Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis, but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model

Background: In bone tissue engineering (BTE), extensive research into vascular endothelial growth factor A (VEGFA)-mediated angiogenesis has yielded inconsistent results. The aim of this study was to investigate the influence on angio- and osteogenesis of adenoviral-mediated delivery of VEGFA alone or in combination with bone morphogenetic protein 2 (BMP2) in bone marrow stromal cells (BMSC) seeded onto a recently developed poly(LLA-co-CL) scaffold. Methods: Human BMSC were engineered to express VEGFA alone or in combination with BMP2 and seeded onto poly(LLA-co-CL) scaffolds. Changes in angiogenic and osteogenic gene and protein levels were examined by quantitative reverse-transcription polymerase chain reaction (RT-PCR), PCR array, and alkaline phosphatase assay. An in vivo subcutaneous mouse model was used to investigate the effect on angio- and osteogenesis of VEGFA alone or in combination with BMP2, using microcomputed tomography (μCT), histology, immunohistochemistry, and immunofluorescence. Results: Combined delivery of a lower ratio (1:3) of VEGFA and BMP2 (ad-BMP2 + VEGFA) led to upregulation of osteogenic and angiogenic genes in vitro at 3 and 14 days, compared with mono-delivery of VEGFA (ad-VEGFA) and other controls. In vivo, in a subcutaneous mouse model, both ad-VEGFA and ad-BMP2 + VEGFA scaffold explants exhibited increased angiogenesis at 2 weeks. Enhanced angiogenesis was largely related to the recruitment and differentiation of mouse progenitor cells to the endothelial lineage and, to a lesser extent, to endothelial differentiation of the implanted BMSC. μCT and histological analyses revealed enhanced de novo bone formation only in the ad-BMP2 + VEGFA group, corresponding at the molecular level to the upregulation of genes related to osteogenesis, such as ALPL, RUNX2, and SPP1. Conclusions: Although BMSC expressing VEGFA alone or in combination with BMP2 significantly induced angiogenesis, VEGFA alone failed to demonstrate osteogenic activity both in vitro and in vivo. These results not only call into question the use of VEGFA alone in bone regeneration, but also highlight the importance in BTE of appropriately formulated combined delivery of VEGFA and BMP2.publishedVersio

MicroRNA-138 abates fibroblast motility with effect on invasion of adjacent cancer cells

Background: Recent studies have shown aberrant expression of micro-RNAs in cancer-associated fibroblasts (CAFs). This study aimed to investigate miR-138 dysregulation in CAFs in oral squamous cell carcinoma (OSCC) and its effects on their phenotype and invasion of adjacent OSCC cells. Methods: Expression of miR-138 was first investigated in OSCC lesions (n = 53) and OSCC-derived CAFs (n = 15). MiR-138 mimics and inhibitors were used to functionally investigate the role of miR-138 on CAF phenotype and the resulting change in their ability to support OSCC invasion. Results: Expression of miR-138 showed marked heterogeneity in both OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression reduced fibroblasts’ motility and collagen contraction ability and suppressed invasion of suprajacent OSCC cells, while its inhibition resulted in the opposite outcome. Transcript and protein examination after modulation of miR-138 expression showed changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFβ1 signaling pathway. Conclusions: Despite its heterogeneous expression, miR-138 in OSCC-derived CAFs exhibits a tumor-suppressive function.publishedVersio

Establishment of a novel cancer cell line derived from vulvar carcinoma associated with lichen sclerosus exhibiting a fibroblast-dependent tumorigenic potential

