Activin A target genes are differentially expressed between normal and neoplastic adult human testes: clues to gonocyte fate choice.
Human testicular germ cell tumours (TGCT) arise from germ cell neoplasia in situ (GCNIS) cells that originate from foetal germ cell precursors. Activin A is central to normal foetal testis development, and its dysregulation may contribute to TGCT aetiology.
(i) To test whether the expression profiles of activin A targets in normal and neoplastic human testes indicates functional links with TGCT progression. (ii) To investigate whether activin A levels influence MMP activity in a neoplastic germ cell line.
(1) Bouin's fixed, paraffin-embedded human testes were utilized for PCR-based transcript analysis and immunohistochemistry. Samples (n = 5 per group) contained the following: (i) normal spermatogenesis, (ii) GCNIS or (iii) seminoma. CXCL12, CCL17, MMP2 and MMP9 were investigated. (2) The human seminoma-derived TCam-2 cell line was exposed to activin A (24 h), and target transcripts were measured by qRT-PCR (n = 4). ELISA (n = 4) and gelatin zymography (n = 3) showed changes in protein level and enzyme activity, respectively.
(i) Cytoplasmic CXCL12 was detected in Sertoli and other somatic cells, including those surrounding seminoma cells. Anti-CCL17 labelled only the cytoplasm of Sertoli cells surrounding GCNIS, while anti-MMP2 and anti-MMP9 labelled germline and epithelial-like cells in normal and neoplastic testes. (ii) Exposing TCam-2 cells to activin A (50 ng/mL) elevated MMP2 and MMP9 transcripts (fourfold and 30-fold), while only MMP2 protein levels were significantly higher after activin A (5 ng/mL and 50 ng/mL) exposure. Importantly, gelatin zymography revealed activin A increased production of activated MMP2.
Detection of CCL17 only in GCNIS tumours may reflect a change in Sertoli cell phenotype to a less mature state. Stimulation of MMP2 activity by activin A in TCam-2 cells suggests activin influences TGCT by modulating the tumour niche.
This knowledge provides a basis for understanding how physiological changes that influence activin/TGF-β superfamily signalling may alter germ cell fate.
Szarek M
，Bergmann M
，Konrad L
，Schuppe HC
，Kliesch S
，Hedger MP
，Loveland KL
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WNT signalling in the normal human adult testis and in male germ cell neoplasms.
Is WNT signalling functional in normal and/or neoplastic human male germ cells?
Regulated WNT signalling component synthesis in human testes indicates that WNT pathway function changes during normal spermatogenesis and is active in testicular germ cell tumours (TGCTs), and that WNT pathway blockade may restrict seminoma growth and migration.
Regulated WNT signalling governs many developmental processes, including those affecting male fertility during early germ cell development at embryonic and adult (spermatogonial) ages in mice. In addition, although many cancers arise from WNT signalling alterations, the functional relevance and WNT pathway components in TGCT, including germ cell neoplasia in situ (GCNIS), are unknown.
The cellular distribution of transcripts and proteins in WNT signalling pathways was assessed in fixed human testis sections with normal spermatogenesis, GCNIS and seminoma (2-16 individuals per condition). Short-term (1-7 h) ligand activation and long-term (1-5 days) functional outcomes were examined using the well-characterised seminoma cell line, TCam-2. Pathway inhibition used siRNA or chemical exposures over 5 days to assess survival and migration.
The cellular localisation of WNT signalling components was determined using in situ hybridisation and immunohistochemistry on Bouin's- and formalin-fixed human testis sections with complete spermatogenesis or germ cell neoplasia, and was also assessed in TCam-2 cells. Pathway function tests included exposure of TCam-2 cells to ligands, small molecules and siRNAs. Outcomes were measured by monitoring beta-catenin (CTNNB1) intracellular localisation, cell counting and gap closure measurements.
Detection of nuclear-localised beta-catenin (CTNNB1), and key WNT signalling components (including WNT3A, AXIN2, TCF7L1 and TCF7L2) indicate dynamic and cell-specific pathway activity in the adult human testis. Their presence in germ cell neoplasia and functional analyses in TCam-2 cells indicate roles for active canonical WNT signalling in TGCT relating to viability and migration. All data were analysed to determine statistical significance.
No large-scale datasets were generated in this study.
As TGCTs are rare and morphologically heterogeneous, functional studies in primary cancer cells were not performed. Functional analysis was performed with the only well-characterised, widely accepted seminoma-derived cell line.
This study demonstrated the potential sites and involvement of the WNT pathway in human spermatogenesis, revealing similarities with murine testis that suggest the potential for functional conservation during normal spermatogenesis. Evidence that inhibition of canonical WNT signalling leads to loss of viability and migratory activity in seminoma cells suggests that potential treatments using small molecule or siRNA inhibitors may be suitable for patients with metastatic TGCTs.
This study was funded by National Health and Medical Research Council of Australia (Project ID 1011340 to K.L.L. and H.E.A., and Fellowship ID 1079646 to K.L.L.) and supported by the Victorian Government's Operational Infrastructure Support Program. None of the authors have any competing interests.
