Endometrial small extracellular vesicles reprogram trophectoderm cell proteome towards a pro-invasive phenotype

To provide insights into the molecular changes in trophectoderm/trophoblast cells that support their sEV-driven invasion, we performed proteomic profiling of hTSCs following single-dose sEV treatment (Figure 2A). A total of 2,318 proteins were identified (Supplementary Table S4) of which 108 were either uniquely identified (55) or displayed significantly higher abundance (53, p < .05) in hTSCs treated with EP-sEVs compared to E-sEVs (Supplementary Table S5A/5B). Gene Ontology enrichment analysis (Biological Processes) of proteins distinct between EP-sEVs and E-sEVs identified significantly (p < .05) enriched terms such as “TGF-beta receptor signaling pathway; p < 4.62E-04”, “transmembrane receptor protein serine/threonine kinase signaling pathway; p < 5.11E-04,” “BMP signaling pathway; p < 6.65E-04,” “regulation of cell-substrate adhesion; p < 7.89E-04,” “keratinocyte cell migration; p < 3.59E-05”; processes that are implicated in cell invasion (Figure 2B, Supplementary Table S6). These proteins include migratory factors DPP4, ADAM9, and RALB, cell adhesion/invasive regulators CCN1, COL1A1, and THBS1, and cell motility signaling regulator NRP1 (Supplementary Table S7). Kinase Enrichment Analysis 3 (KEA3) (Kuleshov et al., 2021) was employed to identify upstream kinases whose substrates in hTSCs following EP-sEVs treatment were enriched, including EGFR, AKT1, and MAPK3 (Figure 2D). Human kinome regulatory network was used to highlight the top-ranked kinases with all kinases labelled by WGCNA modules, including various interaction networks associated with MAPK3 (Figures 2E, F).

Open in a separate window

FIGURE 2

Endometrial small extracellular vesicles reprogram trophoblast proteome towards invasive phenotype. (A) Experimental workflow of hTSC proteome remodeling following treatment with E-/EP-sEV at global and cell surface proteome, with quantified proteins for each group shown. (B) Gene Ontology enrichment analysis (DAVID) for biological processes enriched in TSCs treated with either EP-sEVs or E-sEVs. (C) Gene Ontology enrichment analysis (DAVID) for biological processes enriched in proteins abundant in TSC cell surfaceome following treatment with E-/EP-sEVs (and UT). (D–H) Kinase Enrichment Analysis 3 (KEA3) (Kuleshov et al., 2021) was employed to identify upstream kinases whose substrates are overrepresented in differentially abundant proteins. For higher abundant proteins following EP-sEVs treatment (D) bar chart displays the top-ranking kinases across different libraries whose putative substrates are overrepresented (E) Human kinome regulatory network highlighting the top-ranked kinases with all kinases labelled by WGCNA modules and (F) Kinase co-regulatory networks constructed from top-ranked kinase enrichment results for various kinase-substrate interaction libraries. For higher abundant surface proteins in following EP-sEVs treatment, (G) bar chart displays the top-ranking kinases across different libraries whose putative substrates are overrepresented, (H) Human kinome regulatory network highlighting the top-ranked kinases with all kinases labelled by WGCNA modules and (I) Kinase co-regulatory networks constructed from top-ranked kinase enrichment results for various kinase-substrate interaction libraries.

Of note, 307 (from 2,318) proteins, based on cell surface databases CSPA/SURFY as cell surface proteins (plasma membrane, surface) (e.g., LAMP2, ADAM9, LTBP1, STT3A, DPP4, ITGA2/AV, ERO1A) (Supplementary Table S8) are known to interact with the ECM during invasion. Due to their essential role in ECM remodeling during invasion, we next ascertained their surface localization in hTSCs. For this, we employed cell membrane-impermeant biotin to capture surface proteins that were then identified and quantified using MS, similar to our previous report (Rai et al., 2021c) (Figure 2A, Supplementary Table S9).

