PDPN⁺LTBP1⁺ cancer-associated fibroblasts induce a liver pre-metastatic niche in gastric cancer via PDPN/YAP/LTBP1 and CCL11/CCR3 axis

Abstract

As a key component of the tumor microenvironment, cancer-associated fibroblasts (CAFs) exhibit substantial heterogeneity and contribute significantly to tumor growth and progression. However, their involvement in shaping the pre-metastatic niche remains insufficiently characterized. This study demonstrates that extracellular vesicles (EVs) regulated by YAP signaling in podoplanin (PDPN)⁺LTBP1⁺ CAFs activate hepatic stellate cells (HSCs), thereby enhancing gastric cancer (GC) cell colonization in the liver. Mass spectrometry profiling of EVs from PDPN⁺ and PDPN⁻ CAFs identified latent transforming growth factor beta-binding protein 1 (LTBP1) as a key mediator driving the phenotypic conversion of HSCs into CAF-educated HSCs (CEHs). Exposure to LTBP1-deficient EVs resulted in attenuated CEH-induced malignancy in HGC27 and AGS GC cells. Integrated RNA sequencing and cytokine array analyses further revealed that LTBP1-containing EVs activated TGF-β signaling in HSCs, leading to CCL11 secretion. This chemokine, in turn, recruited CCR3⁺ metastatic cells to the liver microenvironment. Using a GC liver metastasis model in combination with PET–CT imaging, inhibition of the CCL11/CCR3 axis was shown to suppress CEH-driven tumor growth and metastatic potential. These findings identify LTBP1-enriched EVs from PDPN⁺LTBP1⁺ CAFs as a viable therapeutic target to impede GC liver metastasis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-025-02379-6.

Introduction

Metastasis remains the primary challenge in the management of advanced gastric cancer (GC), positioning GC among the deadliest malignancies worldwide [1]. Mounting evidence indicates that primary tumor-derived exosomes contribute to establishing pre-metastatic niches in distant organs, thereby promoting metastatic colonization [2–5]. Among the tumor stroma, cancer-associated fibroblasts (CAFs) represent the predominant cell population and exert broad influence over tumor progression, including therapy resistance and metastatic dissemination [6, 7]. Nonetheless, the involvement of CAFs in shaping the pre-metastatic niche within GC remains unclear.

CAFs exhibit considerable heterogeneity, with phenotypic subtypes that diverge in biological function [8, 9]. Although many CAF subsets are associated with tumor promotion, specific populations correlate with improved clinical outcomes [10]. For instance, CAFs with elevated type I collagen expression impose biomechanical restrictions on tumor expansion, thereby impeding disease progression. These results highlight the necessity of delineating CAF heterogeneity in GC to inform targeted therapeutic strategies. Podoplanin (PDPN), a highly O-glycosylated transmembrane mucin-type glycoprotein, is functionally expressed in CAFs and has been implicated in various oncogenic processes [11]. Acting as a scaffold for signal transduction, PDPN contributes to tumor aggressiveness and unfavorable prognosis in multiple malignancies, including breast and lung cancers [12, 13]. Prior work has shown that PDPN⁺ CAFs markedly enhance the migratory capacity of GC cells [14]. However, their role in the formation of a pre-metastatic microenvironment has not yet been defined.

EVs are increasingly acknowledged as integral mediators of both proximal and distal intercellular communication across diverse cell populations [15, 16], primarily through the lateral transfer of functional biomolecules such as DNA, RNA, and proteins. Mounting evidence supports their indispensable role in establishing pre-metastatic niches, which serve as favorable microenvironments primed for the seeding and outgrowth of disseminated tumor cells—a key phase in the metastatic cascade [4, 17, 18]. Notably, CAF-derived EVs (CAF-EVs) have been shown to modulate signal transduction within the tumor microenvironment (TME), thereby accelerating tumor progression [19]. Nevertheless, limited investigations have addressed the specific involvement of CAF-EVs in pre-metastatic niche formation. Considering the heterogeneity among CAF subsets in their pro-metastatic capacities and the decisive influence of pre-metastatic niches on metastatic dissemination, it is essential to delineate which CAF populations are responsible for generating EVs that initiate this process. Clarifying these mechanistic underpinnings is fundamental to the development of targeted anti-metastatic interventions.

In this study, LTBP1-containing EVs, modulated by the PDPN/YAP/LTBP1 signaling cascade in CAFs, were found to activate hepatic stellate cells (HSCs) through TGF-β signaling. The resulting activation of HSCs led to increased CCL11 secretion, establishing a liver microenvironment conducive to the recruitment and engraftment of CCR3⁺ GC cells. This investigation provides new insight into the functional heterogeneity of the CAF subset and delineates its role in the development of pre-metastatic niches, offering potential therapeutic implications for liver metastasis in GC.

Materials and methods

Patients and specimens

To assess PDPN⁺LTBP1⁺ CAF expression in GC patients with hepatic metastases, primary tumor specimens were obtained exclusively from individuals with GC metastasis who had not received neoadjuvant therapy, thereby constituting an independent dataset. Patients were stratified into four cohorts based on metastatic distribution: Group 1 (no metastases, n = 7), Group 2 (liver-only metastases, n = 6), Group 3 (peritoneal metastases, n = 7), and Group 4 (ovarian metastases, n = 6). Ethical clearance was granted by the Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center (FUSCC; Shanghai, China), and informed consent was secured from all participants.

Cell lines and primary fibroblast isolation

CAFs and normal fibroblasts (NFs) were isolated from GC tissues and adjacent non-tumorous gastric tissues. After excision of necrotic and hemorrhagic regions, tumor specimens were mechanically minced into small fragments under sterile conditions, followed by enzymatic digestion with Collagenase IV at 37 °C for 2 h. Digestion was terminated by the addition of fetal bovine serum (FBS). The resulting cell suspension was centrifuged at 1000 r/min for 5 min, and the pellet was seeded into culture dishes. Medium was replaced every 48 h. Fibroblasts were enriched after two sequential digestions and subsequently employed in downstream analyses [20].

The human GC cell lines HGC27, AGS, MKN45, and the HSC line LX2 were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Since AGS cells lack tumorigenic capacity in BALB/C nude mice, animal studies were conducted using HGC27 and MKN45. In vitro assays were performed using HGC27 and AGS, as MKN45 cells, being suspension-type, are unsuitable for such experiments. All cell lines tested negative for mycoplasma and were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) under standard conditions of 37 °C and 5% CO₂.

