The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue

Abstract

Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell communication. To study the role of EVs released by cancer-associated adipose tissue in breast cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs and maintains their functional characteristics is required. We implemented differential ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation (dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western blot analysis identified EVs that are enriched in flotillin-1, CD9 and CD63, and sized between 20 and 200 nm with a density of 1.076-1.125 g/ml. The lack of protein aggregates and cell organelle proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically intact and biologically active.

Precipitation‐based extracellular vesicle isolation from rat plasma co‐precipitate vesicle‐free microRNAs

“…To study the precipitation‐isolated EV pellet in more detail, we imaged it with TEM and compared it to results of the SEC EV‐isolation from plasma. TEM imaging of the precipitation‐isolated EV pellet showed several round particles, some of which were cup‐shaped, which is consistent with EVs [23,28,29]. Most of the round particles, however, had a morphology comparable to that of lipoproteins [23,30].…”

“…In a recent “Viewpoints” article, Simonsen estimated that one milliliter of plasma contains 10 7 –10 9 EV particles and up to 10 16 lipoprotein particles [7]. Our TEM images of the precipitation‐isolated EV pellet revealed particles with a mainly round shape, consistent with a lipoprotein morphology [23,30], and only very few EVs with a distinct cup‐shaped morphology [23,28,29], suggesting that the plasma lipoproteins were not removed during precipitation‐based EV isolation.…”

Extracellular vesicles derived from MSCs activates dermal papilla cell in vitro and promotes hair follicle conversion from telogen to anagen in mice

“…In this study, we have successfully isolated EVs from MSC cell culture media, which are consistent with the reported size and shape of EVs in previous studies 11 , 15 , 34 . EVs expressed the EV-specific surface markers ALIX and CD63, and cell compartment markers like GM130, cytochrome c and calnexin were not detected in EVs, confirming the isolation of pure EVs without contamination with cell organelles and apoptotic bodies 11 , 35 . EV interaction and uptake, which is necessary for the delivery of biomaterials to recipient cells, involves direct fusion to the plasma membrane via ligand-receptor interactions or lipids such as phosphatidylserine 36 , followed by release of EV contents into the cytoplasm.…”

Biological material collection to advance translational research and treatment of children with CNS tumours: position paper from the SIOPE Brain Tumour Group

“…To maximise the advantages of biomaterial collection and obtain a comprehensive biological understanding of tumours and host-related factors, different types of biomaterials should be collected (table). [65][66][67][68] Adequate amounts of tumour tissue, considering the safety of the patient, should be collected according to the following three criteria: (1) as unfixed, snap-frozen tissue;…”