A total of 1,346 surface proteins were found in hTSCs (vehicle-treated (UT), E-sEV-treated, EP-sEV-treated; identified in at least 2/3 biological replicates for each group, including 328 identified in CSPA/SURFY (Supplementary Table S10). Of the proteins identified as cell surface from CSPA/SURFY, differential analysis revealed 264 and 220 proteins significantly (p < .05) higher abundance in hTSCs stimulated with EP- or E-sEVs compared to PBS-treated hTSCs (Supplementary Tables S9, S10).

Gene Ontology enrichment analysis (Biological Processes) of proteins distinct between each cluster (EP-sEVs vs. UT; 264 proteins, E-sEVs vs. UT; 220 proteins, and EP-sEVs vs. E-sEVs; 185 proteins; Supplementary Table S10) identified significantly (p < .05) enriched terms such as “wound healing,” “cell-substrate adhesion,” and “substrate adhesion-dependent cell spreading” for both EP-sEV and E-sEVs (vs. UT) groups (Figure 2C). For EP-sEV treatment (vs. E-sEVs) processes significantly enriched include “mRNA processing”; p < 3.21E-05, and “extracellular matrix organization”; p < 4.92E-05” (Figure 2C). For EP-sEV treatment (vs. E-sEVs and UT) specific proteins were shown implicated in regulation of antigen presentation (PSMA5, PSMD11, PSMA2), crosslinking of collagen fibrils (BMP1, LOXL1), NOTCH and VEGF signalling (NOTCH2, PSMD11, PSMA2/NCKAP1, NRAS, ROCK2, PXN), and EPHB-mediated forward signalling (ROCK2, ARPC3, EPHB4), important processes regulating cell invasion (Supplementary Table S9/11). These findings suggest EP-sEVs promote remodeling of cell surface proteome in hTSCs, with changes in proteome landscape associated with regulators of implantation and cell invasion (Table 2).

Kinase Enrichment Analysis 3 (KEA3) (Kuleshov et al., 2021) further identified upstream kinases whose substrates from cell surface hTSCs following EP-sEVs treatment were enriched, including cell invasive regulators MAPK3 and AKT1 (Figures 2G–I). We further identify specific growth factor and kinase receptors, including EPHB4/B2, ERBB2, STRAP, EGFR, and PDGFRA, and upregulated expression of pro-invasive regulators associated with focal adhesions (NRP1, PTPRK, ROCK2, TEK) and embryo implantation (FBLN1, NIBAN2, BSG) (Supplementary Table S9) in response to EP-sEV treatment on hTSC surfaceome remodelling. Of note, we report “positive regulation of MAPK cascade”; p < 4.68E-04 (Table 2). This highlights proteome reprogramming of trophoblasts by sEVs towards an invasive phenotype.

Trophectoderm cell culture and spheroid generation

The human trophectodermal stem cells T3-TSC (hTSC) derived from individual blastomeres of donated human embryos (Zdravkovic et al., 2015) were maintained as described (Poh et al., 2021). Briefly, cells were cultured in DMEM/F-12 supplemented with 1% (v/v) Pen/Strep, 10% (v/v) FBS, 10 nM fibroblast growth factor (FGF) (bFGF, R&D Systems) and 10 nM SB431542 (#1614, Tocris Bioscience), in flasks pre-coated with 0.5% (w/v) gelatin, and maintained at 37°C, 5% CO₂.

hTSC spheroids were generated as described (Evans et al., 2019) with modifications. hTSCs (~1500 cells) in 100 µl of hTSC media were cultured in round bottom ultra-low attachment 96-well plates (Costar) for 72 h at 37°C, 5% CO₂ (1 spheroid/well). Spheroids were observed using bright field microscopy and deformed spheroids were excluded from functional use.