Single-cell analysis

The scRNA-seq datasets analyzed in this study were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE163558, GSE167297, and GSE183904. Data normalization was conducted using the Seurat package (v5.0) in R (v4.4). Cells were excluded if they exhibited fewer than 200 or more than 3500 detected genes, mitochondrial gene content above 7%, hemoglobin gene content exceeding 3%, total RNA counts below 1000, or fell within the upper 3% of RNA counts. Doublets were identified and eliminated using the DoubletFinder algorithm. To address batch effects and inter-individual variation, the Harmony algorithm was employed following data integration. For dimensionality reduction and subtype delineation, the top 2000 highly variable genes were selected via the FindVariableFeatures function, followed by data scaling. Principal component analysis (PCA) was subsequently performed on these genes. The FindNeighbors function generated nearest neighbor graphs using the top 50 principal components, and clustering was executed using the FindClusters function. Cluster distributions were visualized using the UMAP algorithm. Annotation of cell clusters was guided by established cell-type-specific markers, including markers for T cells CD3 (delta subunit of T-cell receptor complex [CD3D] and CD3 epsilon subunit of T-cell receptor complex [CD3E]), natural killer (NK) cells (natural killer cell granule protein 7 [NKG7], granulysin [GNLY], and Fc gamma receptor IIIa [FCGR3A]), B cells (CD19, membrane spanning 4-domains A1 [MS4A1], CD79A, and marginal zone B and B1 cell specific protein [MZB1]), plasma cells (marker of proliferation Ki-67 [MKI67], baculoviral IAP repeat containing 5 [BIRC5], and cyclin dependent kinase 1 [CDK1]), mast cells (tryptase alpha/beta 1 [TPSAB1] and tryptase beta 2 [TPSB2]), myeloid cells (S100 calcium binding protein A8 [S100A8], S100 calcium binding protein A9 [S100A9], CD14, and CD68), fibroblasts (collagen type I alpha 2 chain [COL1A2], decorin [DCN], collagen type III alpha 1 chain [COL3A1], and collagen type VI alpha 1 chain [COL6A1]), endothelial cells (platelet and endothelial cell adhesion molecule 1 [PECAM1] and von Willebrand factor [VWF]), and epithelial cells (epithelial cell adhesion molecule [EPCAM], keratin 19 [KRT19], mucin 5AC [MUC5AC], and trefoil factor 1 [TFF1]).

Fluorescence-activated cell sorting (FACS)

Fibroblasts were initially incubated at room temperature for 5–10 min with 5 µL of Human Fc Receptor Blocking Solution (Catalog #422301; BioLegend) per 100 µL of cell suspension. Following blockade, 5 µL of FITC anti-human antibody or control IgG was added and the mixture was incubated on ice for 20 min in the dark. After staining, cells underwent centrifugation at 1000 r for 5 min in Cell Staining Buffer to remove unbound reagents. The pellet was resuspended in 0.5 mL of Cell Staining Buffer for subsequent analysis via FACS.

Conditioned medium (CM)

Fibroblasts were cultured to 70–90% confluence, at which point the medium was replaced with fresh serum-free RPMI-1640. After 24 h of incubation, CM was harvested and centrifuged at 1000 ×g for 1 min to remove cellular debris.

Reagents and antibodies

SIS3 (10 µM; Catalog #HY-13013) were obtained from MedChemExpress (Monmouth Junction, NJ, USA). The following antibodies were utilized for western blotting: anti-α-SMA (1:1000; Catalog #19245; CST), anti-Fibronectin (1:1000; Catallog #250073; zenbio), anti-LTBP1 (1:1000; Catalog #26855-1-AP; Proteintech), anti-CD9 (1:100; Catalog #sc-13118; Santa Cruz), anti-CD63 (1:100; Catalog #sc-5275; Santa Cruz), anti-TSG101 (1:100; Catalog #sc-7964; Santa Cruz), and anti-Alix (1:100; Catalog #sc-53540; Santa Cruz). Additional antibodies included anti-FAP (Catalog #66562; CST), anti-α-SMA (Catalog #BM0002; Boster), anti-vimentin (Catalog #5741; CST), and FITC-conjugated anti-human PDPN (Catalog #337026; Biolegend). The ELISA kit was procured from R&D Systems.

Multiple immunofluorescence staining

Following deparaffinization and rehydration of tissue sections, antigen retrieval and endogenous peroxidase inhibition were performed. Samples were subsequently blocked with 3% BSA and incubated overnight with the first primary antibody. After three PBS washes, HRP-conjugated secondary antibodies were applied and incubated at room temperature for 50 min in darkness. After this step, the slides were exposed, and a second round of primary antibody staining was conducted, followed by application of the corresponding HRP-linked secondary antibody. FITC-TSA development and microwave treatment were then carried out, followed by DAPI counterstaining.

Isolation of EVs

FBS was subjected to ultracentrifugation at 120,000 ×g for 12 h at 4℃ to produce EV-free serum. Once cells reached 70–80% confluence, they were washed twice with pre-warmed PBS, followed by the addition of EV-free serum. After 48 h, the supernatant was collected and centrifuged at 300 ×g for 10 min at 4℃ to remove dead cells. The supernatant was then further centrifuged at 10,000 ×g for 30 min at 4℃ to eliminate any remaining precipitates, leaving only the clarified supernatant. This supernatant was transferred to ultracentrifuge tubes and subjected to ultracentrifugation at 120,000 ×g for 70 min. The supernatant was discarded, and the pellet was resuspended in sterile PBS. After refilling the ultracentrifuge tubes with PBS, a second ultracentrifugation at 120,000 ×g for 70 min was performed. Finally, the liquid was completely removed, and the pellet was resuspended in an appropriate volume of sterile PBS for storage at −80℃.

Transmission electron microscopy (TEM)

EVs were resuspended in 2% paraformaldehyde and placed on an electron microscope grid, with excess liquid removed by aspiration. The specimen was stained with 1% uranyl acetate and allowed to air dry after the residual liquid was absorbed with filter paper. Images were captured using an 80 kV transmission electron microscope.

Nanoparticle tracking analysis (NTA)

Particle size and concentration of EVs were measured using NTA at VivaCell Biosciences, employing the ZetaView PMX 110 instrument (Particle Metrix, Meerbusch, Germany) and the ZetaView 8.04.02 software. EV samples were isolated and diluted with 1× PBS solution (Biological Industries, Israel) to ensure accurate measurements. NTA was conducted at 11 distinct positions. The ZetaView system was calibrated with 110 nm polystyrene particles, and the measurement temperature was maintained between 23℃ and 30℃.

Exosome uptake assay

Dil (red, Beyotime, Catalog #C1991S) or DiO (green, Beyotime, Catalog #C1993S) staining enhancers were diluted 400-fold with staining buffer, following the instructions for the cell membrane fluorescence kit, to prepare the cell membrane staining working solution. An appropriate volume of the working solution was mixed with EVs in a 1:1 ratio and incubated in the dark at 37 °C for 20 min. After incubation, 10 mL of sterile PBS was added to resuspend the mixture, and the EV extraction procedure was repeated to obtain fluorescently labeled EVs. Subsequently, 10 µg/mL of EVs were added to the supernatant of LX2 cells and incubated for 12 h. Following incubation, immunofluorescence staining was performed by fixing and permeabilizing the cells. The cells were then washed three times with PBS, stained with DAPI for 15 min, and mounted for observation.

