Exosomal S100A9 Promotes Lung Metastasis of Adenoid Cystic Carcinoma via Activating Cancer-Associated Fibroblasts

Exosomal S100A9 Promotes Lung Metastasis of Adenoid Cystic Carcinoma via Activating Cancer-Associated Fibroblasts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exosomal S100A9 Promotes Lung Metastasis of Adenoid Cystic Carcinoma via Activating Cancer-Associated Fibroblasts Bin Cheng, Chuwen Chen, Shenrong zhang, Yumeng Yan, Kuangwu Pan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7171055/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Cell Death Discovery → Version 1 posted 4 You are reading this latest preprint version Abstract Salivary adenoid cystic carcinoma (SACC) exhibits a high incidence of lung metastasis, which primarily contributes to patient mortality. The tumor microenvironment (TME) plays a critical role in facilitating tumor progression, yet its specific contribution to ACC metastasis remains unclear. In this study, we used single-cell transcriptomic analysis of primary ACC tumors and adjacent normal salivary gland to reveal strong intercellular communication between epithelial cells and cancer-associated fibroblasts (CAFs). We demonstrate that S100A9, a calcium-binding protein of the S100 family, is upregulated in a subset of ACC cells and their derived exosomes. Exosomes enriched with S100A9 reprogram normal fibroblasts into activated CAFs with elevated fibroblast activation protein (FAP) and α-smooth muscle actin (α-SMA) expression, enhanced migration, and increased contractility. RNA sequencing of fibroblasts treated with S100A9-enriched exosomes revealed activation of IL-17, TNF, and NF-κB signaling pathways, which are known to drive inflammation, extracellular matrix remodeling, and tumor-stroma interactions. Furthermore, activated CAFs promote epithelial-mesenchymal transition in ACC cells and facilitate lung metastasis through IL-17 signaling. These findings demonstrate tumor-derived exosomal S100A9 as a key mediator of intercellular communication between ACC cells and fibroblasts, identifying S100A9 and S100A9-enriched exosomes as potential therapeutic targets for modulating ACC lung metastasis. Biological sciences/Cancer/Cancer microenvironment Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Salivary Adenoid cystic carcinoma (SACC) is a carcinoma of secretory glands, frequently arising in salivary glands. Adenoid cystic carcinoma (ACC) has also been observed in other head and neck regions including the trachea, larynx, nasal and paranasal sinus, and lacrimal glands( 1 , 2 ). SACC accounts for 28% of the total incidence of malignant salivary gland tumors( 3 ) and 50% of malignant tumors in the minor glands ( 4 ). Distant metastases, most commonly to the lung, is the leading cause of reduced survival in ACC. The 5-year survival rate is around 69.7%, and the 10-year survival rate drops to 29%( 5 ). Surgical intervention with radiotherapy provides effective local control but do not effectively treat metastatic ACC, approved chemotherapies or targeted agents remains unclear( 6 ). Elucidating the biological molecular mechanisms of ACC lung metastasis, especially the interaction in the tumor microenvironment (TME), is imperative in improving the current situation. S100A9 is a calcium-binding protein of the S100 family, initially known as a proinflammatory mediator released by neutrophils and monocytes in response to cell damage, infection, or inflammation( 7 ). Previous work has shown that S100A9 is crucial in establishing the premetastatic niche, chemotherapy resistance, and subsequent metastasis in breast cancer and melanoma( 8 ). S100A9 inhibitors have been shown to decrease myocardial infarct size, and alleviate myocardial fibrosis( 9 ). Moreover, a large randomized phase II trial demonstrated that, in patients with metastatic castration-resistant prostate cancer, S100A9 inhibitors significantly prolonged radiographic and symptomatic progression-free survival and were associated with improved overall survival( 10 ). These findings highlight the potential of S100A9 as a biomarker and a therapeutic target. Nevertheless, it is unclear whether S100A9 contributes to lung metastasis in ACC. Exosomes are 40–150 nm, extracellular, phospholipid vesicles, secreted by variety types of cells. They play a crucial role in intercellular communication by carrying bioactive molecules such as proteins, metabolites, and nucleic acids( 11 , 12 ). In contrast to autocrine, paracrine, and juxtacrine signaling mechanisms, greater amounts of functional biomolecules can be transferred to extracellular matrix and circulation via encapsulating into exosomes, known as exosomecrine mechanism( 13 , 14 ). The continuous crosstalk between cancer cells and local/distant microenvironments effectively drives tumor migration, metastasis, and therapy resistance. Among the main actors of the interplay are the exosomes, which taken up by stromal cells. Cancer-associated fibroblasts (CAFs), activated by signals from the tumor or its microenvironment, constitute the most abundant cells in the tumor stroma and dynamically regulate extracellular matrix remodeling, angiogenesis, and immune evasion, especially pre-metastatic niche formation( 15 , 16 ). S100A9 can be packed into exosomes and act as an extracellular matrix mediator to promote chronic lymphocytic leukemia progression( 17 ). However, the role of exosomal S100A9 in regulation of the TME and the premetastatic niche during lung metastasis in ACC remains unclear. Here, we investigated the correlation between S100A9 expression and lung metastasis formation in ACC. Furthermore, we elucidated the underlying mechanisms by which S100A9 modulates the tumor microenvironment to facilitate pulmonary metastasis through exosomecrine. Materials and methods Tissue samples and cell lines ACC (n = 56) and normal gland (NG, n = 6) tissue samples were collected from patients undergoing radical resection at the Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Sun Yat-sen University between 2015 and 2024. None of the patients had received chemotherapy or radiotherapy. Histopathological examination was used to confirm all diagnoses. The SACC-83 cell line was derived from the sublingual gland of a patient. SACC-LM cells, with a high capacity for lung metastasis, were isolated in vivo after tail vein injection of SACC-83 cells into immunodeficient mice( 18 , 19 ). Both cell lines were validated by STR profiling, confirmed Mycoplasma-free by PCR after the last experiment, and used within 15 passages post-thaw. SACC cells were cultured in RPMI-1640 medium with 10% FBS (Gibco, USA). Normal fibroblasts (NFs) were isolated from normal gland tissue while cancer-associated fibroblasts (CAFs) were isolated from human ACC tumor tissues from the same patient according to previously described protocols( 20 ). Fibroblasts were maintained in DMEM/F12 medium supplemented with 10% FBS, 10 000 U/mL penicillin, and 10 000 µg/mL streptomycin (Gibco, USA) at 37°C in 5% CO₂. Conditioned media from exosome-treated NFs were referred to as S100A9-exo-NF-CM or Vector-exo-NF-CM. Overexpression and siRNAs The lentiviral HBLV-h-S100A9-3xflag-ZsGreen-PURO (used to construct the S100A9 overexpression vector), the HBLV-ZsGreen-PURO vector (used to construct the S100A9-expression vector) and HBLV-LUC-PURO were purchased from Hanbio (Hanbio Biotechnology, China). Transfection of siRNAs and plasmids was performed using the Lipofectamine 2000 Kit (Invitrogen, USA), according to the manufacturer's instructions. The S100A9 siRNA was designed and synthesized by RiboBio (Guangzhou, China) and are listed in Supplementary Table 1. Immunohistochemistry (IHC) and multiplex immunohistochemistry staining (mIHC) Paraffin-embedded ACC tumor sections were deparaffinized, rehydrated, and subjected to antigen retrieval in sodium citrate buffer. For standard immunohistochemistry, slides were incubated overnight at 4°C with primary antibodies, followed by secondary antibody incubation for 30 min at room temperature. For multiplex staining, a multi-labeled kit (Panovue, Beijing, China) was used according to the manufacturer’s protocol. HRP conjugates and five fluorescent wavelengths (520, 570, 620, 690, and 440 nm) enabled simultaneous detection of multiple targets. Nuclei were counterstained with DAPI (ZLI-9556, ZSGB-Bio, Beijing, China). Slides were scanned using the Aperio AT2 system (Leica, Germany), and images were analyzed using ImageJ. Antibody details are listed in Supplementary Table 2 . Immunofluorescence Fibroblasts grown on glass coverslips with different exosomes treated for 48–72 h were fixed with 4% paraformaldehyde at room temperature. For staining of paraffin-embedded tumor samples, formalin fixation was dewaxed at room temperature for 30 min. BSA (5%) in PBS with mouse IgG blocking reagent was used. After 1 h, target proteins were captured by each primary antibody listed in Supplementary Table 2 during incubation for 18 h at 4°C. Captured proteins were incubated with fluorescently labeled IgG in a dark room for 1 h before treated with medium containing DAPI (cat. no. ZLI-9556, ZSGB-Bio, Beijing, China) and imaged with a confocal microscope (FV3000, Olympus Corporation, Japan) or an inverted fluorescence microscope (U-TBI90, Olympus, Japan). Microarray expression profiling and mRNA High-throughput Sequencing Total RNA extracted from SACC-83 and SACC-LM cells was analyzed by Shbio (Shanghai, China) using the SBC Human 4 ×180 K ceRNA microarray. Total RNA was extracted from Vector-exo-NF and S100A9-exo-NF, SACC-83 cells co-cultured with exosomes/fibroblasts, and sequenced on the Illumina platform by Shbio (Shanghai, China). RNA preparation and qPCR RNA was extracted using TRIzol reagent (Invitrogen, USA), according to the manufacturer's instructions, and the extracted RNA was reverse transcribed into cDNA using a cDNA Reverse Transcription Kit (Takara, Japan). The SYBR Green-based qPCR analysis was conducted with the Light-Cycler 96 system (Roche). All RT-qPCR primer sequences are listed in Supplementary Table 3. Exosome proteomics mass Spectrometry Exosome proteomics services were supported by Wayen Biotechnologies following a standard protocol. Max-Quant 1.5.8.3 (Max-Planck Institute for Biochemistry) was used as a quantitative proteomics software package for analyzing large mass spectrometry data sets. Metascape ( http://metascape.org ) and cluster profiler package o were used for gene annotation and enrichment analysis. Terms with P -value 1.5 were collected and grouped into clusters based on their membership similarities. Exosomes isolation, labeling, and uptaken Exosomes isolation from cell supernatants was performed using ultracentrifugation and sucrose cushion( 13 ). Supernatants were cleared by centrifugation, concentrated using 100K centrifugal filters (Millipore, USA), and subjected to gradient centrifugation at 100 000 × g for 70 minutes (Optima L-90K, Beckman Coulter). The exosome-enriched fraction was resuspended in PBS and stored at − 80°C. Protein concentration was measured using the Bradford assay (Beyotime, China). Exosomes and fibroblasts were labeled with PKH26 (Sigma-Aldrich, USA) and Actin-Tracker Green (phalloidin-FITC, Beyotime, China), respectively; nuclei were stained with DAPI (ZSGB-Bio, China). Fibroblasts were incubated with labeled exosomes for 24 h, and then visualized using a confocal microscope (FV3000, Olympus, Japan). Colony formation, cell viability and proliferation assays For colony formation, 2 mL of cell medium containing 500 cells was seeded in 6-well plates and cultivated for 10 days. Then, the cells were fixed by using 4% paraformaldehyde (cat. no. BL539A, Biosharp, China) for 30 minutes and stained with 0.1% crystal violet (cat. no. RBG1019-100 mL, Roles-Bio, Guangzhou, China). The colony numbers of each well were counted with ImageJ. Cell Counting Kit 8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) and EdU (cat. no. KGA9602-100, KeyGEN BioTECH, Nanjing, China) assay were used to assess cell viability and proliferation according to manufacturer’s methods. Western blot analysis Proteins were extracted from cultured cells with RIPA lysis buffer (Beyotime Technology). The protein concentrations of cells were measured using BCA protein assay kits. The protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS‒PAGE) and subjected to immunoblot analysis. The antibody information is listed in Supplementary Table 2. Antibody-bound proteins were detected using an enhanced chemiluminescence (ECL) Western blot kit (CWBio, China). Fibroblasts were treated with IKK inhibitor BAY-117082 and prepared with Nuclear and Cytoplasmic Extraction Reagents (Beyotime, P0028). Cells were stimulated with recombinant human IL-17A (Pepro-Tech, London, UK, 200 − 17) and immunoblotting was followed. Transwell assays For the Transwell assay, transwell filter inserts (Costar, Corning, USA) with or without Matrigel (BD Biosciences, USA) coating were used according to the manufacturer's instructions. The cells that passed through the membrane were fixed, stained, and counted under a light microscope. Mouse model of lung metastasis All animal procedures followed institutional regulations and the ARRIVE guidelines. Female NOD/SCID mice (20–22 g, 4–6 weeks old) were stratified by body weight and randomly assigned to control or treatment groups. Mice received daily intravenous injections of either Vector-exo or S100A9-exo (70 µg, n = 8 per group) for 7 days. On day 8, lungs were harvested for immunohistochemistry and immunofluorescence analysis. The remaining mice (n = 7 per group) received 1 × 10⁶ firefly luciferase-labeled SACC-83 cells injection intravenously on day 8. After 8 weeks, lung metastases were evaluated by bioluminescence imaging (Caliper Life Sciences), followed by lung collection for H&E staining. Additionally, 1 × 10⁶ luciferase-labeled SACC-83 cells co-cultured with exosomes/fibroblasts were injected into NOD/SCID mice (n = 7 per group) via the tail vein. After 8 weeks, lung metastases were assessed by imaging and confirmed by H&E staining. Collagen contraction assays After treated with exosomes or plasmids for 48–72 h, fibroblasts were harvested and mixed with 400 µl of collagen mix containing 186.46 µl dH2O, 4.23 µl 1 N NaOH, 40 µl 10 × PBS and 169.31 µl Type 1 Rat Tail Collagen (cat. no. 354249, Corning, NY, USA). Then, the mix was added to 24-well plate and allowed to solidify for 30 min at 37°C. Later, 500 µl DMEM-F12 with 10% FBS was added to the plate for 16 h until the gels were photographed. ImageJ software was employed to measure gel area and evaluate contraction. ELISA The conditioned medium of fibroblasts was incubated with IgG antibody (R&D Systems Inc., Minneapolis, MN, USA) or IL-17A-neutralizing antibody (IL-17A Ab; R&D Systems) (all used at 10µg/ml). The IL-17A concentrations in cell culture supernatant were measured by using the Human IL-17A ELISA Kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China; H014-2) according to the manufacturer’s instructions. Single-Cell Preparation and Library Construction Fresh tumor tissues from five SACC patients and normal salivary gland tissues from two non-SACC individuals were collected immediately after surgery, stored in Tissue Storage Solution (Miltenyi Biotec, Germany) on ice, and transferred to the laboratory. Single-cell suspensions were loaded onto the 10X Genomics Chromium platform to generate Gel Bead-In-Emulsions (GEMs), and libraries were prepared using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Gene Denovo, Guangzhou, China). Each GEM contained primers with an Illumina® R1 sequence, a 16-nt 10x barcode, a 10-nt UMI, and a poly-dT sequence for capturing polyadenylated mRNAs, enabling the synthesis of barcoded full-length cDNA. Data Processing and Quality Control Raw sequencing data were processed using Seurat (v4.1.2) in R (v4.3.0). Cells expressing fewer than 200 or more than 6 000 genes were removed, as were cells with > 15% mitochondrial or > 1% hemoglobin gene content. Mitochondrial genes were excluded from downstream steps. Doublets were identified using DoubletFinder (v2.0.3) and removed. After stringent quality control, 51,128 high-quality cells were retained for analysis. Integration, Clustering, and Cell Type Annotation All samples were normalized and variance-stabilized with SCTransform (v0.3.2), followed by integration using IntegrateData. PCA was performed on the top 2,000 highly variable genes, and 20 principal components were selected using the ElbowPlot function. Uniform Manifold Approximation and Projection (UMAP) was used for visualization. Cell clustering was carried out using the Louvain algorithm (FindClusters, resolution = 0.8). Clusters were annotated based on DEGs (identified with FindAllMarkers) and known cell type markers ( Supplementary Table 4 ). To refine annotations of major cell types, each population was extracted, reprocessed with SCTransform, reintegrated, and re-clustered. Subclusters were defined using DEGs and inferred biological functions. CellChat communication CellChat R package (version 1.6.1) was utilized to investigate the intercellular communication between epithelial cells and fibroblasts. CellChat enables the systematic analysis of ligand-receptor interactions based on scRNA-seq data. Statistical analysis The bioinformatics and statistical analyses were conducted using R 4.4.2, SPSS 27 and GraphPad Prism 8.0 softwires. All in vitro experiments were confirmed in at least three independent experiments and all numerical data represented mean ± standard deviation (SD) unless otherwise stated. The data from one representative experiment were shown. Data were statistically analyzed by two-tailed Student’s t test, Mann Whitney test, Pearson correlation analysis and χ² test. P < 0.05 was considered to indicate a statistically significant difference. Results Figure 1 S100A9 is upregulated in ACC and associated with lung metastasis To investigate the dysregulated genes in ACC progression, we analyzed mRNAs from a pair of microarrays for two ACC cell lines( 18 ) (Fig. 1 A). SACC-LM, with enhanced lung metastatic potential, was derived from the parental low-metastatic SACC-83 line. Considering the prometastatic roles of exosome, we isolated corresponding exosomes (SACC-83-exo/SACC-LM-exo). Their characteristic cup-shaped morphology, size, and concentration were subsequently confirmed (Fig.S.1A and B), and exosomal markers (Hsp70, TSG101, CD63, CD9, CD81) were validated by western blot (Fig.S.1C). Proteomic profiling revealed 149 proteins upregulated and 398 downregulated in SACC-LM-exo (fold change ≥ 2 or ≤ 0.5, P < 0.05) (Fig. 1 B). Integration with transcriptomic data identified S100A9 as consistently elevated in both tumor cells and exosomes, which was further validated by western blot (Fig. 1 C). Gene Ontology (GO) analysis between SACC-83-exo and SACC-LM-exo showed enrichment in vesicle-mediated transport, component biogenesis, and protein localization (Fig. 1 D), while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed to highlight cancer-related and ECM-receptor interaction pathways (Fig. 1 E). IHC analysis demonstrated elevated expression of S100A9, fibroblast activation protein (FAP) and α-smooth muscle actin (α-SMA) in the primary ACC tumor tissues versus normal salivary gland, with higher distribution in patients with lung metastasis (Fig. 1 F). Correlation analysis showed that S100A9 positively associated with FAP and α-SMA in 56 ACC samples and 6 NG samples (Fig. 1 G), suggesting a potential role of S100A9 in the tumor-CAF cross talk. Clinically, high S100A9 expression correlated with advanced stage and lung metastasis ( Table 1 ). However, due to the relatively long survival time of ACC patients and the limited number of available samples, no significant difference in OS was observed, whereas patients with high S100A9 expression exhibited significantly shorter disease-free survival (DFS) compared to those with low expression (Fig. 1 H). Figure 2 Single cell analysis reveals CAF diversity and potential crosstalk with ACC cells in Tumor Microenvironment To investigate tumor-stroma interaction in ACC TME, we analyzed single-cell transcriptomic data from five ACC tumors and two normal salivary glands (Fig. 2A), capturing 67 007 cells and clustering them into 16 distinct cell types (Fig. S.2A and B), with annotations shown in Fig. 2B. A total of 27 663 epithelial cells were re-analyzed and visualized as subclusters (Fig. 2C and Fig. S.2C). Subsequently, inference of copy number alterations (CNAs) identified malignant cells (Fig. 2D and Fig.S.2D), among which a subset with high EMT scores was defined as “EMT-state epithelial cells.” (Fig. 2E, F and Fig.S.2E) CellChat analysis revealed frequent and strong communication between epithelial cells and fibroblasts (Fig. 2G). Given the pivotal role of fibroblasts in EMT-driven tumor metastasis, we classified fibroblasts into antigen-presenting CAFs (apCAFs), matrix CAFs (mCAFs), inflammatory CAFs (iCAFs), vascular CAFs (vCAFs), and non-specific CAFs (nCAFs) (Fig. 2H, I and Fig.S.2F). FAP and α-SMA were expressed across several CAF subsets, but their expression levels were markedly higher in the mCAF population (Fig. 2J and Fig.S.2G, H). Subsequent cell–cell communication analysis revealed that FAP⁺α-SMA⁺CAFs displayed frequent and robust interactions with EMT-state epithelial cells (Fig. 2K). This key finding was further confirmed in an external scRNA-seq dataset, thereby significantly strengthening its validity (GSE216852, n = 6, total cells = 51 128) (Fig.S.3). Multiplex immunohistochemistry (mIHC) of ACC tissues further supported spatial proximity and potential interactions between stromal and EMT-state epithelial populations (Fig.S.4). Collectively, these findings highlight strong interactions between specific CAF subpopulations—particularly FAP⁺α-SMA⁺CAFs—and EMT-state tumor cells, underscoring the importance of identifying key regulatory factors mediating fibroblast–tumor crosstalk in ACC. Figure 3 S100A9 in exosomes mediates fibroblasts activation To assess the relationship between S100A9 and fibroblast activation, primary CAFs and matched NFs were isolated from ACC tumors and adjacent normal salivary gland (Fig.S.5A). Western blot analysis confirmed higher expression levels of fibrosis (FAP, α-SMA) in CAFs versus NFs (Fig.S.5B). Treatment with SACC-LM-exo enhanced the expression of FAP and α-SMA, contraction ability and migration of NFs (Fig. 3 A-C). Exosomes uptake was confirmed by PKH26-labeling and confocal microscopy (Fig.S.5C). To further explore the role of S100A9 in CAFs activation, we established SACC-83 with S100A9 overexpression. Subsequent analyses confirmed elevated S100A9 levels in both the transfected cells and their derived exosomes (Fig.S.6A and B). NFs were transiently transfected with high S100A9 expressing vectors, the transfection effect was verified by qPCR and western blot (Fig.S.6C and D). Across the two experimental groups, exosomal S100A9 consistently led to elevated levels of FAP and α-SMA, greater collagen gel contraction, and increased migration ability (Fig. 3 D-F). S100A9-exo treatment upregulated expression levels of fibrosis, pro-inflammatory cytokines IL-6, IL-8, IL-1β as well as matrix metalloproteinases MMP2 and MMP9 in RNA level (Fig. 3 G), which are molecules contributing to a tumor-promoting microenvironment. S100A9 knockdown in SACC-LM cells via siRNA (Fig.S.6E and F) reduced the fibroblast-activating capacity of their exosomes, evidenced by the reduction in FAP and α-SMA expression via immunofluorescence, along with a diminished collagen contraction capacity (Fig. 3 H, I). Similarly, an S100A9 inhibitor paquinimod suppressed fibroblast activation induced by S100A9-exo (Fig. 3 J, K). Together, these findings support a potential role of S100A9-enriched exosomes in promoting fibroblast activation and stromal remodeling. Figure 4 Exosomal S100A9 potentiate lung metastasis and activate fibroblasts via IL-17-TNF-NF-κB axis Lung pre-metastatic model was established to evaluate the contribution of S100A9-exo on organotropic metastasis (Fig. 4 A). In vivo, intravenously injection of S100A9-exo into NOD/SCID mice showed elevated level of fibroblast-genesis genes (FAP and α-SMA) in lung tissue (Fig. 4 B), yet formed a pre-metastatic microenvironment with higher level of Fibronectin, MMP9 and VEGFR1 (Fig. 4 C). The intravenous injection of exosomes from S100A9-overexpressing cells into immunodeficient mice significantly increased ACC lung metastases (Fig. 4 D, E). We collected vector-exo/S100A9-exo treated NFs and profiled them