PRSS3/Mesotrypsin from Scirrhous Gastric Cancer Cells and Stanniocalcin-1 from Cancer-associated Fibroblasts Mutually Promote Invasion Activity

PRSS3/Mesotrypsin from Scirrhous Gastric Cancer Cells and Stanniocalcin-1 from Cancer-associated Fibroblasts Mutually Promote Invasion Activity | 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 PRSS3/Mesotrypsin from Scirrhous Gastric Cancer Cells and Stanniocalcin-1 from Cancer-associated Fibroblasts Mutually Promote Invasion Activity Takashi Sakuma, Masakazu Yashiro, Hongdong Gao, Dongheng Ma, Tomoya Sano, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9424453/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: Scirrhous gastric cancer (SGC) is characterized by rapid cancer-cell infiltration with extensive stromal fibrosis and has a poor prognosis compared to the other types of gastric cancer (GC). SGC cells and cancer-associated fibroblasts (CAFs) might interact with each other in the tumor microenvironment and contribute to progressive invasion. We investigated these cells' interaction with functional CAFs markers in SGC. Methods: Four pairs of CAFs and normal fibroblasts (NFs) and three GC cell lines were used. We examined the CAF marker by RNA sequencing and evaluated the effects of CAFs on SGC cells by invasion assay and morphologic analysis. Results: In silico analysis indicated that stanniocalcin-1 (STC-1) was a CAF marker. The STC-1 mRNA expression in CAFs was significantly higher than in NFs, and was significantly upregulated by the supernatant of an SGC cell line (OCUM-12 cells). STC-1 from CAFs was associated with invasion activity and epithelial-mesenchymal transition of SGC cells. Mesotrypsin (encoded by PRSS3 ) from OCUM-12 cells activated protease-activated receptor 2 (PAR-2) and upregulated STC-1 expression in CAFs via signal transducer and activator of transcription 3 (STAT-3) pathway. In turn, PRSS3 /mesotrypsin was upregulated by STC-1. High mesotrypsin expression contributed to significantly poorer survival in GC patients. Conclusions: PRSS3 /mesotrypsin from SGC cells and the PAR-2/STAT-3/STC-1 axis in CAFs might contribute to the mutual interaction between SGC cells and CAFs that promotes SGC invasion. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Scirrhous-type gastric cancer (SGC), defined as Borrmann type 4, is characterized by a rapid infiltration of cancer cells with extensive stromal fibrosis. Clinically, SGC induces a high incidence of peritoneal metastasis and has a quite poor prognosis, with a 5-year overall survival rate < 20%.[ 1 , 2 ] It has been speculated that the crosstalk between SGC cells and stromal cells in the cancer microenvironment might play an important role in the rapid progression of this type of cancer.[ 3 ] One of the main stromal cell types in cancer microenvironments is cancer-associated fibroblasts (CAFs), which are a crucial and abundant cellular component in SGC microenvironment. Our research group's studies have revealed that ( i ) CAFs stimulate the proliferation activity of SGC cells,[ 4 , 5 ] and ( ii ) factors from SGC cells stimulate the proliferation of CAFs.[ 6 ] Clarifying the molecular interactions between SGC cells and CAFs will help improve the therapeutic management of SGC. Several molecules have been reported to be a CAFs-specific marker for gastric cancer (GC).[ 7 – 10 ] Our group has demonstrated that secretion of fibroblast growth factor 7 (FGF7) by gastric CAFs is associated with the remarkable proliferation of SGC cells via FGF receptor type 2 (FGFR2).[ 11 ] The expression frequency of FGF7/FGFR2 in SGC is only 20%,[ 12 ] and molecular mechanism of SGC remains unknown. It is also not clear how these molecules affect the characteristics of SGC, or how a potential chain reaction underlying the interaction between cancer cells and CAFs affects the pathogenesis of SGC. We conducted the present study to clarify the molecules that are associated with the mutual stimulating interaction between SGC cells and CAFs. Materials and Methods Patients and cell culture. Three GC cell lines (OCUM-12,[ 13 ] NUGC-3, MKN74), and four pairs of CAFs (CAF128, CAF129, CAF130, CAF174) and normal