Label-free mass spectrometry (MS)

Protein concentration was quantified using the Bradford assay. The peptide eluate was then vacuum-dried and stored at −20 °C for later use. LC-MS/MS data acquisition was performed on an Orbitrap Exploris 480 mass spectrometer, interfaced with an Easy-nLC 1200 system. For DDA mode analysis, each scan cycle included a full-scan mass spectrum (R = 60 K, AGC = 300%, max IT = 20 ms, scan range = 350–1500 m/z), followed by 20 MS/MS events (R = 15 K, AGC = 100%, max IT = auto, cycle time = 2 s). The resulting MS raw data were analyzed using MaxQuant (version 1.6.6) with the Andromeda search algorithm. Spectra were matched against the UniProt protein database, excluding decoy hits, contaminants, and site-only identifications. Only the valid identifications were utilized for subsequent quantification analysis.

RNA sequencing

Total RNA was extracted from cells using TRNzol (Catalog #DP424; Tiangen). Quality control, library preparation, RNA sequencing for differential expression analysis, and KEGG enrichment analysis were performed by Wuhan IGENEBOOK Biotechnology. Three biological replicates were used for both control and experimental groups. Differentially expressed genes were identified with a significance threshold of adjusted P ≤ 0.05.

Secretome array

Supernatants collected from fibroblasts were centrifuged for 1 min at 1000 ×g and 4 °C to eliminate excess cellular fragments and debris. Cytokine levels in the supernatants were quantified following the manufacturer’s guidelines outlined in the Human Cytokine Antibody Arrays V kit (Catalog #AAH-CYT-5; RayBiotech). The microarray was then scanned using the InnoScan Cy3 channel laser scanner (Innopsys). Grey values were determined through background subtraction and normalization to the positive control.

Animal model of tumor metastasis

BALB/C nude mice (4 weeks old) were intravenously administered 10 µg of CAF-EVs every two days as reported [4]. After a 21-day period, tumor cells were injected either sub-splenically or via the tail vein to establish liver and lung metastasis models, respectively. Following a six-week observation period, the mice were euthanized for metastatic evaluation across the different experimental groups. All animal procedures were approved by the Ethics Committee of the Experimental Animal Center at FUSCC (Shanghai, China).

Micro-positron emission tomography–computed tomography (PET/CT) imaging

Micro-PET/CT imaging using 18 F-FDG was conducted to assess tumor dynamics following various treatments. Mice bearing tumors underwent a 6-hour fasting period at specified intervals. Following fasting, 18 F-FDG (110 µCi/100 µL) was administered intravenously via the tail vein. Imaging was performed using the Inveon Acquisition Workplace System (Siemens). The Standardized Uptake Value (SUV) was calculated by dividing the decay-corrected activity (kBq) per milliliter of tissue by the injected 18 F-FDG activity (kBq) per gram of body weight.

Statistical analysis

Statistical analyses were carried out using GraphPad Prism software. Comparisons between two groups were performed with unpaired Student’s t-tests. A P-value of < 0.05 was considered statistically significant.

Results

PDPN⁺LTBP1⁺ CAF correlated to GC liver-specific pre-metastatic niches

In a previous study, we demonstrated that PDPN⁺ CAFs contributed to the migration of GC cells [14]. Since EVs from primary tumors can reach target organs, establishing pre-metastatic niches and enhancing metastatic cell colonization, we hypothesized that PDPN⁺ CAFs might release EVs that alter the hepatic microenvironment and promote metastasis. To test this hypothesis, we established a murine model of tumor metastasis, as described previously. Primary CAFs were isolated from GC tumors of patients with liver metastasis (Fig. 1A), and PDPN⁺ and PDPN⁻ CAFs were separated via FACS (Fig. 1B). EVs were then isolated from both PDPN⁺ and PDPN⁻ CAFs and characterized using western blotting for EV markers, NTA, and TEM (Fig. 1C–E). These EVs were administered to mice via tail vein injections every two days for 21 days to condition the mice (Fig. 1F). The injected EVs were subsequently localized to the liver (Fig. 1G). After the EV conditioning period, HGC27 cells were introduced via sub-spleen or tail vein injections to generate liver and lung metastasis models, respectively. Mice were euthanized three weeks after injection. Mice conditioned with PDPN⁺ CAF-derived EVs exhibited significantly larger liver metastases compared to those conditioned with PDPN⁻ CAF-derived EVs (Fig. 1H). No notable effect on lung metastasis was observed in either group (Fig. 1I). These data indicate that PDPN⁺ CAF-derived EVs can establish a liver-specific pre-metastatic niche that promotes the colonization of disseminated GC cells. Interestingly, immunofluorescent staining revealed no significant differences in the expression of α-SMA and FAP, commonly used CAF markers, between PDPN⁺ and PDPN⁻ CAFs, emphasizing the relevance of PDPN as a CAF subset marker (Supplementary Figure S1).

Fig. 1.

PDPN⁺LTBP1⁺CAF correlated to the liver pre-metastasis of gastric cancer. A Schematic representation of primary CAF isolation from gastric tissues of GC patients with liver metastasis. B CAFs were incubated with FITC-conjugated anti-human PDPN antibody and sorted via FACS. C Immunoblotting for the expression of EV markers TSG101, Alix, CD63, and CD9 in PDPN⁻CAF-EVs and PDPN⁺CAF-EVs. D Size and concentration distribution of PDPN⁻CAF-EVs and PDPN⁺CAF-EVs as assessed by NTA. E Representative TEM images of PDPN⁻CAF-EVs and PDPN⁺CAF-EVs (scale bar = 100 nm). F Schematic diagram depicting EV education in mice via tail vein injections every two days for 21 days to establish lung and liver metastasis models. G Fluorescent microscopy images showing Dio-labeled EV incorporation (green) in the livers of educated mice (scale bar = 50 μm). H Tumor cells were administered via sub-spleen injection to establish a liver metastasis model. After six weeks, the mice were euthanized to evaluate liver metastasis across experimental groups. I Tumor cells were administered through tail vein injections to establish a lung metastasis model. After six weeks, the mice were euthanized to evaluate lung metastasis across experimental groups. J Heatmap displaying the top 20 upregulated and top 20 downregulated differentially expressed proteins in EVs derived from PDPN⁺CAF, NFs, and PDPN⁻CAF, as determined by MS. K Immunoblot for LTBP1 expression in EVs derived from PDPN⁻CAF, NFs, and PDPN⁺CAF. L Immunoblot for LTBP1 expression in PDPN⁺CAF transfected with LTBP1 shRNAs. Immunoblot for LTBP1 expression in EVs derived from PDPN⁺CAF transfected with LTBP1 shRNAs. M Mice were educated with LTBP1-containing EVs and LTBP1-depleted EVs from PDPN⁺CAFs. Tumor cells were injected via sub-spleen to establish a liver metastasis model. After six weeks, the mice were euthanized to assess liver metastasis in the different experimental groups. N PET/CT imaging and double immunofluorescent staining for LTBP1 expression in metastatic organs (liver, peritoneal, and ovarian metastases) of GC patients (scale bar = 100 μm). White arrow indicates tumor sites. The proportion of LTBP1⁺ cells/all cells in metastatic organs of GC patients was shown for liver-confined metastases (n = 6), peritoneal metastases (n = 7), and ovarian metastases (n = 6). (*P < 0.05, **P < 0.01, ***P < 0.001)

