HB-EGF-loaded nanovesicles enhance trophectodermal spheroid attachment and invasion

The ability of trophectodermal cells (outer layer of the embryo) to attach to the endometrial cells and subsequently invade the underlying matrix are critical stages of embryo implantation during successful pregnancy establishment. Extracellular vesicles (EVs) have been implicated in embryo-maternal crosstalk, capable of reprogramming endometrial cells towards a pro-implantation signature and phenotype. However, challenges associated with EV yield and direct loading of biomolecules limit their therapeutic potential. We have previously established generation of cell-derived nanovesicles (NVs) from human trophec-todermal cells (hTSCs) and their capacity to reprogram endometrial cells to enhance adhesion and blastocyst outgrowth. Here, we employed a rapid NV loading strategy to encapsulate potent implantation molecules such as HB-EGF (NVHBEGF). We show these loaded NVs elicit EGFR-mediated eﬀects in recipient endometrial cells, activating kinase phosphorylation sites that modulate their activity (AKT S124/129, MAPK1 T185/Y187), and downstream signalling pathways and processes (AKT signal transduction, GTPase activity). Importantly, they enhanced target cell attachment and invasion. The phosphoproteomics and proteomics approach highlight NVHBEGF-mediated short-term signalling patterns and long-term reprogramming capabilities on endometrial cells which functionally enhance trophectodermal-endometrial interactions. This proof-of-concept study demonstrate feasibility in enhancing the potency of NVs in the context of embryo attachment and establishment.


INTRODUCTION
Embryo implantation is a multi-step process comprising blastocyst apposition and attachment to the maternal endometrial epithelium by its outer trophectodermal layer and its subsequent invasion into the underlying tissue for intrauterine development [1][2][3].Its failure accounts for ∼35% of unsuccessful pregnancy outcomes in Assisted Reproductive Technologies (ART) [1][2][3][4], presenting a significant hurdle for human reproduction.A complex and reciprocal molecular dialogue between the implanting blastocyst and receptive endometrium determines implantation success, directed by the molecules they express or release into the extracellular space.Secreted and surface-expressed molecules initiate ligand-receptor interactions with recipient cells at the embryo-endometrial interface [5], supporting blastocyst tethering to the endometrium [6][7][8]; downstream, they induce receptor conformational changes activating enzymatic or ion channel activity that propagate the signal through the target cell [9,10].This signal transduction initiates a range of molecular and functional changes downstream, although cellular responses are dependent on specific signalling molecules received [11][12][13][14].Paramount for successful implantation, reciprocal embryo-maternal communication [15,16] mediated by secreted signalling players [17][18][19] such as hormones (hCG [20]), cytokines (LIF [21], IL-18 [22]), and growth factors (GM-CSF [23], G-CSF [24]) remains an ongoing topic of investigation in reproductive biology, with efforts to develop them as diagnostic markers of uterine receptivity [25] or therapeutic supplements to enhance implantation success, extending into clinical trials.
Of increasing interest as a signalling modality are extracellular vesicles (EVs) [26,27]; membrane-bound nanosized (30-1000 nm) vesicles that transport and deliver bioactive lipids, proteins, and genetic material to recipient cells, reprogramming and altering their molecular signature and phenotype [28][29][30][31].Indeed, EVs from human embryos and trophectodermal cells (hTSCs) harbour critical regulators of implantation that reprogram recipient endometrial proteome to enhance embryo-endometrial attachment [29,32].However, their low abundance and associated isolation/purification procedures are tedious and time-consuming, prompting investigation into an EV-like alternative; nanovesicles (NVs), generated by serial extrusion of parental cells [33][34][35][36][37].We have demonstrated that from hTSCs, generated NVs displayed similar biophysical and functional properties to natural EVs, functionally capable of significantly promoting trophectodermendometrial attachment and embryo outgrowth [38].As extrusion is recognised as an effective approach for drug loading into nanocarriers such as EVs and liposomes (4-fold higher than passive methods) [39], this methodology enables opportunities for NV cargo modification.
Indeed, loaded EVs and NVs are increasingly explored as fertility therapeutics [40,41].For example, human chorionic gonadotropin (hCG), a potent embryonic signal, was loaded into uterine fluid EVs (UF-EV hCG ) and treated onto endometrial cells, enhancing their expression of receptivity markers [41].Similarly, enrichment of NVs with known regulators of implantation may enhance or confer specific functions while retaining certain influential characteristics of parental cells, such as surface-expressed molecules that facilitate interaction with target