using high-throughput RNA-sequencing. Analysis of the fibroblasts revealed significant activation of IL-17 signaling pathway, chemokine receptor binding, TNF signaling pathway and NF-κB signaling pathway (Fig. 4 F, G). Previous studies have consistently identified NF-κB activation as a pivotal regulatory part in the IL-17 signaling cascade, acting synergistically with TNF-α( 21 , 22 ). Treatment with S100A9-exo significantly altered the mRNA levels of IL-17 and TNF-α in educated NFs (Fig. 4 H). The IKK inhibitor BAY-117082 inhibited NF-κB activation (Fig. 4 I), and blocked the effect of S100A9-exo on fibroblasts (Fig. 4 J). There was an enhanced lipid metabolism and nucleotide metabolism been observed in S100A9-exo group (Fig. 4 K). Therefore, the results showed that S100A9-exo stimulate the production of IL-17 and TNF-α, thereby enhancing NF-κB signaling and activating fibroblasts. Figure 5 Activated fibroblasts promote ACC cell epithelial–mesenchymal transition and lung metastasis by secreting IL-17 To determine whether S100A9-exo-treated-NFs promote SACC-83 development, we co-cultured SACC-83 with exosomes-treated fibroblasts. After 48 h treatment with exosomes, these activated fibroblasts were set on the upper chamber, meanwhile SACC-83 cells were set on the bottom chamber (Fig. 5 A). SACC-83 cells exhibited increased migration, invasion, viability, proliferation, and colony formation ability, along with altered expression of E-cadherin and N-cadherin upon co-cultured with S100A9-exo/fibroblasts (Fig. 5 B-F). Furthermore, it greatly increased the metastatic potential of SACC-83 cells in mouse models (Fig. 5 G, H). Taken together, the results from both S100A9-exo (Fig. 4 D, E) and co-culturation with S100A9-exo/Fibroblasts treatment (Fig. 5 G, H) consistently underscore that exosomal S100A9 significantly promotes lung metastasis of ACC, highlighting its pivotal role in metastatic progression. GO enrichment and KEGG pathway enrichment was conducted on SACC-83 co-cultured with exosomes/fibroblast through RNA sequencing. IL-17 signaling pathway was the most significant pathway, which was also the top enriched pathway between Vector-exo and S100A9-exo group (Fig. 5 I, J). We next examined the expression of IL-17 in the conditioned medium of fibroblasts through ELISA and treated S100A9-exo-NF-CM with IL-17 neutralizing antibody (IL-17 Ab) which evidently reduced IL-17 levels (Fig. 5 K). Notably, western blot analysis exhibited the expression of N-cadherin, E-cadherin, MMP9 and MMP2 in SACC-83 co-cultured with S100A9-exo/Fibroblast with IL-17 Ab (Fig. 5 L). The rhIL-17 significantly induced EMT in a dose-dependent manner in SACC-83 cells (Fig. 5 M). The above results indicate that S100A9-exosomes-treated NFs regulate SACC-83 cells epithelial–mesenchymal transition and lung metastasis by secreting IL‐17. Discussion Despite significant therapeutic advance in ACC, the long-term prognosis of patients with ACC remains unsatisfactory due to our insufficient understanding of its underlying lung metastasis mechanisms( 23 ). Studies confirmed that the interplay between malignant tumor cells and their TME plays a critical role in promoting tumor progression( 24 ) and contributing to drug resistance( 25 ), which are provided by cell-to-cell interactions, soluble factors (cytokines, chemokines) or exosomes( 26 ). In ACC, exosomes have shown to regulate pre-metastatic niche formation by driving angiogenesis and increasing vascular permeability in lung endothelial cells( 13 , 14 ). We hereby report that exosomes derived from different ACC cell line display distinct proteomic profiles, which aligns with established evidence suggesting that exosomal functional properties are dynamically modifiable and hence influence tumor dynamics as the tumors progress to a more aggressive phenotype( 27 ). Compared to SACC-83-exo, differentially expressed proteins in SACC-LM-exo were significantly associated with pathway in cancer and extracellular matrix (ECM) organization. To identify the most relevant protein in ACC lung metastasis, we focused on the ones that were constantly over-expressed in both the aggressive cell line and its exosomes. This implicates that these proteins not only function intracellularly but can also be secreted via exosomes to exert its effects extracellularly. We observed an upregulation of S100A9 in SACC-LM cells and their exosomes through both mRNA microarray and proteomic analyses. Although S100A9 is mainly found in heterodimers with S100A8 (S100A8/A9, also known as calprotectin), it also exists association with tumor development with its own functions( 28 ). Considering the fact that S100A8 exhibited no such increase in our data, and accumulating evidence confirms the association of S100A9 with chronic inflammation and tumor promotion( 29 ), we put our emphasis on S100A9. In our study, high S100A9 expression was found associated with a high incidence of lung metastasis and a more advanced clinical stage. Exosomal S100A9 was confirmed to involved in tumor progression, such as activate the NF-κB pathway in Chronic Lymphocytic Leukemia( 17 ), enhance Colorectal Cancer (CRC) cell stemness and is linked to both CRC occurrence and recurrence( 19 ), implying its potential capacity as a biomarker and therapeutic target. In this study, we injected S100A9-overexpressing cell-derived exosomes into mice via tail vein. The results showed that these exosomes significantly increased ACC lung metastasis in immunodeficient mice compared to vector cell-derived exosomes, indicating that overexpression of S100A9 can promote ACC lung metastasis in an exosomecrine manner. Recent single-cell transcriptomic analyses have pointed that characterizing the ACC stromal microenvironment may contribute to tumor biology and prognosis( 30 ).A single-cell analysis of ACC indicated that the most prevalent cell populations in the ACC stroma were fibroblasts, followed by myeloid cells and T cells( 31 ). Since tumor cell can reprogram the subtype of CAFs and S100A9 has the potential to convert healthy donor-derived monocytes into myeloid-derived suppressor cells( 32 ), we hypothesize that tumor-derived exosomes might influence fibroblasts by their specific S100A9 cargoes which contribute to efficient intercellular communication and tumor-boosting behaviors. As a tumor-supportive myofibroblast phenotype, CAFs are characterized by high fibroblast-genesis genes and ECM protein expression, including FAP, α-SMA, collagens and MMPs( 33 ), and known to remodel the extracellular matrix, induce metabolic reprogramming, create a microenvironment promoting tumor invasion, metastasis and immune evasion( 34 ). In pancreatic ductal adenocarcinoma (PDAC), CAFs that have undergone a fibroblast-to-myofibroblast transition could form metastatic myoCAF-PDAC clusters and hijack cancer cells to distant organ metastases( 35 ). In our analysis of sc-sequencing data, we observed that EMT-state epithelial cells interact strongly with CAF through CellChat. Meanwhile, our investigation showed that SACC-LM-exo and S100A9-exo contains the ability to activate fibroblasts. To further explore the potential mechanism by which S100A9-enriched exosomes activate fibroblasts, our RNA sequencing data showed the differentially expressed genes enriched in IL-17, TNF-α and NF-κB signaling pathway in S100A9-exo-treated NFs. The process of NFs to activated CAFs can be induced by NF-κB signaling in bladder, breast, colorectal, and pancreatic cancer, indicating that NF-κB-driven CAF is common during cancer development( 36 ). IL-17 is a cytokine mostly from Th17 cells which is involved in inflammation, stromal-tumor interactions and immune evasion( 37 ). In PDAC, IL-17A facilitates the differentiation of fibroblasts into iCAF phenotype, suggesting its role in promoting terminal fibroblast activation( 38 ). NF-κB is an indisputable key player in the IL-17 signaling cascade( 39 ); IL-17 treatment could activate the NF-κB and Wnt/β-catenin pathways, which in turn stimulate the release of cytokines CXCL16( 22 ) and CXCL12( 40 ). Previous studies have shown cooperative effects by TNF-α plus IL-17, in which TNF-α induces transcription of target genes while IL-17 stabilizes their mRNAs( 41 ). In our study, we observed that S100A9 exosomes stimulate the expression of IL-17 and TNF-α, leading to enhanced NF-κB signaling and subsequent activation of myoCAFs in ACC. Next, we found the co-culturation of SACC-83 and S100A9/Fibroblasts can enhance the EMT and lung metastasis of SACC-83. Remarkably, KEGG pathway analysis of SACC-83 co-cultured with exosomes/fibroblasts have also highlighted the IL-17 signaling pathway. Above evidence shows IL-17 modulates the tumor microenvironment via CAF, likely driving tumor progression in ACC. Researches showed IL-17 secreted from activated CAFs increased macrophage recruitment to activate IKKβ/NF-κB signaling, facilitating tumor cell proliferation and invasion( 42 ). Also, pancreatic tumor cells co-cultured with IL-17A-iCAF displayed enhanced tumor proliferation and metabolism( 38 ). Our results indicated that rhIL-17 in conditioned medium promoted EMT in ACC tumor cells. However, the conditioned medium from S100A9-exo-NF may also include additional factors that further aggravate tumor progression. To sum up, disrupting this vicious cycle between cancer cells and CAFs may help reshape the tumor microenvironment, potentially suppressing tumor progression. In addition, S100A9 has shown potential to serve as biomarker for tumor diagnosis and prognosis in Acute Myeloid Leukemia and CRC( 19 , 43 ). Using the S100A9 inhibitor Paquinimod, we observed a diminished activation profile in CAFs. Therefore, we propose that further investigation into the role of S100A9 and IL-17 in lung metastasis of ACC holds great promise. In summary, our results suggested show a positive feedback loop in the TME of ACC ( Graphical abstract ). Tumor-derived exosomal S100A9 induce the transformation of NFs to CAFs, remodeling the TME; while ‘CAFs-like’ cells promoted EMT and metastasis of ACC cells via secreting IL-17. Such kinds of positive feedback loop in TME ultimately contribute to the progression of ACC. These results indicate that targeting S100A9 may offer promising therapeutic strategies for ACC lung metastasis. Declarations Author Contributions S.-R.Zhang and C.-W.Chen, contributed to conception and design, data acquisition and analysis, drafted and critically revised the manuscript; Y.-M.Yan, K.-W.Pan and F.-R.Ou contributed to data acquisition, analysis; K.Su and B.Cheng contributed to interpretation, drafted and critically revised the manuscript. All authors gave final approval and agreed to be accountable for all aspects of the work. Acknowledgment We strongly acknowledge the invaluable support of patients and their families, clinicians, and technicians. We would like to acknowledge Xianyue Ren for the time and support. Declaration of Comflicting Interests The authors declare no competing interests. Funding This study was funded by the National Natural Science Foundation of China (No. 82103555), China Postdoctoral Science Foundation (No.2021M703688, No.2022T150753). Ethics approval and consent to participate This study was conducted in compliance with the principles of the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations. Human tissues and primary human cells were provided by the participants with written-informed consent. Ethics approval for human subjects numbered KQEC-2024-140-01 was provided from the Ethics Committee of Hospital of Stomatology, Sun Yat-sen University. All animal procedures were approved by Permit number #JENNIO-IACUC-2023-A068. 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Wu F, Yang J, Liu J, Wang Y, Mu J, Zeng Q, et al. Signaling pathways in cancer-associated fibroblasts and targeted therapy for cancer. Signal Transduct Target Ther. 2021;6(1):218. Li X, Bechara R, Zhao J, McGeachy MJ, Gaffen SL. IL-17 receptor-based signaling and implications for disease. Nat Immunol. 2019;20(12):1594-602. Picard FSR, Lutz V, Brichkina A, Neuhaus F, Ruckenbrod T, Hupfer A, et al. IL-17A-producing CD8(+) T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts. Gut. 2023;72(8):1510-22. Amatya N, Garg AV, Gaffen SL. IL-17 Signaling: The Yin and the Yang. Trends Immunol. 