Flash Talk & E-Poster Presentation 33rd Lorne Cancer Conference 2021

Mapping the matrisome of pancreatic tumours using quantitative proteomics (#45)

Brooke A Pereira 1 2 , Shona Ritchie 1 2 , David Herrmann 1 2 , Cecilia R Chambers 1 2 , Morghan Lucas 1 , Astrid Magenau 1 2 , Kendelle J Murphy 1 2 , Max Nobis 1 2 , Michael Papanicolaou 1 2 , Romain Rouet 1 2 , Sunny Wu 1 2 , Julia Yin 1 2 , Young K Lee 1 , Daniel A Reed 1 , Victoria Lee 1 , Anais Zaratzian 1 , Jeff Evans 3 4 , Lorraine A Chantrill 1 5 , Benjamin L Parker 6 , Gus Grey 7 , Takako Sasaki 8 , Alex Swarbrick 1 , Daniel Christ 1 , Jennifer P Morton 3 4 , Marina Pajic 1 , Anthony J Gill 9 10 11 , Thomas R Cox 1 , Paul Timpson 1
  1. Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
  2. St Vincent's Clinical School, University of New South Wales, Sydney, NSW, Australia
  3. Beatson Institute, Cancer Research UK , Glasgow, Scotland, United Kingdom
  4. Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom
  5. St Vincent’s Hospital, Sydney, NSW, Australia
  6. The University of Melbourne, Melbourne, VIC, Australia
  7. Department of Physiology, The University of Auckland, Auckland, New Zealand
  8. Department of Biochemistry, Oita University, Oita, Japan
  9. Kolling Institute of Medical Research, Sydney, NSW, Australia
  10. NSW Health Pathology, Department of Anatomical Pathology, Royal North Shore Hospital, St. Leonards, NSW, Australia
  11. Department of Surgery, Royal North Shore Hospital, St. Leonards, NSW, Australia

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal, with a five-year survival rate of ~9%. PDAC is characterised by stromal activation, leading to pro-tumourigenic extracellular matrix (ECM) deposition. Recently, we have shown that targeting desmoplasia can improve chemotherapy efficacy and impair metastasis in pre-clinical models. As such, we aimed to use proteomics to dissect the matrisomal signatures of pancreatic tumours derived from the highly metastatic KPC (Pdx1-Cre; LSL-K-rasG12D/+; LSL-p53R172H/+) and poorly metastatic KPflC (Pdx1-Cre; LSL-K-rasG12D/+; LSL-p53fl/+) mouse models. We hypothesised that these tumours would have distinct matrisomes, with these changes revealing novel pro-metastatic matrisomal proteins involved in PDAC.

Pancreatic tissue from wildtype, KPC, and KPflC mice were collected at early (6 weeks), mid (10 weeks) and end-stage disease (12+ weeks) and were decellularised. Data-independent acquisition (DIA) liquid-chromatography tandem mass spectrometry (LC-MS/MS) was used to identify differentially abundant proteins.

LC-MS/MS demonstrates an increased abundance of Nidogen 2 (NID2) in KPC tumours at mid stage disease compared to KPflC. NID2 is a 200 kDa basement membrane glycoprotein that closely interacts with laminins, type IV collagen and perlecan to promote the assembly of ternary complexes. Interrogation of single cell-RNASeq murine and human PDAC datasets revealed that NID2 is enriched in PDAC specimens, especially at mid stage disease, mirroring our proteomics results.

Immunofluorescence, western blotting, and qPCR show that NID2 is significantly enhanced in CAFs isolated from KPC tumours. To assess the functional properties of NID2, CRISPR interference (CRISPRi) was employed to reduce the expression of NID2 in KPC CAFs. 3D organotypic collagen matrices seeded with NID2lo CAFs had significantly lower levels of desmoplasia, shown via second harmonic generation (SHG) imaging and Picrosirius Red staining. Ongoing work includes subcutaneous and orthotopic co-seeding experiments using CRISPR-edited NID2 KO KPC CAFs in combination with KPC CCs to assess the role of CAF-derived NID2 in metastasis and chemoresistance in vivo.