Article Text

Download PDFPDF
352 Epigenetic mechanisms of platinum resistance in ovarian cancer: patient-derived organoids as translational in vitro experimental model
  1. Juliane Reichenbach1,
  2. Tabea Bauer2,
  3. Sophia Hierlmayer1,
  4. Tingyu Zhang1,
  5. Fabian Kraus1,
  6. Bastian Czogalla1,
  7. Sven Mahner1,
  8. Philipp Rathert2,
  9. Mirjana Kessler1 and
  10. Fabian Trillsch1
  1. 1Department of Gynecology and Obstetrics, Ludwig-Maximilian-University Munich, Munich, Germany
  2. 2Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany

Abstract

Introduction/Background Patient-derived ovarian cancer organoids (PDOs) reflecting functional tumor properties in viable 3D cell cultures, appear to be suitable models to study the complex epigenetic landscape of platinum resistance mechanisms of high-grade serous ovarian cancer (HGSOC). In search for promising potential mediators of sensitivity and to study their effects we searched for approaches to suppress epigenetic effector proteins in HGSOC PDOs.

Methodology We assessed morphological, functional and transcriptional changes of HGSOC PDOs after short-term in vitro carboplatin treatment based on their organoid forming efficiency (OFE) and immunofluorescence. To model the effects of epigenetic effector suppression on non-genetic platinum resistance in vitro, we conducted lentiviral mediated knockdown of the putative transcription factor FLYWCH1 expression in PDOs from different clinically characterized HGSOC patients. After selection of PDOs with an effective incorporation of short hairpin RNA (shRNA) that silences FLYWCH1 expression on the basis of a puromycin resistance gene containing vector cassette, successful FLYWCH1 knockdown was validated by quantitative PCR (qPCR).

Results Post platinum treatment, HGSOC PDOs show changed morphology as sustained shift in phenotype and elevated DNA damage (y-H2Ax) in immunofluorescence staining. Stable PDOs of two different donors with a validated 50% knockdown of FLYWCH1 expression were generated by lentiviral transduction and stability of the silencing was confirmed by serial passaging.

Conclusion Given the long-term phenotypical and morphological changes of PDOs after in vitro carboplatin exposure, HGSOC PDOs along with their clinical data serve as valuable models to investigate the complex epigenetic landscape of HGSOC as consequence for drug response. Our study highlights the potential of RNAi-mediated gene knockdown experiments by lentiviral transduction to evaluate the molecular regulation, phenotype and drug response of HGSOC through in vitro simulation of clinical scenarios.

Disclosures F.T. received research funding, advisory board, honoraria, and travel expenses from AstraZeneca, Clovis, Eisai, ImmunoGen, Medac, MSD, PharmaMar, Roche, and Tesaro/GSK. S.M. Received Research funding, advisory board, honorary or travel expenses: AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Hubro, Medac, MSD, Novartis, Nykode, Olympus, PharmaMar, Pfizer, Roche, Sensor Kinesis, Teva, Tesaro.

Abstract 352 Figure 1

Epigenetic mechanisms of platinum resistance in ovarian cancer: patient-derived organoids as translational in vitro experimental model a) Morphological, functional and transcriptional changes of HGSOC PDOs after short-term in vitro carboplatin treatment based on their organoid forming efficiency (OFE) and immunofluorescence b) Lentiviral mediated knockdown of FLYWCH1 expression in PDOs from different clinically characterized HGSOC patients. c) Stable PDOs of two different donors with a validated 50% knockdown of FLYWCH1 expression were generated by lentiviral transduction and stability of the silencing was confirmed by serial passaging

Statistics from Altmetric.com

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.