Trated in our previous study (Leca et al, JCI, 2016). Much more importantly, we demonstrated that PRT99 is physically linked for the ANXA6 complicated discovered in our prior study. Then applying PRT99 blocking antibody, we confirmed the implication of PRT99 in EVs uptake by decreasing EVs internalization in pancreatic cancer cells as well as a consequent decreased migratory capability. Preliminary benefits suggest that following ANXA6+ EVs uptake by pancreatic cancer cells in a PRT99-dependent process enhances their migratory ability by means of p38 signalling pathway activation. Summary/conclusion: Our benefits deepen the understanding of EVs internalization mode and demonstrate that PRT99 is usually a vital element of ANXA6+ EVs uptake by cancer cells and their consequent achieve in migratory ability. Limiting or impairing the action of PRT99 offers a new window to limit the oncogenic dialogue between stromal and cancer cells in pancreatic cancer. Funding: INCa PLBIO13-134, ERC, SIRIC.Thursday, 03 MayPT04.GABARAPL1 is necessary for the secretion of pro-angiogenic extracellular vesicles for the duration of IL-1 Receptor Accessory Proteins Formulation hypoxia Tom G.H. Keulers1; Marijke I. Zonneveld1; Sten F.H.M. Libregts2; Marca H. M. Wauben2; Kasper M.A. Rouschop1 Autophagy lab, division of Radiotherapy, Grow – college for Oncology Developmental Biology, Maastricht University, Maastricht, The Netherlands; 2Department of Biochemistry and Cell Biology Faculty of Veterinary Medicine, Utrecht University, Utrecht, The NetherlandsBackground: Hypoxia is a hallmark of solid tumours and is linked with tumour ADAM12 Proteins Recombinant Proteins progression and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to the tumour. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication inside the tumour microenvironment and mediate intercellular communication by shuttling biological info for instance miRNA’s, mRNA, proteins and development elements to recipient cells. Previously, we demonstrated that the expression of GABARAPL1, a member in the LC3/GABARAP protein loved ones, is induced during hypoxia. Now, we demonstrate that GABARAPL1 is necessary for secretion of pro-angiogenic EVs during hypoxia. Approaches: Ht29 and U87 doxycycline-inducible GABARAPL1 knockdown cell lines have been exposed to hypoxia (16 h, 0.02 O2). EVs had been isolated by sucrose density gradient isolation and analysed by western blot, qNANO or high-resolution flow cytometry. Angiogenic prospective of cells was assessed by tube formation assays. Xenografts were implanted subcutaneously at the lateral flanks of NMRInu/nu mice and tumour size was measured by calliper. Outcomes: GABARAPL1 is expressed on the EV surface and may be targeted with antibodies. This results in blockade of pro-angiogenic responses in vitro. Silencing GABARAPL1 with inducible knockdown models perturbs EV secretion and outcomes in decreased tumour development as a result of decreased vascularisation and enhanced necrosis. Moreover, targeting GABARAPL1 straight following tumour irradiation resulted in enhanced tumour regrowth delay. Moreover, we demonstrate that GABARAPL1+ EVs are detectable and elevated in blood of cancer individuals. Summary/conclusion: Here, we reveal that hypoxic tumour cells secrete a one of a kind EV subset, marked by GABARAPL1 expression. These EVs manage tumour progression, are targetable and are for that reason interesting to pursue as biomarker and therapeutic target. Funding: This function was financially supported by the Dutch Can.