Sertoli Zellen dedifferenzieren sich in Ko-Kultur mit Seminomzellen. Ein neues Zellkulturmodell zur Pathogenese testikulärer Keimzelltumore.
Final Report Abstract
Testicular intraepithelial neoplasia (TIN), the non-invasive precursor of Testicular germ cell tumours (TGCTs), has been correlated with impaired Sertoli cells that show aberrant expression of the fetal marker cytokeratin 18 (KRT18) and pluripotency factor SOX2. Furthermore, Sertoli cells in seminiferous tubules containing TIN show loss of connexin43 expression and disruption of the bloodtestis barrier. It is controversially discussed whether these Sertoli cells represent prepubertal cells arrested in their normal maturation, being a contributing factor to the development of TIN, or adult cells that are de-differentiating as a consequence of TIN. To address this issue, we established a coculture model of adult human Sertoli cells (FS1 cell line) with seminoma-like cells (TCam-2 cell line). Prior to co-culture experiments, both cell lines have been further characterized focusing on differentiation markers and BTB components to identify differentially expressed genes/proteins during following co-cultures. After 2 weeks of co-culture, we detected first de-differentiated FS1 cells expressing KRT18 and SOX2 by Immunocytochemistry and Immunofluorescence. We also demonstrated, that the proliferation rate of FS1 cells was about 8 fold higher than the proliferation rate of FS1 cells in monocultures. This finding further supports that tumour cells can revert mature Sertoli cells into a less differentiated and thus more proliferative state. To analyse the molecular mechanisms/factors by which TCam-2 cells lead to de-differentiation processes in FS1cells, we performed RNA Microarrays comparing the gene expression profiles of FS1 and TCam-2 cells in monoculture and in co-culture. Currently we are searching for significantly altered genes that might be involved in the pathogenesis of human TGCTs. In addition, a membranebased antibody array for determination of the relative levels of selected human cytokines and chemokines was done to explore secretory profile of monocultures (FS1, TCam-2) and co-cultures (FS1+TCam-2 and FS1+eBM-MSC for control). Cystatin-C, EMMPRIN, IGFBP-2, GDF-15, CD14 and Angiogenin showed significant upregulation in FS1+TCam-2 co-culture conditioned medium. To find out if these cytokines play a role in induction of KRT18 and/or SOX2 expression on FS1 cells, in vitro treatment of FS1 cells with these cytokines will be conducted. In order to realise Western blot and RT-qPCR analyses after direct co-culture, we detected specific surface markers for each cell line and performed Fluorescence-activated cell sorting (FACS). The morphology of Sertoli cells in TIN tubules, assessed by light and electron microscopy corresponds to adult Sertoli cells in normal spermatogenesis (nsp). Additionally, we demonstrated that Sertoli cells in TIN tubules co-express the fetal marker KRT18 and the adult marker androgen receptor. Comparing human testicular biopsies with varying degrees of TIN, we found that in most sections containing TIN tubules together with tubules showing nsp, SOX2 expression is high whereas there is no or only few KRT18 expression. Sections containing only TIN tubules usually show high expression of KRT18 and SOX2. It is already known that the expression of KRT18 in Sertoli cells is in direct correlation with the degree of TIN present in the seminiferous tubule, indicating a progressive process. Our findings suggest that the impaired Sertoli cells first start with SOX2 expression, which is subsequently followed by KRT18 expression. This could point out possible signal cascades involved in the process of de-differentiation. Taken together, our data suggest that the abnormal Sertoli cells associated with TIN represent adult cells that are de-differentiating and that seminoma cells induce de-differentiation in Sertoli cells. Therefore, the impaired Sertoli cells seem to be rather the consequence than the cause of TIN.
Publications
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Pathogenesis of testicular intraepithelial neoplasia: De-differentiation of Sertoli cells in co-culture with seminoma cells. BJVM 2012; 15, 45
Fink C, Weigel R, Dern-Wieloch J, Schumacher V, Schorle H, Schnepel N, Bergmann M, Brehm R
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(2015) Reference gene validation for RT-qPCR, a note on different available software packages. PLoS One
De Spiegelaere W, Dern-Wieloch J, Weigel R, Schumacher V, Schorle H, Nettersheim D, Bergmann M, Brehm R, Kliesch S, Vandekerckhove L, Fink C
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Comparative analysis of tight, adherens and gap junctions in the human Sertoli cell line FS1 and the human seminoma like cell line TCam-2 prior to co-culture experiments. Reprod Dom Anim 2015; 50, 57
Brehm R, Schumacher V, Schorle H, Netterheim D, Ngezahayo A, Begandt D, Dilger N, Schnepel N, Gaehle M, Hambruch N, Wilhelm J, Kliesch S, Weidner W, Bergmann M, Fink C