Significance Statement
Nanosized extracellular vesicles and a plethora of growth factors (i.e., HB-EGF) are critical signalling mediators during embryo implantation to the maternal endometrium-a cardinal event of pregnancy establishment.This study highlights a rapid and scalable cell extrusion method to load HB-EGF into trophectodermal cell-derived nanovesicles (NV HBEGF ).We report, through phosphoproteomics and proteomics analyses, NV HBEGF short-term signalling and long-term reprogramming capabilities on recipient endometrial cells, including but not limited to EGFRmediated phosphorylation patterns, downstream signalling events, and cellular processes intimately associated with embryo implantation and endometrial receptivity.Importantly, the application of NV HBEGF stimulated heightened endometrial-trophectodermal attachment, and trophectodermal invasion-pivotal events in the early stages of pregnancy.We have thus harnessed trophectodermal NVs loaded with HB-EGF to orchestrate multifaceted signalling and cellular events in endometrial cells crucial for pregnancy establishment.Loaded NVs possess immense potential for therapeutic development and warrants further investigation.
Amongst the molecules investigated for facilitating embryomaternal crosstalk that governs successful implantation, heparin-binding EGF-like growth factor (HB-EGF) [43][44][45][46][47] remains one of the longest-standing and well-established.With potent embryotropic and endometrial reprogramming capabilities, HB-EGF is secreted by both the developing blastocyst and the receptive endometrium; importantly, both entities express its cognate receptors [5], and are thus responsive to its role in mediating surface interactions, and downstream signalling cascades.Indeed, EGFR, MAPK, and PI3K-AKT signalling pathways and their associated processes are indispensable for successful embryo and endometrial reprogramming [48][49][50] during implantation and throughout pregnancy.In this study, we modified NVs to activate intracellular pathways in low-receptive state recipient cells involved in endometrial cell activation and function.Here, by employing the extrusion methodology [37,38] to enrich hTSC NVs, we encapsulated HB-EGF (NV HBEGF ) and investigated the response of low-receptive HEC1A endometrial recipient cell to modify cell proteome and phosphorylation signalling landscape [51,52].Further, we assessed NV HBEGF functional capacity to enhance trophectodermal spheroid attachment on stimulated endometrial cells and trophectodermal spheroid invasion into Matrigel.Here, we provide new knowledge on how NVs activate recipient cells and associated NV cargo responsible for the subsequent signalling and functional effect.
Cells were grown on flasks coated with 0.5% gelatin prior to experimental seeding and passaged using Trypsin-EDTA (Gibco).Spheroids were generated as described [32,54] with slight modifications.T3-TSC cells were seeded at 1500 cells per well in an ultra-low adhesion round-bottom 96-well plate in 100 μL of trophectoderm medium and incubated for 72 h.

HEC1A endometrial epithelial cells
Human endometrial carcinoma HEC1A cells (HTB-112) were a kind gift from Professor Lois Salamonsen purchased from American Type Culture Collection (ATCC; Rockville, MD, USA).Endometrial cells were routinely maintained in DMEM/F12 supplemented with 1% P/S, and 5% v/v FCS and incubated at 37 • C with 5% CO 2 .Cells were routinely passaged using 0.5% v/v trypsin-EDTA (Gibco).Prior to treatments used in this study, cells were cultured in basal media overnight comprising DMEM/F12 supplemented with 0.6% insulin transferrin selenium (ITS, Gibco) and 1% v/v P/S.
Briefly, samples were reduced with 10 mM DTT at RT for 1 h (350 rpm), alkylated with 20 mM iodoacetamide (IAA) (Sigma-Aldrich) for 20 min at RT (light protected), and quenched with 10 mM DTT.A Sera-Mag SpeedBead carboxylate-modified magnetic particle mixture (1:1 hydrophilic and hydrophobic mix, #65152105050250, #45152105050250, Cytiva) was added to protein extracts and incubated in 50% v/v ethanol for 10 min (1000 rpm) at RT. Beads were sedimented on a magnetic rack to remove the supernatant.Beads were washed 3 times with 200 μL 80% v/v ethanol, then resuspended in 100 μL 50 mM TEAB pH 8.0 and digested overnight with trypsin (1:50 trypsin: protein ratio; Promega, V5111) at 37 • C, 1000 rpm.The peptide and bead mixture were centrifuged at 20,000 g for 1 min at RT.Samples were then placed on a magnetic rack and the supernatant was collected, acidified to a final concentration of 1.5% formic acid, frozen at −80 • C for 20 min, and dried by vacuum centrifugation.Peptides were resuspended in 0.07% trifluoroacetic acid (TFA), quantified by Fluorometric Peptide Assay (Thermo Fisher Scientific, 23290) as per manufacturer's instructions, and normalised to 0.5 μg/μL with 0.07% TFA.