2017;38(5):310-22. Lu X, Xu X, Zhou M, Ge J, Chen L, Yu W, et al. IL-17A-induced cancer-associated fibroblasts releases CXCL12 to promote lung adenocarcinoma progression via Wnt/β-Catenin signaling pathway. Cytokine. 2024;180:156676. Song X, Dai D, He X, Zhu S, Yao Y, Gao H, et al. Growth Factor FGF2 Cooperates with Interleukin-17 to Repair Intestinal Epithelial Damage. Immunity. 2015;43(3):488-501. Gao F, Chen X, Li X, Deng C, Luo P. The Pro-Migratory and Pro-Invasive Roles of Cancer-Associated Fibroblasts Secreted IL-17A in Prostate Cancer. J Biochem Mol Toxicol. 2025;39(2):e70047. Fan R, Satilmis H, Vandewalle N, Verheye E, De Bruyne E, Menu E, et al. Targeting S100A9 protein affects mTOR-ER stress signaling and increases venetoclax sensitivity in Acute Myeloid Leukemia. Blood Cancer J. 2023;13(1):188. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations There is no conflict of interest Supplementary Files Supplementaryfigurelegends.docx Supplementary figure legends fig.S.1.tif Supplementary figure 1 fig.S.2.tif Supplementary figure 2 fig.S.3.tif Supplementary figure 3 fig.S.4.tif Supplementary figure 4 fig.S.5.tif Supplementary figure 5 fig.S.6.tif Supplementary figure 6 wbfigure.pdf western blot figure Supplementarytable1.xlsx Supplementary table 1 Supplementarytable2.xlsx Supplementary table 2 Supplementarytable3.xlsx Supplementary table 3 Supplementarytable4.xlsx Supplementary table 4 Table1.xlsx Table 1 Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Cell Death Discovery → Version 1 posted Editorial decision: revise 20 Oct, 2025 Submission checks completed at journal 03 Oct, 2025 Editor assigned by journal 02 Oct, 2025 First submitted to journal 02 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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11:56:51","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136054,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/c5ee2c36330f8f6497c855c4.html"},{"id":92802278,"identity":"b1feb37f-0ea4-4693-9289-7252a6f29e17","added_by":"auto","created_at":"2025-10-05 11:40:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10716631,"visible":true,"origin":"","legend":"\u003cp\u003eS100A9 is highly expressed in SACC-LM and SACC-LM-exo and is correlated with lung metastasis in ACC. \u003cstrong\u003eA\u003c/strong\u003e The heatmap and hierarchical clustering of eleven selected mRNAs differentially expressed in SACC-LM cells compared with SACC-83 cells are shown. Red, high relative expression; green, low relative expression. \u003cstrong\u003eB\u003c/strong\u003e Heat\u003cstrong\u003e \u003c/strong\u003emap and hierarchical clustering of ten selected proteins differentially expressed in SACC-83-exo and SACC-LM-exo. \u003cstrong\u003eC\u003c/strong\u003e Western blot analysis of the S100A9 expression level in SACC-83, SACC-LM and their corresponding exosomes. \u003cstrong\u003eD\u003c/strong\u003e and \u003cstrong\u003eE \u003c/strong\u003eThe GO (\u003cstrong\u003eD\u003c/strong\u003e), KEGG (\u003cstrong\u003eE\u003c/strong\u003e) pathway enrichment of differentially expressed proteins between SACC-83-exo and SACC-LM-exo based on the 4D mass spectrometry proteomics data. BP: biological process; CC: cellular component; MF: molecular function. \u003cstrong\u003eF\u003c/strong\u003e Representative images of S100A9, FAP and α-SMA expression and quantification in 56 human ACC tissue samplesand 6 normal salivary gland tissue samples. The data points represent the individual patient sample scored. Scale bar = 50 μm. n = 62. \u003cstrong\u003eG \u003c/strong\u003eThe correlation analysis of FAP and α-SMA with S100A9 in all ACC tissues. AOD: average optical density. Data are represented as the mean ± SD. S100A9 and FAP group were analyzed by Kruskal-Wallis test while α-SMA group was analyzed by one-way ANOVA for (\u003cstrong\u003eF\u003c/strong\u003e). P values were calculated using Pearson correlation analysis for (\u003cstrong\u003eG\u003c/strong\u003e). \u003cstrong\u003eH \u003c/strong\u003eOverall survival (OS) and disease-free survival (DFS) in ACC patients after surgery stratified by S100A9 expression. (cutoff: median) *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/57c48bf27d0e6cd57eb14c31.png"},{"id":92802273,"identity":"a3940f79-5260-4233-b0bf-6650e05503a7","added_by":"auto","created_at":"2025-10-05 11:40:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8591046,"visible":true,"origin":"","legend":"\u003cp\u003eScRNA-seq profiling and cell-cell communication in ACC samples. \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e UMAP visualizations of five sample origins (\u003cstrong\u003eA\u003c/strong\u003e) and all cells (\u003cstrong\u003eB\u003c/strong\u003e) led to the identification of 16 cell clusters based on cellular identity, and all cell types are defined by known marker genes (see \u003cstrong\u003eSupplementary Table 4\u003c/strong\u003e).\u003cstrong\u003eC\u003c/strong\u003e UMAP visualizations of 14 cell clusters in ACC epithelial cells.\u003cstrong\u003e D\u003c/strong\u003eInferCNV of ACC epithelial cells. \u003cstrong\u003eE\u003c/strong\u003e EMT scores of ACC epithelial cells. \u003cstrong\u003eF\u003c/strong\u003e UMAP visualizations of non-malignant epithelial cell and malignant epithelial heterogeneity\u003cstrong\u003e \u003c/strong\u003ein ACC. \u003cstrong\u003eG\u003c/strong\u003e Cellcell interactions between each cell subgroup and others. \u003cstrong\u003eH\u003c/strong\u003e and \u003cstrong\u003eI\u003c/strong\u003e UMAP visualizations of 8 clusters (\u003cstrong\u003eH\u003c/strong\u003e) and all cells (\u003cstrong\u003eI\u003c/strong\u003e) led to the identification based on cellular identity (see \u003cstrong\u003eSupplementary Table 4\u003c/strong\u003e).\u003cstrong\u003e J\u003c/strong\u003e Distribution of FAP⁺α-SMA⁺CAFs in fibroblast clusters. \u003cstrong\u003eK\u003c/strong\u003e Cellcell interactions between EMT-state epithelial cells and FAP⁺α-SMA⁺CAFs. The number represents the interaction strength.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/5e4ec17da0cf9bbd30496c79.png"},{"id":92802275,"identity":"2b37ed2f-8852-4aeb-9c9a-5bae4d9d394a","added_by":"auto","created_at":"2025-10-05 11:40:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3528342,"visible":true,"origin":"","legend":"\u003cp\u003eExosomal S100A9 mediates fibroblasts activation.\u003cstrong\u003e A \u003c/strong\u003eRepresentative IF images displayed the expression of α-SMA and FAP of NFs, CAFs and NFs treated with different exosomes.\u003cstrong\u003e \u003c/strong\u003eScale bar = 50 μm.\u003cstrong\u003e B \u003c/strong\u003eRepresentative collagen contraction images of NFs, CAFs and NFs treated with different exosomes. Scale bar =2 mm. \u003cstrong\u003eC\u003c/strong\u003e Migration of NFs, CAFs and NFs treated with different exosomes were accessed by transwell assay. Scale bar = 50 μm. \u003cstrong\u003eD \u003c/strong\u003eRepresentative IF images displayed the expression of α-SMA and FAP of NFs treated with different exosomes and plasmid. Scale bar = 50 μm\u003cstrong\u003e. E\u003c/strong\u003e Representative collagen contraction images of NFs treated with different exosomes and plasmid. Scale bar =2 mm. \u003cstrong\u003eF\u003c/strong\u003e Migration of NFs treated with different exosomes and plasmid were accessed by transwell assay. Scale bar= 50 μm. \u003cstrong\u003eG\u003c/strong\u003eThe mRNA levelof FAP, α-SMA and inflammatory markers in fibroblasts after incubation with according exosomes. \u003cstrong\u003eH \u003c/strong\u003eand \u003cstrong\u003eI \u003c/strong\u003eThe expression of α-SMA and FAP, the contraction ability in fibroblasts cultivating with exosomes of SACC-LM treated with siS100A9. Scale bar = 50 μm \u003cstrong\u003e(H)\u003c/strong\u003e, and 2 mm \u003cstrong\u003e(I)\u003c/strong\u003e. \u003cstrong\u003eJ \u003c/strong\u003eand\u003cstrong\u003e K\u003c/strong\u003e The expression of α-SMA and FAP, the contraction ability inS100A9-exo treated NFs with or without Paquinimod. Scale bar = 50 μm \u003cstrong\u003e(J)\u003c/strong\u003e, and 2 mm \u003cstrong\u003e(K)\u003c/strong\u003e. Data are presented as the mean ± SD. P values were calculated using two-tailed Student’s t test for (\u003cstrong\u003eA-K\u003c/strong\u003e), *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/82cca5e583aea3f4611094f6.png"},{"id":92802972,"identity":"36d7ebf9-1359-496e-86ff-94d0e03bcfbe","added_by":"auto","created_at":"2025-10-05 11:48:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10820701,"visible":true,"origin":"","legend":"\u003cp\u003eExosomal S100A9 actives fibroblasts through NF-κB signaling pathway. \u003cstrong\u003eA\u003c/strong\u003e NOD/SCID mice were pre-treated with exosomes (n = 8 per group) for 7 days, then the remaining mice from each group were tail injected with SACC-83 cells (n = 7 per group) for 8 weeks. \u003cstrong\u003eB \u003c/strong\u003eIHC analysis of FAP and α-SMA in lung tissues from NOD/SCID mice 7 days after exosome stimulation (n = 8 per group). Scale bar = 200 μm. \u003cstrong\u003eC \u003c/strong\u003eIF staining of Fibronectin, MMP9, and VEGFR1 in serial sections of the same cohort (n = 8 per group). Scale bar = 200 μm. \u003cstrong\u003eD\u003c/strong\u003e Formation of lung metastases in mice after stimulation with Vector-exo or S100A9-exo, as represented by bioluminescence signals (left) and normalized photon flux (right). \u003cstrong\u003eE\u003c/strong\u003e Representative images (left) and quantification of the metastatic tumor colony (right) in lung sections from NOD/SCID mice. Scale bar = 100 μm. (n = 7 per group) \u003cstrong\u003eF \u003c/strong\u003eGO annotation of different expressing mRNAs with the top 20 enrichment factors. \u003cstrong\u003eG\u003c/strong\u003e KEGG pathway enrichment of the differential expressing mRNAs by RNA sequencing. \u003cstrong\u003eH\u003c/strong\u003eThe relative IL-17 and TNF-α mRNA level in fibroblasts after incubation with according exosomes. \u003cstrong\u003eI \u003c/strong\u003eThe effect of Vector-exo, S100A9-exo or BAY-117082 in combination with exosomes on the cytoplasmic and nuclear protein levels of p65, p-p65 in NFs after incubation. \u003cstrong\u003eJ \u003c/strong\u003eThe effect of S100A9-exo and BAY-117082 in combination with S100A9-exo on the mRNA expression of FAP and α-SMA, inflammatory markers and cytokines in fibroblasts after incubation.\u003cstrong\u003eK\u003c/strong\u003e Overview of pathway alteration regarding S100A9-exo applied using Metabolomics data. Data are presented as the mean ± SD for (\u003cstrong\u003eH, J\u003c/strong\u003e), as the mean ± SEM for (\u003cstrong\u003eB right, C right,\u003c/strong\u003e \u003cstrong\u003eD right\u003c/strong\u003e). P values were calculated using two-tailed Student’s t test for (\u003cstrong\u003eB right\u003c/strong\u003e, \u003cstrong\u003eC right\u003c/strong\u003e, \u003cstrong\u003eD right, H, J\u003c/strong\u003e), and Mann Whitney test (\u003cstrong\u003eE right\u003c/strong\u003e). **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/b028f726d89df62e60b64bb7.png"},{"id":92802290,"identity":"e0b6c529-04d7-4c6a-a260-8069464b53d7","added_by":"auto","created_at":"2025-10-05 11:40:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3269657,"visible":true,"origin":"","legend":"\u003cp\u003eActivated fibroblasts promote lung metastasis in ACC.\u003cstrong\u003e A\u003c/strong\u003e Schematic diagram of Exo-modulated fibroblast cells regulated tumor cells, and generated a feedback loop for adjacent tumor cells. The fibroblasts were pre-treated with Vector-exo or S100A9-exo for 48h for activation, these activated fibroblasts were next co-cultured with SACC-83 for follow-up experiments. \u003cstrong\u003eB\u003c/strong\u003eCCK-8 cell assay of SACC-83 co-cultured exosomes/fibroblastswere determined by every 24h. \u003cstrong\u003eC \u003c/strong\u003eMigration and invasion of these co-cultured samples were accessed by transwell assay. Scale bar = 200 μm.