Vulvar squamous cell carcinoma associated with lichen sclerosus (VLS-VSCC) are rare tumors but with higher recurrence and worse prognosis than other types of VSCC. Lack of experimental models has limited the search for better understanding of the biology and development of treatment modalities. In this study, we isolated and characterized primary cells from VSCC (n = 7) and normal vulvar tissue adjacent to tumor (n = 7). Detailed characterization of the novel spontaneously immortalized cell line, VCC1 revealed a characteristic epithelial morphology in vitro and a well-differentiated keratinizing SCC histology in vivo, closely resembling the tumor of origin. VCC1 expressed higher levels of epithelial-mesenchymal transition markers and higher clonogenic properties as compared to other established non VLS-VSCC cell lines. In vitro 3D organotypic assays and in vivo xenografts revealed a prominent role of cancer-associated fibroblasts in VCC1 invasion and tumor formation. In conclusion, VCC1 mirrored several major VLS-VSCC features and provided a robust experimental tool for further elucidation of VLS-related oncogenesis and drug testing.publishedVersio

Functional and Prognostic Role of MicroRNAs in Cancer Associated Fibroblasts and Stroma in Oral Squamous Cell Carcinoma

Nyere studier har vist at forandringer i bindevevscellene som omgir kreftsvulsten er viktige for utviklingen av munnhulekreft. Kreft stroma kan regulere fenotypen av kreftcellene, og på den måten kan påvirke klinisk forløpet av kreftsykdommen. Målet med denne studien var å undersøke dereguleringen av mikro-RNA (miRNAs) i kreft assosierte fibroblaster (KAF) i oralt plateepitelkarsinom (OPK). KAF og normale orale fibroblaster (NOF) ble isolert fra biopsier av pasienter med OPK og friske individer etter informert samtykke. Globalt miR-uttrykk ble profilert ved bruk av Illumina versjon 2-paneler. Uovervåket gruppering av KAF og NOF etter globalt miR-uttrykk viste separate klynger for KAF og NOF, og SAM analysen påviste 12 miRNAs som uttrykes ulikt i KAF og NOF. Den funksjonelle rollen av to ulikt uttrykte miRNA-er, miR-204 og miR138, ble vurdert i 2D- og 3D-organotypiske ko- kulturer ved bruk av miRNA-mimik og hemmere. Ved å modulere miR-204- ekspresjon i KAF ble deres morfologi og motilitet endret, og dette påvirket invasjonen av kreftcellene i 3D-organotypiske ko-kulturmodeller. Videre, viste 3'UTR miRNA-målreporteranalyse at ITGA11 var et direkte mål for miR-204. Disse funnene påviste at miR-204 har en tumorundertrykkende funksjon ved å hemme migrasjonen av fibroblaster gjennom modulering av uttrykket av flere forskjellige molekyler og ved direkte målretting av ITGA11. I tillegg, ble miR-204 i bindevevet identifisert som en uavhengig prognostisk biomarkør for OPK. Uttrykk av miR-138 viste markert heterogenitet både i OPK vev fra pasienter og i dyrkete fibroblaster. Ektopisk utrykk av miR-138 reduserte fibroblastenes motilitet og sine evner til å kontrahere kollagen, og undertrykket invasjonen av nærliggende OPK celler. Hemming av miR-138 resulterte i motsatt resultat. Transkripsjon og proteinundersøkelsene viste at antitumor effekten av miR-138 i KAF var assosiert med endringer av KAF-fenotypespesifikke molekyler, fokal adhesjonskinaseakse og TGFβ1-signalveien. Samlet, viser resultatene av denne avhandlingen betydelige endringer av miRNA utrykk i bindevevet av OPK samenlignet med fibroblaster fra normal munnslimhinne og identifiserer miR-204 og miR-138 som har tumorundertrykkende funksjon ved å endre KAF sin fenotype og motilitet.Recent studies have shown that in addition to the transformation of epithelial cells, dysfunction of stroma is crucial in oral carcinogenesis, and cancer-associated stroma can regulate the phenotype of cancer cells and influence the clinical outcome. The aim of this study was to investigate the role of micro RNAs (miRNAs) dysregulation in cancer-associated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). CAF and normal oral fibroblasts (NOF) were isolated from biopsies of OSCC patients and healthy individuals after informed consent. Global miRNA expression was first profiled using Illumina version 2 panels. Unsupervised clustering of global miRNA expression showed two separate clusters for CAF and NOF, and SAM analysis revealed significant differential expression of 12 miRNAs between CAF and NOF. Functional impact of two significantly Differentially expressed miRNAs between CAF and NOF, miR-204 and miR138, was assessed in 2D and 3D-organotypic co-cultures using miRNA mimic and inhibitors. Modulation of miR-204 expression in CAF resulted in alteration of their motility with effect on suprajacent OSCC cell invasion in 3D- organotypic co-culture models. Furthermore, 3 ́UTR miRNA target reporter assay showed ITGA11 as a direct target of miR-204 in CAF. In conclusion, these results showed that miR-204 has a tumor suppressive function through inhibition of fibroblast migration by modulating the expression of several different molecules and by directly targeting ITGA11. Moreover, stromal miR-204 was identified as an independent prognostic biomarker for OSCC patient survival. On the other hand, expression of miR- 138 showed marked heterogeneity both in OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression altered fibroblasts’ morphology, reduced their motility and collagen contraction ability, and suppressed invasion of suprajacent OSCC cells in 3D models. Inhibition of miR-138 resulted in the opposite outcome. Transcript and protein examination showed that the anti-tumour effect of miR-138 in CAFs was associated to changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFβ1 signaling pathway. Taken together, results of this thesis demonstrate significant alteration of miRNAs in the stromal compartment of OSCC and identify miR-204 and miR-138 with a tumour suppressive function when expressed in CAF.Doktorgradsavhandlin