fibroblasts (NFs) (NF128, NF129, NF130, NF174), were used. OCUM-12 and NUGC-3 were derived from diffuse-type (SGC cell lines), and MKN-74 from intestinal-type (non-SGC cell lines). Four CAFs were from tumoral gastric serosa, and NFs from non-tumoral. CAF128, CAF130, CAF174 were established from each three patient with poorly differentiated adenocarcinoma of GC, and CAF129 from signet ring cell. GC cell lines were incubated in a culture medium consisting of Dulbecco's modified Eagle medium (DMEM; Wako, Osaka, Japan) with 5 % ftal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan), 100 IU/mL penicillin (Wako), 100 mg/mL streptomycin (Wako), and 0.5 mM sodium pyruvate (Wako). CAFs and NFs were cultured under similar conditions except that 10 % FS was used. 11 cells were cultured in 21 % O at 37°C. Next-generation sequencing. Total RNA was isolated from three pairs of CAFs and NFs 128–130 with RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany), and was subjected to RNA sequencing using Ion GeneStudio S5 Sequencer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer’s protocol. Collection of conditioned medium. Conditioned medium (CM) was collected from semi-confluent GC cell lines or CAFs after incubation in serum-free DMEM for 72 hours. The supernatant was stored at -20°C until use. The effects of various molecules or CM on each cell. GC cells were cultured with culture medium as control, or 50% of CM from CAF174 with/without 100 ng/mL of anti-stanniocalcin-1 (STC-1) antibody (Proteintech, Rosemont, Illinois, USA) as neutralization, or 100 ng/mL of recombinant human STC-1 (CLOUD-CLONE CORP., Houston, Texas, USA). Each fibroblast was also educated with culture medium as control, or 50% of CM from GC cell lines with/without 150 ng/mL of anti-mesotrypsin neutralizing antibody (MAB3710, R&D Systems, Minneapolis, Minnesota, USA), or various concentration (30, 100, 300 ng/mL) of recombinant human mesotrypsin (CLOUD-CLONE CORP.) with/without 20 nM of AZ-3451 (MedChemexpress, Monmouth, NJ, USA) for protease-activated receptor 2 (PAR-2) inhibitor. After 72 hours incubation, mRNA or protein was extracted for subsequent analyses. Concentration of STC-1 recombinant protein was determined by proliferation assay for GC cell lines, and each antibody and inhibitor was by IC 50 values. Bioinformatic analysis. All raw FASTQ files for RNA sequencing were analyzed against ENSEMBL cDNA transcriptomes [Human assembly hg38 (GRCh38), release 94][ 14 ] to quantify transcript expression for each sample for indexing with the number of bootstraps set to 100 using kallisto (v0.46.2).[ 15 ] Gene expression between CAFs and NFs was compared using sleuth.[ 14 ] Differentially expressed genes (DEGs) satisfied p < 0.05. Volcano plots were generated to display DEGs within the overall gene expression levels. All DEGs were input into Metascape database [ https://metascape.org ][ 16 ] for Gene Ontology (GO) process enrichment analysis, with q < 0.05. Reverse transcription-polymerase chain reaction (RT-PCR). Total cellular RNA was extracted with RNeasy Plus Mini Kit (QIAGEN). The quality of RNA was analyzed using NanoDrop 2000c (Thermo Fisher Scientific). Subsequently, complementary DNA (cDNA) was synthesized from 1 µg of RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) and DNA was subjected to 28 PCR cycles with each primer (Sigma-Aldrich, Burlington, Massachusetts, USA) (Supplementary Table 1) using AmpliTaq Gold 360 DNA polymerase (Thermo Fisher Scientific) under gel electrophoresis. The PCR conditions were as follows: annealing for 30 sec at 60°C, extension for 1 min at 72°C, and final incubation for 7 min at 72°C. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used to normalize mRNA levels for differences. Gel electrophoresis. TAE buffer in 50×TAE (NIPPON GENE, Tokyo, Japan) and agarose S (NIPPON GENE) was allowed to melt and solidify, and GelRed™ (Biotium, Fremont, California, USA) were added for staining. 