\u003cstrong\u003e D \u003c/strong\u003eRepresentative images of colonies formed by these co-cultured samples after 10 days. Colonies were fixed and stained with crystal violet. \u003cstrong\u003eE \u003c/strong\u003eRepresentative fluorescent images displayed EdU incorporation (green) in these co-cultured samples. Nuclei were counterstained with DAPI (blue). Scale bar = 100µm. \u003cstrong\u003eF\u003c/strong\u003e E-cadherin and N-cadherin expression in these co-cultured samples. \u003cstrong\u003eG\u003c/strong\u003e Formation of lung metastases in mice after injection with SACC-83 co-cultured with exosome/fibroblasts, as represented by bioluminescence signals (left) and normalized photon flux (right) (n = 7 per group). \u003cstrong\u003eH \u003c/strong\u003eRepresentative images (left) and quantification of the metastatic tumor colony (right) in HE staining of lung sections from NOD / SCID mice (n = 7 per group). Scale bar = 100 μm. \u003cstrong\u003eI \u003c/strong\u003eand\u003cstrong\u003e J \u003c/strong\u003eGO \u003cstrong\u003e(I)\u003c/strong\u003e, KEGG \u003cstrong\u003e(J)\u003c/strong\u003e enrichment analysis of SACC-83 co-cultured with exosome/fibroblastsby RNA sequencing. \u003cstrong\u003eK\u003c/strong\u003eThe relative IL-17 protein level in the supernatant of fibroblasts after incubation with according exosomes or IL-17 Ab were measured by ELISA.\u003cstrong\u003e L \u003c/strong\u003eE-cadherin, N-cadherin, E-cad and N-cad expression in co-cultured samples with or without the presence of IL-17 Ab, with GAPDH serving as the loading control.\u003cstrong\u003e M \u003c/strong\u003eThe effect of different concentration of rhIL-17 on the protein level of EMT markers in SACC-83.\u003cstrong\u003e \u003c/strong\u003eData are presented as the mean ± SD for (\u003cstrong\u003eC right, D, E right, K\u003c/strong\u003e), as the mean ± SEM for (\u003cstrong\u003eG right\u003c/strong\u003e). 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11:40:51","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3103112,"visible":true,"origin":"","legend":"Supplementary figure 4","description":"","filename":"fig.S.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/ae9d28dbf5e02c2abfabcf51.tif"},{"id":92802966,"identity":"1c3c8e1a-e67d-460c-b8fe-1fc0a7a8a263","added_by":"auto","created_at":"2025-10-05 11:48:51","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":5946768,"visible":true,"origin":"","legend":"Supplementary figure 5","description":"","filename":"fig.S.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/f792622d18d92f1f7dec1090.tif"},{"id":92802314,"identity":"8646601e-3fdc-4474-9894-861726831b30","added_by":"auto","created_at":"2025-10-05 11:40:52","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1993900,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 6\u003c/p\u003e","description":"","filename":"fig.S.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/eab5982ecd54c0b9abf8f1ab.tif"},{"id":92802968,"identity":"ae09c37a-2e16-49d1-b989-8d2cb8d733d7","added_by":"auto","created_at":"2025-10-05 11:48:51","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":791928,"visible":true,"origin":"","legend":"\u003cp\u003ewestern blot figure\u003c/p\u003e","description":"","filename":"wbfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/1b525010c1382fa95fe81114.pdf"},{"id":92802970,"identity":"c7e51618-71c4-4381-bb91-c033563b919f","added_by":"auto","created_at":"2025-10-05 11:48:51","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":9611,"visible":true,"origin":"","legend":"Supplementary table 1","description":"","filename":"Supplementarytable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/1c1f572710b593f3d84314c0.xlsx"},{"id":92802963,"identity":"8cfdd913-8a91-404f-9515-894efd7cbfb4","added_by":"auto","created_at":"2025-10-05 11:48:50","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":11171,"visible":true,"origin":"","legend":"Supplementary table 2","description":"","filename":"Supplementarytable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/43db718bf6bf44ff2bee0b62.xlsx"},{"id":92802276,"identity":"263f7e35-9dfa-4064-bec8-64e66c996846","added_by":"auto","created_at":"2025-10-05 11:40:51","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":10251,"visible":true,"origin":"","legend":"Supplementary table 3","description":"","filename":"Supplementarytable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/a6f9947e68bb32b378072dec.xlsx"},{"id":92802973,"identity":"a4680df8-6baf-42e4-8ebd-fd24da4c0086","added_by":"auto","created_at":"2025-10-05 11:48:51","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":11816,"visible":true,"origin":"","legend":"Supplementary table 4","description":"","filename":"Supplementarytable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/6baa23011d07482c5c723e69.xlsx"},{"id":92802969,"identity":"b1d6fe2f-c73b-4ed6-866c-c144d0a4bd4f","added_by":"auto","created_at":"2025-10-05 11:48:51","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":13816,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7171055/v1/0844f3316b56b3c5b867e9e2.xlsx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Exosomal S100A9 Promotes Lung Metastasis of Adenoid Cystic Carcinoma via Activating Cancer-Associated Fibroblasts","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSalivary Adenoid cystic carcinoma (SACC) is a carcinoma of secretory glands, frequently arising in salivary glands. Adenoid cystic carcinoma (ACC) has also been observed in other head and neck regions including the trachea, larynx, nasal and paranasal sinus, and lacrimal glands(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). SACC accounts for 28% of the total incidence of malignant salivary gland tumors(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and 50% of malignant tumors in the minor glands (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Distant metastases, most commonly to the lung, is the leading cause of reduced survival in ACC. The 5-year survival rate is around 69.7%, and the 10-year survival rate drops to 29%(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Surgical intervention with radiotherapy provides effective local control but do not effectively treat metastatic ACC, approved chemotherapies or targeted agents remains unclear(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Elucidating the biological molecular mechanisms of ACC lung metastasis, especially the interaction in the tumor microenvironment (TME), is imperative in improving the current situation.\u003c/p\u003e\u003cp\u003eS100A9 is a calcium-binding protein of the S100 family, initially known as a proinflammatory mediator released by neutrophils and monocytes in response to cell damage, infection, or inflammation(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Previous work has shown that S100A9 is crucial in establishing the premetastatic niche, chemotherapy resistance, and subsequent metastasis in breast cancer and melanoma(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). S100A9 inhibitors have been shown to decrease myocardial infarct size, and alleviate myocardial fibrosis(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Moreover, a large randomized phase II trial demonstrated that, in patients with metastatic castration-resistant prostate cancer, S100A9 inhibitors significantly prolonged radiographic and symptomatic progression-free survival and were associated with improved overall survival(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). These findings highlight the potential of S100A9 as a biomarker and a therapeutic target. Nevertheless, it is unclear whether S100A9 contributes to lung metastasis in ACC. Exosomes are 40\u0026ndash;150 nm, extracellular, phospholipid vesicles, secreted by variety types of cells. They play a crucial role in intercellular communication by carrying bioactive molecules such as proteins, metabolites, and nucleic acids(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In contrast to autocrine, paracrine, and juxtacrine signaling mechanisms, greater amounts of functional biomolecules can be transferred to extracellular matrix and circulation via encapsulating into exosomes, known as exosomecrine mechanism(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The continuous crosstalk between cancer cells and local/distant microenvironments effectively drives tumor migration, metastasis, and therapy resistance. Among the main actors of the interplay are the exosomes, which taken up by stromal cells. Cancer-associated fibroblasts (CAFs), activated by signals from the tumor or its microenvironment, constitute the most abundant cells in the tumor stroma and dynamically regulate extracellular matrix remodeling, angiogenesis, and immune evasion, especially pre-metastatic niche formation(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). S100A9 can be packed into exosomes and act as an extracellular matrix mediator to promote chronic lymphocytic leukemia progression(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, the role of exosomal S100A9 in regulation of the TME and the premetastatic niche during lung metastasis in ACC remains unclear.\u003c/p\u003e\u003cp\u003eHere, we investigated the correlation between S100A9 expression and lung metastasis formation in ACC. Furthermore, we elucidated the underlying mechanisms by which S100A9 modulates the tumor microenvironment to facilitate pulmonary metastasis through exosomecrine.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTissue samples and cell lines\u003c/h2\u003e\u003cp\u003eACC (n\u0026thinsp;=\u0026thinsp;56) and normal gland (NG, n\u0026thinsp;=\u0026thinsp;6) tissue samples were collected from patients undergoing radical resection at the Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Sun Yat-sen University between 2015 and 2024. None of the patients had received chemotherapy or radiotherapy. Histopathological examination was used to confirm all diagnoses.\u003c/p\u003e\u003cp\u003eThe SACC-83 cell line was derived from the sublingual gland of a patient. SACC-LM cells, with a high capacity for lung metastasis, were isolated \u003cem\u003ein vivo\u003c/em\u003e after tail vein injection of SACC-83 cells into immunodeficient mice(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Both cell lines were validated by STR profiling, confirmed Mycoplasma-free by PCR after the last experiment, and used within 15 passages post-thaw. SACC cells were cultured in RPMI-1640 medium with 10% FBS (Gibco, USA).\u003c/p\u003e\u003cp\u003eNormal fibroblasts (NFs) were isolated from normal gland tissue while cancer-associated fibroblasts (CAFs) were isolated from human ACC tumor tissues from the same patient according to previously described protocols(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Fibroblasts were maintained in DMEM/F12 medium supplemented with 10% FBS, 10 000 U/mL penicillin, and 10 000 \u0026micro;g/mL streptomycin (Gibco, USA) at 37\u0026deg;C in 5% CO₂. Conditioned media from exosome-treated NFs were referred to as S100A9-exo-NF-CM or Vector-exo-NF-CM.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eOverexpression and siRNAs\u003c/h3\u003e\n\u003cp\u003eThe lentiviral HBLV-h-S100A9-3xflag-ZsGreen-PURO (used to construct the S100A9 overexpression vector), the HBLV-ZsGreen-PURO vector (used to construct the S100A9-expression vector) and HBLV-LUC-PURO were purchased from Hanbio (Hanbio Biotechnology, China).\u003c/p\u003e\u003cp\u003eTransfection of siRNAs and plasmids was performed using the Lipofectamine 2000 Kit (Invitrogen, USA), according to the manufacturer's instructions. The S100A9 siRNA was designed and synthesized by RiboBio (Guangzhou, China) and are listed in \u003cb\u003eSupplementary Table\u0026nbsp;1.