S100 genes drive metastasis in salivary gland carcinoma

S100 genes drive metastasis in salivary gland carcinoma | 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 Research Article S100 genes drive metastasis in salivary gland carcinoma Hee-Weon Lee, Minho Kang, Tae Woo Kim, Uk Yeol Moon, Han-Sin Jeong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6868185/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background High-grade salivary gland carcinoma (SGC) is an aggressive malignancy that demands a multidisciplinary treatment approach. However, its metastatic mechanisms remain poorly understood due to the rarity and heterogeneity of SGC and the lack of suitable tumor models. This study aimed to explore key oncogenic drivers of distant metastasis by comparing two SGC subtypes: salivary duct carcinoma (SDC) and adenoid cystic carcinoma (ACC). Methods We conducted a comparative analysis of SDC and ACC to identify potential oncogenes related to metastasis. A cDNA-mediated Annealing, Selection, Extension, and Ligation (DASL) assay was used to evaluate S100 gene expression in patient samples. In vivo studies were performed using orthotopic xenograft mouse models (WR21, A253, and SCA9), and functional studies employed WR21 and NIH3T3 cell lines with S100 gain- or loss-of-function to assess proliferation, migration, and invasion. Statistical analyses were applied to evaluate gene expression patterns and biological effects. Results SDC demonstrated more aggressive behavior and higher metastatic potential than ACC, accompanied by upregulation of S100 genes associated with epithelial-mesenchymal transition (EMT). SGC tumors exhibited increased expression of S100A4, S100A8, and S100A9 in both primary and metastatic lesions. Functional assays confirmed that S100 overexpression promotes EMT-related transcriptomic changes and enhances tumor cell proliferation and migration. Conditioned media from S100-expressing fibroblasts also stimulated these phenotypes, suggesting paracrine interactions in the tumor microenvironment. Conclusions S100 family proteins play a critical role in high-grade SGC progression by promoting EMT and paracrine-mediated metastasis. Hence, S100 proteins have potential as prognostic biomarkers and therapeutic targets of SGC. salivary gland carcinoma salivary duct carcinoma adenoid cystic carcinoma S100 epithelial-mesenchymal transition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Salivary gland cancer (SGC) comprises 0.3% of new cancer incidence and 0.2% of all cancer mortalities worldwide [ 1 ]. While SGCs account for only about 5% of all head and neck malignancies, high-grade SGC is an extremely aggressive cancer characterized by a strong propensity for local invasion and distant metastasis [ 2 , 3 ]. Although some patients are successfully treated with surgery and adjuvant radiotherapy or chemoradiotherapy, 20–50% experience recurrence and distant metastases [ 4 ]. With 21 distinct histological subtypes, SGC is typically classified into three groups based on their immune microenvironment and neoantigen landscape [ 5 ]. The most aggressive SGC subtypes include salivary duct carcinoma (SDC), high-grade adenocarcinoma, and poorly differentiated carcinoma [ 6 ]. Adenoid cystic carcinoma (ACC) is the most common SGC subtype, accounting for approximately 10% of all cases [ 7 ]. Notably, T-cell dysfunction is more severe in SDC than in ACC, with greater T-cell infiltration and upregulation of T-cell checkpoints, including PD-L1, CTLA-4, LAG3, TIM3, TIGIT, and VISTA [ 5 ]. The most common metastatic site of SGC is the lung, although this manifests differently between SDC and ACC; distant metastases have a 53% incidence in SDC, whereas ACC exhibits late, indolent courses of systemic metastasis [ 8 ]. Patients with high-grade pathology were 7.5 times more likely to develop distant metastases than those with low-grade pathology [ 8 ]. The stromal microenvironment interacts closely with tumor cells, initiating cancer growth, invasion, and metastasis [ 9 , 10 ]. An important mediator of this interaction is the S100 Ca + -binding protein [ 11 ], part of the highly conserved vertebrate S100 protein family, with well-characterized roles in tumor growth, metastasis, angiogenesis, and immune evasion [ 12 ]. This protein family has 25 known subtypes [ 13 ], most forming symmetrical dimers, although the S100A8/S100A9 heterodimer is an exception. S100 proteins contain two Ca 2+ -binding domains that increase in affinity for Ca 2+ upon binding to the target protein. Downstream effects include activating pathways involved in cytokine production, the cell cycle, apoptosis, and migration [ 12 ]. S100 protein expression level is cell type-specific, but exerts widespread intra- and extracellular effects, being secreted in the serum, plasma, urine, saliva, and cerebrospinal fluid [ 14 , 15 ]. Research on S100 member S100A4 shows that overexpression promotes metastases in various cancer models, whereas knockouts limit metastasis to the lungs [ 16 – 18 ]. However, the expression and role of S100 proteins have never been thoroughly explored in SGC or SGC metastasis. Owing to the rarity of SGC and its various histological subtypes, the molecular characteristics of distant metastases remain largely unknown. Therefore, this study aimed to examine the characteristics of two SGC subtypes (SDC and ACC), analyzing the expression patterns of genes putatively involved in tumor regulation and identifying factors associated with distant metastasis. We hypothesized that S100 proteins regulate SGC migration and invasion, contributing to metastasis. 2. Materials and Methods Patient information and IRB approval The study included patients with SDC or ACC (N = 26 and 9) from the authors’ institutional salivary gland cancer registry. Eligible participants had complete clinical data and preserved tumor samples in the tissue bank. Written patient consent was obtained for use of de-identified clinical information, samples, and genomic analysis results. The protocol was approved by the institutional review board of Samsung Medical Center (approval no. 2015-12-13). Tumor samples (formalin-fixed paraffin-embedded, FFPE) were collected, and 11 samples were excluded from subsequent analyses after quality checking extracted RNA. Finally, 26 SDC and 9 ACC tumor samples were analyzed with cDNA-mediated annealing, selection, extension, and ligation (DASL) assays (Supplementary Table 1). DASL assay Total RNA was extracted from FFPE samples for analysis using an Illumina Whole-Genome DASL® HT Assay for Expression Profiling (Illumina, San Diego, CA) with MCS4 cDNA synthesis reagents and Illumina DASL BeadChip [ 19 ]. The Illumina Whole-Genome DASL® HT assay can generate reproducible expression profiles [ 20 ]. Fluorescence intensity data collected from the Bead Array platform were analyzed using proprietary Illumina software. Gene set variation analysis (GSVA) The R package “GSVA” was utilized to determine relative enrichment scores per patient of 50 cancer hallmark gene sets from MSigDB (h.all.v7.5.1.symbols), including genes related to epithelial-mesenchymal transition (EMT) [ 21 ]. The score was an estimation of cancer hallmark pathway activity in SDC and ACC samples. Relationships were analyzed using Pearson’s correlation. All statistical analyses were conducted in R (version 3.2.2). Significance was set at p < 0.05 unless otherwise noted. Cell lines and in-vivo models Three SGC cell lines and a fibroblast cell line were selected for experiments: SCA9 (mouse, CRL-1734, ATCC, Manassas, VA, USA, RRID: CVCL_JY08), A253 (human, HTB-41, ATCC, Manassas, VA, USA, RRID: CVCL_1060), WR21 (mouse, CRL-2189, ATCC, Manassas, VA, USA, RRID: CVCL_3623), and NIH3T3 (mouse, 30-2020, ATCC, Manassas, VA, USA, RRID: CVCL_0594). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM: Gibco ThermoFisher Scientific Korea, Seoul, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco ThermoFisher Scientific Korea) and 1% penicillin/streptomycin (Gibco ThermoFisher Scientific Korea) and maintained at 37℃ with 5% CO₂. Human cell line has been authenticated using STR (or SNP) profiling within the last three years.All experiments were performed with mycroplasma free cells. Female BALB/c nude mice (4–6 weeks old) were obtained from Orient Bio (Seongnam, Korea). Animals were housed in temperature- and light-controlled (12 h light/dark cycle) conditions, with food and water available ad libitum. The three SGC lines were counted (5.0 × 10 5 cells), re-suspended in 50 µL media, and injected into the submandibular gland of mice. Tumor growth was monitored twice per week. Upon attaining a gross tumor volume of 1500 mm 3 , tumor and lung tissue (with signs of metastasis) were collected for analysis. Mice were anesthetized by a single intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg), and then placed in a chamber where they were euthanized by exposure to CO₂ gas at a flow rate of 21 L/min for approximately 5 minutes. The whole tumor was excised, washed in sterile PBS, and processed with 4% paraformaldehyde (Biosesang, Yongin, Korea) for fixation and sectioning. Protocol approval of animal experiments The study followed international and institutional guidelines for animal treatment and complied with relevant legislation. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Samsung Medical Center (No. 20151122001). Western blotting Cells were rinsed with phosphate-buffered saline (PBS), suspended in homogenization lysis buffer, and centrifuged for 5 min at 12,000 × g and 4°C. Supernatant was collected to determine the protein concentration using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). The standard was BSA. Whole cell lysates were separated using 6–15% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline containing 0.2% Tween 20 (TBST) at 20–22°C and then probed with the appropriate primary (1:500) and secondary (1:5000) antibodies (see Supplementary Table 4). Blots were developed using an enhanced chemiluminescence kit to obtain density ratios of relative band intensity. The control was β-actin. Migration and invasion assay To measure cell migration, WR21-RFP cells (2 × 10 5 in 100–200 µL serum-free medium) were seeded alone into the upper chamber of a 24-well transwell plate (ECM509, Merck Millipore), or else seeded with NIH3T3-CM into the bottom chamber. The plate was then incubated for 5–7 days at 37°C, and 5% CO 2 . Total cell count on the insert and migrated cells on the bottom were counted under a fluorescence microscope. To measure cell invasion, WR21 shRNA cells were seeded with NIH3T3-CM into the upper chamber of an ECMatrix™ coated 24-well transwell plate (ECM550 8 µm, Merck Millipore) and incubated for 5 days under the same conditions as the migration assay. The total number of invaded cells was determined. Quantitative real time-PCR To synthesize cDNA, total RNA was isolated from cultured cells using an RNA extraction kit (Kusatsu, St. Shiga, Japan). Next, 10 µL of SYBR green premix (BioRad), 8 µL of sterile water, and 1 µL each of forward and reverse primer were mixed for a total reaction volume to 20 µL. Fluorescence was measured at each cycle. For PCR primer sequences used to analyze S100 expression, see Supplementary Table 2. Construction of an RNA interference cell line To generate stable S100 shRNA-expressing cells, shRNA oligos of S100s (Supplementary Table 3) were cloned into pGreenPuro TM shRNA lentivectors (SBI System Biosciences, Mountain View, CA, USA). Cells in six-well culture plates were transfected with S100A4, S100A8, and S100A9 shRNA using shRNA transfection reagent (Invitrogen Biotechnology, CA, USA), following manufacturer protocol. After selection with puromycin, resistant cells were expanded for at least one month and tested for efficiency using western blotting. Construction of an over-expression cell line S100A4, S100A8, and S100A9 expression vectors were constructed via cDNA ligation into pIRES-mCherry (addgene, MA, USA). The S100 cDNA region was PCR-amplified from S100A4, S100A8, and S100A9 clones (Sino Biological Inc, Tx, USA). Cells in six-well culture plates were transfected with S100 expression vectors. Transduced cells were selected with neomycin and expanded for at least one month before being tested for efficiency using western blotting. Immunofluorescence assay Immunohistochemistry was performed on prepared paraffin sections (7 um), as described previously [ 22 ]. Briefly, tissue sections were deparaffinized and rehydrated through an ethanol series and distilled water. After 30 min of antigen retrieval with boiling citrate buffer, sections were stained with antibodies (see supplementary table 4). Statistical analyses Results from fluorescence intensity analysis, cell migration assays, and cell invasion assays were analyzed using Student’s t -tests. Significance was set at p < 0.05. 3. Results 3.1. Differential gene expression between SDC and ACC Patients with SDC and ACC expressed hallmark genes belonging to different transcriptomic subtypes (Fig. 1 A). We observed more upregulation of cancer metastasis- and development-associated genes in SDC than in ACC; notable pathways included EMT, interferon-alpha (IFN-α) and interferon-gamma (IFN-γ) response, mTORC1 signaling, IL2 signaling, and KRAS signaling (Fig. 1 A). Hence, SDC was more aggressive than ACC and had elevated cancer metastasis signals. Volcano and violin plots visualizing results from principal component analysis of hallmark gene scores revealed markedly higher EMT transcriptomic expression in SDC than in ACC (Fig. 1 B–D). Interestingly, SDC also exhibited significant upregulation of IFN-α and IFN-γ responses. Violin plots show different hallmark scores for the two SGC subtypes (Fig. 1 D), while volcano plots depict differentially expressed S100 genes between SDC and ACC, as well as the correlation between S100 genes and EMT (Fig. 1 E and 1 F). In addition, most S100 genes were highly expressed in SDC, with 12 SDC-specific genes being significantly upregulated (Fig. 1 G). These results demonstrate differential expression of cancer-related genes between SDC and ACC. In particular, the upregulation of specific metastasis-associated genes in SDC highlights its aggressive nature. 3.2. S100 expression in human SDC tissues To investigate S100 protein expression, we performed immunofluorescence analysis using primary and metastatic tumor tissue samples from human SDC. S100A4, S100A8/S100A9, and S100P were expressed in SDC primary tumors (Fig. 2 A, 2 B). Furthermore, S100 expression was also elevated in metastatic SDC tumors. S100A4 , S100A8 , and S100A9 expression also increased significantly, with high S100A7 and S100P upregulation that varied considerably across tumors (Fig. 2 C). The results demonstrate that SDC is associated with increased S100 expression in both primary and metastatic tumors. 3.3 S100 expression in tissues from orthotopic xenograft mice generated by SGC cancer cell lines At 2 ~ 4 weeks, mice implanted with salivary gland cell lines (SCA9, A253 and WR21 cells) had elevated S100 expression in most primary tumors, according to immunofluorescence results. S100A4 , S100A8 , and S100A9 expression was upregulated in all tumors, whereas S100B expression was not (Fig. 3 C). Consistent with gene expression, western blot analysis of WR21 primary tumors revealed a significant increase in S100A4 and S100A9. In contrast, S100A8 showed increased gene expression but decreased protein expression (Fig. 3 C, 3 D). S100A4, S100A9, and S100B expression increased significantly in metastatic lung tumors (Fig. 3 E, 3 F), whereas S100A8 expression did not. S100A4 , S100A8 , and S100A9 expression was upregulated in metastatic WR21 tumors, indicating that S100A8 and S100B gene expression did not match their protein expression (Fig. 3 F, 3 G). Notably, S100A4 and S100A9 expression was consistently upregulated at both the gene and protein levels in WR21 primary and metastatic tumors. Overall, S100 expression is associated with SGC in mice. 3.4 NIH-3T3 cells regulate WR21 cell proliferation and migration After co-culturing WR21-RFP and NIH-3T3 cells in a transwell migration chamber, we verified the influence of NIH-3T3 cells on WR21 cell migration and proliferation (Fig. 4 A). Co-culturing with NIH-3T3 cells increased WR21-RFP cell proliferation but not cell migration. Interestingly, WR21 cell migration was significantly increased when NIH-3T3 cells were cultured at the bottom, suggesting that NIH-3T3 cells had secreted molecules, such as exosomes or inflammatory cytokines, which regulated WR21-RFP cell migration and induced cell recruiting. (Fig. 4 B). When we added conditioned medium (CM) from S100-overexpressing NIH-3T3 cells, WR21 cell migration to the transwell bottom also increased (Fig. 4 C). Hence, the CM contained S100-upregulating factors that further enhanced WR21 migration, implying that intracellular S100 proteins of NIH-3T3 cells are critical regulators of SGC migration and proliferation. 3.5 S100 knockdown in WR21 cells and CM from S100-knockdown NIH-3T3 cells reduce WR21 cell invasion Transwell invasion assays of WR21 cells expressing S100A4, S100A8, and S100A9 shRNA with normal NIH-3T3 CM demonstrated that WR21 cell invasion was suppressed (Fig. 5 A). Additionally, when WR21-shRNA empty cells were cultured with CM from S100A8- and S100A9-knockdown NIH-3T3 cells, WR21 cell invasion also decreased (Fig. 5 B). The results confirm that intracellular and extracellular S100A4, S100A8, and S100A9 are strongly associated with WR21 cell invasion. These findings suggest that the S100 family plays a crucial role in cancer cell migration and invasion signaling pathways, and that inhibiting S100-related signaling can block SGC invasion. 