To investigate the mechanisms underlying the regulation of HSC biological behavior by PDPN⁺ CAF-EVs, a proteomic analysis was performed to compare protein expression profiles between PDPN⁺ CAF-EVs, PDPN⁻ CAF-EVs, and NF-EVs. A total of 186 proteins were upregulated, and 161 were downregulated in PDPN⁺ CAF-EVs (Fig. 1J). Among the top five upregulated proteins, LTBP1 was significantly more abundant in PDPN⁺ CAF-EVs than in PDPN⁻ CAF-EVs and NF-EVs (Fig. 1K). To further assess the role of LTBP1, silencing of LTBP1 in PDPN⁺ CAFs was achieved using two distinct shRNAs, leading to decreased LTBP1 expression in the EVs (Fig. 1L). This reduction was associated with a marked decrease in tumor metastatic nodes in HGC27 cell-injected animals (Fig. 1M). These observations confirm the involvement of PDPN⁺LTBP1⁺ CAF-EVs in GC liver-specific metastasis.

To explore the clinical relevance of PDPN⁺LTBP1⁺ CAFs in GC metastasis, immunofluorescence staining for LTBP1 was conducted on metastatic sites. Patients were classified into three groups: Group 1 (liver-confined metastases), Group 2 (peritoneal metastases), and Group 3 (ovarian metastases). LTBP1 expression was significantly elevated in liver metastases compared to peritoneal and ovarian metastases (Fig. 1N). In conclusion, LTBP1-containing EVs derived from PDPN⁺LTBP1⁺ CAFs may contribute to the formation of a liver-specific microenvironment, thereby promoting the establishment of GC liver pre-metastatic niches.

LTBP1 secretion was modulated by YAP signaling in PDPN⁺ LTBP1⁺ CAFs

To comprehensively investigate the expression profiles of PDPN and LTBP1 in tumors, three scRNA-seq datasets (GSE163558, GSE167297, and GSE183904) including 39 gastric tumor samples were integrated (Fig. 2A). The transcriptomic analysis included 55,032 cells, which were clustered into 12 distinct groups and classified into seven major cell types based on established single-cell markers (Fig. 2B). Specifically, 10,919 B cells were identified, marked by CD19, MS4A1, CD79A, and MZB1; 1,367 mast cells were identified by TPSAB1 and TPSB2; 7,322 myeloid cells were marked by S100A8, S100A9, CD14, and CD68; 4,515 fibroblasts by COL1A2, DCN, COL3A1, and COL6A1; 1,967 endothelial cells by PECAM1 and VWF; 3,864 epithelial cells by EPCAM, KRT19, MUC5AC, and TFF1; and 25,078 T/NK cells, including T cells identified by CD3D and CD3E, and NK cells marked by NKG7, GNLY, and FCGR3A (Fig. 2C). The analysis revealed that LTBP1 and PDPN were primarily expressed in fibroblasts (Fig. 2D-E). Additionally, high levels of LTBP1 were detected within the stromal compartment, particularly in areas enriched with PDPN⁺ CAFs (Fig. 2F). Importantly, a significant correlation between PDPN mRNA expression and LTBP1 levels was observed in tumor tissues from 407 gastric cancer patients in the TCGA dataset (R = 0.562, P < 0.001) (Fig. 2G). The relationship between the presence of PDPN⁺ LTBP1⁺ CAFs and patient prognosis in gastric cancer was also assessed. Patients in the PDPN⁺ LTBP1⁺ CAFs group showed markedly poorer overall survival compared to those in the PDPN⁻ LTBP1⁻ CAFs group [P = 0.001, HR = 1.550 (1.117–2.151)] (Fig. 2H).

Fig. 2.

PDPN⁺LTBP1⁺CAF promote gastric liver metastasis via YAP-mediated LTBP1 secretion. A Schematic diagram illustrating the collection and processing of GC scRNA-seq data. B UMAP plot displaying the seven primary cell types identified in the scRNA-seq analysis. C Dot plot showing the expression of classical molecular markers associated with the identified cell types in the scRNA-seq datasets. D UMAP plots highlighting the expression patterns of LTBP1 and PDPN across various cell types in the scRNA-seq datasets. E Dot plot illustrating the expression levels of LTBP1 and PDPN across different cell types in the scRNA-seq datasets. F Representative immunofluorescent images of PDPN and LTBP1 in tumor samples from GC patients (scale bar = 50 μm). G Correlation analysis of LTBP1 and PDPN mRNA expression in tumor tissues from 407 GC patients in the TCGA cohort (R = 0.562, P < 0.001). H Kaplan-Meier survival curve showing overall survival (OS) of GC patients stratified by levels of PDPN⁺LTBP1⁺ CAFs in the TCGA cohort (n = 377). I Heatmap presenting the top differentially expressed genes between PDPN⁺LTBP1⁺ CAFs and PDPN⁻LTBP1⁻ CAFs in the scRNA-seq datasets. J KEGG pathway enrichment analysis of differentially expressed genes, highlighting pathways significantly enriched in PDPN⁺LTBP1⁺ CAFs and PDPN⁻LTBP1⁻ CAFs in the scRNA-seq datasets. K Immunoblot analysis of PDPN, YAP, and LTBP1 expression in PDPN⁻CAF and PDPN⁺CAF samples. L Immunofluorescent staining showing the expression of PDPN, YAP, and LTBP1 in PDPN⁻CAF and PDPN⁺CAF samples. M Immunoblot analysis further assessing YAP and LTBP1 expression in PDPN⁺ CAFs treated with or without Verteporfin (20 µmol/L) for 24 h. N Immunofluorescent staining visualizing YAP and LTBP1 expression in CAFs treated with or without Verteporfin (20 µmol/L) for 24 h, with LTBP1 in green, YAP in red, and nuclei stained with DAPI (blue). O Immunofluorescent staining examining PDPN, YAP, and LTBP1 expression in PDPN⁺CAFs transfected with PDPN siRNAs or control siRNA. (*P < 0.05, **P < 0.01, ***P < 0.001)