Endometrial small extracellular vesicle isolation and purification

E- and EP-primed CM from Ishikawa cells were centrifuged at 500 g, 5 min and 2,000 g, 10 min at 4°C, with supernatant then centrifuged at 10,000 g, 30 min, 4°C (SW28 rotor; Optima L-90K Ultracentrifuge). The resulting supernatant was centrifuged at 100,000 g, 1 h, 4°C to pellet crude sEVs which were washed in 1 ml PBS at 100,000 g, 1 h, 4°C. sEVs from Ishikawa cells were purified using OptiPrep™ density gradient-based separation as described (Rai et al., 2021c). Briefly, 1 ml volumes of 40%, 20%, 10%, and 0.6 ml of 5% iodixanol solution were layered sequentially in a polypropylene tube (11 × 60 mm). Dilutions of iodixanol solution were made in 0.2 M sucrose/1× PBS solution. sEVs (200 µl) were overlaid and centrifuged at 100,000 g, 18 h, 4°C (SW60 rotor, Optima L-90K Ultracentrifuge). Twelve 300 µl fractions were collected, diluted in 1 ml PBS and subjected to a wash spin at 100,000 g, 1 h, 4°C. Pellets were re-suspended in 20–100 µl PBS; fractions six to eight were pooled (termed purified E-sEVs or EP-sEVs) and stored at −80°C. The density of each fraction was determined using a control OptiPrep™ gradient overlaid with 200 µl PBS; each fraction was diluted 1:10,000 in MilliQ and absorbance measured at 244 nm using Nanodrop 2000c; absorbance of fractions were converted to density as described (Rai et al., 2021c).

sEV particle size was determined using nanoparticle tracking analysis (NTA, NS300 NanoSight system) as described (Rai et al., 2021c). Syringe pump speed was set to 100 camera detection threshold was at least 10 and temperature set to 25°C. Three technical replicates (60 s video) were recorded and analyzed using NTA software (3.1.45).

Experimental parameters are submitted to EV-TRACK knowledgebase (EV-TRACK ID: EV220403) (Consortium et al., 2017).

Western blotting

Samples were lysed in western blot buffer [4% (w/v) SDS, 20% (v/v) glycerol and 0.01% bromophenol blue, 0.125 M Tris-hydrochloride, pH 6.8] with 100 mM dithiothreitol (DTT, Thermo Fisher Scientific) as described (Rai et al., 2021c). Membranes were incubated with primary mouse or rabbit antibodies against ALIX (1:1,000 dilution) (3A9; Cell Signaling Technology), TSG101 (1:1,000 dilution) (622696; BD Biosciences), GAPDH (1:1,000 dilution) (D4C6R; Cell Signaling Technology), estrogen receptor alpha (ERα) (1:500 dilution) (D6R2W; Cell Signaling Technology), progesterone receptor A/B (PR A/B) (1:500 dilution) (D8Q2J; Cell Signaling Technology), p-MAPK (9101; Cell signaling), MAPK (9102; Cell signaling), in Tween-PBS (TPBS), overnight at 4°C. Membranes were rinsed with TPBS and incubated with secondary antibodies (1:15,000); IRDye 800CW goat anti-mouse antibody or IRDye 680RD goat anti-rabbit antibody (Li-COR Biosciences), for 1 h while shaking at RT. Membranes were rinsed with TPBS and imaged using Odyssey Infrared Imaging System (Li-COR Biosciences, Nebraska United States), measuring 700 nm and 800 nm wavelengths. Protein-based densitometry to quantify p-MAPK/MAPK relative expression was performed using ImageJ (v1.53c).

Lipophilic dye labelling of small extracellular vesicles and cell uptake assay

E- and EP-sEVs (10 µg) were incubated with 1 µM DiI (Invitrogen) staining solution at RT, 10 min sEVs (and PBS dye control) were overlaid on a 100 µl cushion of 10% OptiPrep™ (in PBS) and centrifuged at 100,000 g, 1 h, 4°C. Supernatants were removed and pellets resuspended in PBS. hTSCs were cultured to ~70% confluency as droplets in 8-well glass chamber slides (Sarstedt) pre-coated with 0.5% (w/v) gelatin, at 37°C, 5% CO₂. Cells were incubated with 100 μg/ml E- or EP-sEVs (or PBS dye control) at 37°C, 2 h. Cells were washed in DMEM/F-12 media. Nuclei were stained with Hoechst 33,342 stain (Thermo Fisher Scientific) (10 μg/ml) for 10 min, washed with DMEM/F-12 and fixed with 4% formaldehyde at RT, prior to imaging by Nikon A1R confocal microscope equipped with resonant scanner, using a Plan Fluor 20× MImm (DIC N2, Nikon, Tokyo, Japan). Images were sequentially acquired and are representative of three biological replicates.