4. Discussion This study confirmed that S100 proteins are involved in SGC cell migration and metastasis through both autocrine and paracrine signal pathways. The aggressive SDC subtype exhibits the highest mortality among SGC [ 6 ]. In addition to local invasive growth, the clinical course of SDC involves early lymphatic and systemic metastasis. In contrast, ACC has an indolent clinical progression and less lymphatic metastasis, but high incidence of local infiltration, perineural spread, and systemic spread. However, even after lung metastasis, patients with ACC survive longer than 4–5 years [ 23 ]. Because SDC and ACC have distinct dissemination patterns, we conducted a comparative gene expression analysis using samples from both cancers. The DASL assay revealed that SDC tumors overexpress cytokines (IFN-α and γ) and EMT-related genes, with the S100 family being particularly associated with EMT level. Corroborating our results, previous research has also found upregulation of IFN-α and IFN-γ response gene sets in patients with SGC subtypes of poor prognosis [ 24 ]. Additionally, our findings support the hypothesis that S100 proteins regulate SGC migration and invasion, contributing to metastasis. Mouse experiments indicated that S100A4, S100A8, and S100A9 were upregulated in both primary and metastatic tumors, largely consistent with observations in human SDC tissues. However, while S100A4 RNA and protein expression was upregulated in human tumors, this outcome was not fully replicated in mouse primary SGC tumors. In mice, upregulated S100A4 expression was localized, and the overall expression was low. Serum and tumor S100B expression is diagnostic for glioma, breast cancer, and melanoma [ 25 – 27 ]. In particular, upregulated S100B serves as marker for a positive breast cancer prognosis, as it regulates the migration of ER-negative breast cancer cells [ 25 ]. Additionally, recombinant S100B promotes E-cadherin expression, inhibiting cell migration and facilitating an epithelial phenotype [ 25 , 26 ]. In contrast, serum and tumor S100B levels are markers of poor prognosis in malignant melanoma, as S100B upregulation reflects tumor load, stage, metastasis, and lower survival rates [ 28 ]. However, downregulation indicates a good response to therapy. Hence, S100B appears to exert a dual effect and exhibits diverse activities depending on the cancer or context [ 29 , 30 ]. Similarly, our study found that S100B expression differed significantly between human SGC and mouse primary tumors. In human SGC, S100B expression did not significantly increase in primary or metastatic tumors, whereas mouse tumors (A253 and WR21) exhibited high S100B protein expression in both primary and metastatic tumors. Therefore, while S100B appears to influence SGC cell proliferation and metastasis, the effect varies depending on tumor context or host. Another important cancer metastasis marker in the S100 family is S100P [ 31 ]. High S100P expression was observed in the intracapsular and extracapsular components, as well as in malignant subtypes of carcinoma ex pleomorphic adenoma (CXPA) of the salivary gland [ 32 ]. In this study, S100P expression was significantly upregulated in human metastatic SGC tumors. However, we could not validate this finding in mice, because S100P is absent in most rodents [ 33 ]. S100A8 and S100A9 expression is tissue-specific, primarily occurring in bone marrow-derived cells such as monocytes, neutrophils, myeloid-derived suppressor cells, and cancer cells [ 34 , 35 ]. Typically, this protein family is expressed both intracellularly and extracellularly. The secreted S100 protein has a cytokine-like function and contributes to pre-metastatic niche (PMN) formation via binding to extracellular receptors, including receptors for advanced glycation end products (RAGE), Toll-like receptor 4, ErbB4, dopamine D2, and FGFR1 [ 36 – 38 ]. Regulation of these receptors enhances cell invasion and migration [ 39 , 40 ]. For example, S100A8/A9 regulates TLR9 and RAGE activity in multiple inflammatory pathways to induce inflammatory cytokines, a key part of PMN [ 41 ]. In rodents, both proteins act as chemo-attractants that activate phagocytes and facilitate the homing of tumor cells to the PMN [ 42 , 43 ]. Human S100A9 enhances THP-1 cell migration through RAGE-dependent signaling pathways such as MEK/ERK and PI3K [ 44 ]. Also known as metastasin, calvasculin, mts-1, and fibroblast-specific protein 1, S100A4 expression is similarly associated with malignancies [ 45 , 46 ]. Of the cell lines used in our in-vivo orthotopic study, WR21 cells exhibited the most aggressive behavior, including rapid tumor formation, metastasis, and high S100 expression. However, the S100 family did not always show consistent RNA and protein expression. S100A4, S100A8, and S100A9 RNA increased dramatically in WR21 primary tumors, but S100A8 protein levels were lower in the tumor than in the normal salivary gland (control). In contrast, S100A9 RNA and protein expression was elevated in both primary and metastatic SGC tumors samples from human SGC. S100A9 expression was also significantly upregulated in metastatic tumors derived from A253 and WR21 cells. These data strongly suggest that S100A9 plays a crucial role in cancer cell migration and metastasis. Because S100A8 protein expression was far lower than its RNA levels, this protein may not form a heterodimer and may be uniquely exerting extracellular effects on receptors as a single protein. Cancer-associated fibroblasts are another group of effector cells in the stromal compartment that express S100 proteins [ 47 , 48 ]. Tumor-secreted exosomes directly or indirectly mediate metastasis via upregulating S100s in fibroblasts within pre-metastatic regions, initiating PMN formation [ 49 ]. In this study, we observed that S100A4 RNA and protein levels significantly increased in both human and mouse SGC tumors. Furthermore, we highlighted both autocrine and paracrine functions of S100s in fibroblasts. Conditioned media culturing S100-overexpressing NIH-3T3 cells promoted WR21 cell migration, and in particular, S100A9-overexpressing NIH-3T3 cells significantly enhanced WR21 cell migration. Silencing experiments demonstrated the crucial role of S100 gene expression in invasion, as inhibiting S100A4, S100A8, and S100A9 in WR21 cells significantly lowered invasion activity. We further confirmed the role of S100 genes in invasion when we observed that CM from shRNA-S100 NIH3T3 cells reduced WR21 cell invasion. The crucial role of S100-expressing fibroblasts in cancer cell metastasis was consistent with previous findings [ 50 ]. To summarize, S100-overexpressing NIH3T3 cells promote the secretion of S100-mediated inflammatory cytokines or S100 proteins, contributing to WR21 migration. As WR21 cells migrate to the metastatic site, their expression of S100 proteins may further enhance their invasive activity. In conclusion, our study demonstrated the importance of S100 proteins in promoting the progression and metastasis of SDC, an extremely aggressive SGC subtype. The most notable S100 proteins were S100A4, S100A8, and S100A9, all upregulated in both primary and metastatic SGC. Moreover, the significant correlation of S100 proteins with EMT highlighted the role of this protein family in SGC cell migration and invasion. Therefore, these proteins may be markers of poor prognosis in SGC and potential therapeutic targets to limit metastasis. Abbreviations ACC adenoid cystic carcinoma EMT epithelial-mesenchymal transition SDC salivary duct carcinoma SGC salivary gland carcinoma Declarations Ethics approval and consent to participate This study was performed according to the guidelines of the Declaration of Helsinki. Human tissue was obtained based on the study approval protocol by the institutional review board of Samsung Medical Center (approval no. 2015-12-13). Consent for publication Not applicable Availability of data and materials The raw microarray data generated in this study is available in GEO under accession number GSE293801. The data set analysed and generated in this study are available from the corresponding author upon request. Conpeting interests The authors declare that they have no conpeting interest. Funding This work was supported by National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF-2016R1A6A3A11931072). Conflict of interest The authors declare no conflict of interest. Author contributions Hee-Weon Lee: data curation, writing original draft, writing review editing. Minho Kang: data curation, methodology, data analysis, writing original draft. Tae Woo Kim: data curation, investigation. Uk Yeol Moon: supervision, conceptualization, data curation, writing original draft, writing review & editing. Han-Sin Jeong: supervision, conceptualization, writing original draft. Acknowledgements Not applicable References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229–63. Shen Y, Shan J. Chemoradiotherapy versus radiotherapy in high risk salivary gland cancer. 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Chromosomal mapping, differential origin and evolution of the S100 gene family. Genet Sel Evol. 2008;40(4):449–64. Eisenblaetter M, Flores-Borja F, Lee JJ, Wefers C, Smith H, Hueting R, Cooper MS, Blower PJ, Patel D, Rodriguez-Justo M, et al. Visualization of Tumor-Immune Interaction - Target-Specific Imaging of S100A8/A9 Reveals Pre-Metastatic Niche Establishment. Theranostics. 