(A) Cell-surface HS staining in untreated (Ctrl) and HS lyase treated U87 MG cells as control for Figure 7B. (B) Bodipy-FA specifically labels vesicular lipid membranes of U87 MG cells as donors of BodipyFA-EVs applied in Figure 7C. (C) Quantification and visualization of BodipyFA-EV uptake in relation to PKH-labelled EVs by flow cytometry (left panel) and confocal microscopy (right panel), respectively. (D) Free Bodipy-FA does not directly label LDs. Hypoxic U87 MG cells were allowed to form LDs by incubation with EVs for 48 h, and were then stained with LipidTox (red) and Bodipy-FA (yellow). White square indicates zoomed in area for detailed view. Scale bars=20 μm.
Funding
Cancerfonden
Vetenskapsrådet
Swedish Childhood Cancer Foundation
Mrs. Berta Kamprad Foundation
Skane County Council's Research and Development Foundation
ARTICLE ABSTRACT
As an adaptive response to hypoxic stress, aggressive tumors rewire their metabolic phenotype into increased malignant behavior through extracellular lipid scavenging and storage in lipid droplets (LD). However, the underlying mechanisms and potential lipid source retrieved in the hypoxic tumor microenvironment remain poorly understood. Here, we show that exosome-like extracellular vesicles (EV), known as influential messengers in the tumor microenvironment, may also serve anabolic functions by transforming hypoxic, patient-derived human glioblastoma cell lines into the LD+ phenotype. EVs were internalized via a hypoxia-sensitive, endocytic mechanism that fueled LD formation through direct lipid transfer, and independently of fatty acid synthase activity. EVs can enter cells through multiple and yet ill-defined pathways. On a mechanistic level, we found that hypoxia-mediated EV uptake depends on increased heparan sulfate proteoglycan (HSPG) endocytosis that preferentially followed the lipid raft pathway. The functional relevance of HSPG was evidenced by the reversal of EV-mediated LD loading by targeting of HSPG receptor function.
Together, our data extend the multifaceted role of EVs in cancer biology by showing their LD-inducing capacity in hypoxic glioma cells. Moreover, these findings highlight a potential function for HSPG-mediated endocytosis as a salvage pathway for EV retrieval during tumor stress conditions.