5 µl of each amplicon and 50bp DNA Ladder RTU (GeneDireX, Inc., Zuhan, Taiwan) were loaded. The gel was run in a gel electrophoresis apparatus at 85 Volts for 30 min, and then observed under a UV illuminator. Quantitative real-time RT-PCR. Reverse transcription to cDNA was performed as above. Real-time RT-PCR was performed on ABI Prism 7000 (Applied Biosystems, Waltham, Massachusetts, USA) according to manufacturer’s protocol using TaqMan Fast Advanced Master Mix (2×) (Thermo Fisher Scientific). Total reaction volume was 20 µl. The amplification parameters were set at 95°C for 3 sec and 60°C for 30 sec (40 cycles total). Hypoxanthine phosphoribosyltransferase 1 ( HPRT1 ) was used to normalize mRNA levels for each gene. The threshold cycle (Ct) values were used for calculation of the relative expression ratios between control and treated cells using the formula described by Pfaffl.[ 17 ] All quantitative PCR reactions were performed three times. Morphology. OCUM-12 cells under CM from CAF174, or under CM from CAF174 with/without 100 ng/mL of anti-STC-1 antibody as neutralization, were cultured for 48 hours. Cell morphological changes were observed microscopically and continuously. Epithelial-mesenchymal transition (EMT) was determined when spindle-shaped cancer cells were found compared to OCUM-12 cells under normal condition, by phase-contrast microscopy. Invasion assay. The in vitro invasiveness was measured by two-chamber Matrigel invasion assay as previously reported.[ 3 ] GC cell lines (2 × 10 3 cells per chamber) were seeded in the upper chamber, with 100 ng/mL of STC-1 recombinant protein, or CM from CAF174, or CM from CAF174 and 100 ng/mL of anti-STC-1 antibody as neutralization. After 72 hours, cancer cells invading the lower surface of the membrane through a filter were stained with Diff-Quik (Sysmex, Hyogo, Japan), and were manually counted at ×200 magnification. Six randomly chosen fields and the mean of six fields were calculated. Western blot analysis. Cell lysates from each cell was collected and 2 µg of total protein were subjected on 4–20% Mini Protean TGX™ Precast Gel (#4561096, Bio-Rad, Hercules, California, USA). The protein bands were transferred to Trans-Blot Turbo Transfer Pack (#1704156, Bio-Rad). Primary antibodies were as follows: β-actin (ACTB) (1:1000; Sigma-Aldrich), signal transducer and activator of transcription 3 (STAT-3) (1:1000; Proteintech), phosphorylation of STAT-3 (p-STAT3) (1:1000; Abcepta, San Diego, California, USA), PAR-2 (1:1000; Santa Cruz Biotechnology, Dallas, Texas, USA), STC-1 (1:1000), mesotrypsin (1:1000; Abcepta). Patients with GC. A total of 1,205 patients, who underwent gastrectomy with regional lymph node dissection for GC at Osaka Metropolitan University Hospital from 2001 and 2019, were retrospectively enrolled. None of patients had received radiotherapy or chemotherapy before the surgery. This study was approved by Osaka Metropolitan University Ethics Committee (approval number 0924, 2022-077). The study protocol conformed to the ethical guidelines of the Declaration of Helsinki, and informed consent was obtained from all patients. Immunohistochemical staining of mesotrypsin on GC tissue. The tissue samples were deparaffinized, and slides were heated. After endogenous peroxidase activity was blocked, the samples were incubated with anti-mesotrypsin antibody (1:100; Abcepta) for 1 hour at room temperature. The samples were incubated with biotinylated secondary antibody (Nichirei Bioscience Inc.) for 1 hour at room temperature, then treated with streptavidin-peroxidase reagent and counterstained with Mayer’s hematoxylin. The expression level of mesotrypsin in cancer cells was analyzed by evaluating the staining intensity and the percentage of positively stained areas. Intensity was rated 0–2 as follows (0 + = no staining, 1 + = weak staining, 2 + = strong staining), and positivity was also graded on a scale 0–3 (0 = 0–19%, 1 = 20–39%, 2 = 40–59%, and 3 = 60–100%). Two scores were added to gain the final result of 0–6 points. Expression was considered positive when the mesotrypsin score was ≥ 2. The evaluations were conducted by two double-blinded independent observers who were unaware of patients’ clinical data and outcomes. When a different evaluation between the two independent observers was found, the evaluation was rechecked and discussed. Statistical