2017;7(9):2392–401. Chen Y, Ouyang Y, Li Z, Wang X, Ma J. S100A8 and S100A9 in Cancer. Biochim Biophys Acta Rev Cancer. 2023;1878(3):188891. Schmidt AM, Hofmann M, Taguchi A, Yan SD, Stern DM. RAGE: a multiligand receptor contributing to the cellular response in diabetic vasculopathy and inflammation. Semin Thromb Hemost. 2000;26(5):485–93. Huttunen HJ, Kuja-Panula J, Sorci G, Agneletti AL, Donato R, Rauvala H. Coregulation of neurite outgrowth and cell survival by amphoterin and S100 proteins through receptor for advanced glycation end products (RAGE) activation. J Biol Chem. 2000;275(51):40096–105. Srikrishna G, Nayak J, Weigle B, Temme A, Foell D, Hazelwood L, Olsson A, Volkmann N, Hanein D, Freeze HH. Carboxylated N-glycans on RAGE promote S100A12 binding and signaling. J Cell Biochem. 2010;110(3):645–59. Nurmenniemi S, Kuvaja P, Lehtonen S, Tiuraniemi S, Alahuhta I, Mattila RK, Risteli J, Salo T, Selander KS, Nyberg P, et al. Toll-like receptor 9 ligands enhance mesenchymal stem cell invasion and expression of matrix metalloprotease-13. Exp Cell Res. 2010;316(16):2676–82. Yin C, Li H, Zhang B, Liu Y, Lu G, Lu S, Sun L, Qi Y, Li X, Chen W. RAGE-binding S100A8/A9 promotes the migration and invasion of human breast cancer cells through actin polymerization and epithelial-mesenchymal transition. Breast Cancer Res Treat. 2013;142(2):297–309. Wang S, Song R, Wang Z, Jing Z, Wang S, Ma J. S100A8/A9 in Inflammation. Front Immunol. 2018;9:1298. Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis. Nat Cell Biol. 2006;8(12):1369–75. Rafii S, Lyden D. S100 chemokines mediate bookmarking of premetastatic niches. Nat Cell Biol. 2006;8(12):1321–3. Chen B, Miller AL, Rebelatto M, Brewah Y, Rowe DC, Clarke L, Czapiga M, Rosenthal K, Imamichi T, Chen Y, et al. S100A9 induced inflammatory responses are mediated by distinct damage associated molecular patterns (DAMP) receptors in vitro and in vivo. PLoS ONE. 2015;10(2):e0115828. Boye K, Maelandsmo GM. S100A4 and metastasis: a small actor playing many roles. Am J Pathol. 2010;176(2):528–35. Ebralidze A, Tulchinsky E, Grigorian M, Afanasyeva A, Senin V, Revazova E, Lukanidin E. Isolation and characterization of a gene specifically expressed in different metastatic cells and whose deduced gene product has a high degree of homology to a Ca2+-binding protein family. Genes Dev. 1989;3(7):1086–93. Zeisberg EM, Potenta S, Xie L, Zeisberg M, Kalluri R. Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts. Cancer Res. 2007;67(21):10123–8. Hu D, Li Z, Zheng B, Lin X, Pan Y, Gong P, Zhuo W, Hu Y, Chen C, Chen L, et al. Cancer-associated fibroblasts in breast cancer: Challenges and opportunities. Cancer Commun (Lond). 2022;42(5):401–34. Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, Molina H, Kohsaka S, Di Giannatale A, Ceder S, et al. Tumour exosome integrins determine organotropic metastasis. Nature. 2015;527(7578):329–35. Schmidt-Hansen B, Klingelhofer J, Grum-Schwensen B, Christensen A, Andresen S, Kruse C, Hansen T, Ambartsumian N, Lukanidin E, Grigorian M. Functional significance of metastasis-inducing S100A4(Mts1) in tumor-stroma interplay. J Biol Chem. 2004;279(23):24498–504. Additional Declarations No competing interests reported. Supplementary Files SGCS100manuscriptsupplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 22 Jul, 2025 Reviewers agreed at journal 14 Jul, 2025 Reviewers invited by journal 14 Jul, 2025 Editor assigned by journal 09 Jul, 2025 Editor invited by journal 18 Jun, 2025 Submission checks completed at journal 18 Jun, 2025 First submitted to journal 18 Jun, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6868185","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486146486,"identity":"200d7f59-a75e-4075-906f-6e8ebcf1a73c","order_by":0,"name":"Hee-Weon Lee","email":"","orcid":"","institution":"Daegu Gyeongbuk Medical Innovation Foundation","correspondingAuthor":false,"prefix":"","firstName":"Hee-Weon","middleName":"","lastName":"Lee","suffix":""},{"id":486146487,"identity":"624765c2-8e1f-490c-9ac2-c19dc72234af","order_by":1,"name":"Minho Kang","email":"","orcid":"","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Minho","middleName":"","lastName":"Kang","suffix":""},{"id":486146488,"identity":"58eb606b-3e7a-4e51-b744-612768aa33ab","order_by":2,"name":"Tae Woo Kim","email":"","orcid":"","institution":"Daegu Gyeongbuk Medical Innovation Foundation","correspondingAuthor":false,"prefix":"","firstName":"Tae","middleName":"Woo","lastName":"Kim","suffix":""},{"id":486146489,"identity":"2ca78bf4-cf9e-4091-b8e5-5d5a90729eac","order_by":3,"name":"Uk Yeol Moon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACA3YILScBJCQSGxgSQDzGBnxamCG0MelaEmeAtDASo8Wcmcfw0c22w+kzpx0+eOPhjsN5DNKHDzDO3INbi2Uzj7Fxbtvh3NnSackWiWcOFzPwpSUwbniGx2GHecykQVrmSeeYSSS2HU5s4OExYHxwAK8W899ALely0vnfoFr4PxDSYsYM1JIgLZ3DBrOFgXEDHi2WzWzF0jnn0g1nzk4ztkhsSy9m42EzODgDjxZz9uaNn3PKrOUlbic/vPmzzTqPn4f54cMePFqgoBnBZANiwhoYGOqIUDMKRsEoGAUjFgAAFodSE6yLAAkAAAAASUVORK5CYII=","orcid":"","institution":"Daegu Gyeongbuk Medical Innovation Foundation","correspondingAuthor":true,"prefix":"","firstName":"Uk","middleName":"Yeol","lastName":"Moon","suffix":""},{"id":486146490,"identity":"80fe4be9-1d4b-48df-a27c-7d33fb06aa65","order_by":4,"name":"Han-Sin Jeong","email":"","orcid":"","institution":"Samsung Medical Center, Sungkyunkwan University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Han-Sin","middleName":"","lastName":"Jeong","suffix":""}],"badges":[],"createdAt":"2025-06-11 05:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6868185/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6868185/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87270474,"identity":"525e2180-cfb2-4ad2-8414-e40f497b8812","added_by":"auto","created_at":"2025-07-22 08:19:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":775674,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of differentially expressed genes between SDC and ACC using DASL assay. (A) Heatmap shows SDC and ACC hallmarks belonging to different transcriptomic subtypes. (B) Principal component analysis of hallmark scores based on transcriptomic data in two SGC subtypes. (C) Volcano plot of different hallmarks between SDC and ACC. Red and blue dots represent high and low hallmarks, respectively (p \u0026lt; 0.01). Gray dots represent genes that were not differentially expressed. Black dots indicate the top three hallmarks. (D) Violin plot of distinct hallmark scores in two SGC subtypes (Student’s \u003cem\u003et\u003c/em\u003e-test). (E) Volcano plot of differently expressed genes between SDC and ACC. Red and blue dots represent up- and downregulated genes, respectively (p \u0026lt; 0.01, |log2FC| \u0026gt; 1). Gray dots represent genes that were not differentially expressed. Black dots are upregulated S100 family genes. (F) Correlation coefficients (R) and p-value (P) between EMT scores and the expression of six S100 mRNAs in SGC. (G) Differential expression of S100 genes between ADC and ACC. ****p \u0026lt; 0.0001, ***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figures1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6868185/v1/02d84cea84c825ed29f5e6c6.jpg"},{"id":87268840,"identity":"69439073-0c8a-45d9-adf0-b5ad87bd6550","added_by":"auto","created_at":"2025-07-22 08:11:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":571092,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of S100 family expression in human SDC. (A) Immunofluorescence staining of S100A4, S100A8, S100A9, S100B, and S100P in primary and metastatic human SDC tumors. (B) Fluorescence intensity analysis of S100 family. (C) Differential expression of S100 genes in primary human SDC tumors. **p \u0026lt; 0.01, *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figures2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6868185/v1/89200843b961594901d32817.jpg"},{"id":87268834,"identity":"f8fb3754-99c4-4beb-a0d2-79535a102892","added_by":"auto","created_at":"2025-07-22 08:11:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":973612,"visible":true,"origin":"","legend":"\u003cp\u003eS100 expression in orthotopic xenograft mice generated from SGC cancer cell lines (SCA9, A253 and WR21). SGC cells were implanted into mouse salivary glands to induce tumor formation. Immunofluorescence staining of primary (A) and metastatic (E) tumor samples revealed the fluorescent intensity of S100 family proteins (B and F). S100 gene expression levels were analyzed in primary (C) and metastatic (G) tumors. S100 protein levels were analyzed in primary tumors with western blotting (D). **p \u0026lt; 0.01, *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figures3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6868185/v1/2821d334af412044cf588314.jpg"},{"id":87270476,"identity":"ff88e304-06b7-4d15-a57f-fe0c4f953254","added_by":"auto","created_at":"2025-07-22 