Phosphopeptide enrichment
Peptide digests from each HEC1A cell treatment group (n = 3) were lyophilised by vacuum centrifugation and reconstituted in Binding/Equilibration Buffer for phosphopeptide enrichment [52] using High-Select TiO 2 Phosphopeptide Enrichment kit (Thermo Fisher Scientific, A32993), as described [51].Briefly, peptide digests were transferred to a pre-equilibrated TiO 2 spin tip and centrifuged twice at 1000 g, 5 min.The column was washed twice with binding/equilibration buffer and subsequent wash buffer at 3000 g, 2 min, then with MSgrade water at 3000 g, 2 min.Phosphopeptides were eluted in 100 μL phosphopeptide elution buffer by centrifugation at 1000 g, 5 min, dried by vacuum centrifugation, and reconstituted in 0.07% TFA before quantification by Colorimetric Peptide Assay (ThermoFisher Scientific, #23275) as per manufacturer's instructions.

Data processing and bioinformatics
Peptide identification and quantification were performed as described previously [29,38,51]  algorithm in MaxQuant was used to obtain quantification intensity values and processed using Perseus as described [63].Cytoscape [64] (v3.9.1) with STRING and EnrichmentMap plugins were used for functional enrichment analyses (KEGG, Reactome, Gene Ontology (GO) biological process) of proteins and to generate protein-protein interaction networks.The kinase-substrate database from PhosphoSite Plus (https://www.phosphosite.org/)was used to identify upstream kinases for phosphorylated proteins.

Statistical analysis
Data clean up and analysis were performed using Perseus (MaxQuant computational platform) and Excel.Protein intensities were log 2 transformed and subjected to one-way ANOVA followed by Post hoc Tukey's HSD test to identify significant differences between treatment groups.
For stimulated HEC1A endometrial cells, proteins identified in ≥2

Generation of HB-EGF-loaded NVs from human trophectodermal cells
Cell-derived NVs were generated by serial extrusion of hTSCs (6.25 × 10 6 ) suspended in PBS through microfilters of decreasing pore size (10-5-1 μm) as described [37].Compared to EV isolation from conditioned media (∼14 days), NVs can be generated in 6 h [38].To generate NVs loaded with HB-EGF (NV HBEGF ), we serially extruded hTSCs in PBS containing 50 ng/mL of HB-EGF (Figure 1A).NVs were then isolated using density gradient separation [37] (Figure 1A).NVs and NV HBEGF displayed similar buoyant densities of 1.10-1.20 g/cm 3 , and cryo electron microscopy revealed that NVs were spherical in shape and morphologically intact (Figure 1B), ranging 20-250 nm in diameter (mean 104.2 nm) (Figure 1C), consistent with NVs [37] generated previously.We next questioned whether HB-EGF is successfully incorporated into NVs.We subjected NVs (NVs and NV HBEGF , n = 3) to mass spectrometry-based proteomic profiling, noting differential enrichment of HF-EGF in NV HBEGF (identified in all biological replicates) compared to unloaded NV controls (Figure 1D).Based on stringent peptide and protein identification criteria we quantified HB-EGF and various other EGF-associated and CD44 protein markers in NV HBEGF , compared to NVs.We orthogonally validated loading of HB-EGF into NVs using a monoclonal antibody specific to human HB-EGF protein by Western blotting (Figure 1E).