analysis. Statistical analysis for RNA sequencing and GO analysis for DEGs was performed as above. As for gene expression data and invasion assay, the unpaired Student’s t -test was used to determine the significance of differences between groups. Survival rates of overall survival (OS) and recurrence free survival (RFS) were calculated by Kaplan-Meier method, and survival curves were compared with the log-rank test. Univariate and multivariate analyses were also performed using the Cox proportional hazards model. P < 0.05 was considered as statistically significant. SPSS software (SPSS Japan) was applied for all analysis. RESULTS RNA sequencing data and enrichment analysis with three pairs of CAFs and NFs derived from GC tissue, and identification of STC-1 RNA sequencing with CAFs and NFs 128–130 revealed that 223 of the total 40,453 genes satisfied the criteria of p < 0.05. In 55 of the 223 genes, the log 2 fold change of the expression level (i.e., the number of CAFs divided by the number of NFs) was ≥ 1.5-fold ( Fig. 1 A ) . We considered these 55 genes differentially expressed genes (DEGs). GO process enrichment analysis of 55 DEGs indicated that the ratio of genes with the annotation "epithelial cell development" was significantly higher than that of the background gene population ( Fig. 1 B ) . We further extracted ten DEGs with annotations related to "epithelial cell," i.e., cancer cells, from the database (Supplementary Table 2) . Four of these genes were subsequently excluded because of the type of receptor or the absence of expression of NFs in the RNA sequencing data. We examined the remaining six genes as candidate CAFs-specific functional markers: a disintegrin and metalloprotease 17 ( ADAM17 ), a nnexin A3 ( ANXA3 ), Fraser extracellular matrix complex subunit 1 ( FRAS1 ), netrin-1 ( NTN-1 ), SRY-box transcription factor 9 ( SOX9 ), and STC-1 . To determine the final single molecule of CAFs, we compared mRNA expression levels of these six genes in another pair of fibroblast lines, CAF174 and NF174 ( Fig. 1 C ) , and we eventually identified STC-1 as the molecule of CAFs because STC-1 expression was also higher in CAF174 than in NF174. The results of the quantitative RT-PCR revealed that the expression level of STC-1 was significantly higher in CAF174 at passage 3 (P3) compared to NF174 at P3 (p = 0.030). Moreover, after 72 hours with CM from OCUM-12 cells (an SGC cell line), STC-1 mRNA expression in CAF174 was significantly increased, and it was also significantly higher than that obtained with CM from MKN-74 cells (a non-SGC cell line) ( Fig. 1 D ) . The effects of STC-1 from CAFs on the invasion activity and EMT of GC cell lines We evaluated the effects of STC-1 from CAFs on SGC cell lines. Matrigel invasion assay revealed that in OCUM-12 cells, both STC-1 recombinant protein and CM from CAF174 significantly increased the number of invading cells, whereas the addition of both CM from CAF174 and anti-STC-1 antibody significantly decreased the number of invading cells (Fig. 2 A). Representative images of OCUM-12 cells that passed through 12-µm-pore membrane filter in invasion assay are provided in Fig. 2 B. Regarding EMT markers, we observed that stimulation by STC-1 decreased E-cadherin ( E-cad ) mRNA expression and increased the expressions of N-cadherin ( N-cad ), Vimentin ( Vim ), and Snail Family Transcriptional Repressor 2 ( slug ) mRNA (Fig. 2 C). Figure 2 D is a representative by phase-contrast microscopy image of OCUM-12 cells. After 48 hours of culturing with CM from CAF174, OCUM-12 cells showed a morphology that was more spindle-shaped than that of control cells, but when both the same CM and anti-STC-1 antibody were applied, no morphological changes occurred. Together these results indicated that STC-1 from CAFs stimulated invasion activity and EMT of SGC cells. The factors derived from SGC cells and the receptors on CAFs that upregulated STC-1 from CAFs We identified the factors derived from SGC cells that upregulate STC-1 from CAFs. We first observed that ( i ) Protease serine 3 ( PRSS3 ) was expressed more than the other two isoforms, i.e., Protease serine 1 ( PRSS1 ) and Protease serine 2 ( PRSS2 ) in the GC cell lines, and ( ii ) PRSS3 was expressed at a considerably higher level in the SGC cell lines (OCUM-12 cells and NUGC-3 cells) than in the non-SGC cell line (MKN-74 cells) ( Fig. 3 A ) . Regarding the receptor on the fibroblasts, PAR-2 , encoded by F2R-like trypsin receptor 1 ( F2RL1 ), was expressed at a significantly higher level in CAF174 than in NF174, as also shown by the RNA sequencing data of CAFs and NFs 128–130 ( Fig. 3 B ) . The interaction between mesotrypsin from SGC cells and the PAR-2/STAT-3/STC-1 axis in CAFs Mesotrypsin affected the expression levels of PAR-2 and STC-1 in CAF174. CAF174 cells were cultured with various mesotrypsin concentrations (30, 100, 300 ng/mL). With 100 ng/mL of mesotrypsin recombinant protein, the expressions of both PAR-2 and STC-1 mRNA were significantly higher than the control values. Furthermore, the expression of PAR-2 mRNA was significantly increased by CM from OCUM-12 cells, but anti-mesotrypsin antibody significantly suppressed the increase by the same CM ( Fig. 4 A ) . In western blot analysis, the expression levels of p-STAT-3, PAR-2, and STC-1 were all upregulated by mesotrypsin stimulation in a concentration-dependent manner. However, the addition of both 100 ng/mL of recombinant human mesotrypsin and 20 nM PAR-2 inhibitor (AZ-3451) decreased these expression levels to nearly the same levels as the control values ( Fig. 4 B ) . This result demonstrated that in CAF174, mesotrypsin stimulation induced STAT-3 signaling through activated PAR-2, and no other receptors. The results on the side of GC cells are depicted in Fig. 4 C,D. The mRNA expression level of PRSS3 obtained with STC-1 for each GC cell line is illustrated in Fig. 4 C. The results of western blot analysis clarified that the mesotrypsin protein level in OCUM-12 cells was upregulated by STC-1 stimulation ( Fig. 4 D ) . These results (both the mRNA and protein expression levels) suggested that the level of STC-1 from CAFs was upregulated by mesotrypsin from SGC cells through activated PAR-2 via STAT-3 signaling, and conversely, PRSS3 /mesotrypsin from SGC cells was upregulated by STC-1. The relationship between the expression of mesotrypsin in GC and the clinicopathological characteristics of patients with GC We observed the expression of mesotrypsin in the cytoplasm of GC cells ( Fig. 5 A ) . The correlation between mesotrypsin expression in GC cells and clinicopathological characteristics of the patients with GC are summarized in Table 1 . Among the 1,205 cases of GC patients examined in this study, 378 (31.4%) expressed a high level of mesotrypsin. Mesotrypsin expression in GC cells was significantly correlated with the following characteristics: Borrmann type 4 GC, tumor size (> 50 mm), tumor depth (T2–T4), lymph node metastases, and pathological stage (2–4). Figure 5 B provides OS and RFS curves classified by mesotrypsin expression in the total series of 1,205 GC patients and the 112 patients with Borrmann type 4 GC. The 5-year OS rate was 59.5% in the patients with high mesotrypsin expression in GC cells and 73.8% in the patients with low mesotrypsin expression in GC cells. The 5-year RFS rates in the same groups of patients were 72.0% and 80.4%, respectively. Both OS and RFS of mesotrypsin-positive patients in the total series (n = 378) were significantly poorer than those of the mesotrypsin-negative patients. Among the 112 patients with Borrmann type 4 cancer, mesotrypsin-positive patients (n = 52) had significantly poorer OS. Table 1 Correlation between Mesotrypsin expression in gastric cancer cells and clinicopathological features in 1,205 gastric cancer patients. Variables Mesotrypsin p -value positive (n = 378) negative (n = 827) Age ≥ 60 296 (78.3%) 622 (75.2%) 0.242 Gender Male 246 (65.1%) 568 (68.7%) 0.215 Female 132 (34.9%) 259 (31.4%) Macroscopic type Borrmann type 4 52 (13.8%) 60 (7.26%) < 0.01 Other types 326 (86.2%) 767 (92.2%) Microscopic type Undifferentiated 206 (52.4%) 427 (51.6%) 0.355 Differentiated 172 (47.6%) 400 (48.4%) Tumor size (≥ 50 mm) 198 (52.4%) 306 (37.0%) < 0.01 Tumor depth (T ≥ 2) 278 (73.5%) 480 (58.0%) < 0.01 Lymph node metastases (N ≥ 1) 