Differential gene expression analysis between PDPN⁺LTBP1⁺ CAFs and PDPN⁻LTBP1⁻ CAFs revealed distinct expression profiles, with PDPN⁺LTBP1⁺ CAFs exhibiting markedly elevated levels of pro-tumorigenic factors, such as cathepsin C (CTSC), C-X-C motif chemokine ligand 14 (CXCL14), and periostin (POSTN) (Fig. 2I). KEGG pathway enrichment analysis further highlighted differential activation of key signaling pathways, including Phosphoinositide 3 Kinase/Protein Kinase B (PI3K-AKT), Mitogen-Activated Protein Kinase pathway (MAPK), and Hippo, between these two fibroblast subsets (Fig. 2J). Inhibition of YAP, a central regulator of Hippo signaling, can diminish CAFs’ pro-tumoral capabilities [21–23] and their secretion of matrix proteins like collagen type I alpha 1 chain (COL1A1) [24]. Consequently, the YAP signaling pathway was selected for further investigation. The results demonstrated that both YAP and LTBP1 expression were significantly upregulated in PDPN⁺CAFs compared to PDPN⁻ CAFs (Fig. 2K-L). Furthermore, treatment with the YAP inhibitor Verteporfin (20 µmol/L) effectively reduced LTBP1 expression in PDPN⁺ CAFs (Fig. 2M-N). These results suggest that YAP signaling in PDPN⁺CAFs regulates LTBP1 expression. To explore the role of PDPN, PDPN expression was inhibited using two siRNAs targeting PDPN, which led to a substantial decrease in both YAP and LTBP1 expression (Fig. 2O). Taken together, these results support the notion that the PDPN/YAP/LTBP1 axis regulates LTBP1 production and sustains the phenotype of PDPN⁺LTBP1⁺ CAFs.

LTBP1 within PDPN⁺LTBP1⁺ CAF-EVs induce a fibrotic pre-metastatic niche via HSC activation

Given the ability of cancer-derived EVs to mediate communication between primary tumor cells and the ECM microenvironment of distant organs, thereby supporting metastasis, the potential of PDPN⁺LTBP1⁺ CAF-derived EVs to induce a fibrotic liver microenvironment was further explored. Exposure to PDPN⁺ CAF-EVs resulted in significant remodeling of the liver ECM, as indicated by the upregulation of fibronectin, α-SMA, and collagen I (Figs. 1H and 3A, Supplementary Figure S2A). In vivo experiments confirmed that PDPN⁺ CAF-EVs, when depleted of LTBP1, reduced ECM remodeling in the liver microenvironment (Figs. 1M and 3B, Supplementary Figure S2B). Given the central role of HSCs in hepatic ECM remodeling, the effect of PDPN⁺LTBP1⁺ CAF-EVs on HSC behavior was then examined (Fig. 3C). Immunofluorescence staining demonstrated the efficient uptake of EVs by the LX2 HSC cell line (Fig. 3D) and the protein expression of LTBP1 was significantly up-regulated in HSCs treated with PDPN⁺ CAF-EVs (Supplementary Figure S3A). Further, both immunofluorescence and Western blotting revealed that PDPN⁺ CAF-EVs could induce HSC activation, marked by increased expression of fibronectin, α-SMA, and collagen I, whereas PDPN⁻ CAF-EVs had no such effect (Fig. 3E-F). We also obtained CAF-CM with EVs depleted (CM-d) and found that 303 proteins were up-regulated and 175 proteins were downregulated in PDPN⁺ CAF-CM-d vs. PDPN⁻ CAF-CM-d groups by MS (Fold change > 2, P < 0.05). However, enrichment analysis of differentially expressed proteins associated with liver metastasis is unclear (Supplementary Figure S3B-E). Furthermore, PDPN⁺ CAF-CM-d failed to induce HSC activation, confirmed by expression of α-SMA, and collagen I (Supplementary Figure S3F). It revealed that PDPN⁺ CAFs exert unique functional effects on HSCs and metastatic colonization dependent of EVs.

Fig. 3.

LTBP1 within PDPN⁺LTBP1⁺ CAF-EVs induces HSC activation. A Fluorescent microscopy images showing fibronectin, α-SMA, and collagen I expression in liver tissue following exposure to PBS, EVs from PDPN⁻ CAFs, and PDPN⁺ CAFs (scale bar = 50 μm). B Fluorescent microscopy images illustrating fibronectin, α-SMA, and collagen I expression in the liver after treatment with EVs from PDPN⁺ CAFs transfected with either LTBP1 shRNAs or control shRNA. “shNC” denotes LTBP1-containing EVs, while “shLTBP1” indicates LTBP1-depleted EVs (scale bar = 50 μm). C Schematic representation of EV internalization by HSCs. D Immunofluorescent staining showing internalization of fluorescently-labeled EVs (red) by HSCs (green) (scale bar = 25 μm). E Representative immunofluorescent images for fibronectin, α-SMA, and collagen I expression in HSCs treated with EVs from different CAF subsets (scale bar = 25 μm). F Immunoblot analysis of fibronectin, α-SMA, and collagen I expression in HSCs treated with EVs from various CAF subsets. G Representative immunofluorescent images of fibronectin, α-SMA, and collagen I expression in HSCs pre-cultured with EVs from PDPN⁺ CAFs transfected with LTBP1 shRNAs or control shRNA (scale bar = 25 μm). H Immunoblot analysis of fibronectin, α-SMA, and collagen I expression in HSCs pre-cultured with EVs from PDPN⁺ CAFs transfected with LTBP1 shRNAs or control shRNA. I Wound-healing assay images depicting migration ability of HSCs treated with EVs from different CAF subsets (scale bar = 100 μm). J Wound-healing assay images showing migration capability of HSCs pre-cultured with EVs from PDPN⁺ CAFs transfected with LTBP1 shRNAs or control shRNA (scale bar = 100 μm). K Colony-forming assay of HGC27 and AGS cells pre-cultured in conditioned medium (CM) from HSCs treated with PDPN⁻ CAF-EVs or PDPN⁺ CAF-EVs. L Transwell assays assessing the invasive potential of HGC27 and AGS cells pre-cultured in CM from HSCs treated with PDPN⁻ CAF-EVs or PDPN⁺ CAF-EVs (scale bar = 50 μm). M Colony-forming assay of gastric cancer cells treated with CM from HSCs pre-cultured with EVs from PDPN⁺ CAFs transfected with LTBP1 shRNAs. N Transwell assays evaluating the invasive capacity of HGC27 and AGS cells treated with CM from HSCs pre-cultured with EVs from PDPN⁺ CAFs transfected with LTBP1 shRNAs (scale bar = 50 μm). (*P < 0.05, **P < 0.01, ***P < 0.001)