hTSC spheroid invasion assay into Matrigel™ matrix

Transwell-Matrigel™ invasion assays were performed using growth factor-reduced Matrigel™ matrix (Corning), as previously described (Rai et al., 2019). Briefly, 8-well microscopy chambers (Corning) coated with Matrigel™ were overlaid with hTSC spheroids (harvested after 72 h growth, ~30 spheroids/tube) that were pre-treated with sEVs or PBS alone for 2 h at 37°C. The inserts were nested onto 24-well companion plate (Corning) containing DMEM/F-12 with 1% (v/v) Pen/Strep, 10 nM FGF, 10 nM SB431542 and either E-sEVs or EP-sEVs (50 μg/ml) or PBS alone and incubated for 24 h at 37°C. Subsequently, 50 µl media was removed and mixed 1:1 with Matrigel™ and gently overlaid on spheroids in wells, Matrigel™ allowed to solidify for 30 min at 37°C followed by addition of 200 μl of DMEM/F-12 [10% (v/v) FBS, 1% (v/v) Pen/Strep] to each well. After 48 h, spheroids were imaged using Olympus FSX100 [n = 13–20 spheroids analyzed, with four measurements (mm) taken per spheroid]. The extent (%) of invasion was assessed by calculating [(outer diameter—inner diameter)/(inner diameter) × 100] using a digital ruler. Data are presented as average ±SEM. Where indicated, hTSC spheroids were pre-treated with 14 nM Selumetinib (Sellekchem, AZD6244) (Rai et al., 2019) or DMSO for 30 min at 37°C before treatment with EP-sEVs (100 μg/ml) or PBS for 45 min hTSC spheroids were overlaid onto solidified Matrigel™ to invade for 48 h and imaged as described (n = 7–13 spheroids analyzed).

Sample preparation and mass spectrometry of endometrial sEV and cell proteome

Global mass spectrometry-based proteomics of Ishikawa cells or derived sEVs (n = 3), or hTSCs following sEV treatment (n = 5) (10 μg) was performed as described (Claridge et al., 2021b; Lozano et al., 2022) using single-pot solid-phase-enhanced sample preparation (SP3) method (Hughes et al., 2019). Briefly, Ishikawa cell and sEV derivatives were solubilized in 1% (v/v) SDS, 50 mM tetraethylammonium bromide (TEAB) pH 8.0, while hTSCs were solubilized in 1% (v/v) SDS, 50 mM HEPES pH 8.0. Samples were incubated at 95°C for 5 min, with cell lysates tip probe sonicated (10 s, 23 amplitude) (Misonix—S-4000 Ultrasonic Liquid Processor). Samples were reduced with 10 mM DTT for 50 min at 25°C followed by alkylation with 20 mM iodoacetamide (IAA) for 30 min at 25°C in the dark. The reaction was quenched to a final concentration of 20 mM DTT. Magnetic beads were prepared by mixing SpeedBeads™ magnetic carboxylate modified particles (65152105050250, 45152105050250, Cytiva) at 1:1 (v:v) ratio and washing twice with 200 µl MS-water. Magnetic beads were reconstituted to a final concentration of 100 μg/μl. Magnetic beads were added to the samples at 10:1 beads-to-protein ratio and 100% ethanol (EA043, ChemSupply) added for a final concentration of 50% ethanol (v/v). Protein-bound magnetic beads were washed three times with 200 µl of 80% ethanol and reconstituted in 50 mM TEAB and digested with trypsin (Promega, V5111) at a 1:50 enzyme-to-substrate ratio for 16 h at 37°C with constant shaking (1,000 rpm). The peptide mixture was acidified to a final concentration of 2% formic acid (FA) (pH ~1–2) and centrifuged at 20,000 g for 1 min. The peptide digests were frozen at −80°C and dried by vacuum centrifugation (Savant SPD121P, Thermo Fisher Scientific), reconstituted in 0.07% trifluoroacetic acid (TFA), and quantified by Fluorometric Peptide Assay (23,290, Thermo Fisher Scientific) as per manufacturer’s instruction.