MicroRNAs as important players and biomarkers in oral carcinogenesis

Oral cancer, represented mainly by oral squamous cell carcinoma (OSCC), is the eighth most common type of human cancer worldwide. The number of new OSCC cases is increasing worldwide, especially in the low-income countries, and the prognosis remains poor in spite of recent advances in the diagnostic and therapeutic modalities. MicroRNAs (miRNAs), 18–25 nucleotides long noncoding RNA molecules, have recently gained significant attention as potential regulators and biomarkers for carcinogenesis. Recent data show that several miRNAs are deregulated in OSCC, and they have either a tumor suppressive or an oncogenic role in oral carcinogenesis. This review summarizes current knowledge on the role of miRNAs as tumor promotors or tumor suppressors in OSCC development and discusses their potential value as diagnostic and prognostic markers in OSCC

Combined in situ hybridization and immunohistochemistry on archival tissues reveals stromal microRNA-204 as prognostic biomarker for oral squamous cell carcinoma

Micro-RNAs (miRs) are emerging as important players in carcinogenesis. Their stromal expression has been less investigated in part due to lack of methods to accurately differentiate between tumor compartments. This study aimed to establish a robust method for dual visualization of miR and protein (pan-cytokeratin) by combining chromogen-based in situ hybridization (ISH) and immunohistochemistry (IHC), and to apply it to investigate stromal expression of miR204 as a putative prognostic biomarker in oral squamous cell carcinoma (OSCC). Four different combinations of methods were tested and ImageJ and Aperio ImageScope were used to quantify miR expression. All four dual ISH-IHC methods tested were comparable to single ISH in terms of positive pixel area percentage or integrated optical density of miRs staining. Based on technical simplicity, one of the methods was chosen for further investigation of miR204 on a cohort of human papilloma virus (HPV)-negative primary OSCC (n = 169). MiR204 stromal expression at tumor front predicted recurrence-free survival (p = 0.032) and overall survival (p = 0.036). Multivariate Cox regression further confirmed it as an independent prognostic biomarker in OSCC. This study provides a methodological platform for integrative biomarker studies based on simultaneous detection and quantification of miRs and/or protein and reveals stromal miR204 as a prognostic biomarker in OSCC

Additional file 3: Figure S2. of Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis, but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model

A limited number of blood capillaries, only in ad-VEGFA scaffold explants, were weakly positive for anti-CD31 antibody targeting human CD31 protein. (B–G) No CD31-positive staining was observed in the capillary/vessel-like structures (black arrows) in the entire scaffold explants from all groups both at 2 and 8 weeks, except for a few capillaries in the ad-VEGFA explants at 8 weeks (E, green arrows). (A) Positive control (normal human oral mucosa) showed multiple CD31-positive capillary-like structures. (TIF 14626 kb

