryopreservation of human teeth for future organization of a tooth bank--a preliminary study.

This study was aimed at evaluating whether cryopreserved teeth can be used for future transplantation by examining the viability and differentiation capability of periodontal ligament (PDL) cells and measuring the hardness of dental hard tissue. Fifty-four teeth were divided into two groups, control and frozen teeth. A MTT assay and a TUNEL assay were performed for the examination of the viability and apoptotic death of PDL cells. Immunohistochemical staining for alkaline phosphatase was performed to observe whether the differentiation capability of PDL cells was maintained by the freezing and thawing procedure. Hardness was measured to detect whether dental hard tissue was affected by the freezing conditions. The MTT and TUNEL assays showed no significant difference in the viability of PDL cells between the two groups. The differentiation capability of PDL cells was maintained in frozen teeth as evidenced by alkaline phosphatase staining. The hardness of frozen teeth was not changed, but a longitudinal fracture was found in 25% of the frozen group. The viability and differentiation capability of PDL cells were maintained in a frozen environment; however, it is thought that a new cryopreservation method preventing fracture of dental hard tissue should be developed for clinical application.