220 (58.2%) 375 (45.3%) < 0.01 Stage ≥ 2 253 (66.9%) 412 (49.8%) < 0.01 Table 2 summarizes the results of the univariate and multivariate analyses for OS after surgery in the 1,205 patients with GC. The univariate analysis revealed that OS was significantly correlated with age (≥ 60 yrs), Borrmann type 4, undifferentiated microscopic type, tumor size (> 50 mm), tumor depth (T2–T4), lymph node metastases, the pathological stage (2–4), and mesotrypsin expression in GC cells. The multivariate analysis with the Cox proportional hazards model identified the following as independent predictive parameters for the patients' OS: age (≥ 60 yrs), tumor size (> 50 mm), lymph node metastases, stage (2–4), and mesotrypsin expression in GC cells. Table 2 Univariate and Multivariable analysis for overall survival after surgery in 1,205 gastric cancer patients. Variables Univariate Multivariate Hazard ratio 95% CI p -value Hazard ratio 95% CI p -value Age (≥ 60) 1.307 1.018–1.679 0.036 1.789 1.039–2.880 0.017 Gender (male) 1.113 0.798–1.554 0.528 Macroscopic type (Borrmann type 4) 3.965 3.096–5.078 < 0.01 1.343 0.787–2.292 0.279 Microscopic type (Undifferentiated) 1.440 1.171–1.770 < 0.01 1.027 0.743–1.418 0.874 Tumor size (≥ 50 mm) 3.893 3.138–4.830 < 0.01 1.624 1.143–2.307 < 0.01 Tumor depth (T ≥ 2) 5.138 3.785–6.975 < 0.01 1.239 0.637–2.409 0.528 Lymph node metastases (N ≥ 1) 4.636 3.622–5.933 < 0.01 1.806 1.126–2.897 0.014 Stage ≥ 2 5.950 4.488–7.888 < 0.01 1.368 0.659–2.843 0.400 Mesotrypsin positive 1.839 1.495–2.262 < 0.01 1.471 1.039–2.082 0.030 CI: confidence interval. DISCUSSION Our findings demonstrated that the level of PRSS3 from SGC cells was upregulated by stimulation with STC-1 from CAFs. Figure 6 is a schema of the interaction between SGC cells and CAFs via a mutual activation of mesotrypsin and STC-1. Our analyses revealed that ( i ) STC-1 from CAFs stimulated cell-invasion activity and promoted EMT, especially on SGC cells ( Fig. 6 A ) ; ( ii ) mesotrypsin from SGC cells stimulated PAR-2 activation and an upregulation of STC-1 via STAT-3 signaling in CAFs ( Fig. 6 B ) ; and ( iii ) PRSS3 /mesotrypsin was upregulated by STC-1 ( Fig. 6 C ) . Our search of the relevant literature identified no report regarding the function of STC-1 derived from CAFs for SGC cells; our present study is thus the first to clarify the interaction between PRSS3 /mesotrypsin from SGC cells and PAR-2/STAT-3/STC-1 axis from CAFs. Stanniolcalcin-1 (STC-1) moderates serum calcium and phosphate homeostasis.[ 18 , 19 ] STC-1 has been reported to be overexpressed in tissues of various types of cancer including GC[ 20 ] and to promote malignant phenotypes.[ 19 ] The expression of STC-1 in CAFs was observed to be higher than that in NFs in several types of cancers.[ 21 – 24 ] To the best of our knowledge, the significance of STC-1 in CAF tissues in GC has not been reported. The results of the present study demonstrated that STC-1 expression in CAFs was significantly higher than that in NFs in GC tissues, and STC-1 derived from CAFs promoted invasion activity and was involved in EMT progression of SGC cells by the downregulation of E-cadherin and the upregulation of N-cadherin , Vimentin , and slug . SGC exhibits a rapid infiltration of cancer cells, and the changes in the expression levels of EMT markers suggest both the suppression of cellular adhesion ability and the acquisition of mesenchymal characteristics. We suspect that the change in SGC cell morphology to spindle-shaped might be associated with invasion activity of SGC cells. STC-1 from GC cells (not CAFs) was observed to help promote metastasis via tumor angiogenesis.[ 25 ] We speculated that in light of the effects of STC-1 from CAFs on SGC cells observed in the present study, STC-1 might affect not only SGC cells but also the cancer microenvironment. Our findings revealed that the expression of STC-1 mRNA tended to increase with CM from SGC cells more than with CM from non-SGC cells. These results suggest that the factors from OCUM-12 cells might mediate the conversion of NFs to CAFs. It has been reported that activated STAT-3 signaling induced STC-1 secretion,[ 22 , 26 ] contributing to cancer progression.