MicroRNA-138 abates fibroblast motility with effect on invasion of adjacent cancer cells

Background: Recent studies have shown aberrant expression of micro-RNAs in cancer-associated fibroblasts (CAFs). This study aimed to investigate miR-138 dysregulation in CAFs in oral squamous cell carcinoma (OSCC) and its effects on their phenotype and invasion of adjacent OSCC cells. Methods: Expression of miR-138 was first investigated in OSCC lesions (n = 53) and OSCC-derived CAFs (n = 15). MiR-138 mimics and inhibitors were used to functionally investigate the role of miR-138 on CAF phenotype and the resulting change in their ability to support OSCC invasion. Results: Expression of miR-138 showed marked heterogeneity in both OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression reduced fibroblasts’ motility and collagen contraction ability and suppressed invasion of suprajacent OSCC cells, while its inhibition resulted in the opposite outcome. Transcript and protein examination after modulation of miR-138 expression showed changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFβ1 signaling pathway. Conclusions: Despite its heterogeneous expression, miR-138 in OSCC-derived CAFs exhibits a tumor-suppressive function

Establishment of a novel cancer cell line derived from vulvar carcinoma associated with lichen sclerosus exhibiting a fibroblast-dependent tumorigenic potential

Vulvar squamous cell carcinoma associated with lichen sclerosus (VLS-VSCC) are rare tumors but with higher recurrence and worse prognosis than other types of VSCC. Lack of experimental models has limited the search for better understanding of the biology and development of treatment modalities. In this study, we isolated and characterized primary cells from VSCC (n = 7) and normal vulvar tissue adjacent to tumor (n = 7). Detailed characterization of the novel spontaneously immortalized cell line, VCC1 revealed a characteristic epithelial morphology in vitro and a well-differentiated keratinizing SCC histology in vivo, closely resembling the tumor of origin. VCC1 expressed higher levels of epithelial-mesenchymal transition markers and higher clonogenic properties as compared to other established non VLS-VSCC cell lines. In vitro 3D organotypic assays and in vivo xenografts revealed a prominent role of cancer-associated fibroblasts in VCC1 invasion and tumor formation. In conclusion, VCC1 mirrored several major VLS-VSCC features and provided a robust experimental tool for further elucidation of VLS-related oncogenesis and drug testing

Profiling and functional analysis of microRNA deregulation in cancer-associated fibroblasts in oral squamous cell carcinoma depicts an anti-invasive role of microRNA-204 via regulation of their motility

Background: Knowledge on the role of miR changes in tumor stroma for cancer progression is limited. This study aimed to investigate the role of miR dysregulation in cancer-associated fibroblasts (CAFs) in oral squamous cell carcinoma (OSCC). Methodology: CAF and normal oral fibroblasts (NOFs) were isolated from biopsies of OSCC patients and healthy individuals after informed consent and grown in 3D collagen gels. Total RNA was extracted. Global miR expression was profiled using Illumina version 2 panels. The functional impact of altered miR-204 expression in fibroblasts on their phenotype and molecular profile was investigated using mimics and inhibitors of miR-204. Further, the impact of miR-204 expression in fibroblasts on invasion of adjacent OSCC cells was assessed in 3D-organotypic co-cultures. Results: Unsupervised hierarchical clustering for global miR expression resulted in separate clusters for CAF and NOF. SAM analysis identified differential expression of twelve miRs between CAF and NOF. Modulation of miR-204 expression did not affect fibroblast cell proliferation, but resulted in changes in the motility phenotype, expression of various motility-related molecules, and invasion of the adjacent OSCC cells. 3′ UTR miR target reporter assay showed ITGA11 to be a direct target of miR-204. Conclusions: This study identifies differentially expressed miRs in stromal fibroblasts of OSCC lesions compared with normal oral mucosa and it reveals that one of the significantly downregulated miRs in CAF, miR-204, has a tumor-suppressive function through inhibition of fibroblast migration by modulating the expression of several different molecules in addition to directly targeting ITGA11