Interactions of human MSC with head and neck squamous cell carcinoma cell line PCI-13 reduce markers of epithelia-mesenchymal transition.

OBJECTIVES: Cancer progression is influenced by tumor microenvironment and communication of stromal cells and tumor cells. Interactions may enhance epithelial-mesenchymal transition (EMT) of tumor cells through signaling proteins such as Wnt/beta-catenin and matrix metalloproteinases (MMP), as well as loss of cellular integrity, which affects invasion, progression, and metastasis of head and neck squamous cell carcinoma (HNSCC). In this study, we are testing the hypothesis that interactions of human mesenchymal stromal cells (MSCs) with HNSCC might influence the expression of markers of EMT and tumor progression by co-culturing human MSC with the PCI-13 HNSCC line. MATERIALS AND METHODS: Pooled MSCs were derived from the iliac bone marrow of seven patients and co-cultured in transwell permeable membrane wells with tumor cells of the established HNSCC cell line PCI-13 (UICC: T3, N1, M0). MSCs were characterized through fluorescence-activated cell sorting (FACS) analysis. Expression of Wnt3, E-cadherin, beta-catenin, MMP14, cathepsin b, and ETS1 was assessed by quantitative RT-PCR. RESULTS: We were able to show that co-culture of MSCs and PCI-13 leads to a significantly reduced expression of Wnt3, MMP14, and beta-catenin compared to controls, whereas the expression of cathepsin b and ETS1 was not significantly different between co-cultures and controls. CONCLUSION: Our results suggest that the interaction between MSCs and PCI-13 may suppress EMT in cancer cells. CLINICAL RELEVANCE: The influence of MSCs can suppress the onset of EMT in HNSCC, affecting tumor progression and therapy.

Influence of tumour necrosis factor alpha on epithelial-mesenchymal transition of oral cancer cells in co-culture with mesenchymal stromal cells.

Tumour progression in head and neck squamous cell carcinoma (HNSCC) is influenced by the surrounding stroma and inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). The aim of this study was to test the hypothesis that TNF-α modulates the interactions of HNSCC cell line PCI-13 and bone marrow mesenchymal stromal cells (BMSCs) and influences markers of epithelial-mesenchymal transition (EMT). Following induction with TNF-α, mono- and co-cultures of BMSCs and the established HNSCC cell line PCI-13 were analyzed; protein expression of E-cadherin and vimentin and qRT-PCR expression of Snail, Twist, MMP14, vimentin, E-cadherin, and ß-catenin were examined, and changes in cellular AKT signalling were analyzed. TNF-α induced a significant decrease in E-cadherin (64.5±6.0%, P=0.002) and vimentin (10.4±3.5%, P=0.04) protein expression in co-cultured PCI-13, while qRT-PCR showed a significant increase in ß-catenin (BMSCs P<0.0001; PCI-13 P=0.0005) and Snail (BMSCs P=0.009; PCI-13 P=0.01). TNF-α also resulted in a down-regulation of AKT downstream targets S6 (38.7±20.9%, P=0.01), p70S6 (16.7±12%, P=0.05), RSK1 (23.6±28.8%, P=0.02), and mTOR (27.4±17.5%, P=0.004) in BMSC co-cultures. In summary, while reducing the expression of vimentin and AKT-signalling in PCI-13 and BMSC, respectively, TNF-α introduced an inflammatory-driven tumour-stroma transition, marked by an increased expression of markers of EMT.

Osseointegration of dental implants in ectopic engineered bone in three different scaffold materials.

The in vivo regeneration of bone flaps might be an alternative to autogenous bone grafting. The first human case of mandibular reconstruction using the greater omentum as a bioreactor was reported in 2016. However, whether engineered bone will support the osseointegration of dental implants has not yet been investigated. In this study, bone tissue engineering was performed in the greater omentum of nine miniature pigs using bone morphogenetic protein 2, bone marrow aspirate, and three different scaffolds: hydroxyapatite, biphasic calcium phosphate (BCP), and titanium. After 8 weeks, two implants were placed in each scaffold; after another 8 weeks, the bone blocks were harvested for radiographic, histological, and histomorphometric analysis. All implants exhibited sufficient primary stability, and the success rate was 100%. The bone-to-implant contact ratios (BICs) were 38.2%, 68.5%, and 42.9%; the inter-thread bone densities were 29.4%, 64.9%, and 33.5%; and the peri-implant bone-scaffold densities were 56.4%, 87.6%, and 68.6% in the hydroxyapatite, BCP, and titanium groups, respectively. The BIC showed a strong correlation (r = 0.76) with the peri-implant bone-scaffold density. This study shows that de novo engineered bone leads to successful osseointegration and therefore may allow implant-based prosthodontic rehabilitation.