\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry (IHC) and multiplex immunohistochemistry staining (mIHC)\u003c/h3\u003e\n\u003cp\u003eParaffin-embedded ACC tumor sections were deparaffinized, rehydrated, and subjected to antigen retrieval in sodium citrate buffer. For standard immunohistochemistry, slides were incubated overnight at 4\u0026deg;C with primary antibodies, followed by secondary antibody incubation for 30 min at room temperature. For multiplex staining, a multi-labeled kit (Panovue, Beijing, China) was used according to the manufacturer\u0026rsquo;s protocol. HRP conjugates and five fluorescent wavelengths (520, 570, 620, 690, and 440 nm) enabled simultaneous detection of multiple targets. Nuclei were counterstained with DAPI (ZLI-9556, ZSGB-Bio, Beijing, China). Slides were scanned using the Aperio AT2 system (Leica, Germany), and images were analyzed using ImageJ. Antibody details are listed in \u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eFibroblasts grown on glass coverslips with different exosomes treated for 48\u0026ndash;72 h were fixed with 4% paraformaldehyde at room temperature. For staining of paraffin-embedded tumor samples, formalin fixation was dewaxed at room temperature for 30 min. BSA (5%) in PBS with mouse IgG blocking reagent was used. After 1 h, target proteins were captured by each primary antibody listed in \u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e during incubation for 18 h at 4\u0026deg;C. Captured proteins were incubated with fluorescently labeled IgG in a dark room for 1 h before treated with medium containing DAPI (cat. no. ZLI-9556, ZSGB-Bio, Beijing, China) and imaged with a confocal microscope (FV3000, Olympus Corporation, Japan) or an inverted fluorescence microscope (U-TBI90, Olympus, Japan).\u003c/p\u003e\n\u003ch3\u003eMicroarray expression profiling and mRNA High-throughput Sequencing\u003c/h3\u003e\n\u003cp\u003eTotal RNA extracted from SACC-83 and SACC-LM cells was analyzed by Shbio (Shanghai, China) using the SBC Human 4 \u0026times;180 K ceRNA microarray. Total RNA was extracted from Vector-exo-NF and S100A9-exo-NF, SACC-83 cells co-cultured with exosomes/fibroblasts, and sequenced on the Illumina platform by Shbio (Shanghai, China).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eRNA preparation and qPCR\u003c/h2\u003e\u003cp\u003eRNA was extracted using TRIzol reagent (Invitrogen, USA), according to the manufacturer's instructions, and the extracted RNA was reverse transcribed into cDNA using a cDNA Reverse Transcription Kit (Takara, Japan). The SYBR Green-based qPCR analysis was conducted with the Light-Cycler 96 system (Roche). All RT-qPCR primer sequences are listed in \u003cb\u003eSupplementary Table\u0026nbsp;3.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExosome proteomics mass Spectrometry\u003c/h3\u003e\n\u003cp\u003eExosome proteomics services were supported by Wayen Biotechnologies following a standard protocol. Max-Quant 1.5.8.3 (Max-Planck Institute for Biochemistry) was used as a quantitative proteomics software package for analyzing large mass spectrometry data sets. Metascape (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://metascape.org\u003c/span\u003e\u003cspan address=\"http://metascape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and cluster profiler package o were used for gene annotation and enrichment analysis. Terms with \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u003cem\u003e\u0026lt;\u003c/em\u003e\u0026thinsp;0.05, minimum counts of 3, and enrichment factor\u0026thinsp;\u003cem\u003e\u0026gt;\u003c/em\u003e\u0026thinsp;1.5 were collected and grouped into clusters based on their membership similarities.\u003c/p\u003e\n\u003ch3\u003eExosomes isolation, labeling, and uptaken\u003c/h3\u003e\n\u003cp\u003eExosomes isolation from cell supernatants was performed using ultracentrifugation and sucrose cushion(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Supernatants were cleared by centrifugation, concentrated using 100K centrifugal filters (Millipore, USA), and subjected to gradient centrifugation at 100 000 \u0026times; g for 70 minutes (Optima L-90K, Beckman Coulter). The exosome-enriched fraction was resuspended in PBS and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Protein concentration was measured using the Bradford assay (Beyotime, China).\u003c/p\u003e\u003cp\u003eExosomes and fibroblasts were labeled with PKH26 (Sigma-Aldrich, USA) and Actin-Tracker Green (phalloidin-FITC, Beyotime, China), respectively; nuclei were stained with DAPI (ZSGB-Bio, China). Fibroblasts were incubated with labeled exosomes for 24 h, and then visualized using a confocal microscope (FV3000, Olympus, Japan).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eColony formation, cell viability and proliferation assays\u003c/h2\u003e\u003cp\u003eFor colony formation, 2 mL of cell medium containing 500 cells was seeded in 6-well plates and cultivated for 10 days. Then, the cells were fixed by using 4% paraformaldehyde (cat. no. BL539A, Biosharp, China) for 30 minutes and stained with 0.1% crystal violet (cat. no. RBG1019-100 mL, Roles-Bio, Guangzhou, China). The colony numbers of each well were counted with ImageJ. Cell Counting Kit 8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) and EdU (cat. no. KGA9602-100, KeyGEN BioTECH, Nanjing, China) assay were used to assess cell viability and proliferation according to manufacturer\u0026rsquo;s methods.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\u003cp\u003eProteins were extracted from cultured cells with RIPA lysis buffer (Beyotime Technology). The protein concentrations of cells were measured using BCA protein assay kits. The protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS‒PAGE) and subjected to immunoblot analysis. The antibody information is listed in \u003cb\u003eSupplementary Table\u0026nbsp;2.\u003c/b\u003e Antibody-bound proteins were detected using an enhanced chemiluminescence (ECL) Western blot kit (CWBio, China). Fibroblasts were treated with IKK inhibitor BAY-117082 and prepared with Nuclear and Cytoplasmic Extraction Reagents (Beyotime, P0028). Cells were stimulated with recombinant human IL-17A (Pepro-Tech, London, UK, 200\u0026thinsp;\u0026minus;\u0026thinsp;17) and immunoblotting was followed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTranswell assays\u003c/h2\u003e\u003cp\u003eFor the Transwell assay, transwell filter inserts (Costar, Corning, USA) with or without Matrigel (BD Biosciences, USA) coating were used according to the manufacturer's instructions. The cells that passed through the membrane were fixed, stained, and counted under a light microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMouse model of lung metastasis\u003c/h2\u003e\u003cp\u003e All animal procedures followed institutional regulations and the ARRIVE guidelines. Female NOD/SCID mice (20\u0026ndash;22 g, 4\u0026ndash;6 weeks old) were stratified by body weight and randomly assigned to control or treatment groups.\u003c/p\u003e\u003cp\u003eMice received daily intravenous injections of either Vector-exo or S100A9-exo (70 \u0026micro;g, n\u0026thinsp;=\u0026thinsp;8 per group) for 7 days. On day 8, lungs were harvested for immunohistochemistry and immunofluorescence analysis.\u003c/p\u003e\u003cp\u003eThe remaining mice (n\u0026thinsp;=\u0026thinsp;7 per group) received 1 \u0026times; 10⁶ firefly luciferase-labeled SACC-83 cells injection intravenously on day 8. After 8 weeks, lung metastases were evaluated by bioluminescence imaging (Caliper Life Sciences), followed by lung collection for H\u0026amp;E staining.\u003c/p\u003e\u003cp\u003eAdditionally, 1 \u0026times; 10⁶ luciferase-labeled SACC-83 cells co-cultured with exosomes/fibroblasts were injected into NOD/SCID mice (n\u0026thinsp;=\u0026thinsp;7 per group) via the tail vein. After 8 weeks, lung metastases were assessed by imaging and confirmed by H\u0026amp;E staining.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCollagen contraction assays\u003c/h2\u003e\u003cp\u003eAfter treated with exosomes or plasmids for 48\u0026ndash;72 h, fibroblasts were harvested and mixed with 400 \u0026micro;l of collagen mix containing 186.46 \u0026micro;l dH2O, 4.23 \u0026micro;l 1 N NaOH, 40 \u0026micro;l 10 \u0026times; PBS and 169.31 \u0026micro;l Type 1 Rat Tail Collagen (cat. no. 354249, Corning, NY, USA). Then, the mix was added to 24-well plate and allowed to solidify for 30 min at 37\u0026deg;C. Later, 500 \u0026micro;l DMEM-F12 with 10% FBS was added to the plate for 16 h until the gels were photographed. ImageJ software was employed to measure gel area and evaluate contraction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eELISA\u003c/h2\u003e\u003cp\u003eThe conditioned medium of fibroblasts was incubated with IgG antibody (R\u0026amp;D Systems Inc., Minneapolis, MN, USA) or IL-17A-neutralizing antibody (IL-17A Ab; R\u0026amp;D Systems) (all used at 10\u0026micro;g/ml). The IL-17A concentrations in cell culture supernatant were measured by using the Human IL-17A ELISA Kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China; H014-2) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eSingle-Cell Preparation and Library Construction\u003c/h2\u003e\u003cp\u003eFresh tumor tissues from five SACC patients and normal salivary gland tissues from two non-SACC individuals were collected immediately after surgery, stored in Tissue Storage Solution (Miltenyi Biotec, Germany) on ice, and transferred to the laboratory. Single-cell suspensions were loaded onto the 10X Genomics Chromium platform to generate Gel Bead-In-Emulsions (GEMs), and libraries were prepared using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Gene Denovo, Guangzhou, China). Each GEM contained primers with an Illumina\u0026reg; R1 sequence, a 16-nt 10x barcode, a 10-nt UMI, and a poly-dT sequence for capturing polyadenylated mRNAs, enabling the synthesis of barcoded full-length cDNA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eData Processing and Quality Control\u003c/h2\u003e\u003cp\u003eRaw sequencing data were processed using Seurat (v4.1.2) in R (v4.3.0). Cells expressing fewer than 200 or more than 6 000 genes were removed, as were cells with \u0026gt;\u0026thinsp;15% mitochondrial or \u0026gt;\u0026thinsp;1% hemoglobin gene content. Mitochondrial genes were excluded from downstream steps. Doublets were identified using DoubletFinder (v2.0.3) and removed. After stringent quality control, 51,128 high-quality cells were retained for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eIntegration, Clustering, and Cell Type Annotation\u003c/h2\u003e\u003cp\u003eAll samples were normalized and variance-stabilized with SCTransform (v0.3.2), followed by integration using IntegrateData. PCA was performed on the top 2,000 highly variable genes, and 20 principal components were selected using the ElbowPlot function. Uniform Manifold Approximation and Projection (UMAP) was used for visualization. Cell clustering was carried out using the Louvain algorithm (FindClusters, resolution\u0026thinsp;=\u0026thinsp;0.8). Clusters were annotated based on DEGs (identified with FindAllMarkers) and known cell type markers (\u003cb\u003eSupplementary Table\u0026nbsp;4\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo refine annotations of major cell types, each population was extracted, reprocessed with SCTransform, reintegrated, and re-clustered. Subclusters were defined using DEGs and inferred biological functions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eCellChat communication\u003c/h2\u003e\u003cp\u003eCellChat R package (version 1.6.1) was utilized to investigate the intercellular communication between epithelial cells and fibroblasts. CellChat enables the systematic analysis of ligand-receptor interactions based on scRNA-seq data.