[ 27 ] In contrast, our present study's RNA sequencing data revealed that F2RL1 was significantly expressed in CAFs ( Fig. 1 A, Supplementary Table 2) , and this gene encodes PAR-2, which is one type of G protein-coupled receptor (GPCR). In addition, endogenous serine proteases such as trypsin and tryptase specifically activate PAR-2 by cleaving the extracellular N-terminus to expose its tethered ligand domain.[ 28 ] These findings might indicate that STC-1 from CAFs is regulated by PAR-2 on CAFs via activated STAT-3 signaling, and that serine protease derived from SGC cells might upregulate STC-1 from CAFs through PAR-2. There are several serine proteases, and we focused on Protease serine 3 ( PRSS3 ), a component of a trypsin mRNA isoform. Unlike cationic trypsinogen encoded by PRSS1 and anionic trypsinogen encoded by PRSS2 , mesotrypsin encoded by PRSS3 contributes only 3%–10% of the trypsin activity in human pancreatic fluid.[ 29 ] Mesotrypsin has been recognized as a molecule that is associated with tumor progression in several types of cancer,[ 30 ] but little is known about the regulation of mesotrypsin expression, downstream signaling, and receptor selectivity. Especially, trypsin activates PAR-2 by cleaving,[ 31 ] but the question of whether mesotrypsin acts on PAR-2 remains a matter of discussion.[ 30 , 32 , 33 ] This contradiction might be due to multiple factors such as the differences in PAR-2 expression, cell types used, and the effects of the extracellular matrix. The results of our present investigation indicate that STC-1 from CAFs is upregulated by mesotrypsin from SGC cells through activated PAR-2 on CAFs via STAT-3 signaling, and conversely, PRSS3 /mesotrypsin from SGC cells is upregulated by STC-1. PRSS3 /mesotrypsin and PAR-2/STAT-3/STC-1 might interact with each other. It has been reported that STAT-3 is related to the proliferation and invasion of tumor cells through cyclin D1[ 34 ] or mircoRNA-499-5p activation.[ 35 ] STAT-3 is frequently activated in CAFs in various types of cancer including GC.[ 36 ] A positive activating interaction between cancer cells and CAFs via STAT-3 signaling might be essential to the invasion of cancer cells. Our immunohistochemical examination of 1,205 cases of GC showed that high mesotrypsin expression contributed to a significantly poor prognosis (both OS and RFS). Corresponding to the report by Wang et al. ,[ 29 ] our study demonstrated high mesotrypsin expression and lymph node metastases were common independent prognostic factors for OS. It has been observed that PRSS3 /Mesotrypsin promoted the proliferation, invasion, and migration activities of GC cells, displaying properties of an oncogene.[ 29 ] We observed that mesotrypsin contributes to not only the maintenance of stemness of GC cells and the promotion of autonomic tumor progression such as lymph node metastases; it also indirectly enhances invasion activity of SGC cells via an upregulation of STC-1. These findings suggested that mesotrypsin might be a crucial factor for the invasion of cancer cells by affecting the whole SGC microenvironment along with stromal tissues. STC-1 in CAFs and/or mesotrypsin in SGC cells might be a promising therapeutic target for SGC. We should acknowledge some study limitations. First, STC-1 has been reported to act on receptors such as VEGF-R2 and Notch-1.[ 19 , 37 ] However, VEGF-A2 did not affect the expression level of OCUM-12 cells in our study (data not shown). Trypsin effectively prevents autolysis and preserves esterase activity for a prolonged time under Ca 2+ binding conditions.