NV HBEGF uptake by recipient endometrial HEC1A cells
Previously, we have shown that hTSC NVs can be taken up by endometrial HEC1A cells to enhance their attachment to hTSC cell spheroids [38].Here, we questioned whether loading of HB-EGF into NVs impacts their uptake.For this, NV HBEGF were labelled with fluorescent lipophilic DiI dye (red) and incubated with HEC1A cells over a 2-h period.Confocal fluorescence microscopy revealed that NV HBEGF , similar to unloaded NVs, were readily taken up by HEC1A cells (Figure 2A).
Imaging along the z-axis showed that NV HBEGF were internalised and appeared as punctate structures, typical of vesicle uptake by recipient cells [29,52] (Figure 2B).
For insights into whether the HB-EGF loaded into NVs are functional in recipient HEC1A cells, we stimulated HEC1A cells with NV HBEGF and NVs (5 min treatment) and performed phosphoproteomics analysis (Figure 3A, Table S1).Further, to investigate the dynamic cellular signalling events initiated by NV HBEGF ; Erlotinib [69], an EGFR inhibitor; was used as a pre-treatment to suppress NV HBEGF -mediated EGFR signalling in HEC1A cells (ErloNV HBEGF ) (Figure 3A).
From this profiling analysis we demonstrate that NVs loaded with HB-EGF can mediate rapid (5 min) and dynamic changes in the phosphorylation landscape of HEC1A endometrial cells, including regulators of intracellular signal transduction and EGFR signalling networks, as well as known regulators of endometrial receptivity.

NV HBEGF treatment with trophectodermal spheroids significantly enhances their invasive capacity into Matrigel matrix
Trophoblast invasion and outgrowth into the endometrium is a hallmark of successful implantation and placentation [84,[94][95][96] and assessed in vitro using the Matrigel matrix invasion assay [25,32,52] (Figure 5C).Here, trophectodermal spheroids were incubated with corresponding treatments for 2 h prior to seeding into Matrigel.A second dose of treatment in media was supplemented after 24 h and the level of invasion monitored across 72 h using light microscopy (Figure 5E,F, Figure S2).Increase in invasion was measured by subtracting the area of the original spheroid from the final measured area of invasion (Figure 5E,F).NV HBEGF treatment displayed the highest significant increase in spheroid invasion (%) at 248.7 ± 75.1 -approximately 1.5-times higher than PBS (185 ± 32.6, p < 0.0005), while NV (237.9 ± 76.9, p > 0.05) and HB-EGF (210.5 ± 79.5, p < 0.05) treatment performed similarly (Figure 5D).From our observations with ErloNV HBEGF (80.9 ± 36.4,p < 0.0005), EGFR inhibition with erlotinib diminished the invasive capacity of spheroids which could not be restored by subsequent NV HBEGF treatment (Figure 5D).
Our findings demonstrate the enhanced functional impact of HB-EGF loading into NVs by demonstrating increased (i) attachment of low receptive endometrial cells to trophectodermal spheroids and (ii) invasion of trophectodermal spheroids into Matrigel matrix, compared to unmodified NVs.In doing so, we highlight EGFR signalling as a critical mediator of NV HBEGF function.