08:19:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":490159,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NIH-3T3 cells on WR21 cell proliferation and migration. (A) WR21 cells were co-cultured with NIH-3T3 cells in a transwell migration assay chamber. Cells remaining on the insert and cells that migrated to the chamber bottom were counted. (B) Effect of distantly located NIH3T3 cells on WR21 cells. WR21-RFP cells were seeded in the transwell insert and NIH3T3 cells were seeded on the chamber bottom. WR21 cells on the insert and the bottom were counted. (C) Effect of S100 genes in NIH-3T3 cells on WR21 migration. Conditioned media (CM) from NIH-3T3 cells expressing S100A4, S100A8, and S100A9 were added to WR21-RFP cells. After 5 and 7 days, WR21 cells on the chamber bottom was counted\u003cstrong\u003e. \u003c/strong\u003e*p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figures4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6868185/v1/3d4ee1190851d01da3ec65c4.jpg"},{"id":87270477,"identity":"6e0bbc14-1e44-4d1c-9475-f631018a7816","added_by":"auto","created_at":"2025-07-22 08:19:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":634722,"visible":true,"origin":"","legend":"\u003cp\u003eIntracellular and extracellular effects of the S100 family on WR21 cell invasion. (A) \u003cem\u003eS100\u003c/em\u003e-knockdown WR21 cells were seeded in a transwell invasion assay chamber and incubated for 5 days with CM from normal NIH3T3 cells, when invaded cells were quantified. (B) Normal WR21 cells were incubated with CM from \u003cem\u003eS100\u003c/em\u003e-knockdown\u003cem\u003e \u003c/em\u003eNIH3T3 cells, and invaded cells were quantified after 5 days.\u003cstrong\u003e \u003c/strong\u003e*p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figures5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6868185/v1/168cb7da29fc5cb643ac51f1.jpg"},{"id":87273080,"identity":"b8ec7908-070d-4541-b896-28a5e7e58bac","added_by":"auto","created_at":"2025-07-22 08:35:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4113068,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6868185/v1/f8a3e0c1-c6a6-45cd-b6a0-c574b74fae50.pdf"},{"id":87268831,"identity":"e3e54a54-4114-4aa9-aa8d-bcc84fbd9562","added_by":"auto","created_at":"2025-07-22 08:11:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":33748,"visible":true,"origin":"","legend":"","description":"","filename":"SGCS100manuscriptsupplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6868185/v1/932e38cab4252ae9491f568b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"S100 genes drive metastasis in salivary gland carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSalivary gland cancer (SGC) comprises 0.3% of new cancer incidence and 0.2% of all cancer mortalities worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While SGCs account for only about 5% of all head and neck malignancies, high-grade SGC is an extremely aggressive cancer characterized by a strong propensity for local invasion and distant metastasis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although some patients are successfully treated with surgery and adjuvant radiotherapy or chemoradiotherapy, 20\u0026ndash;50% experience recurrence and distant metastases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. With 21 distinct histological subtypes, SGC is typically classified into three groups based on their immune microenvironment and neoantigen landscape [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The most aggressive SGC subtypes include salivary duct carcinoma (SDC), high-grade adenocarcinoma, and poorly differentiated carcinoma [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Adenoid cystic carcinoma (ACC) is the most common SGC subtype, accounting for approximately 10% of all cases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Notably, T-cell dysfunction is more severe in SDC than in ACC, with greater T-cell infiltration and upregulation of T-cell checkpoints, including PD-L1, CTLA-4, LAG3, TIM3, TIGIT, and VISTA [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The most common metastatic site of SGC is the lung, although this manifests differently between SDC and ACC; distant metastases have a 53% incidence in SDC, whereas ACC exhibits late, indolent courses of systemic metastasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Patients with high-grade pathology were 7.5 times more likely to develop distant metastases than those with low-grade pathology [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe stromal microenvironment interacts closely with tumor cells, initiating cancer growth, invasion, and metastasis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. An important mediator of this interaction is the S100 Ca\u003csup\u003e+\u003c/sup\u003e-binding protein [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], part of the highly conserved vertebrate S100 protein family, with well-characterized roles in tumor growth, metastasis, angiogenesis, and immune evasion [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This protein family has 25 known subtypes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], most forming symmetrical dimers, although the S100A8/S100A9 heterodimer is an exception. S100 proteins contain two Ca\u003csup\u003e2+\u003c/sup\u003e-binding domains that increase in affinity for Ca\u003csup\u003e2+\u003c/sup\u003e upon binding to the target protein. Downstream effects include activating pathways involved in cytokine production, the cell cycle, apoptosis, and migration [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. S100 protein expression level is cell type-specific, but exerts widespread intra- and extracellular effects, being secreted in the serum, plasma, urine, saliva, and cerebrospinal fluid [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Research on S100 member S100A4 shows that overexpression promotes metastases in various cancer models, whereas knockouts limit metastasis to the lungs [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the expression and role of S100 proteins have never been thoroughly explored in SGC or SGC metastasis.\u003c/p\u003e\u003cp\u003eOwing to the rarity of SGC and its various histological subtypes, the molecular characteristics of distant metastases remain largely unknown. Therefore, this study aimed to examine the characteristics of two SGC subtypes (SDC and ACC), analyzing the expression patterns of genes putatively involved in tumor regulation and identifying factors associated with distant metastasis. We hypothesized that S100 proteins regulate SGC migration and invasion, contributing to metastasis.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cem\u003ePatient information and IRB approval\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe study included patients with SDC or ACC (N\u0026thinsp;=\u0026thinsp;26 and 9) from the authors\u0026rsquo; institutional salivary gland cancer registry. Eligible participants had complete clinical data and preserved tumor samples in the tissue bank. Written patient consent was obtained for use of de-identified clinical information, samples, and genomic analysis results. The protocol was approved by the institutional review board of Samsung Medical Center (approval no. 2015-12-13). Tumor samples (formalin-fixed paraffin-embedded, FFPE) were collected, and 11 samples were excluded from subsequent analyses after quality checking extracted RNA. Finally, 26 SDC and 9 ACC tumor samples were analyzed with cDNA-mediated annealing, selection, extension, and ligation (DASL) assays (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003cem\u003eDASL assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from FFPE samples for analysis using an Illumina Whole-Genome DASL\u0026reg; HT Assay for Expression Profiling (Illumina, San Diego, CA) with MCS4 cDNA synthesis reagents and Illumina DASL BeadChip [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The Illumina Whole-Genome DASL\u0026reg; HT assay can generate reproducible expression profiles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Fluorescence intensity data collected from the Bead Array platform were analyzed using proprietary Illumina software.\u003c/p\u003e\u003cp\u003e\u003cem\u003eGene set variation analysis (GSVA)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe R package \u0026ldquo;GSVA\u0026rdquo; was utilized to determine relative enrichment scores per patient of 50 cancer hallmark gene sets from MSigDB (h.all.v7.5.1.symbols), including genes related to epithelial-mesenchymal transition (EMT) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The score was an estimation of cancer hallmark pathway activity in SDC and ACC samples.\u003c/p\u003e\u003cp\u003eRelationships were analyzed using Pearson\u0026rsquo;s correlation. All statistical analyses were conducted in R (version 3.2.2). Significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 unless otherwise noted.