Human Dupuytren's &lt;em&gt;Ex Vivo&lt;/em&gt; Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Karkampouna, Sofia; Kloen, Peter; Obdeijn, Miryam C.; Riester, Scott M.; Wijnen, André J. van; Julio, Marianna Kruithof-de

doi:10.3791/52534

Cited by 6 publications

(5 citation statements)

References 27 publications

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“…Tissues from HCC PDX or HepG2‐CRIPTO‐derived tumours were maintained in ex vivo cultures. Tissue slices (150–200 μm) were cultured using Transwell plates with an attached nitrocellulose membrane (ThinCert #662640 inserts for 24‐well plates, 0.4 μm pore size; Greiner Bio‐One, Kremsmünster, Austria) that allowed contact of the tissue with the growth medium but not the plastic in a manner that prevented alteration of the tissue . Culture plates were placed in a sealed container saturated with oxygen, 40–50%, at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma

Karkampouna

Helm

Gray

et al. 2018

The Journal of Pathology

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Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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Section: Methodsmentioning

confidence: 99%

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma

Karkampouna

Helm

Gray

et al. 2018

The Journal of Pathology

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“…We utilized the previously described dermal ex vivo organoid culture technique [15]. The human biological samples were sourced ethically, and their research use was in accord with the terms of informed consent under an IRB/EC-approved protocol.…”

Section: Human Skin Organoid Culture Ex Vivomentioning

confidence: 99%

The role of the oncostatin M/OSM receptor β axis in activating dermal microvascular endothelial cells in systemic sclerosis

et al. 2020

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Background Scleroderma (SSc) is a rare autoimmune disease characterized by vascular impairment and progressive fibrosis of the skin and other organs. Oncostatin M, a member of the IL-6 family, is elevated in SSc serum and was recognized as a significant player in various stages of fibrosis. The goal of this study was to assess the contribution of the OSM/OSMRβ pathway to endothelial cell (EC) injury and activation in SSc. Methods IHC and IF were used to assess the distribution of OSM and OSMRβ in SSc ( n = 14) and healthy control ( n = 7) skin biopsies. Cell culture experiments were performed in human dermal microvascular endothelial cells (HDMECs) and included mRNA and protein analysis, and cell migration and proliferation assays. Ex vivo skin organoid culture was used to evaluate the effect of OSM on perivascular fibrosis. Results OSMRβ protein was elevated in dermal ECs and in fibroblasts of SSc patients. Treatments of HDMECs with OSM or IL-6+sIL-6R have demonstrated that both cytokines similarly stimulated proinflammatory genes and genes related to endothelial to mesenchymal transition (EndMT). OSM was more effective than IL-6+sIL-6R in inducing cell migration, while both treatments similarly induced cell proliferation. The effects of OSM were mediated via OSMRβ and STAT3, while the LIFR did not contribute to these responses. Both OSM and IL-6+sIL-6R induced profibrotic gene expression in HDMECs, as well as expansion of the perivascular PDGFRβ + cells in the ex vivo human skin culture system. Additional studies in HDMECs showed that siRNA-mediated downregulation of FLI1 and its close homolog ERG resulted in increased expression of OSMRβ in HDMECs. Conclusions This work provides new insights into the role of the OSM/OSMRβ axis in activation/injury of dermal ECs and supports the involvement of this pathway in SSc vascular disease.

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“…We utilized the previously described dermal ex vivo organoid culture technique [15]. The human biological samples were sourced ethically, and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol.…”

Section: Gomori's Trichrome Stainingmentioning

confidence: 99%

The role of the Oncostatin M/ OSM receptor β axis in activating dermal microvascular endothelial cells in Systemic Sclerosis

Marden

Wan

Wilks

et al. 2020

Preprint

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Abstract Background: Scleroderma (SSc) is a rare autoimmune disease characterized by vascular impairment and progressive fibrosis of the skin and other organs. Oncostatin M, a member of the IL-6 family, is elevated in SSc serum and was recognized as a significant player in various stages of fibrosis. The goal of this study was to assess the contribution of the OSM/OSMRβ pathway to endothelial cell (EC) injury and activation in SSc. Methods: IHC and IF were used to assess the distribution of OSM and OSMRβ in SSc (n=14) and healthy control (n=7) skin biopsies. Cell culture experiments were performed in human dermal microvascular endothelial cells (HDMECs) and included mRNA and protein analysis, and cell migration and proliferation assays. Ex vivo skin organoid culture was used to evaluate the effect of OSM on perivascular fibrosis.Results: OSMRβ protein was elevated in dermal ECs and in fibroblasts of SSc patients. Treatments of HDMECs with OSM or IL-6 +sIL-6R have demonstrated that both cytokines similarly stimulated proinflammatory genes and genes related to endothelial-to mesenchymal transition ((EndMT). OSM was more effective than IL-6+sIL-6R in inducing cell migration, while both treatments similarly induced cell proliferation. The effects of OSM were mediated via OSMRβ and STAT3, while the LIFR did not contribute to these responses. Both, OSM and IL-6+sIL-6R induced profibrotic gene expression in HDMECs, as well as expansion of the perivascular PDGFRβ+ cells in the ex vivo human skin culture system. Additional studies in HDMECs showed that siRNA-mediated downregulation of FLI1 and its close homolog ERG resulted in increased expression of OSMRβ in HDMECs.Conclusions: This work provides new insights into the role of the OSM/OSMRβ axis in activation/injury of dermal ECs and supports the involvement of this pathway in SSc vascular disease.

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Human Dupuytren's <em>Ex Vivo</em> Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Cited by 6 publications

References 27 publications

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma

The role of the oncostatin M/OSM receptor β axis in activating dermal microvascular endothelial cells in systemic sclerosis

The role of the Oncostatin M/ OSM receptor β axis in activating dermal microvascular endothelial cells in Systemic Sclerosis

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