Poster Presentation NSW State Cancer Conference 2023

Deciphering the exosomal RNA cargo involved in pancreatic cancer-related diabetes using transcriptomics: a novel approach for early detection of pancreatic cancer (#104)

Helen B Binang 1 2 , Wilson Wong 2 3 , Tanzila Khan 1 2 , Anandwardhan Hardikar 2 3 , Zhihong Xu 1 2 , Marco Falasca 4 , Jerry Greenfield 5 6 7 , Ron Pirola 1 2 , Jeremy Wilson 1 2 , Chamini Perera 1 2 , Minoti Apte 1 2
  1. Pancreatic Research Group, South Western Sydney Clinical Campuses, School of Clinical Medicine, Faculty of Medicine and Health, UNSW Sydney, Sydney 2052, NSW, Australia
  2. Ingham Institute for Applied Medical Research, Sydney 2170, NSW, Australia
  3. Western Sydney University, Sydney, NSW, Australia
  4. Curtin University, Perth, WA 6102, Australia
  5. St Vincent Clinical School, Faculty of Medicine and Health, UNSW Sydney, Sydney 2052, NSW, Australia
  6. Healthy Ageing, Garvan Institute of Medical Research, Darlinghurst 2830, NSW, Australia
  7. Department of Diabetes and Endocrinology, St Vincent's Hospital, Darlinghurst 3065, NSW, Australia

Background and aim: Pancreatic cancer (PC) is a dismal condition with very poor survival rates. A subset (30%) of patients with PC is diagnosed with diabetes 3-5 years before their PC diagnosis. This diabetes, termed pancreatic cancer-related diabetes or PCRD, could be a harbinger of asymptomatic PC.  

Pancreatic stellate cells (PSCs) are found around the earliest PC lesions known as pancreatic intra-epithelial neoplasms (PanINs), and also around islets. PSCs produce the collagenous stroma of PC and interact with cancer cells to promote disease progression. PSC-PC cell interactions may lead to secretion of factors (proteins, RNA, lipids) that are carried by exosomes to influence the function of recipient cells. We hypothesise that such exosomal cargo could impair islet cell function and peripheral insulin signalling leading to PCRD. Specifically, we aimed to identify the RNA cargo within PC/stroma-derived exosomes that may play a role in PCRD. 

Methods: Mouse PC cell line (KPC, n=4) and mouse PSCs (n=5) were cultured either alone or together (KPC + PSC) (n=4). Exosomes were isolated using ultracentrifugation and morphologically characterised by TEM. Total RNA was extracted, and sequencing was carried out using Illumina Stranded Total RNA Ribo-Zero Plus workflow with >10ng of exosomal RNA input. RNA was reversed transcribed to cDNA, followed by library amplification and sequencing on the Illumina sequencing platform.  

Results: Exosomes from all 3 culture types were within 40-160nm in size, exhibited typical cup-shaped morphology and expressed exosome specific markers (Alix and/or TSG101). Both coding and non-coding RNAs (such as scaRNAs, snRNAs, lncRNAs, and miRNAs) were expressed in all groups. Interestingly, 35 miRNAs were differentially expressed (p<0.05) between co-cultures and PSCs. Among them, fifteen miRNAs (miR143, miRNA10a, miR30d, miR99b, miR411, miR134, miR30a, miR382, miR337, miR186, miR29a, miR150, miR668, miR484, and miR206) showed strong association with the insulin signalling pathway and regulation of insulin secretion. Also identified were RNAs of the mirlet7 family (let7a-1, let7a-2, let7b, let7c-1, let7c-2, let7e and let7i) which have been shown to block glucose-induced insulin secretion.  

Conclusion: Co-cultured KPCs and PSCs produce exosomes that carry specific miRNAs that are known to modulate pathways known to initiate diabetes. Detailed characterisation of these miRNAs may identify novel biomarkers/therapeutic targets of PC.