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell lines and in-vivo models\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThree SGC cell lines and a fibroblast cell line were selected for experiments: SCA9 (mouse, CRL-1734, ATCC, Manassas, VA, USA, RRID: CVCL_JY08), A253 (human, HTB-41, ATCC, Manassas, VA, USA, RRID: CVCL_1060), WR21 (mouse, CRL-2189, ATCC, Manassas, VA, USA, RRID: CVCL_3623), and NIH3T3 (mouse, 30-2020, ATCC, Manassas, VA, USA, RRID: CVCL_0594). Cells were grown in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM: Gibco ThermoFisher Scientific Korea, Seoul, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco ThermoFisher Scientific Korea) and 1% penicillin/streptomycin (Gibco ThermoFisher Scientific Korea) and maintained at 37℃ with 5% CO₂. Human cell line has been authenticated using STR (or SNP) profiling within the last three years.All experiments were performed with mycroplasma free cells.\u003c/p\u003e\u003cp\u003eFemale BALB/c nude mice (4\u0026ndash;6 weeks old) were obtained from Orient Bio (Seongnam, Korea). Animals were housed in temperature- and light-controlled (12 h light/dark cycle) conditions, with food and water available ad libitum. The three SGC lines were counted (5.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells), re-suspended in 50 \u0026micro;L media, and injected into the submandibular gland of mice. Tumor growth was monitored twice per week. Upon attaining a gross tumor volume of 1500 mm\u003csup\u003e3\u003c/sup\u003e, tumor and lung tissue (with signs of metastasis) were collected for analysis. Mice were anesthetized by a single intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg), and then placed in a chamber where they were euthanized by exposure to CO₂ gas at a flow rate of 21 L/min for approximately 5 minutes. The whole tumor was excised, washed in sterile PBS, and processed with 4% paraformaldehyde (Biosesang, Yongin, Korea) for fixation and sectioning.\u003c/p\u003e\u003cp\u003e\u003cem\u003eProtocol approval of animal experiments\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe study followed international and institutional guidelines for animal treatment and complied with relevant legislation. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Samsung Medical Center (No. 20151122001).\u003c/p\u003e\u003cp\u003e\u003cem\u003eWestern blotting\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCells were rinsed with phosphate-buffered saline (PBS), suspended in homogenization lysis buffer, and centrifuged for 5 min at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C. Supernatant was collected to determine the protein concentration using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). The standard was BSA. Whole cell lysates were separated using 6\u0026ndash;15% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline containing 0.2% Tween 20 (TBST) at 20\u0026ndash;22\u0026deg;C and then probed with the appropriate primary (1:500) and secondary (1:5000) antibodies (see Supplementary Table\u0026nbsp;4). Blots were developed using an enhanced chemiluminescence kit to obtain density ratios of relative band intensity. The control was β-actin.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMigration and invasion assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo measure cell migration, WR21-RFP cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e in 100\u0026ndash;200 \u0026micro;L serum-free medium) were seeded alone into the upper chamber of a 24-well transwell plate (ECM509, Merck Millipore), or else seeded with NIH3T3-CM into the bottom chamber. The plate was then incubated for 5\u0026ndash;7 days at 37\u0026deg;C, and 5% CO\u003csub\u003e2\u003c/sub\u003e. Total cell count on the insert and migrated cells on the bottom were counted under a fluorescence microscope.\u003c/p\u003e\u003cp\u003eTo measure cell invasion, WR21 shRNA cells were seeded with NIH3T3-CM into the upper chamber of an ECMatrix\u0026trade; coated 24-well transwell plate (ECM550 8 \u0026micro;m, Merck Millipore) and incubated for 5 days under the same conditions as the migration assay. The total number of invaded cells was determined.\u003c/p\u003e\u003cp\u003e\u003cem\u003eQuantitative real time-PCR\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo synthesize cDNA, total RNA was isolated from cultured cells using an RNA extraction kit (Kusatsu, St. Shiga, Japan). Next, 10 \u0026micro;L of SYBR green premix (BioRad), 8 \u0026micro;L of sterile water, and 1 \u0026micro;L each of forward and reverse primer were mixed for a total reaction volume to 20 \u0026micro;L. Fluorescence was measured at each cycle. For PCR primer sequences used to analyze S100 expression, see Supplementary Table\u0026nbsp;2.\u003c/p\u003e\u003cp\u003e\u003cem\u003eConstruction of an RNA interference cell line\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo generate stable S100 shRNA-expressing cells, shRNA oligos of S100s (Supplementary Table\u0026nbsp;3) were cloned into pGreenPuro\u003csup\u003eTM\u003c/sup\u003eshRNA lentivectors (SBI System Biosciences, Mountain View, CA, USA). Cells in six-well culture plates were transfected with S100A4, S100A8, and S100A9 shRNA using shRNA transfection reagent (Invitrogen Biotechnology, CA, USA), following manufacturer protocol. After selection with puromycin, resistant cells were expanded for at least one month and tested for efficiency using western blotting.\u003c/p\u003e\u003cp\u003e\u003cem\u003eConstruction of an over-expression cell line\u003c/em\u003e\u003c/p\u003e\u003cp\u003eS100A4, S100A8, and S100A9 expression vectors were constructed via cDNA ligation into pIRES-mCherry (addgene, MA, USA). The S100 cDNA region was PCR-amplified from S100A4, S100A8, and S100A9 clones (Sino Biological Inc, Tx, USA). Cells in six-well culture plates were transfected with S100 expression vectors. Transduced cells were selected with neomycin and expanded for at least one month before being tested for efficiency using western blotting.\u003c/p\u003e\u003cp\u003e\u003cem\u003eImmunofluorescence assay\u003c/em\u003e\u003c/p\u003e\u003cp\u003eImmunohistochemistry was performed on prepared paraffin sections (7 um), as described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, tissue sections were deparaffinized and rehydrated through an ethanol series and distilled water. After 30 min of antigen retrieval with boiling citrate buffer, sections were stained with antibodies (see supplementary table 4).\u003c/p\u003e\u003cp\u003e\u003cem\u003eStatistical analyses\u003c/em\u003e\u003c/p\u003e\u003cp\u003eResults from fluorescence intensity analysis, cell migration assays, and cell invasion assays were analyzed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests. Significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Differential gene expression between SDC and ACC\u003c/h2\u003e\u003cp\u003ePatients with SDC and ACC expressed hallmark genes belonging to different transcriptomic subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We observed more upregulation of cancer metastasis- and development-associated genes in SDC than in ACC; notable pathways included EMT, interferon-alpha (IFN-α) and interferon-gamma (IFN-γ) response, mTORC1 signaling, IL2 signaling, and KRAS signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Hence, SDC was more aggressive than ACC and had elevated cancer metastasis signals.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eVolcano and violin plots visualizing results from principal component analysis of hallmark gene scores revealed markedly higher EMT transcriptomic expression in SDC than in ACC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;D). Interestingly, SDC also exhibited significant upregulation of IFN-α and IFN-γ responses. Violin plots show different hallmark scores for the two SGC subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), while volcano plots depict differentially expressed \u003cem\u003eS100\u003c/em\u003e genes between SDC and ACC, as well as the correlation between \u003cem\u003eS100\u003c/em\u003e genes and EMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In addition, most S100 genes were highly expressed in SDC, with 12 SDC-specific genes being significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). These results demonstrate differential expression of cancer-related genes between SDC and ACC. In particular, the upregulation of specific metastasis-associated genes in SDC highlights its aggressive nature.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e3.2. S100 expression in human SDC tissues\u003c/h2\u003e\u003cp\u003eTo investigate S100 protein expression, we performed immunofluorescence analysis using primary and metastatic tumor tissue samples from human SDC. S100A4, S100A8/S100A9, and S100P were expressed in SDC primary tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, S100 expression was also elevated in metastatic SDC tumors. \u003cem\u003eS100A4\u003c/em\u003e, \u003cem\u003eS100A8\u003c/em\u003e, and \u003cem\u003eS100A9\u003c/em\u003e expression also increased significantly, with high S100A7 and S100P upregulation that varied considerably across tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The results demonstrate that SDC is associated with increased S100 expression in both primary and metastatic tumors.