Peptides were analyzed on a Dionex UltiMate NCS-3500RS nanoUHPLC coupled to a Q-Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer equipped with nanospray ion source in positive mode as described (Greening et al., 2021; Kompa et al., 2021). Peptides were loaded (Acclaim PepMap100 C18 3 μm beads with 100 Å pore-size, Thermo Fisher Scientific) and separated (1.9 µm particle size C18, 0.075 × 250 mm, Nikkyo Technos Co. Ltd.,) with a gradient of 2%–28% acetonitrile containing 0.1% FA over 95 min at 300 nl·min⁻¹ followed by 28–80% from 95–98 min at 300 nL·min⁻¹ at 55°C (butterfly portfolio heater, Phoenix S&T). An MS1 scan was acquired from 350 to 1,650 m/z [60,000 resolution, 3 × 10⁶ automatic gain control (AGC), 128 m injection time] followed by MS/MS data-dependent acquisition (top 25) with collision-induced dissociation and detection in the ion trap (30,000 resolution, 1 × 10⁵ AGC, 60 m injection time, 28% normalized collision energy, 1.3 m/z quadrupole isolation width). Unassigned, 1, six to eight precursor ions charge states were rejected and peptide match disabled. Selected sequenced ions were dynamically excluded for 30 s. Data was acquired using Xcalibur software v4.0 (Thermo Fisher Scientific). All MS-based proteomics data (cellular, sEV, phosphoproteome, surfaceome) have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository and are available via ProteomeXchange with identifier (PXD027642). Therefore, for this study, biological replicates were performed as indicated: global proteome analysis (n = 5) and cell surfaceome analysis (n = 3).

Sample preparation and mass spectrometry of hTSC phosphoproteome

hTSCs (~20,000) were seeded in 96-well plates pre-coated with 0.5% (w/v) gelatin and cultured to 100% confluency. Cells were serum-starved for 24 h prior to treatment with 100 μg/ml purified EP-sEVs or PBS control for 15 min at 37°C, 5% CO₂ (n = 4). Cells were immediately washed 2× in ice-cold PBS and lysed in-well with MS lysis buffer [1% (v/v) SDS, 1:100 HALT protease phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78,442), 50 mM HEPES pH 8] on ice for 5 min, heat treated at 95°C for 5 min prior to SP3 sample preparation. Cell lysates were reduced, alkylated, quenched and trypsin digested as described, with the resulting peptide mixture acidified to a final concentration of 2% FA, and centrifuged at 20,000 g for 1 min. For phosphoproteomic analysis by tandem mass tag (TMT) multiplexing, peptides were labelled with 10-plex TMT labels according to the manufacturer’s instructions (Thermo Fisher Scientific, 90406/A34807, lot UG287488/278919). A list of the sample labelling strategy is available in PRIDE proteomeXchange (PXD027642). Peptides were labelled in a final concentration of 50% acetonitrile for 90 min at RT followed by de-acylation with 0.25% hydroxylamine for 15 min at RT and quenching with 0.1% TFA. Peptides from each 10-plex experiment were pooled and digests lyophilised by vacuum centrifugation and reconstituted in Binding/Equilibration Buffer for phosphopeptide enrichment, using the High-Select™ TiO2 Phosphopeptide Enrichment workflow, as per manufacter’s instructions (Thermo Fisher Scientific, A32993). Briefly, TMT-labelled peptide digests were transferred to a pre-equilibrated TiO₂ spin tip and centrifuged twice at 1,000 g, 5 min. The column was washed twice with binding/equilibration buffer and subsequent wash buffer at 3,000 g, 2 min, followed by a wash with MS-grade water at 3,000 g, 2 min. Phosphopeptides were eluted in 100 µl phosphopeptide elution buffer by centrifugation at 1,000 g, 5 min, dried by vacuum centrifugation and reconstituted in 0.07% TFA, before quantification by Colorimetric Peptide Assay (Thermo Fisher Scientific, 23,275) as per manufacturer’s instructions.

