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Background and purpose: Hypoxia is a common feature of tumours, associated with poor prognosis due to increased resistance to radio- and chemotherapy and enhanced metastasis development. Previously we demonstrated that GABARAPL1 is required for the secretion of extracellular vesicles (EV) with pro-angiogenic properties during hypoxia. Here, we explored the role of GABARAPL1+ EV in the metastatic cascade. Materials and methods: GABARAPL1 deficient or control MDA-MB-231 cells were injected in murine mammary fat pads. Lungs were dissected and analysed for human cytokeratin 18. EV from control and GABARAPL1 deficient cells exposed to normoxia (21% O2) or hypoxia (O2 < 0.02%) were isolated and analysed by immunoblot, nanoparticle tracking analysis, high resolution flow cytometry, mass spectrometry and next-generation sequencing. Cellular migration and invasion were analysed using scratch assays and transwell-invasion assays, respectively. Results: The number of pulmonary metastases derived from GABARAPL1 deficient tumours decreased by 84%. GABARAPL1 deficient cells migrate slower but display a comparable invasive capacity. Both normoxic and hypoxic EV contain proteins and miRNAs associated with metastasis development and, in line, increase cancer cell invasiveness. Although GABARAPL1 deficiency alters EV content, it does not alter the EV-induced increase in cancer cell invasiveness. Conclusion: GABARAPL1 is essential for metastasis development. This is unrelated to changes in migration and invasion and suggests that GABARAPL1 or GABARAPL1+ EV are essential in other processes related to the metastatic cascade.

Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro-angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+ EVs with GABARAPL1 targeting antibodies results in blockade of pro-angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+ EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target.

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