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.3 \u003cem\u003eS100 expression in tissues from orthotopic xenograft mice generated by SGC cancer cell lines\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eAt 2\u0026thinsp;~\u0026thinsp;4 weeks, mice implanted with salivary gland cell lines (SCA9, A253 and WR21 cells) had elevated S100 expression in most primary tumors, according to immunofluorescence results. \u003cem\u003eS100A4\u003c/em\u003e, \u003cem\u003eS100A8\u003c/em\u003e, and \u003cem\u003eS100A9\u003c/em\u003e expression was upregulated in all tumors, whereas \u003cem\u003eS100B\u003c/em\u003e expression was not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Consistent with gene expression, western blot analysis of WR21 primary tumors revealed a significant increase in S100A4 and S100A9. In contrast, \u003cem\u003eS100A8\u003c/em\u003e showed increased gene expression but decreased protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). S100A4, S100A9, and S100B expression increased significantly in metastatic lung tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), whereas S100A8 expression did not. \u003cem\u003eS100A4\u003c/em\u003e, \u003cem\u003eS100A8\u003c/em\u003e, and \u003cem\u003eS100A9\u003c/em\u003e expression was upregulated in metastatic WR21 tumors, indicating that \u003cem\u003eS100A8\u003c/em\u003e and \u003cem\u003eS100B\u003c/em\u003e gene expression did not match their protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Notably, S100A4 and S100A9 expression was consistently upregulated at both the gene and protein levels in WR21 primary and metastatic tumors. Overall, S100 expression is associated with SGC in mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.4 NIH-3T3 cells regulate WR21 cell proliferation and migration\u003c/h2\u003e\u003cp\u003eAfter co-culturing WR21-RFP and NIH-3T3 cells in a transwell migration chamber, we verified the influence of NIH-3T3 cells on WR21 cell migration and proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Co-culturing with NIH-3T3 cells increased WR21-RFP cell proliferation but not cell migration. Interestingly, WR21 cell migration was significantly increased when NIH-3T3 cells were cultured at the bottom, suggesting that NIH-3T3 cells had secreted molecules, such as exosomes or inflammatory cytokines, which regulated WR21-RFP cell migration and induced cell recruiting. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). When we added conditioned medium (CM) from S100-overexpressing NIH-3T3 cells, WR21 cell migration to the transwell bottom also increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Hence, the CM contained S100-upregulating factors that further enhanced WR21 migration, implying that intracellular S100 proteins of NIH-3T3 cells are critical regulators of SGC migration and proliferation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.5 S100 knockdown in WR21 cells and CM from S100-knockdown NIH-3T3 cells reduce WR21 cell invasion\u003c/h2\u003e\u003cp\u003eTranswell invasion assays of WR21 cells expressing S100A4, S100A8, and S100A9 shRNA with normal NIH-3T3 CM demonstrated that WR21 cell invasion was suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Additionally, when WR21-shRNA empty cells were cultured with CM from S100A8- and S100A9-knockdown NIH-3T3 cells, WR21 cell invasion also decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The results confirm that intracellular and extracellular S100A4, S100A8, and S100A9 are strongly associated with WR21 cell invasion. These findings suggest that the S100 family plays a crucial role in cancer cell migration and invasion signaling pathways, and that inhibiting S100-related signaling can block SGC invasion.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study confirmed that S100 proteins are involved in SGC cell migration and metastasis through both autocrine and paracrine signal pathways. The aggressive SDC subtype exhibits the highest mortality among SGC [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition to local invasive growth, the clinical course of SDC involves early lymphatic and systemic metastasis. In contrast, ACC has an indolent clinical progression and less lymphatic metastasis, but high incidence of local infiltration, perineural spread, and systemic spread. However, even after lung metastasis, patients with ACC survive longer than 4\u0026ndash;5 years [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Because SDC and ACC have distinct dissemination patterns, we conducted a comparative gene expression analysis using samples from both cancers. The DASL assay revealed that SDC tumors overexpress cytokines (IFN-α and γ) and EMT-related genes, with the \u003cem\u003eS100\u003c/em\u003e family being particularly associated with EMT level. Corroborating our results, previous research has also found upregulation of IFN-α and IFN-γ response gene sets in patients with SGC subtypes of poor prognosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, our findings support the hypothesis that S100 proteins regulate SGC migration and invasion, contributing to metastasis. Mouse experiments indicated that S100A4, S100A8, and S100A9 were upregulated in both primary and metastatic tumors, largely consistent with observations in human SDC tissues. However, while S100A4 RNA and protein expression was upregulated in human tumors, this outcome was not fully replicated in mouse primary SGC tumors. In mice, upregulated S100A4 expression was localized, and the overall expression was low.\u003c/p\u003e\u003cp\u003eSerum and tumor S100B expression is diagnostic for glioma, breast cancer, and melanoma [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In particular, upregulated S100B serves as marker for a positive breast cancer prognosis, as it regulates the migration of ER-negative breast cancer cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, recombinant S100B promotes E-cadherin expression, inhibiting cell migration and facilitating an epithelial phenotype [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In contrast, serum and tumor S100B levels are markers of poor prognosis in malignant melanoma, as S100B upregulation reflects tumor load, stage, metastasis, and lower survival rates [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, downregulation indicates a good response to therapy. Hence, S100B appears to exert a dual effect and exhibits diverse activities depending on the cancer or context [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similarly, our study found that S100B expression differed significantly between human SGC and mouse primary tumors. In human SGC, S100B expression did not significantly increase in primary or metastatic tumors, whereas mouse tumors (A253 and WR21) exhibited high S100B protein expression in both primary and metastatic tumors. Therefore, while S100B appears to influence SGC cell proliferation and metastasis, the effect varies depending on tumor context or host. Another important cancer metastasis marker in the S100 family is S100P [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. High S100P expression was observed in the intracapsular and extracapsular components, as well as in malignant subtypes of carcinoma ex pleomorphic adenoma (CXPA) of the salivary gland [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study, S100P expression was significantly upregulated in human metastatic SGC tumors. However, we could not validate this finding in mice, because S100P is absent in most rodents [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eS100A8 and S100A9 expression is tissue-specific, primarily occurring in bone marrow-derived cells such as monocytes, neutrophils, myeloid-derived suppressor cells, and cancer cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Typically, this protein family is expressed both intracellularly and extracellularly. The secreted S100 protein has a cytokine-like function and contributes to pre-metastatic niche (PMN) formation via binding to extracellular receptors, including receptors for advanced glycation end products (RAGE), Toll-like receptor 4, ErbB4, dopamine D2, and FGFR1 [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Regulation of these receptors enhances cell invasion and migration [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. For example, S100A8/A9 regulates TLR9 and RAGE activity in multiple inflammatory pathways to induce inflammatory cytokines, a key part of PMN [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In rodents, both proteins act as chemo-attractants that