MS-based proteomic analysis of TMT-labelled phosphopeptides was performed on a Dionex UltiMate NCS-3500RS nanoUHPLC coupled to a Q-Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer equipped with nanospray ion source in positive mode. TMT labelled peptides were separated (1.9 µm particle size C18, 0.075 × 250 mm, Nikkyo Technos Co. Ltd.,) with a gradient of 4%–28% ACN containing 0.1% FA over 224 min at 300 nl/min at 55°C (butterfly portfolio heater, Phoenix S&T). The MS1 scan was acquired from 300 to 1650 m/z (60,000 resolution, 3e6 AGC, 128 m injection time) followed by MS/MS data-dependent acquisition of the top 15 ions with HCD (30,000 resolution, 1e5 AGC, 60 ms injection time, 33 NCE, 0.8 m/z isolation width). Unassigned, 1, six to eight precursor ions charge states were rejected and peptide match disabled. Selected sequenced ions were dynamically excluded for 30 s. Data was acquired using Xcalibur software v4.0. Therefore, for this study, biological replicates of the phosphoproteome analysis (n = 4).

Sample preparation and mass spectrometry of hTSC cell surfaceome

hTSCs (~80,000) were seeded in 24-well plates pre-coated with 0.5% (w/v) gelatin and cultured to ~80% confluency, serum-starved for 24 h prior to treatment with E- or EP-sEVs (50 μg/ml) or PBS for 48 h (n = 3). Cell surface proteins were biotinylated using Pierce™ Cell Surface Biotinylation (A44390, Thermo Fisher Scientific) as described (Rai et al., 2021c). Briefly, cells were washed twice with PBS and labeled with 0.25 mg/ml EZ-Link Sulfo-NHS-SS-Biotin for 10 min at RT. Cells were washed twice with ice-cold Tris-buffered saline and lysed (30 min on ice) as per manufacturer’s instructions. Biotin-labelled proteins were captured onto NeutrAvidin Agarose slurry (30 min at RT with end-over-end mixing on a rotor). Samples were loaded onto epTIPS (Eppendorf, 200 μl) fitted with 20 μm nylon net (NY2004700, Merck Millipore), washed 3× as per manufacturers instruction, and reduced in 10 mM DTT in 100 mM TEAB for 45 min at 25°C. Eluted proteins were alkylated, quenched and trypsin digested as described, with resultant peptides acidified (pH ~2.0) to a final concentration of 2% FA, peptides desalted using SDB-RPS Stage-Tips (Rai et al., 2021a) followed by elution with 30%–80% acetonitrile, 0.1% TFA, and dried by vacuum centrifugation. Peptides were reconstituted in 0.07% TFA and quantified by Fluorometric Peptide Assay. Peptides were analysed on a Dionex UltiMate NCS-3500RS nanoUHPLC coupled to a Q-Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer equipped with nanospray ion source in positive data-dependent acquisition mode over 95 min gradient, as described (Rai et al., 2021c).

Data processing and database analyses

Raw mass spectrometric data were processed in MaxQuant (v1.6.14.0) with its built-in search engine Andromeda (Cox and Mann, 2008) to perform peptide identification and quantification as described (Kompa et al., 2021; Lozano et al., 2022). Database search was with the Andromeda search engine against the Human-only canonical sequence database (2020) with a contaminants database employed. Cysteine carbamidomethylation was set as a fixed modification and acetyl (Protein N-term) and methionine oxidations as variable modifications. Differential search parameters included: TMT tags on peptide N terminus/lysine and carbamidomethylation of cysteine set as a fixed modification; for phosphoproteome analysis phosphorylation of serine, threonine and tyrosine were set as variable modifications; for biotin surface proteome analysis additional Thioacyl (DSP) {[C (3)H (4)OS]} (Rai et al., 2021c) was employed. Enzyme specificity was set as C-terminal to arginine and lysine using trypsin protease, and a maximum of two missed cleavages allowed. Match between runs options were enabled, with the matching time window set to 1 min, with label-free protein quantitation (LFQ) performed where applicable. False discovery rate (FDR) at the PSM, protein, and site levels were each 1%. Peptides were identified with an initial precursor mass deviation of up to 7 ppm and a fragment mass deviation of 20 ppm. A maximum of two missed cleavages were allowed. Protein group or phosphorylation site tables were imported into Perseus (v1.6.7) for analysis, with contaminants and reverse peptides removed.