Proteomic Comparison of Malignant Human Germ Cell Tumor Cell Lines

Abstract

Malignant germ cell tumors (GCT) are the most common malignant tumors in young men between 18 and 40 years. The correct identification of histological subtypes, in difficult cases supported by immunohistochemistry, is essential for therapeutic management. Furthermore, biomarkers may help to understand pathophysiological processes in these tumor types. Two GCT cell lines, TCam-2 with seminoma-like characteristics, and NTERA-2, an embryonal carcinoma-like cell line, were compared by a quantitative proteomic approach using high-resolution mass spectrometry (MS) in combination with stable isotope labelling by amino acid in cell culture (SILAC). We were able to identify 4856 proteins and quantify the expression of 3936. 347 were significantly differentially expressed between the two cell lines. For further validation, CD81, CBX-3, PHF6, and ENSA were analyzed by western blot analysis. The results confirmed the MS results. Immunohistochemical analysis on 59 formalin-fixed and paraffin-embedded (FFPE) normal and GCT tissue samples (normal testis, GCNIS, seminomas, and embryonal carcinomas) of these proteins demonstrated the ability to distinguish different GCT subtypes, especially seminomas and embryonal carcinomas. In addition, siRNA-mediated knockdown of these proteins resulted in an antiproliferative effect in TCam-2, NTERA-2, and an additional embryonal carcinoma-like cell line, NCCIT. In summary, this study represents a proteomic resource for the discrimination of malignant germ cell tumor subtypes and the observed antiproliferative effect after knockdown of selected proteins paves the way for the identification of new potential drug targets.

CDK4/6 inhibition presents as a therapeutic option for paediatric and adult germ cell tumours and induces cell cycle arrest and apoptosis via canonical and non-canonical mechanisms

“…Immunohistochemical reactions were performed on 4-μm formalin-fixed and paraffin-embedded tissue sections as published previously. 24 Antigen retrieval was carried out at 98 °C in ethylenediaminetetraacetic acid buffer (pH 9; 20 min). Primary antibodies were incubated for 30 min at RT.…”

The detection of isochromosome i(12p) in malignant germ cell tumours and tumours with somatic malignant transformation by the use of quantitative real‐time polymerase chain reaction

“…IHC reactions were performed on 2‐μm FFPE testicular tissue sections, as described previously 21 . Antigen retrieval was carried out at 97°C in citrate buffer (pH 6) or EDTA buffer (pH 9).…”

Liquid Biopsies in the Clinical Management of Germ Cell Tumor Patients: State-of-the-Art and Future Directions

“…In fact, most proteomic-based studies in this area have focused on tissues instead of liquid biopsies and have disclosed putative biomarkers up- or down-regulated in specific histological subtypes, such as GSTM3, GCP6, CDK10, STARD7, DMTR1, PIWIL1, TMPRSS12 and PAK4, most being implicated in cell cycle regulation, apoptosis, and metabolism [ 170 , 171 , 172 , 173 ]. Additionally, studies in representative cell lines may be informative for guiding further work in patient samples [ 174 ]. There is an opportunity to apply these methodologies to serum investigations as well, as performed by Strenziok and collaborators [ 175 ], who through surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) disclosed a cluster of proteins differentially present in TGCT patients compared to healthy subjects.…”