3 minute lightning oral presentation (and poster) ESA-SRB 2023 in conjunction with ENSA

RNA-seq to identify activin and TGFβ target genes in Testicular Germ Cell Tumours (#207)

Sarah C Moody 1 2 , Benedict Nathaniel 1 , Ciara Conduit 3 , Nathan Lawrentschuk 4 5 6 , Ben Tran 3 7 , 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. Department of Medical Oncology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  4. Department of Urology, Royal Melbourne Hospital, Melbourne, VIC, Australia
  5. EJ Whitten Centre for Prostate Cancer Research, Epworth Healthcare, Melbourne, VIC, Australia
  6. Department of Urology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  7. Division of Personalised Medicine, Walter and Eliza Hall Institute, Parkville, VIC, Australia

Testicular germ cell tumours (TGCTs) are the most common solid tumour affecting young men (19-44 yo). TGCTs arise when fetal germ cells fail to differentiate, forming germ cell neoplasia in situ (GCNIS) and transforming into seminomas or non-seminomas around puberty. As in many cancers, altered TGFβ superfamily has been implicated. To identify activin A and TGFβ superfamily target genes, fresh TGCT and adjacent tissue was obtained from two consented patient orchidectomies (non-seminoma, HTCa8; seminoma, HTCa9). Each was divided in half (A and B), then further divided for histology (4% PFA), transcript analysis (snap-frozen) at collection (T=0) and culture (1 mm3 fragments, 48 hours, 30 µL drops (0.1% BSA/DMEM:F12 + ITS + PS) with 50ng/mL activin A, 10µM SB431542 (TGFβ/activin/Nodal inhibitor) or vehicle controls (n=3-5).  By immunohistochemistry, HTCa8 tumour appeared heterogeneous (classical non-seminoma); HTCa8 adjacent contained GCNIS (OCT4+). HTCa9 tumours displayed classic homogeneous seminoma (OCT4+), with immune infiltrates (CD68+); HTCa9 adjacent tubules were abnormal. Bulk RNA-sequencing and DAVID analysis was performed on HTCa8 adjacent and HTCa9 tumour fragment cultures. HTCa8A and HTCa8B exhibited distinct transcript profiles (A: normal germ cells, DDX4, TNP1, minimal GCNIS; B: GCNIS-rich, POU5F1), and were analysed separately. HTCa9A and B fragments consistently expressed seminoma markers (SOX17, POU5F1, TFAP2C) and were combined for analysis. Changes following culture in untreated samples (vs T=0) included downregulation of cholesterol and steroid biosynthesis-associated genes in HTCa8, and downregulation in chemokine-related transcripts in HTCa9 (FDR<0.05, LogFC>1). Activin A and SB431542-treated samples each displayed differentially expressed genes (DEGs; vs vehicle; p<0.05, LogFC>1), with reciprocal expression of some between these opposing treatments, and DEGs common across HTCa8A, HTCa8B and HTCa9 tissue following activin A or SB431542 treatment. Interrogation of DEGs is ongoing, informing future culture experiments in conjunction with spatial transcriptomics to delineate cell targets and outcomes associated with disrupted pathway activity.