Depletion of LTBP1 in PDPN⁺ CAF-EVs significantly reduced the expression of these markers, suggesting that LTBP1 in PDPN⁺LTBP1⁺ CAF-derived EVs contributes to HSC activation (Fig. 3G-H). Moreover, PDPN⁺ CAF-EVs enhanced the migration of HSCs (Fig. 3I), and LTBP1 depletion markedly diminished this migratory response (Fig. 3J). These results indicate that LTBP1-containing EVs are key mediators in the interaction between PDPN⁺LTBP1⁺ CAFs and HSCs. Additionally, HSCs activated by PDPN⁺LTBP1⁺ CAF-EVs exhibited significantly enhanced colony formation and invasion capabilities compared to non-educated HSCs (Fig. 3K-L). Moreover, LTBP1 depletion markedly reduced the pro-tumorigenic effects of HSCs influenced by PDPN⁺LTBP1⁺ CAF-EVs (Fig. 3M-N). In accordance with this, overexpression of LTBP1 in HSCs stimulated the activation and the migration of HSCs, thereby exhibiting significantly enhanced colony formation and invasion capabilities (Supplementary Figure S4). Conclusively, the data further support the notion that LTBP1 from PDPN⁺LTBP1⁺ CAF-EVs activates HSCs to adopt a pro-tumorigenic phenotype, potentially fostering a fibrotic liver pre-metastatic microenvironment that facilitates GC liver metastasis.

LTBP1-containing EVs activate HSCs via transforming growth factor beta (TGF-β) signaling pathway

To investigate the mechanism by which LTBP1-containing EVs influence HSCs, RNA sequencing was performed to compare differential mRNA expression in LX2 cells co-cultured with LTBP1-containing or LTBP1-depleted EVs derived from PDPN⁺LTBP1⁺ CAFs (Fig. 4A). The analysis revealed significant gene expression changes, with 36 downregulated and 30 upregulated genes in HSCs exposed to LTBP1-depleted EVs compared to those treated with LTBP1-containing EVs (two-fold change, P < 0.05; Fig. 4B). GO enrichment analysis identified several biological processes potentially involved (Fig. 4C), among which the TGF-β pathway, integral to CAF function, was selected for further exploration. The results demonstrated that PDPN⁺LTBP1⁺ CAF-EVs significantly activated the TGF-β signaling pathway in HSCs, an effect that was diminished when LTBP1 was depleted from the CAF-EVs (Fig. 4D). Treatment of CEHs with SIS3, a TGF-β pathway inhibitor at 10 µM, effectively reversed the activation induced by PDPN⁺LTBP1⁺ CAF-EVs on HSCs (Fig. 4E), highlighting the central role of TGF-β signaling in HSC activation.

Fig. 4.

HSCs are activated by LTBP1-containing EVs via TGF-β signaling pathway. A Schematic representation of RNA sequencing in HSCs treated with EVs. B A heatmap depicting significantly regulated genes in HSCs treated with LTBP1-containing EVs compared to LTBP1-depleted EVs, based on RNA sequencing (n = 3). shLTBP1 EVs: LTBP1-depleted EVs; shNC-EVs: LTBP1-containing EVs. C Gene Ontology (GO) enrichment analysis of differentially expressed genes in HSCs treated with LTBP1-containing EVs versus LTBP1-depleted EVs. D Immunoblot analysis for smad2/3, p-smad2/3, smad4, TGF-βR1, TGF-βR2 expression in HSCs treated with PBS, LTBP1-containing EVs, or LTBP1-depleted EVs. shLTBP1 EVs: LTBP1-depleted EVs; shNC-EVs: LTBP1-containing EVs. E Representative immunofluorescence images showing fibronectin, α-SMA, and collagen I expression in CEHs treated with PBS or the TGF-β inhibitor SIS3 (10 µM) (scale bar = 25 μm). F Schematic diagram of the in vivo animal experiment. G Tumor images and tumor weights on day 14 following inoculation in each group. PBS + HSC: HGC27 cells and HSCs; EVs + HSC: HGC27 cells, EVs, and HSCs; SIS3 + EVs + HSC: HGC27 cells, EVs, and HSCs pretreated with SIS3. H Representative images of H&E and IHC staining for Ki-67 in xenograft tumors from the indicated groups (scale bar = 100 μm). (*P < 0.05, **P < 0.01, ***P < 0.001)

To further confirm whether LTBP1 promotes HSC activation through TGF-β signaling in vivo, HSCs were injected subcutaneously with different treatments into the flank of BALB/c nude mice, along with HGC27 cells (Fig. 4F). The results showed that HSCs pretreated with PDPN⁺LTBP1⁺ CAF-EVs contributed to significantly larger tumors, and the pro-tumor effect of CEHs was markedly diminished by the TGF-β pathway inhibitor SIS3 (Fig. 4G-H). In conclusion, the results indicate that LTBP1 within PDPN⁺LTBP1⁺ CAF-EVs promotes the formation of CEHs via TGF-β signaling pathway activation.

Increased CCL11 secretion by CEHs modulates liver colonization in CCR3⁺ GC cells

To investigate the mechanism through which CEHs promote the colonization of GC cells in the liver, antibody microarrays were employed for a comparative analysis of the cytokine profiles in CEHs and HSCs. The results indicated a marked increase in several cytokines in the supernatant from CEHs compared to HSCs, with CCL11 showing the highest expression levels (Fig. 5A). Furthermore, HSCs exhibited an enhanced production of CCL11 when exposed to PDPN⁺ CAF-EVs, as opposed to PDPN⁻ CAF-EVs (Fig. 5B). Depletion of LTBP1 in PDPN⁺ CAF-EVs abolished their ability to stimulate CCL11 secretion in HSCs (Fig. 5C). Additionally, inhibition of TGF-β signaling with SIS3 significantly reduced CCL11 production by CEHs (Fig. 5D). The data confirm that LTBP1-containing EVs from PDPN⁺ LTBP1⁺ CAFs activate the TGF-β pathway in HSCs, leading to the induction of CCL11 secretion. To further examine the role of CCL11 in CEH-mediated GC cell colony formation, a CCL11-neutralizing antibody was used to block CCL11 in the conditioned medium (CM) from CEHs/tumor cell models (Fig. 5E). The results revealed that CCL11 blockade significantly diminished the effects of CEHs on colony formation and invasion of HGC27 and AGS cells (Fig. 5F-G). Moreover, treatment with human CCL11 alone promoted colony formation and invasion of GC cells (Fig. 5H-I). These results highlight the critical role of CCL11 secreted by CEHs in driving the proliferation and invasion of GC cells.

