Poster Presentation ESA-SRB-APEG-NZSE 2022

Delineating cell-cell communications in testicular germ cell tumours (#368)

Sarah C Moody 1 2 , Benedict Nathaniel 1 , Daniela Fietz 3 , Hans-Christian Schuppe 4 , Mark Frydenberg 5 6 , Ben Tran 7 8 , Kate L Loveland 1 2
  1. Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, VIC, Australia
  2. Department of Molecular and Translational Sciences, School of Clinical Sciences, Monash University, Clayton, VIC, Australia
  3. Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University Giessen, Giessen, Germany
  4. Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Giessen, Germany
  5. Department of Srugery, Monash University, Clayton, VIC, Australia
  6. Department of Urology, Cabrini Institute, Cabrini Health, Melbourne, VIC, Australia
  7. Department of Medical Oncology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  8. Division of Personalised Medicine, Walter and Eliza Hall Institute, Parkville, VIC, Australia

The incidence of testicular germ cell tumours (TGCTs), the most common tumour in men aged 19-44, is increasing. Precursor germ cell neoplasia in situ (GCNIS) cells arise during fetal life, eventually forming seminoma or non-seminoma tumours. What controls their progression is not understood, though both genetic and environmental factors contribute. This study combines spatial whole transcriptomics on four individual tumour samples with hanging-drop cultures to identify signalling mechanisms, cell profiles and cell-cell interactions within the tumour microenvironment. Fresh testis tissue from orchidectomies were obtained by consent (three non-seminoma and one seminoma, including tumour-free regions). Tissues were 4% PFA-fixed and paraffin-embedded or snap-frozen. Nanostring GeoMx Spatial Whole Transcriptome analysis was performed on fixed sections, with 95 regions of interest (ROI) selected following immunodetection of CD45, PanCK, and a nuclear stain. ROIs included normo- and hypospermatogenic tubules, interstitium, immune cell infiltrates, GCNIS, non-seminoma and seminoma. Following data normalisation, we mapped the expression profile of signalling pathway components and their downstream targets, identifying transcripts robustly linked with specific phenotypes, including activin/TGFβ signalling pathway components (ACVR1B, TGFRB3). Additional tissue from the non-seminomas were cut (1-2 mm3 pieces) for 48-hour hanging drop cultures in 30µL media (0.1% BSA/DMEM:F12/ITS/Pen-Strep) containing 5 or 50 ng/mL activin A, 10µM SB431542 (activin/Nodal/TGFβ inhibitor), or vehicle. Fragments (n=2-3 per treatment) were analysed by immunohistochemistry (OCT4 for GCNIS, CD68 for macrophages) and transcripts measured by qRT-PCR. Cultured fragment histology was consistent with uncultured tissue, retaining macrophages and GCNIS. Transcripts including OCT4, SOX2, SOX17, INHBA and KIT were not significantly altered by activin or SB431542 exposure; variation within treatment groups highlighted non-seminoma heterogeneity. However, MMP9 increased ~30-fold, and LEFTY2 decreased following culture. Identification of activin A target genes is ongoing in conjunction with continued sample collection. These approaches will enable identification of cell communication networks within TGCTs and mechanisms governing tumour phenotypes.