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe bioinformatics and statistical analyses were conducted using R 4.4.2, SPSS 27 and GraphPad Prism 8.0 softwires. All \u003cem\u003ein vitro\u003c/em\u003e experiments were confirmed in at least three independent experiments and all numerical data represented mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) unless otherwise stated. The data from one representative experiment were shown. Data were statistically analyzed by two-tailed Student\u0026rsquo;s t test, Mann Whitney test, Pearson correlation analysis and χ\u0026sup2; test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003eS100A9 is upregulated in ACC and associated with lung metastasis\u003c/h2\u003e\u003cp\u003eTo investigate the dysregulated genes in ACC progression, we analyzed mRNAs from a pair of microarrays for two ACC cell lines(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). SACC-LM, with enhanced lung metastatic potential, was derived from the parental low-metastatic SACC-83 line. Considering the prometastatic roles of exosome, we isolated corresponding exosomes (SACC-83-exo/SACC-LM-exo). Their characteristic cup-shaped morphology, size, and concentration were subsequently confirmed (Fig.S.1A and B), and exosomal markers (Hsp70, TSG101, CD63, CD9, CD81) were validated by western blot (Fig.S.1C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eProteomic profiling revealed 149 proteins upregulated and 398 downregulated in SACC-LM-exo (fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 or \u0026le;\u0026thinsp;0.5, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Integration with transcriptomic data identified S100A9 as consistently elevated in both tumor cells and exosomes, which was further validated by western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Gene Ontology (GO) analysis between SACC-83-exo and SACC-LM-exo showed enrichment in vesicle-mediated transport, component biogenesis, and protein localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed to highlight cancer-related and ECM-receptor interaction pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eIHC analysis demonstrated elevated expression of S100A9, fibroblast activation protein (FAP) and α-smooth muscle actin (α-SMA) in the primary ACC tumor tissues versus normal salivary gland, with higher distribution in patients with lung metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Correlation analysis showed that S100A9 positively associated with FAP and α-SMA in 56 ACC samples and 6 NG samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), suggesting a potential role of S100A9 in the tumor-CAF cross talk. Clinically, high S100A9 expression correlated with advanced stage and lung metastasis (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). However, due to the relatively long survival time of ACC patients and the limited number of available samples, no significant difference in OS was observed, whereas patients with high S100A9 expression exhibited significantly shorter disease-free survival (DFS) compared to those with low expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eFigure 2\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eSingle cell analysis reveals CAF diversity and potential crosstalk with ACC cells in Tumor Microenvironment\u003c/h2\u003e\u003cp\u003eTo investigate tumor-stroma interaction in ACC TME, we analyzed single-cell transcriptomic data from five ACC tumors and two normal salivary glands (Fig.\u0026nbsp;2A), capturing 67 007 cells and clustering them into 16 distinct cell types (Fig. S.2A and B), with annotations shown in Fig.\u0026nbsp;2B.\u003c/p\u003e\u003cp\u003eA total of 27 663 epithelial cells were re-analyzed and visualized as subclusters (Fig.\u0026nbsp;2C and Fig. S.2C). Subsequently, inference of copy number alterations (CNAs) identified malignant cells (Fig.\u0026nbsp;2D and Fig.S.2D), among which a subset with high EMT scores was defined as \u0026ldquo;EMT-state epithelial cells.\u0026rdquo; (Fig.\u0026nbsp;2E, F and Fig.S.2E)\u003c/p\u003e\u003cp\u003eCellChat analysis revealed frequent and strong communication between epithelial cells and fibroblasts (Fig.\u0026nbsp;2G). Given the pivotal role of fibroblasts in EMT-driven tumor metastasis, we classified fibroblasts into antigen-presenting CAFs (apCAFs), matrix CAFs (mCAFs), inflammatory CAFs (iCAFs), vascular CAFs (vCAFs), and non-specific CAFs (nCAFs) (Fig.\u0026nbsp;2H, I and Fig.S.2F). FAP and α-SMA were expressed across several CAF subsets, but their expression levels were markedly higher in the mCAF population (Fig.\u0026nbsp;2J and Fig.S.2G, H).\u003c/p\u003e\u003cp\u003eSubsequent cell\u0026ndash;cell communication analysis revealed that FAP⁺α-SMA⁺CAFs displayed frequent and robust interactions with EMT-state epithelial cells (Fig.\u0026nbsp;2K). This key finding was further confirmed in an external scRNA-seq dataset, thereby significantly strengthening its validity (GSE216852, n\u0026thinsp;=\u0026thinsp;6, total cells\u0026thinsp;=\u0026thinsp;51 128) (Fig.S.3). Multiplex immunohistochemistry (mIHC) of ACC tissues further supported spatial proximity and potential interactions between stromal and EMT-state epithelial populations (Fig.S.4).\u003c/p\u003e\u003cp\u003eCollectively, these findings highlight strong interactions between specific CAF subpopulations\u0026mdash;particularly FAP⁺α-SMA⁺CAFs\u0026mdash;and EMT-state tumor cells, underscoring the importance of identifying key regulatory factors mediating fibroblast\u0026ndash;tumor crosstalk in ACC.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eS100A9 in exosomes mediates fibroblasts activation\u003c/h2\u003e\u003cp\u003eTo assess the relationship between S100A9 and fibroblast activation, primary CAFs and matched NFs were isolated from ACC tumors and adjacent normal salivary gland (Fig.S.5A). Western blot analysis confirmed higher expression levels of fibrosis (FAP, α-SMA) in CAFs versus NFs (Fig.S.5B). Treatment with SACC-LM-exo enhanced the expression of FAP and α-SMA, contraction ability and migration of NFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Exosomes uptake was confirmed by PKH26-labeling and confocal microscopy (Fig.S.5C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further explore the role of S100A9 in CAFs activation, we established SACC-83 with S100A9 overexpression. Subsequent analyses confirmed elevated S100A9 levels in both the transfected cells and their derived exosomes (Fig.S.6A and B). NFs were transiently transfected with high S100A9 expressing vectors, the transfection effect was verified by qPCR and western blot (Fig.S.6C and D). Across the two experimental groups, exosomal S100A9 consistently led to elevated levels of FAP and α-SMA, greater collagen gel contraction, and increased migration ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). S100A9-exo treatment upregulated expression levels of fibrosis, pro-inflammatory cytokines IL-6, IL-8, IL-1β as well as matrix metalloproteinases MMP2 and MMP9 in RNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), which are molecules contributing to a tumor-promoting microenvironment.\u003c/p\u003e\u003cp\u003eS100A9 knockdown in SACC-LM cells via siRNA (Fig.S.6E and F) reduced the fibroblast-activating capacity of their exosomes, evidenced by the reduction in FAP and α-SMA expression via immunofluorescence, along with a diminished collagen contraction capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I). Similarly, an S100A9 inhibitor paquinimod suppressed fibroblast activation induced by S100A9-exo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, K). Together, these findings support a potential role of S100A9-enriched exosomes in promoting fibroblast activation and stromal remodeling.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eExosomal S100A9 potentiate lung metastasis and activate fibroblasts via IL-17-TNF-NF-κB axis\u003c/h2\u003e\u003cp\u003eLung pre-metastatic model was established to evaluate the contribution of S100A9-exo on organotropic metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In vivo, intravenously injection of S100A9-exo into NOD/SCID mice showed elevated level of fibroblast-genesis genes (FAP and α-SMA) in lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), yet formed a pre-metastatic microenvironment with higher level of Fibronectin, MMP9 and VEGFR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The intravenous injection of exosomes from S100A9-overexpressing cells into immunodeficient mice significantly increased ACC lung metastases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). We collected vector-exo/S100A9-exo treated NFs and profiled them using high-throughput RNA-sequencing. Analysis of the fibroblasts revealed significant activation of IL-17 signaling pathway, chemokine receptor binding, TNF signaling pathway and NF-κB signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G). Previous studies have consistently identified NF-κB activation as a pivotal regulatory part in the IL-17 signaling cascade, acting synergistically with TNF-α(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Treatment with S100A9-exo significantly altered the mRNA levels of IL-17 and TNF-α in educated NFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). The IKK inhibitor BAY-117082 inhibited NF-κB activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), and blocked the effect of S100A9-exo on fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). There was an enhanced lipid metabolism and nucleotide metabolism been observed in S100A9-exo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Therefore, the results showed that S100A9-exo stimulate the production of IL-17 and TNF-α, thereby enhancing NF-κB signaling and activating fibroblasts.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eActivated fibroblasts promote ACC cell epithelial\u0026ndash;mesenchymal transition and lung metastasis by secreting IL-17\u003c/h2\u003e\u003cp\u003eTo determine whether S100A9-exo-treated-NFs promote SACC-83 development, we co-cultured SACC-83 with exosomes-treated fibroblasts. After 48 h treatment with exosomes, these activated fibroblasts were set on the upper chamber, meanwhile SACC-83 cells were set on the bottom chamber (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). SACC-83 cells exhibited increased migration, invasion, viability, proliferation, and colony formation ability, along with altered expression of E-cadherin and N-cadherin upon co-cultured with S100A9-exo/fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-F). Furthermore, it greatly increased the metastatic potential of SACC-83 cells in mouse models (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H). Taken together, the results from both S100A9-exo (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E) and co-culturation with S100A9-exo/Fibroblasts treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H) consistently underscore that exosomal S100A9 significantly promotes lung metastasis of ACC, highlighting its pivotal role in metastatic progression. GO enrichment and KEGG pathway enrichment was conducted on SACC-83 co-cultured with exosomes/fibroblast through RNA sequencing. IL-17 signaling pathway was the most significant pathway, which was also the top enriched pathway between Vector-exo and S100A9-exo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, J). We next examined the expression of IL-17 in the conditioned medium of fibroblasts through ELISA and treated S100A9-exo-NF-CM with IL-17 neutralizing antibody (IL-17 Ab) which evidently reduced IL-17 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Notably, western blot analysis exhibited the expression of N-cadherin, E-cadherin, MMP9 and MMP2 in SACC-83 co-cultured with S100A9-exo/Fibroblast with IL-17 Ab (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). The rhIL-17 significantly induced EMT in a dose-dependent manner in SACC-83 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). The above results indicate that S100A9-exosomes-treated NFs regulate SACC-83 cells epithelial\u0026ndash;mesenchymal transition and lung metastasis by secreting IL‐17.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite significant therapeutic advance in ACC, the long-term prognosis of patients with ACC remains unsatisfactory due to our insufficient understanding of its underlying lung metastasis mechanisms(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Studies confirmed that the interplay between malignant tumor cells and their TME plays a critical role in promoting tumor progression(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and contributing to drug resistance(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), which are provided by cell-to-cell interactions, soluble factors (cytokines, chemokines) or exosomes(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In ACC, exosomes have shown to regulate pre-metastatic niche formation by driving angiogenesis and increasing vascular permeability in lung endothelial cells(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe hereby report that exosomes derived from different ACC cell line display distinct proteomic profiles, which aligns with established evidence suggesting that exosomal functional properties are dynamically modifiable and hence influence tumor dynamics as the tumors progress to a more aggressive phenotype(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Compared to SACC-83-exo, differentially expressed proteins in SACC-LM-exo were significantly associated with pathway in cancer and extracellular matrix (ECM) organization. To identify the most relevant protein in ACC lung metastasis, we focused on the ones that were constantly over-expressed in both the aggressive cell line and its exosomes. This implicates that these proteins not only function intracellularly but can also be secreted via exosomes to exert its effects extracellularly.