Fig. 5.

LTBP1-activated HSCs endow malignant property to GC cells via CCL11/CCR3 axis. A. Cytokine arrays displaying cytokine production by HSCs and CEHs. CCL11 was the most significantly elevated cytokine in the supernatant of CEHs compared to that of HSCs (fold change = 6.22). B ELISA quantification of CCL11 in the supernatant of HSCs treated with either PDPN⁻ CAF-EVs or PDPN⁺ CAF-EVs. C ELISA analysis of CCL11 in the supernatant of HSCs treated with LTBP1-containing EVs or LTBP1-depleted EVs. shNC-EV: LTBP1-containing EVs; sh-LTBP1 EV: LTBP1-depleted EVs. D ELISA measurement of CCL11 in the supernatant of CEHs treated with PBS or SIS3. E Schematic representation of cancer cells co-cultured with CEHs. F Colony-forming assay of HGC27 and AGS cells precultured in CM from CEHs or CM from CEHs combined with a CCL11 neutralizing antibody. G Transwell assays evaluating the invasive capacity of HGC27 and AGS cells precultured in CM from CEHs or CM from CEHs with a CCL11 neutralizing antibody. H Colony-forming assay of HGC27 and AGS cells precultured in 1640 medium or 1640 medium with CCL11. I Transwell assays assessing the invasive capacity of HGC27 and AGS cells precultured in 1640 medium or 1640 medium with CCL11. J Schematic diagram of Colony-forming assay and Transwell assays of cancer cells transfected with CCR3 shRNAs or control shRNA. K Immunoblotting for CCR3 expression in HGC27 and AGS cells transfected with CCR3 shRNAs or control shRNA. L Colony-forming assay of HGC27 and AGS cells transfected with CCR3 shRNAs and cultured in CM from CEHs. M Transwell assays assessing the invasive capacity of HGC27 and AGS cells transfected with CCR3 shRNAs and cultured in CM from CEHs. (*P < 0.05, **P < 0.01, ***P < 0.001)

CCR3, as the primary receptor for CCL11 in cancer cells [25, 26], was further assessed to determine its essential role in CCL11-induced colony formation in GC cells (Fig. 5J). Initially, CCR3 expression in HGC27 and AGS cells was silenced using two shRNAs targeting CCR3 (Fig. 5K). As shown in Fig. 3K-L, the presence of conditioned medium (CM) from CEHs enhanced tumor cell colony formation and invasion. However, this effect was abolished upon CCR3 knockdown in cancer cells (Fig. 5L-M), indicating that CCR3 is integral to the crosstalk between CEHs and tumor cells. These results suggest that CEHs secrete CCL11, which subsequently promotes proliferation and invasion in CCR3⁺ GC cells. To explore the therapeutic potential of targeting the CCL11/CCR3 axis in liver metastasis of GC, a comparative analysis was performed by intrasplenic injection of tumor cells into EVs-educated mice (Fig. 6A). In alignment with in vitro data, the number of liver metastatic tumors was significantly reduced following CCR3 knockdown (Fig. 6B), reinforcing the critical role of the CCL11/CCR3 signaling pathway in supporting liver colonization by GC cells.

Fig. 6.

CCR3 inhibition in GC cells suppresses LTBP1-containing EV-induced liver metastasis. A Schematic representation of the in vivo animal experiment. B Representative PET/CT scan images of GC liver metastasis in mice. Macroscopic liver tissue images, H&E staining, and IHC staining for a-SMA and CCR3 expression are shown (scale bar = 100 μm). Arrows indicate metastatic tumors in the liver (*** P < 0.001, n = 6/group). C Diagram illustrating the mechanism by which PDPN⁺LTBP1⁺ CAFs promote the transformation of HSCs into CEHs, thereby supporting tumor progression. LTBP1-containing EVs, regulated by YAP in PDPN⁺LTBP1⁺ CAFs, trigger the transformation of HSCs into CEHs. Mechanistically, LTBP1 induces CCL11 production by activating the TGF-β signaling pathway in CEHs. CCL11, secreted by CEHs, subsequently influences the liver colonization of GC cells. CAF: cancer-associated fibroblast; HSCs: hepatic stellate cells; CEH: CAF-educated HSCs; EVs: extracellular vesicles. The diagram was partially created using Figdraw (www.figdraw.com)

In conclusion, the data demonstrate that LTBP1-containing EVs derived from YAP-mediated PDPN⁺LTBP1⁺CAFs stimulate HSC conversion into CEHs via TGF-β signaling activation. This cascade induces CCL11 secretion, thereby enhancing liver metastasis in CCR3⁺ GC cells (Fig. 6C).

Discussion

CAFs, a prevalent component of the TME, are activated fibroblasts that mediate interactions between the stroma and tumors during GC progression [27–29]. Increasing evidence indicates that CAFs do not represent a homogeneous entity solely involved in promoting tumor growth by secreting pro-tumorigenic factors [30]. Instead, functionally distinct subsets of CAFs exist within the GC microenvironment, with some promoting cancer progression and others inhibiting it [31, 32]. Additionally, certain CAF subsets can modulate immune cells and influence anti-tumor immunity, particularly in breast and pancreatic cancers [33, 34]. Therefore, a non-selective approach to targeting CAFs may inadvertently accelerate cancer progression. Understanding the heterogeneity of these subsets could inform more precise therapeutic strategies. PDPN has been identified as a marker of a CAF subset linked to tumor growth, chemoresistance, and metastasis across multiple cancer types [11, 13, 35, 36]. Although a transcriptional signature of mouse PDPN⁺ gastric CAFs has been associated with aggressive GC phenotypes and poor prognosis, whether PDPN⁺ CAFs contribute to the initiation of a pre-metastatic niche remains unclear [37]. This study clarifies the mechanism by which EVs mediate the interaction between primary gastric PDPN⁺ CAFs and resident HSCs, establishing a permissive pre-metastatic niche before GC cells reach the liver.