Influence of TGF-ß1 on tumor transition in oral cancer cell and BMSC co-cultures.

OBJECTIVES: TGF-ß1 signaling modulates epithelial mesenchymal transitions (EMT) of head and neck squamous cell carcinoma (HNSCC). Bone marrow mesenchymal stromal cells (BMSC) are able to exert a regulating influence on the expression of markers of EMT in HNSCC cells. It was thus the aim of this study to test the hypothesis that TGF-ß1 modulates the interactions of tumor transition between BMSCs and HNSCC, affecting the expression of E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin. Furthermore, we analyzed alterations in the AKT-signaling of tumor and stroma cells. MATERIALS AND METHODS: BMSCs were isolated from iliac bone marrow aspirates and co-cultured in trans-well permeable membrane wells with tumor cells of the established HNSCC cell line PCI-13. Following the induction with TGF-ß1 under serum free conditions the expression of Vimentin and E-Cadherin was assessed via immunofluorescence. A quantitative RT-PCR analysis of tumor transition markers E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin was performed. Changes in AKT-Signaling were identified via protein analysis. RESULTS: In non-induced co-cultures, BMSC were able to suppress Vimentin in PCI-13 as a marker of tumor transition. In TGF-ß1 induced co-cultures PCI-13 significantly increased the expression of Vimentin, Twist, Snail, MMP14, GSK3a, PRAS40, 4E-BP1, and AMPKa compared to monolayer controls. TGF-ß1 co-cultured BMSC demonstrated a significant increase of Snail, PRAS40, mTOR, GSK3a/b, Bad, PDK1 and 4E-BP1. CONCLUSIONS: TGF-ß1 was able to attenuate the modulating influence of BMSC in co-culture and drive the co-culture towards a progressive tumor transition, affecting the expression of markers of EMT, AKT-Signaling and proliferative checkpoints.

Stem cell-derived chondrocytes for regenerative medicine.

The regenerative capacity of cartilage is limited. Transplantation methods used to treat cartilage lesions are based mainly on primary cultures of chondrocytes, which dedifferentiate during cultivation in vitro and lose their functional properties. Stem cells are considered as an alternative source to generate cells for two reasons: first, they can almost indefinitely divide in culture, and second, they are able to differentiate into various mature cell types. Herein, we asked the question whether chondrocytes could be differentiated from mouse embryonic stem (ES) cells to a state suitable for regenerative use. When cultivated as embryoid bodies (EBs), murine ES cells differentiate into mesenchymal progenitor cells, which progressively develop into mature, hypertrophic chondrocytes and transdifferentiate into calcifying cells recapitulating all of the cellular processes of chondrogenesis. Chondrocytes isolated from EBs exhibit a high regenerative capacity. They dedifferentiate initially in culture, but later reexpress stable characteristics of mature chondrocytes. However, in cultures of chondrocytes isolated from EBs, additional mesenchymal cell types can be observed. Mesenchymal stem (MS) cells from bone marrow have already been used in tissue engineering settings. We compared the chondrogenic differentiation of MS and ES cells.

In vivo matrix-guided human mesenchymal stem cells.

Microfracture of subchondral bone results in intrinsic repair of cartilage defects. Stem or progenitor cells from bone marrow have been proposed to be involved in this regenerative process. Here, we demonstrate for the first time that mesenchymal stem (MS) cells can in fact be recovered from matrix material saturated with cells from bone marrow after microfracture. This also introduces a new technique for MS cell isolation during arthroscopic treatment. MS cells were phenotyped using specific cell surface antibodies. Differentiation of the MS cells into the adipogenic, chondrogenic and osteogenic lineage could be demonstrated by cultivation of MS cells as a monolayer, as micromass bodies or mesenchymal microspheres. This study demonstrates that MS cells can be attracted to a cartilage defect by guidance of a collagenous matrix after perforating subchondral bone. Protocols for application of MS cells in restoration of cartilage tissue include an initial invasive biopsy to obtain the MS cells and time-wasting in vitro proliferation and possibly differentiation of the cells before implantation. The new technique already includes attraction of MS cells to sites of cartilage defects and therefore may overcome the necessity of in vitro proliferation and differentiation of MS cells prior to transplantation.