Brain metastases-derived extracellular vesicles induce binding and aggregation of low-density lipoprotein

Abstract

Background Cancer cell-derived extracellular vesicles (EVs) have previously been shown to contribute to pre-metastatic niche formation. Specifically, aggressive tumors secrete pro-metastatic EVs that travel in the circulation to distant organs to modulate the microenvironment for future metastatic spread. Previous studies have focused on the interface between pro-metastatic EVs and epithelial/endothelial cells in the pre-metastatic niche. However, EV interactions with circulating components such as low-density lipoprotein (LDL) have been overlooked. Results This study demonstrates that EVs derived from brain metastases cells (Br-EVs) and corresponding regular cancer cells (Reg-EVs) display different interactions with LDL. Specifically, Br-EVs trigger LDL aggregation, and the presence of LDL accelerates Br-EV uptake by monocytes, which are key components in the brain metastatic niche. Conclusions Collectively, these data are the first to demonstrate that pro-metastatic EVs display distinct interactions with LDL, which impacts monocyte internalization of EVs.

A Simple and Quick Method for Loading Proteins in Extracellular Vesicles

“…EVs were separated from other medium components through tangential flow filtration. Consistent with previously published studies [ 45 , 46 , 65 ], tangential flow filtration resulted in separation and concentration of nanosized EVs ( Figure 1 b, Supplementary Figure S1 ) that exhibited a negative zeta potential as expected ( Figure 1 c). Isolated EVs contained Hsc70, annexin V, CD63, and CD9 ( Figure 1 d), which are characteristic markers [ 12 ] and were negative for calnexin, an endoplasmic reticulum protein ( Figure 1 d) typically used as an indicator of intracellular vesicle contaminants [ 12 ].…”

“…Such agents could potentially exhibit tumorigenicity or lead to accelerated growth of existing tumors. Additionally, EVs from cancer cells may display abnormal interactions with circulating components [ 65 ]. Despite the potential safety concerns of cancer cell-derived EVs, several initial clinical trials have demonstrated that such EVs have acceptable safety profiles [ 79 ].…”

“…The medium harvested from MDA-MB-231 cells in culture was centrifuged (800× g ; 30 min; Sotvall ST 16R centrifuge, Thermo Scientific, Waltham, MA, USA) to pellet down floating cells and large apoptotic bodies. Following a published protocol [ 45 , 46 , 65 ], MDA-MB-231 EVs, referred to as EVs throughout the text, were isolated using a KrosFlo Research 2i Tangential Flow Filtration System (Spectrum Labs, San Francisco, CA, USA). Briefly, MDA-MB-231 conditioned medium (0.5–0.8 L) was processed using sterile hollow fiber modified-polyethersulfone membranes designed with 0.65 μm (D02-E65U-07-S; Spectrum Labs, San Francisco, CA) and sterile hollow fiber polysulfone membranes 500 kDa (D02-S500-05-S; Spectrum Labs, San Francisco, CA, USA) molecular weight cut-off pores to remove larger components (~> 650 nm), such as cell debris and small components (~< 30 nm), such as proteins.…”

“…EV sample size and concentration were determined by nanoparticle tracking analysis (NTA), as previously reported [ 45 , 46 , 65 ]. EV samples were diluted (1:100 or 1:50) in cell culture grade PBS (1 mL) and measured with a NanoSight NS300 (Malvern Panalytical, Malvern, Westborough, MA, USA) (60-s measurement; 3 capture replicates).…”

“…Cells were also electroporated with four different doses of Cas9 (1, 0.2, 0.1, or 0.01 µg/10 5 cells) to promote protein intracellular delivery. Based on our previous studies, the delivery efficiency of EVs reaches a saturation point at 2 × 10 4 EVs/cell [ 45 , 65 ], which was, therefore, used as the highest dose in this study. The maximum EV dose was then decreased 5, 10 and 100 times to assess dose-responses.…”