DISCUSSION
Nanosized EVs and a plethora of growth factors (i.e., HB-EGF) are critical signalling mediators during embryo implantation to the maternal endometrium-a cardinal event of pregnancy establishment.This study highlights a rapid and scalable cell extrusion method to load the implantation regulator HB-EGF into trophectodermal cell-derived nanovesicles (NV HBEGF ).Our study employs phosphoproteomics and proteomics analysis to demonstrate NV HBEGF short-term signalling and long-term reprogramming capabilities on recipient low receptive HEC1A human endometrial cells.We highlight that NV HBEGF elicit EGFR-mediated effects in recipient endometrial cells.Importantly, these protein phosphorylation activities and signalling patterns, including the activation of kinases and phosphorylation sites that regulate their function (i.e., AKT1 S124 [75] and S129 [76], MAPK1 T185 and Y187 [77]); and signalling processes (i.e., AKT signal transduction, GTPase activity) downstream of EGFR activation; induce functional changes in recipient cells to enhance endometrial attachment to the trophectoderm, and trophectodermal invasion into Matrigel matrix.
Indeed, we show that hTSC NV HBEGF and NVs, enriched in these molecules, are effective supplements for promoting endometrial adhesion to trophectodermal cells and trophectodermal invasion into Matrigel (Figure 5).For example, they may be paired with clinically available supplements such as EmbryoGlue, a viscous embryo transfer media which minimises embryo mobility [111,112], supports embryo health [113], and which hyaluronan content promotes interactions with CD44 [114], purported to enhance implantation [112,115] and live birth [116][117][118][119] rates by 7%-9%.Like gelatin methacryloyl-rich hydrogels [120][121][122], they may serve to prolong the stability and function of NVs.Similarly in various applications, NV composition can be tailored to suit various therapeutic purposes, such as the selection of macrophages for spinal cord [42] or tumour [33] targeting, stem cells for their regenerative properties [35][36][37], and insulin-producing cells for diabetes management [123].However, the parental cells' natural composition can often limit their function, requiring dose titrations and functional assays [27,124] to determine an effective dose, although selection of the appropriate functional assays and their standardisation remains an area of active discussion [27].
Modifying NV composition is a method of fine-tuning their function; for example loading of chemotherapeutic drugs [33,125] for cancer therapy or drug-specific investigations, or antioxidative enzymes [39,126] for oxidative stress-related diseases; it may thus be explored further to achieve a range of outcomes in different contexts.The extrusion strategy described in this study, for example, can be amended to load other factors to enhance implantation, such as those explored in clinical trials (i.e., hCG (NCT01786252 [127], NCT01030393 [128])), without genetically modifying parental hTSCs [129].While HB-EGF was selected for enrichment into NVs for its indispensable roles in pregnancy establishment [8, 10, 44-47, 49, 130, 131], its well-researched mechanism of action makes it a suitable target for functional validation and for dissecting the embryo-maternal interface.HB-EGF interacts with RTKs EGFR and ERBB4 expressed on target cells to initiate multiple downstream signalling cascades [48,132] (i.e., MAPK, PI3K-AKT/PIP, small GTPase) (reviewed [133]).Furthermore, HB-EGF may perform synergistically with the high expression of heparan sulfate proteoglycans [134] expressed in NVs from their trophectodermal source, as this enhances their binding to high-affinity receptors (i.e., ERBB4 [8]), potentially augmenting its influence in recipient cells.
However, given the variety of signalling patterns initiated by EGFR, this can induce variable phenotypic responses and outcomes in cells [11][12][13][14]; for example, GTPase activity regulates cytoskeletal remodelling and cell polarity [135,136] in endometrial cells to enhance their adhesive capacity [103,137,138]; in embryos, however, it influences transcription activity and signalling (CREB, WNT, JNK) [139,140] to modulate cell differentiation [140] and embryo size [139].We have thus assessed the temporal effects of NV HBEGF treatment; from the early phosphorylation-mediated signalling events occurring in recipient cells, to its molecular landscape and function at the approximate time of embryo attachment (1 to 2 days [80]).
Upon binding to compatible ligands, CD44 phosphorylates GAB1 [145] to initiate AKT signalling, and activates downstream effectors including RhoGTPases [146][147][148], to induce cytoskeletal reorganisation and cell migration and adhesion.Interestingly, despite EGFR inhibition, NV HBEGF induced the phosphorylation of GAB1 (S163) (Figure 3B), and other proteins implicated in the regulation of GTPase activity, supporting NV HBEGF -CD44 interaction as another pivotal driver of endometrial reprogramming.At the site of embryo implantation, GTPase activity exerts influence on PI3K-AKT signalling and RhoA in mouse embryos to mediate their implantation [149], endometrial cell contraction/migration [136,150], and focal adhesion [135,[150][151][152]; it is thus an indispensable mediator of embryo-endometrial interactions [103,137,138].Compared to endometrial cells, hTSCs and their derived EVs were enriched in GTPases [29]; the latter's treatment onto recipient endometrial cells upregulated cytoskeletal organisation and cell polarity processes, potentially through GTPase activity as a trophectoderm-mediated signalling strategy.Indeed, supplementation of our unloaded NVs significantly augmented the adhesive capacity of HEC1A endometrial cells to trophectodermal spheroids, as well as the invasive capacity of trophectodermal cells (Figure 5).Whether the latter observation is attributed to PI3K-AKT signalling [149] still warrants investigation.
We have demonstrated marked functional influence of NV HBEGF on HEC1A endometrial cells compared to HB-EGF and NVs; which significantly augmented their adhesion to trophectoderm cells by ∼40% from baseline (PBS)-double the capacity of HB-EGF and NVs (Figure 5).Given that NV HBEGF and HB-EGF share a higher proportion of upregulated proteins in endometrial cells compared to NVs, and the well-studied role of HB-EGF [8, 10, 44-47, 49, 130, 131] and ERBB/EGFR [12,[153][154][155] signalling at the embryo-maternal interface, we posit that the latter has substantial influence on our functional observations.Indeed, with the erlotinib targeted inhibition of EGFR [156], NV HBEGF treatment could not restore endometrial or trophectodermal cell function to baseline (PBS) levels.Moreover, amongst the phosphorylation of kinases and expression of their corresponding proteins downregulated by EGFR inhibition (Figure 4D), the most dysregulated proteins include those upregulated at implantation sites [81,157] (Figure 4E).Even so, the functional capacity of HB-EGF was inconsistent, and at best comparable to NVs; a similar phenomenon was observed in hCG-loaded EVs from human uterine fluid [158], which demonstrated the enhanced capacity to induce expression of receptivity markers in recipient endometrial cells compared to hCG alone, EVs alone, or co-supplementation of hCG with EVs.Prior attempts to develop signalling mediators (i.e., hCG [20], LIF [21] and G-CSF [24]) with strong links to fertility and endometrial receptivity as fertilityenhancing supplements have also been unsuccessful in clinical trials.
Taken together, these observations allude to a multi-faceted signalling mechanism by engineered EVs or NVs that encompass properties of their enriched molecule and their biological source, thereby enhancing their functional benefit and potential therapeutic utility.NVs thus represent a feasible and adaptable method of large-scale generation of therapeutic vesicles for tuning endometrial phenotype and function and their therapeutic potential may be heightened when paired with clinically available diagnostic tools including the endometrial receptivity array (ERA) [78,159].For example, LIF and EGFR are molecules typically expressed in the endometrium during receptivity [160][161][162][163], selecting LIF or HB-EGF that binding to target receptors can be an effective way of activating these signalling pathways.Moreover, these molecules may be loaded into or conjugated on the surface of NVs and administered via intraperitoneal injection or vaginally to improve implantation outcomes [164,165].This proof-of-concept study demonstrate feasibility in enhancing the potency of NVs in the context of embryo attachment and pregnancy establishment.Whether these loaded NVs improve implantation rate in vivo warrants future investigation.
68] ERBB2/4 and F I G U R E 1 Production and characterisation of NV HBEGF .(A) NV HBEGF were generated by serial extrusion (10, 5, 1 μm filters, 13 times per membrane) of human trophectodermal cells (T3-TSCs) with either 50 ng/mL of HB-EGF or PBS and purified using density-cushion ultracentrifugation to obtain 7 fractions (F1-7) of increasing density.NV-containing fraction (F5) was obtained.(B) Cryo-electron microscopic image of NV HBEGF displayed spherical and morphologically intact structures; scale 100 nm.(C) Size distribution of NV HBEGF based on cryo-electron microscopic images (n = 4) reveal enrichment of particles 50-150 nm in diameter.(D) Abundance of HB-EGF using mass spectrometry analysis; normalized LFQ intensities (log 2 ) of HB-EGF between NV HBEGF and NVs generated using the same workflow from hTSCs and mouse embryonic fibroblasts.Additional marker proteins associated with EGFR, HSPG2, and CD44 were also compared, noting similarly expression in both NV groups.(E) Western blot display of HB-EGF enrichment in NV HBEGF compared to NVs (n = 3).NV HBEGF , HB-EGF-loaded NVs; NVs, nanovesicles.

