Platelet-derived extracellular vesicles inhibit ferroptosis and promote distant metastasis of nasopharyngeal carcinoma by upregulating ITGB3

Nasopharyngeal carcinoma (NPC) is a malignancy with high metastatic and invasive nature. Distant metastasis contributes substantially to treatment failure and mortality in NPC. Platelets are versatile blood cells and the number of platelets is positively associated with the distant metastasis of tumor cells. However, the role and underlying mechanism of platelets responsible for the metastasis of NPC cells remain unclear. Here we found that the distant metastasis of NPC patients was positively correlated with the expression levels of integrin β3 (ITGB3) in platelet-derived extracellular vesicles (EVs) from NPC patients (P-EVs). We further revealed that EVs transfer occurred from platelets to NPC cells, mediating cell-cell communication and inducing the metastasis of NPC cells by upregulating ITGB3 expression. Mechanistically, P-EVs-upregulated ITGB3 increased SLC7A11 expression by enhancing protein stability and activating the MAPK/ERK/ATF4/Nrf2 axis, which suppressed ferroptosis, thereby facilitating the metastasis of NPC cells. NPC xenografts in mouse models further confirmed that P-EVs inhibited the ferroptosis of circulating NPC cells and promoted the distant metastasis of NPC cells. Thus, these findings elucidate a novel role of platelet-derived EVs in NPC metastasis, which not only improves our understanding of platelet-mediated tumor distant metastasis, but also has important implications in diagnosis and treatment of NPC.


Generation of ITGB3 and SLC7A11 knockout cells
Both ITGB3 and SLC7A11 knockout (KO) 6-10B and 5-8F cells were generated using the CRISPR/Cas9 system, as described previously [1]. Briefly, guide RNA (gRNA) targeting ITGB3 or SLC7A11 was constructed into the lentiCRISPR v2 plasmid. 6-10B and 5-8F cells were infected with lentiviruses obtained from HEK293T cells cotransfected with lentiCRISPR v2 coding for the respective sgRNA, psPAX2, and pMD2.G. After selection with 1 µg/ml puromycin for 3 days, single cells were seeded into 96-well plates and cultured for 3 weeks, followed by harvesting for ITGB3 and SLC7A11 KO testing using western blot analysis. The gRNA sequences targeting ITGB3 and SLC7A11 used in the study have been published previously [2,3].

Western blot and co-immunoprecipitation (Co-IP)
Total was applied to determine protein expression levels. Detailed information on the antibodies is summarized in Table S1. For Co-IP, whole-cell lysates were incubated with anti-ITGB3 antibodies for 6 h and followed by capture with protein A/G-magnetic beads for 16 h. The bound proteins were then washed, eluted, separated by SDS-PAGE, and detected by western blotting.
Briefly, the purified EVs were diluted in PBS solution to achieve a concentration range of 10 7 -10 8 particles per ml. Data were obtained by reading three 1-min videos with parameters set at a camera level of 12 and a detection threshold of 3. The average particle size distribution and concentration were analyzed using NTA software (version 3.2 Build 16) and graphs were plotted using GraphPad Prism 5.

Cell migration and invasion assay
Cell migration and invasion assays were performed as described previously [1]. Briefly, Culture-Insert (Ibidi) was used to perform a wound healing assay to measure cell migration. Culture-Insert was removed to form scratches after NPC cells were seeded

Transendothelial migration assay
The transendothelial migration assay was performed as previously reported [5]. Briefly, mCherry-transfected HMEC-1 cells were seeded into a μ-Dish (Ibidi) to achieve a monolayer of cells. P-EVs (5 µg/ml)-treated 6-10B-GFP and 5-8F-GFP stable cells were then overlaid onto the endothelial layer and incubated for 2 h. Cells were fixed in 4% paraformaldehyde, and z-stack confocal images were acquired from top to bottom of the HMEC-1 cell layer.

3D spheroid cultures
NPC cells were resuspended in RPMI-1640 medium containing 30% FBS at a concentration of 1×10 6 cells/ml. Cells were mixed with VitroGel Hydrogel Matrix (TheWell, Bioscence, USA) at a 1:2 ratio and 300 µL of the hydrogel/cell mixture was pipetted into a 24-well plate and incubated for 15 min at room temperature. Complete culture medium (300 µL) was added to the top of the hydrogel, and 3D cell cultures were maintained for 10 days. Spheroids were recovered from the hydrogel using VitroGel Cell Recovery Solution (TheWell, Bioscience) according to the manufacturer's instructions for subsequent western blotting and immunofluorescence analysis.