activate phagocytes and facilitate the homing of tumor cells to the PMN [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Human S100A9 enhances THP-1 cell migration through RAGE-dependent signaling pathways such as MEK/ERK and PI3K [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Also known as metastasin, calvasculin, mts-1, and fibroblast-specific protein 1, S100A4 expression is similarly associated with malignancies [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOf the cell lines used in our in-vivo orthotopic study, WR21 cells exhibited the most aggressive behavior, including rapid tumor formation, metastasis, and high S100 expression. However, the S100 family did not always show consistent RNA and protein expression. S100A4, S100A8, and S100A9 RNA increased dramatically in WR21 primary tumors, but S100A8 protein levels were lower in the tumor than in the normal salivary gland (control). In contrast, S100A9 RNA and protein expression was elevated in both primary and metastatic SGC tumors samples from human SGC. S100A9 expression was also significantly upregulated in metastatic tumors derived from A253 and WR21 cells. These data strongly suggest that S100A9 plays a crucial role in cancer cell migration and metastasis. Because S100A8 protein expression was far lower than its RNA levels, this protein may not form a heterodimer and may be uniquely exerting extracellular effects on receptors as a single protein.\u003c/p\u003e\u003cp\u003eCancer-associated fibroblasts are another group of effector cells in the stromal compartment that express S100 proteins [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Tumor-secreted exosomes directly or indirectly mediate metastasis via upregulating S100s in fibroblasts within pre-metastatic regions, initiating PMN formation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In this study, we observed that S100A4 RNA and protein levels significantly increased in both human and mouse SGC tumors. Furthermore, we highlighted both autocrine and paracrine functions of S100s in fibroblasts. Conditioned media culturing S100-overexpressing NIH-3T3 cells promoted WR21 cell migration, and in particular, S100A9-overexpressing NIH-3T3 cells significantly enhanced WR21 cell migration. Silencing experiments demonstrated the crucial role of S100 gene expression in invasion, as inhibiting S100A4, S100A8, and S100A9 in WR21 cells significantly lowered invasion activity. We further confirmed the role of S100 genes in invasion when we observed that CM from shRNA-S100 NIH3T3 cells reduced WR21 cell invasion. The crucial role of S100-expressing fibroblasts in cancer cell metastasis was consistent with previous findings [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. To summarize, S100-overexpressing NIH3T3 cells promote the secretion of S100-mediated inflammatory cytokines or S100 proteins, contributing to WR21 migration. As WR21 cells migrate to the metastatic site, their expression of S100 proteins may further enhance their invasive activity.\u003c/p\u003e\u003cp\u003eIn conclusion, our study demonstrated the importance of S100 proteins in promoting the progression and metastasis of SDC, an extremely aggressive SGC subtype. The most notable S100 proteins were S100A4, S100A8, and S100A9, all upregulated in both primary and metastatic SGC. Moreover, the significant correlation of S100 proteins with EMT highlighted the role of this protein family in SGC cell migration and invasion. Therefore, these proteins may be markers of poor prognosis in SGC and potential therapeutic targets to limit metastasis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACC\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; adenoid cystic carcinoma\u003c/p\u003e\n\u003cp\u003eEMT\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; epithelial-mesenchymal transition\u003c/p\u003e\n\u003cp\u003eSDC \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;salivary duct carcinoma\u003c/p\u003e\n\u003cp\u003eSGC \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;salivary gland carcinoma\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed according to the guidelines of the Declaration of Helsinki. Human tissue was obtained based on the study approval protocol by the institutional review board of Samsung Medical Center (approval no. 2015-12-13).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw microarray data generated in this study is available in GEO under accession number GSE293801. The data set analysed and generated in this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConpeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conpeting interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF-2016R1A6A3A11931072).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHee-Weon Lee: data curation, writing original draft, writing review editing. Minho Kang: data curation, methodology, data analysis, writing original draft. Tae Woo Kim: data curation, investigation. Uk Yeol Moon: supervision, conceptualization, data curation, writing original draft, writing review \u0026amp; editing. Han-Sin Jeong: supervision, conceptualization, writing original draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen Y, Shan J. Chemoradiotherapy versus radiotherapy in high risk salivary gland cancer. World J Surg Oncol. 2024;22(1):181.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLorini L, Ronchi S, Nevens D, Klinghammer K, Orlandi E, Bossi P, Szturz P. 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J Biol Chem. 2004;279(23):24498\u0026ndash;504.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"salivary gland carcinoma, salivary duct carcinoma, adenoid cystic carcinoma, S100, epithelial-mesenchymal transition","lastPublishedDoi":"10.21203/rs.3.rs-6868185/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6868185/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eHigh-grade salivary gland carcinoma (SGC) is an aggressive malignancy that demands a multidisciplinary treatment approach. However, its metastatic mechanisms remain poorly understood due to the rarity and heterogeneity of SGC and the lack of suitable tumor models. This study aimed to explore key oncogenic drivers of distant metastasis by comparing two SGC subtypes: salivary duct carcinoma (SDC) and adenoid cystic carcinoma (ACC).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe conducted a comparative analysis of SDC and ACC to identify potential oncogenes related to metastasis. A cDNA-mediated Annealing, Selection, Extension, and Ligation (DASL) assay was used to evaluate S100 gene expression in patient samples. In vivo studies were performed using orthotopic xenograft mouse models (WR21, A253, and SCA9), and functional studies employed WR21 and NIH3T3 cell lines with S100 gain- or loss-of-function to assess proliferation, migration, and invasion. Statistical analyses were applied to evaluate gene expression patterns and biological effects.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSDC demonstrated more aggressive behavior and higher metastatic potential than ACC, accompanied by upregulation of S100 genes associated with epithelial-mesenchymal transition (EMT). SGC tumors exhibited increased expression of S100A4, S100A8, and S100A9 in both primary and metastatic lesions. Functional assays confirmed that S100 overexpression promotes EMT-related transcriptomic changes and enhances tumor cell proliferation and migration. Conditioned media from S100-expressing fibroblasts also stimulated these phenotypes, suggesting paracrine interactions in the tumor microenvironment.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eS100 family proteins play a critical role in high-grade SGC progression by promoting EMT and paracrine-mediated metastasis. Hence, S100 proteins have potential as prognostic biomarkers and therapeutic targets of SGC.\u003c/p\u003e","manuscriptTitle":"S100 genes drive metastasis in salivary gland carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 08:11:32","doi":"10.21203/rs.3.rs-6868185/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"152293453372868758408541030407950496355","date":"2025-07-22T13:53:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225952152854193417205886512692520814236","date":"2025-07-14T21:35:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T19:50:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-09T11:22:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-18T10:31:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-18T05:56:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-06-18T05:45:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ef9b6d9-f38d-4145-a547-e1072dac66bc","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-22T08:11:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 08:11:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6868185","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6868185","identity":"rs-6868185","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}