For
insights into the downstream cellular processes and signalling pathways affected by EGFR inhibition following NV treatment, we F I G U R E 2 Uptake of NV HBEGF and NVs by HEC1A endometrial cells.(A) Confocal fluorescent microscopy images demonstrating uptake of NV HBEGF or NVs labelled with DiI lipophilic fluorescent dye labelled (red) by HEC1A endometrial cells after a 2-h incubation (n = 3).(B) Fluorescent Z-stack image displaying intracellular distribution of DiI-labelled NV HBEGF (red).Nuclei of HEC1A endometrial cells were stained with Hoechst (blue).Scale bar 10 μm.NV HBEGF , HB-EGF-loaded NVs; NVs, nanovesicles.
Erlotinib treatment disrupted phosphorylation of ERBB/EGFR signalling players; a potential mechanism by which NV HBEGF and NVs reprogram HEC1A cells.We analysed the proteome of HEC1A cells following ErloNV HBEGF treatment and compared with NV HBEGF treatment.Indeed, of 127 proteins downregulated (107 not detected, 20 significantly downregulated) by ErloNV HBEGF treatment compared to NV HBEGF (Figure 4C) included 3 proteins associated with ERBB/EGFR signalling: (i) MTOR, a protein synthesis regulator that forms a positive feedback loop to AKT signalling; (ii) GRB2, upstream regulator of MAPK and PI3K signalling pathways; and (iii) RPS6KA1, a gene expression regulator.Processes associated with the downregulated proteins include vesicle-mediated transport, symbiotic process, organelle organisation and cellular localisation; with 86 proteins categorised as 'KW-0597: phosphoprotein' (Figure 4C, Table

