Abstract 1588: Identification of new signaling pathways between fibroblasts & tumor cells in pancreatic cancer
Authors: Shoukari Ayman Al , Batardiere Maelle , Movahed Nooshin , beaussier Camille , Baig Jumanah , gonzalez Melissa , Moraille Patricia , Bonneil Eric , Rousseau Louise , turcotte Simon , Delgiorno Kathleen , Tan Marcus , Means Anna , Ajibisheh Elham Dianati , Trinh Quoc-huy 
Published: March 2024

We explore the unstudied role of nanoparticles (NPs), recently discovered secreted non-vesicular nanoparticles, in signaling between pre-cancer associated fibroblasts (preCAF), CAFs, and tumor cells. We generated two novel primary lines preCAF PFUM1 from a patient with high-grade intraductal papillary mucinous neoplasms and CAF FUM2 from a patient with pancreatic ductal adenocarcinoma, all tests performed between passages 2 and 3, fibroblast purity validated by RT-PCR and ACTA2 immunofluorescence. Small extracellular vesicles (sEVs), exomeres, and supermeres (the two main forms of NPs) were isolated by the ultracentrifugation method from PF-UM1 and F-UM2 with n=2-3 experiments. To validate the isolation, we used nanoparticle tracking, electron, and fluid-phase atomic force microscopy. Proteins were quantified by BCA assay. Liquid chromatography-mass spectrometry characterized the cargo from each fraction. Human cancer lines MIAPACA2 and SU8686 were treated with 10 μg of each fraction and cell proliferation was tested by phase-contrast images (Cytation 5), analyzed by Ilastik-based 1.4.0 machine-learning. We performed fluorescent Ki-67 and vimentin staining and quantified in QuPath 0.4.3. Non-parametric statistics were performed in Prism GraphPad.

NPs constitute the major fraction of small particles released by fibroblasts, containing 80.5% (PFUM1) and 77.5% (FUM2) of the protein load by quantification. Mass spectrometry reveals enrichment of growth factors, epithelial-to-mesenchymal (EMT) transition, and metabolism-associated proteins in the NPs compared to sEVs. At 72 hours, cell proliferation for Su8686 reveals a proliferative effect for all fractions from both PFUM1 and FUM2 (19-63% increase compared to controls, p=0.049 to &lt;0.0001). For MIAPACA2, only supermeres from PFUM1 and FUM2 consistently increased proliferation (9.5-35% increase, p=0.0051 to &lt;0.0001). Ki-67 expression in MIAPACA2, increased by 9.6-23.0% following treatment by NPs (all p&lt;0.0001) and by 2.9-9.2% with sEVs (p=0.011 to &lt;0.0001). EMT was estimated by vimentin in MIAPACA2, as all preCAF fractions from PFUM1 reduced vimentin expression (p&lt;0.001), while CAF FUM2 NPs increased vimentin expression (p&lt;0.0001) while sEVs had no effect (p=0.075).

In conclusion, the majority of pancreatic tumor-associated fibroblast small secretome is composed of NPs, exhibiting significant functional pro-tumoral effects.

Citation Format: Ayman Al Shoukari, Maelle Batardiere, Nooshin Movahed, Camille beaussier, Jumanah Baig, Melissa gonzalez, Patricia Moraille, Eric Bonneil, Louise Rousseau, Simon turcotte, Kathleen Delgiorno, Marcus Tan, Anna Means, Elham Dianati Ajibisheh, Quoc-huy Trinh. Identification of new signaling pathways between fibroblasts & tumor cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1588.