Young JC
，Kerr G
，Micati D
，Nielsen JE
，Rajpert-De Meyts E
，Abud HE
，Loveland KL
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Specific immune cell and cytokine characteristics of human testicular germ cell neoplasia.
Which immune cells and cytokine profiles are characteristic for testicular germ cell neoplasia and what consequences does this have for the understanding of the related testicular immunopathology?
The unique immune environment of testicular germ cell neoplasia comprises B cells and dendritic cells as well as high transcript levels of IL-6 and other B cell supporting or T helper cell type 1 (Th1)-driven cytokines and thus differs profoundly from normal testis or inflammatory lesions associated with hypospermatogenesis.
T cells are known to be the major component of inflammatory infiltrates associated with either hypospermatogenesis or testicular cancer. It has previously been reported that B cells are only involved within infiltrates of seminoma samples, but this has not been investigated further.
Immunohistochemical characterisation (IHC) of infiltrating immune cells and RT-qPCR-based analysis of corresponding cytokine microenvironments was performed on different testicular pathologies. Testicular biopsies, obtained from men undergoing andrological work-up of infertility or taken during surgery for testicular cancer, were used in this study. Samples were grouped as follows: (i) normal spermatogenesis (n = 18), (ii) hypospermatogenesis associated with lymphocytic infiltrates (n = 10), (iii) samples showing neoplasia [germ cell neoplasia in situ (GCNIS, n = 26) and seminoma, n = 18].
IHC was performed using antibodies against T cells (CD3+), B cells (CD20cy+), dendritic cells (CD11c+), macrophages (CD68+) and mast cells (mast cell tryptase+). Degree and compartmental localisation of immune cells throughout all groups analysed was evaluated semi-quantitatively. RT-qPCR on RNA extracted from cryo-preserved tissue samples was performed to analyse mRNA cytokine expression, specifically levels of IL-1β, IL-6, IL-17a, tumour necrosis factor (TNF)-α (pro-inflammatory), IL-10, transforming growth factor (TGF)-β1 (anti-inflammatory), IL-2, IL-12a, IL-12b, interferon (IFN)-γ (Th1-driven), IL-4, IL-5, IL-13, IL-23a (Th2-driven), CXCL-13, CXCL-10 and CCL-5 (chemokines).
This is the first study showing a direct linkage between the distribution pattern of immune cells in hypospermatogenesis versus testicular cancer and analysis of a wide range of 17 related cyto- and chemokines. A fundamental difference between testicular inflammation patterns associated with different testicular inflammatory conditions either containing or lacking neoplastic cells was demonstrated. In hypospermatogenesis, T cells were detected, whereas B cells and dendritic cells were almost absent. Within GCNIS and seminoma, in addition to T cells, high numbers of dendritic cells and B cells were found, the latter additionally organised in cell clusters, whereas mast cells were absent. Transcripts encoding pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), anti-inflammatory cytokines (TGF-β1), Th1-driven cytokines (IL-2 and IFN-γ) as well as chemokines (CXCL-13, CXCL-10 and CCL-5) were all significantly increased in testicular germ cell neoplasia (P ≤ 0.01), suggesting the presence of a pro-tumorigenic environment. In contrast, Th2-related cytokines (IL-5, IL-13 and IL-23a) characterised the environment within samples showing normal spermatogenesis as well as hypospermatogenesis. One of the most important outcomes is the pivotal role of IL-6 in testicular cancer that opens potential novel diagnostic and/or immune-therapeutic perspective for testis cancer.
Testicular tissue composed of immune cells as well as other somatic cells and germ cells does not allow identification of specific cytokine sources or single cell types, being responsible for establishing the overall cytokine environment. In this study, laser-assisted microdissection did not reach the required efficiency for RT-qPCR analyses. Therefore, in vitro models would be suggested for addressing the above-mentioned issue. Conclusions about cytokine levels in testes with GCNIS are based on a small number of samples.
The unique B cell presence and the significantly increased expression level of IL-6 in testicular germ cell neoplasia (P < 0.001) strengthen its special role in this disease. In line with current knowledge on other types of cancer, these results underline the relevance of further investigating the potential of IL-6 as early biomarker and target for therapeutic intervention in testicular germ cell neoplasia.
This study (and B.K. in person) was supported by the Deutsche Forschungsgemeinschaft (DFG) as part of the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) on 'Molecular pathogenesis on male reproductive disorders'. T.H., H.-C.S. and M.B. were supported by the LOEWE focus group 'MIBIE' (male infertility during infection & inflammation)-an excellence initiative of the German state government of Hessen. From the Australian side, K.L. was supported by NHMRC grants (Fellowship, ID1079646 and Project, ID1081987); K.L., S.I. and M.H. received scholarship (S.I.) and research funding (K.L., M.H.) from Monash University. The project also drew support from the Victorian Government's Operational Infrastructure Support Program. The authors have no competing interests to declare.
Klein B
，Haggeney T
，Fietz D
，Indumathy S
，Loveland KL
，Hedger M
，Kliesch S
，Weidner W
，Bergmann M
，Schuppe HC
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