Total RNA isolation and expression analysis
The two cell types (6-10B and 5-8F) were treated with or without P-EVs (5 μg/ml) for 48 h. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA-seq libraries were prepared, sequenced, and analyzed on an Illumina HiSeq 2500 platform by the Beijing Biomarker Technology Corporation, as described in our previous report [1]. All raw data have been deposited under the Gene Expression Omnibus accession number GSE196879.

Flow cytometry
For flow cytometry analysis of cell surface ITGB3 and SLC7A11, 100 μL NPC cells (1×10 6 ) were digested into a single-cell suspension and incubated with primary antibody followed by corresponding FITC-and Alexa 555-conjugated IgG secondary antibody. Rabbit IgG or mouse IgG was used as isotype control. Cells were then washed 3 times by centrifugation at 400 g and analyzed using a CytoFLEX flow cytometer (Beckman, Germany).

Intracellular ROS detection
Intracellular ROS levels were measured as previously described [4]. Briefly, NPC cells were incubated with a DCFH-DA probe at 1:1000 dilution in serum-free RPMI medium at 37℃ for 30 min, washed with serum-free medium in the dark, and detected by a fluorescent reader (TECAN) at an excitation wavelength of 488 nm.

GSH/GSSG detection
NPC cells were lysed by repeated freeze-thaw cycles and then centrifuged to collect the supernatant. Intracellular GSH and GSSG concentrations were detected using GSH and GSSG Assay Kit (Abbkine), according to the manufacturer's instructions.

Cell viability assay
Growth factor-starved NPC cells were seeded into 96-well plates at a density of 3×10 3 cells/well and treated with P-EVs (5 µg/ml) or various inhibitors or activators for 24 h.

Transmission electron microscopy
Transmission electron microscopy assay was performed as previously described [6]. In brief, NPC cells were collected after P-EVs (5 µg/ml) or RSL3 (10 µM) treatment for 24 h and re-suspended in 2.5% glutaraldehyde (Sigma) at 4 °C for preservation. Fixed cells were washed in 0.1 M PB (pH 7.4) and pre-embedded in 1% agarose solution.
Samples were then fixed with 1% OsO4 and dehydrated by concentration gradient of ethanol. Next, samples were penetrated with resin and kept in 37 °C oven overnight.
Resin blocks were then cut to 60 nm section and fished out to 150 meshed cuprum grids and stained with 2 % uranium acetate and 2.6 % lead citrate solution for observation using JEM-1400Flash transmission electron microscopy (JEOL, Japan).

Mitochondrial membrane potential assay
Mitochondrial membrane potential was measured using the JC-10 dye solution (Solarbio, China) as previously described [7]. Briefly, NPC cells were seeded in 6-well plates and treated with P-EVs (5 µg/ml) or RSL3 (10 µM) for 24 h. The cells were trypsinized and centrifuged at 600 g for 3 min. The pellet was resuspended in 500 µl RPMI-1640 medium and 500 µl JC-10 dye solution at a density of 2 × 10 5 cells/tube and incubated at 37 º C in 5 % CO2 for 20 min (in the dark). The membrane potential was analyzed with a CytoFLEX flow cytometer (Beckman) by measuring the fluorescence with excitation at 488 nm and emission at 530 nm and 585 nm.

Intracellular iron (Fe 2+ ) measurement
Intracellular iron (Fe 2+ ) levels were measured using the fluorescent indicator Phen Green SK (Invitrogen) as previously described [8]. Briefly, NPC cells were seeded in 6-well plates and treated with P-EVs (5 µg/ml) or RSL3 (10 µM) for 24 h. The cells were then incubated with 10 µM Phen Green SK in PBS at 37℃ for 10 min, washed with PBS followed by visualized using Nikon EclipseTi2-E fluorescence microscope (Nikon) or analyzed on a CytoFLEX flow cytometer (Beckman).

Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR)
ChIP-qPCR analysis was performed as previously described [9]. In brief, NPC cells were fixed in 1% formaldehyde for 10 min, and after 5 min, glycine was added at a Normal rabbit IgG was used as the negative control. After capture with protein A/Gmagnetic beads, immunoprecipitated chromatin was de-cross-linked, and coimmunoprecipitated DNA fragments were purified for qPCR analysis using the following primer pairs: human SLC7A11 promoter region, forward, 5′-TTGAGCAACAAGCTCCTCCT-3′, and reverse, 5′-CAAACCAGCTCAGCTTCCTC-3′.