\u003c/p\u003e\u003cp\u003eWe observed an upregulation of S100A9 in SACC-LM cells and their exosomes through both mRNA microarray and proteomic analyses. Although S100A9 is mainly found in heterodimers with S100A8 (S100A8/A9, also known as calprotectin), it also exists association with tumor development with its own functions(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Considering the fact that S100A8 exhibited no such increase in our data, and accumulating evidence confirms the association of S100A9 with chronic inflammation and tumor promotion(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), we put our emphasis on S100A9. In our study, high S100A9 expression was found associated with a high incidence of lung metastasis and a more advanced clinical stage.\u003c/p\u003e\u003cp\u003eExosomal S100A9 was confirmed to involved in tumor progression, such as activate the NF-κB pathway in Chronic Lymphocytic Leukemia(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), enhance Colorectal Cancer (CRC) cell stemness and is linked to both CRC occurrence and recurrence(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), implying its potential capacity as a biomarker and therapeutic target. In this study, we injected S100A9-overexpressing cell-derived exosomes into mice via tail vein. The results showed that these exosomes significantly increased ACC lung metastasis in immunodeficient mice compared to vector cell-derived exosomes, indicating that overexpression of S100A9 can promote ACC lung metastasis in an exosomecrine manner.\u003c/p\u003e\u003cp\u003eRecent single-cell transcriptomic analyses have pointed that characterizing the ACC stromal microenvironment may contribute to tumor biology and prognosis(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).A single-cell analysis of ACC indicated that the most prevalent cell populations in the ACC stroma were fibroblasts, followed by myeloid cells and T cells(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Since tumor cell can reprogram the subtype of CAFs and S100A9 has the potential to convert healthy donor-derived monocytes into myeloid-derived suppressor cells(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), we hypothesize that tumor-derived exosomes might influence fibroblasts by their specific S100A9 cargoes which contribute to efficient intercellular communication and tumor-boosting behaviors. As a tumor-supportive myofibroblast phenotype, CAFs are characterized by high fibroblast-genesis genes and ECM protein expression, including FAP, α-SMA, collagens and MMPs(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), and known to remodel the extracellular matrix, induce metabolic reprogramming, create a microenvironment promoting tumor invasion, metastasis and immune evasion(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In pancreatic ductal adenocarcinoma (PDAC), CAFs that have undergone a fibroblast-to-myofibroblast transition could form metastatic myoCAF-PDAC clusters and hijack cancer cells to distant organ metastases(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In our analysis of sc-sequencing data, we observed that EMT-state epithelial cells interact strongly with CAF through CellChat. Meanwhile, our investigation showed that SACC-LM-exo and S100A9-exo contains the ability to activate fibroblasts.\u003c/p\u003e\u003cp\u003eTo further explore the potential mechanism by which S100A9-enriched exosomes activate fibroblasts, our RNA sequencing data showed the differentially expressed genes enriched in IL-17, TNF-α and NF-κB signaling pathway in S100A9-exo-treated NFs. The process of NFs to activated CAFs can be induced by NF-κB signaling in bladder, breast, colorectal, and pancreatic cancer, indicating that NF-κB-driven CAF is common during cancer development(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIL-17 is a cytokine mostly from Th17 cells which is involved in inflammation, stromal-tumor interactions and immune evasion(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In PDAC, IL-17A facilitates the differentiation of fibroblasts into iCAF phenotype, suggesting its role in promoting terminal fibroblast activation(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). NF-κB is an indisputable key player in the IL-17 signaling cascade(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e); IL-17 treatment could activate the NF-κB and Wnt/β-catenin pathways, which in turn stimulate the release of cytokines CXCL16(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and CXCL12(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Previous studies have shown cooperative effects by TNF-α plus IL-17, in which TNF-α induces transcription of target genes while IL-17 stabilizes their mRNAs(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). In our study, we observed that S100A9 exosomes stimulate the expression of IL-17 and TNF-α, leading to enhanced NF-κB signaling and subsequent activation of myoCAFs in ACC.\u003c/p\u003e\u003cp\u003eNext, we found the co-culturation of SACC-83 and S100A9/Fibroblasts can enhance the EMT and lung metastasis of SACC-83. Remarkably, KEGG pathway analysis of SACC-83 co-cultured with exosomes/fibroblasts have also highlighted the IL-17 signaling pathway. Above evidence shows IL-17 modulates the tumor microenvironment via CAF, likely driving tumor progression in ACC. Researches showed IL-17 secreted from activated CAFs increased macrophage recruitment to activate IKKβ/NF-κB signaling, facilitating tumor cell proliferation and invasion(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Also, pancreatic tumor cells co-cultured with IL-17A-iCAF displayed enhanced tumor proliferation and metabolism(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Our results indicated that rhIL-17 in conditioned medium promoted EMT in ACC tumor cells. However, the conditioned medium from S100A9-exo-NF may also include additional factors that further aggravate tumor progression. To sum up, disrupting this vicious cycle between cancer cells and CAFs may help reshape the tumor microenvironment, potentially suppressing tumor progression.\u003c/p\u003e\u003cp\u003eIn addition, S100A9 has shown potential to serve as biomarker for tumor diagnosis and prognosis in Acute Myeloid Leukemia and CRC(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Using the S100A9 inhibitor Paquinimod, we observed a diminished activation profile in CAFs. Therefore, we propose that further investigation into the role of S100A9 and IL-17 in lung metastasis of ACC holds great promise.\u003c/p\u003e\u003cp\u003eIn summary, our results suggested show a positive feedback loop in the TME of ACC (\u003cb\u003eGraphical abstract\u003c/b\u003e). Tumor-derived exosomal S100A9 induce the transformation of NFs to CAFs, remodeling the TME; while \u0026lsquo;CAFs-like\u0026rsquo; cells promoted EMT and metastasis of ACC cells via secreting IL-17. Such kinds of positive feedback loop in TME ultimately contribute to the progression of ACC. These results indicate that targeting S100A9 may offer promising therapeutic strategies for ACC lung metastasis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.-R.Zhang and C.-W.Chen, contributed to conception and design, data acquisition and analysis, drafted and critically revised the manuscript; Y.-M.Yan, K.-W.Pan and F.-R.Ou contributed to data acquisition, analysis; K.Su and B.Cheng contributed to interpretation, drafted and critically revised the manuscript. All authors gave final approval and agreed to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe strongly acknowledge the invaluable support of patients and their families, clinicians, and technicians. We would like to acknowledge Xianyue Ren for the time and support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Comflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (No. 82103555), China Postdoctoral Science Foundation (No.2021M703688, No.2022T150753).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in compliance with the principles of the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations. Human tissues and primary human cells were provided by the participants with written-informed consent. Ethics approval for human subjects numbered KQEC-2024-140-01 was provided from the Ethics Committee of Hospital of Stomatology, Sun Yat-sen University. All animal procedures were approved by Permit number #JENNIO-IACUC-2023-A068.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDrier Y, Cotton MJ, Williamson KE, Gillespie SM, Ryan RJ, Kluk MJ, et al. An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma. Nat Genet. 2016;48(3):265-72.\u003c/li\u003e\n\u003cli\u003ede Sousa LG, Neto FL, Lin J, Ferrarotto R. Treatment of Recurrent or Metastatic Adenoid Cystic Carcinoma. 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Blood Cancer J. 2023;13(1):188.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7171055/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7171055/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSalivary adenoid cystic carcinoma (SACC) exhibits a high incidence of lung metastasis, which primarily contributes to patient mortality. The tumor microenvironment (TME) plays a critical role in facilitating tumor progression, yet its specific contribution to ACC metastasis remains unclear. In this study, we used single-cell transcriptomic analysis of primary ACC tumors and adjacent normal salivary gland to reveal strong intercellular communication between epithelial cells and cancer-associated fibroblasts (CAFs). We demonstrate that S100A9, a calcium-binding protein of the S100 family, is upregulated in a subset of ACC cells and their derived exosomes. Exosomes enriched with S100A9 reprogram normal fibroblasts into activated CAFs with elevated fibroblast activation protein (FAP) and α-smooth muscle actin (α-SMA) expression, enhanced migration, and increased contractility. RNA sequencing of fibroblasts treated with S100A9-enriched exosomes revealed activation of IL-17, TNF, and NF-κB signaling pathways, which are known to drive inflammation, extracellular matrix remodeling, and tumor-stroma interactions. Furthermore, activated CAFs promote epithelial-mesenchymal transition in ACC cells and facilitate lung metastasis through IL-17 signaling. These findings demonstrate tumor-derived exosomal S100A9 as a key mediator of intercellular communication between ACC cells and fibroblasts, identifying S100A9 and S100A9-enriched exosomes as potential therapeutic targets for modulating ACC lung metastasis.\u003c/p\u003e","manuscriptTitle":"Exosomal S100A9 Promotes Lung Metastasis of Adenoid Cystic Carcinoma via Activating Cancer-Associated Fibroblasts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-05 11:40:45","doi":"10.21203/rs.3.rs-7171055/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-10-20T09:20:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-03T11:29:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-02T10:40:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death Discovery","date":"2025-10-02T10:40:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"045f7662-3009-4de6-b271-c38c880a6cbe","owner":[],"postedDate":"October 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55721641,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":55721642,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2026-03-21T07:10:19+00:00","versionOfRecord":{"articleIdentity":"rs-7171055","link":"https://doi.org/10.1038/s41420-026-02991-6","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2026-02-27 05:00:00","publishedOnDateReadable":"February 27th, 2026"},"versionCreatedAt":"2025-10-05 11:40:45","video":"","vorDoi":"10.1038/s41420-026-02991-6","vorDoiUrl":"https://doi.org/10.1038/s41420-026-02991-6","workflowStages":[]},"version":"v1","identity":"rs-7171055","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7171055","identity":"rs-7171055","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}