EVs are abundant in the TME and are critical in mediating communication between CAFs and tumor cells, influencing various aspects of cancer progression, including chemoresistance and metastasis [38–40]. In GC, for example, CAFs derived from the tumor stroma enhance tumor cell resistance to chemotherapy through the transfer of EVs containing annexin A6 [41]. Additionally, heat shock transcription factor 1 (HSF1) regulates the secretion of inhibin subunit beta A (INHBA) and thrombospondin 2 (THBS2) within CAF-EVs, which in turn promotes aggressive phenotypes of GC within the TME [37]. EVs are also recognized as key contributors to the establishment of pre-metastatic niches. In pancreatic ductal adenocarcinoma, EVs from cancer cells, carrying the CD44v6/complement C1q binding protein (C1QBP) complex, promote liver metastasis by activating HSCs and creating a fibrotic liver microenvironment [42]. Zhang et al. demonstrated that epidermal growth factor receptor (EGFR) carried by EVs from primary GC cells triggered pre-metastatic niche formation, thereby promoting liver-specific metastasis [2]. Furthermore, Kong et al. reported that CAF-EVs could prime the formation of pre-metastatic niches in the lungs by activating the TGF-β signaling pathway in lung fibroblasts, enhancing the metastasis of salivary adenoid cystic carcinoma [43]. However, the potential of CAF-EVs to induce the formation of pre-metastatic niches in the liver remains unclear. This study not only clarified the role of EVs from the PDPN⁺ CAF subset in liver metastasis but also identified LTBP1^high as a key protein profile within these EVs, which primes the formation of a pre-metastatic niche in the liver.

LTBP1, a member of the latent TGF-β binding protein family, regulates the bioavailability of TGF-β family members [44]. Its expression is linked to the transformation of CAFs and tumor progression in esophageal squamous cell carcinoma [45]. Oncogenic signaling activation can promote intercellular communication between stromal and tumor cells through EVs, involving pathways such as focal adhesion kinase (FAK) and TGF-β signaling [46–48]. We confirmed that LTBP1-containing EVs were transferred to HSCs, triggering TGF-β pathway activation. This, in turn, initiated the formation of a fibrotic pre-metastatic niche in the liver. Therefore, these results highlight the critical role of LTBP1-containing EVs secreted by PDPN⁺ CAFs in sustaining the liver pre-metastatic niche.

Supportive media are crucial for maintaining the malignant traits of cancer cells within the TME [49]. CAFs are central in this process, secreting cytokines that promote tumor cell proliferation and preserve their “stemness” [50, 51]. For example, CAFs could alter the malignant properties of liver cancer cells by secreting hepatocyte growth factor (HGF) and interleukin 6 (IL-6) [50]. Moreover, immune cells and CAFs in the TME can jointly regulate gastric Cancer Stem Cells (CSCs) [29, 52].

Elevated CCL11 expression is commonly observed in cancer cells and is associated with disease progression [25]. Increasing evidence suggests that tumor stromal cells also produce CCL11, which binds to its receptor CCR3 on tumor cells, thereby promoting tumor progression [26, 53]. Lin et al. demonstrated that CCL11 secreted by Myeloid-Derived Suppressor Cells (MDSCs) interacted with CCR3 on non-small cell lung cancer (NSCLC) cells, activating the extracellular signal-regulated kinase (ERK)/protein kinase B (AKT) signaling pathway and enhancing NSCLC metastasis [53]. Additionally, recent studies have shown that CAFs can enhance the cancer stem cell properties of head and neck cancer (HNC) by secreting CCL11. The expression levels of CCL11 and CCR3 were significantly correlated with poor overall survival (OS) in HNC patients [26]. The current study expands on these observations by showing that LTBP1-containing EVs activate HSCs, the primary source of paracrine CCL11 in the hepatic pre-metastatic niche. The persistent release of CCL11 from HSCs promotes the colonization of disseminated CCR3⁺ GC cells. Targeting the CCL11/CCR3 axis significantly impedes the supportive role of PDPN⁺ CAFs-EVs in promoting GC liver metastasis.

Limitations of this study include the following aspects: among the top five upregulated proteins in PDPN ⁺ CAF-derived EVs, collagen type VI alpha 3 chain (COL6A3) and collagen type XIV alpha 1 chain (COL14A1) have been reported to be closely associated with tumor metastasis [54–56]. COL6A3 serves as both a metastatic driver and potential therapeutic target in epithelial ovarian cancer through exosome-mediated mechanisms [54]. COL14A1 initiated by circ-CDYL/EEF1A2 activates ERK signaling and enhances EMT and lung metastasis in hepatocellular carcinoma [55]. Although our study revealed the role of LTBP1 on establishing pre-metastatic niches of gastric cancer liver metastasis, other secreted proteins required further investigation. Moreover, the tail vein injection protocol (administered every 2 days for 21 days) employed in this study presents several key limitations. First, this intermittent dosing regimen may not accurately replicate the continuous secretion dynamics of extracellular vesicles (EVs) under physiological conditions, potentially resulting in non-physiological peak-trough fluctuations of EV levels, while the supraphysiological concentrations post-injection could introduce experimental artifacts. Second, the dosing frequency has not been quantitatively correlated with actual EV levels in gastric cancer (GC) patients, nor does it adequately consider the temporal kinetic differences in clinical metastatic progression. Furthermore, high-dose EV administration might activate nonspecific pathways, and the repeated injection procedure itself could alter the host microenvironment by inducing inflammatory responses and other stress reactions. Future studies should compare the pharmacokinetic parameters of injected EVs with patient-derived EV profiles, incorporate dose-escalation experiments to validate threshold effects, and explore more physiologically relevant administration methods such as osmotic pump-based continuous infusion.

In conclusion, this study highlights the critical role of PDPN⁺ CAF-EVs in facilitating liver-tropic metastasis in GC by initiating a pro-metastatic microenvironment within the liver. Mechanistically, LTBP1-containing EVs released by PDPN⁺ CAFs activate the TGF-β pathway in HSCs, triggering the secretion of CCL11, which supports the liver colonization of CCR3⁺ GC cells. These findings reveal a novel mechanism of liver-tropic metastasis in GC and provide potential insights for developing targeted therapies against GC liver metastasis.

Abbreviations

GC

Gastric cancer

CAFs

Cancer-associated fibroblasts

HSCs

Hepatic stellate cells

CEHs

CAF-educated HSCs

EVs

Extracellular vesicles

LTBP1

Latent-transforming growth factor beta-binding protein 1

PDPN

Podoplanin

TME

Tumor microenvironment

CM

Conditioned medium

TGF-β

Transforming growth factor beta 1

YAP

Yes associated protein

Authors’ contributions

Z.Z.: Data curation, Project administration, Investigation. S.X.: Methodology, Software. E. G.: Investigation. H. H.: Resources, Writing – review & editing. Y. Z.: Funding acquisition, Writing original draft.

Funding

This research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No. LQN25H160007 and Zhejiang Cancer Hospital Foundation (PY2024006).

Data availability

The data that support the findings of this study are available on reasonable request.

Declarations

Ethics approval and consent to participate

Ethical approval for this study was obtained from the Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center (FUSCC; Shanghai, China). Written informed consent was acquired from all participants. All animal experiments were approved by the Ethics Committee of the Experimental Animal Center at FUSCC (Shanghai, China).

Consent for publication

All authors have consented to the publication of this study in this journal.

Competing interests

The authors declare no competing interests.