F
I G U R E 3 NV HBEGF remodel the phosphoproteome landscape in HEC1A endometrial cells.(A) Workflow for NV HBEGF and NV treatment onto recipient HEC1A endometrial cells, including a 2-step treatment of erlotinib (EGFR inhibition) followed by NV HBEGF stimulation, and subsequent cell phosphoproteome preparation and analysis.(B) Heatmap expression (log 2 ) of phosphorylated proteins and phosphosites of players of the EGFR signalling pathway, which are downregulated when EGFR is inhibited by erlotinib (white corresponds to missing values).(C) (Top) Erlotinib inhibited the phosphorylation of 421 proteins (compared to PBS), while subsequent NV HBEGF treatment induced phosphorylation of 261 of the inhibited proteins; (Bottom) bubble plot displaying key biological processes and pathways corresponding to the 421 and 261 proteins respectively.NV HBEGF , HB-EGF-loaded NVs; NVs, nanovesicles.
9).Collectively, we highlight the capacity of NV-mediated reprogramming of endometrial cells to modulate proteome dynamics associated with EGFR signalling and changes in the endometrium associated with F I G U R E 4 NV HBEGF remodel the proteome landscape and EGFR signaling network at the time of implantation.(A) Workflow employed for proteomic analysis of stimulated HEC1A endometrial cells.(B) Proteins uniquely identified and significantly upregulated in NV HBEGF -or NV-treated HEC1A cells compared to PBS.(C) Pre-treatment of HEC1A cells with erlotinib followed by NV HBEGF downregulated the expression of 127 proteins compared to NV HBEGF , which are categorised into related biological processes.(D) NV HBEGF -and ErloNV HBEGF -mediated phosphorylation levels of 13 kinases that are matched to downregulated proteins.(E) Comparative analysis of HEC1A cellular proteome treated with NV HBEGF compared to ErloNV HBEGF and PBS, and a two-way scatter plot highlighting top dysregulated proteins in the presence of EGFR inhibitor, erlotinib.(F) Bubble plot display of biological processes and pathways associated with proteins significantly upregulated (including unique) by NV HBEGF treatment and proteins significantly downregulated (including not detected) in NV HBEGF compared to ErloNV HBEGF and PBS.NV HBEGF , HB-EGF-loaded NVs; NVs, nanovesicles.

F I G U R E 5
NV HBEGF enhances attachment to endometrial cells and outgrowth and invasion in Matrigel of trophectodermal spheroids.(A) Experimental workflow for co-culture attachment assay.(B) Box plot indicating percentage of spheroid attachment to HEC1A endometrial cells following treatment with PBS, NV HBEGF , NV, HB-EGF, or ErloNV HBEGF (n = 5), where rate of spheroid attachment (%) is the number of attached spheroids divided by the number of seeded spheroids expressed as a percentage.(C) Experimental workflow for TSC spheroid outgrowth and invasion into Matrigel.(D) Box plot indicating quantified area of TSC spheroid outgrowth and invasion into Matrigel 72 h following treatment with PBS, NV HBEGF , NV, HB-EGF, or ErloNV HBEGF (n = 8).(E) Bright-field microscopic images of TSC spheroids outgrowth and invasion into Matrigel 72 h following treatment with PBS, NV HBEGF , NV, HB-EGF, or ErloNV HBEGF .Scale bar 100 μm.(F) Area of outgrowth extending from spheroid taken for measurements is shaded in grey and quantified using ImageJ.*p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.001.