[ 38 ] There might be a molecular interaction between STC-1 and PRSS3 /mesotrypsin through calcium-associated receptors. Second, we did not perform in vivo experiments to investigate the interaction between PRSS3 /mesotrypsin from SGC cells and STC-1 from CAFs. To clarify the therapeutic effects of mesotrypsin or an STC-1 inhibitor on SGC cells, in vivo experiments using a mouse SGC model might be informative. In conclusion, the results of this study suggest that PRSS3 /mesotrypsin from SGC cells and a PAR-2/STAT-3/STC-1 axis in CAFs might interact and promote invasion activity of SGC cells. Our findings might provide the foundation for a promising therapeutic target for SGC. Abbreviations CAFs cancer associated fibroblasts CM conditioned medium F2RL1 F2R Like Trypsin Receptor 1 GC gastric cancer NFs normal fibroblasts PAR-2 protease-activated receptor 2 PRSS3 protease serine 3 STAT-3 activator of transcription 3 SGC scirrhous-type gastric cancer STC-1 stanniocalcin-1 Declarations Availability of data and materials The RNA sequencing datasets generated during the current study are available in the in the DDBJ Sequenced Read Archive under the accession numbers from DRR963239 to DRR963244. The other data can be obtained from the corresponding author or the Ethics Committee ( [email protected] ) on reasonable request. Additional Information Acknowledgements We appreciate Rachel Feldman of KN International, Inc. for assistance with editing the manuscript for English grammar and usage. Funding information This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors’ contributions TS (Takashi Sakuma) and MY designed and performed the experiments, and co-wrote the manuscript. HG, DM, CF, HN, YY, and HK accumulated the data. TS (Tomoya Sano) and DI prepared and collected GC tissue specimens. KM suggested and co-designed the study. All authors read and approved the final manuscript. Ethics statement This study was approved by the Medical Ethics Committee of Osaka Metropolitan University (approval number 0924, 2022-077). Patients provided written informed consent. This retrospective study was conducted in accordance with the principles of the Declaration of Helsinki. Registry and the Registration No. of the study/trial: 0924, 2022-077. Animal Studies: N/A. Consent for publication Not applicable. Competing Interests The authors have no conflicts of interest to declare regarding this study. References Yook JH, Oh ST, Kim BS. Clinicopathological Analysis of Borrmann Type IV Gastric Cancer. Cancer Res Treat. 2005;37:87–91. Nashimoto A, Akazawa K, Isobe Y, Miyashiro I, Katai H, Kodera Y, Tsujitani S, Seto Y, Furukawa H, Oda I, et al. Gastric cancer treated in 2002 in Japan: 2009 annual report of the JGCA nationwide registry. Gastric Cancer. 2013;16:1–27. Miki Y, Yashiro M, Okuno T, Kitayama K, Masuda G, Hirakawa K, Ohira M. CD9-positive exosomes from cancer-associated fibroblasts stimulate the migration ability of scirrhous-type gastric cancer cells. Br J Cancer. 2018;118:867–77. Kinoshita H, Yashiro M, Fukuoka T, Hasegawa T, Morisaki T, Kasashima H, Masuda G, Noda S, Hirakawa K. Diffuse-type gastric cancer cells switch their driver pathways from FGFR2 signaling to SDF1/CXCR4 axis in hypoxic tumor microenvironments. Carcinogenesis. 2015;36:1511–20. Kushiyama S, Yashiro M, Yamamoto Y, Sera T, Sugimoto A, Nishimura S, Togano S, Kuroda K, Okuno T, Miki Y, et al. Dipeptidyl Peptidase-4 from Cancer-associated Fibroblasts Stimulates the Proliferation of Scirrhous-type Gastric Cancer Cells. Anticancer Res. 2022;42:501–9. Fuyuhiro Y, Yashiro M, Noda S, Kashiwagi S, Matsuoka J, Doi Y, Kato Y, Hasegawa T, Sawada T, Hirakawa K. Upregulation of cancer-associated myofibroblasts by TGF-β from scirrhous gastric carcinoma cells. Br J Cancer. 2011;105:996–1001. Kim KT, Lee MH, Shin SJ, Cho I, Kuk JC, Yun J, Choi YY. Decorin as a key marker of desmoplastic cancer-associated fibroblasts mediating first-line immune checkpoint blockade resistance in metastatic gastric cancer. Gastric Cancer. 2025;28:12–26. Ozmen E, Demir TD, Ozcan G. Cancer-associated fibroblasts: protagonists of the tumor microenvironment in gastric cancer. Frontiers in Molecular Biosciences. 2024;11:1340124. Zhang Y, Chen H, Zhang W, Zhou H. Identification of cancer-associated fibroblast-related Ectodysplasin-A as a novel indicator for prognosis and immune response in gastric cancer. Heliyon. 2024;10:e34005. Nurmik M, Ullmann P, Rodriguez F, Haan S, Letellier E. In search of definitions: Cancer-associated fibroblasts and their markers. International Journal of Cancer. 2020;146:895–905. Nakazawa K, Yashiro M, Hirakawa K. Keratinocyte Growth Factor Produced by Gastric Fibroblasts Specifically Stimulates Proliferation of Cancer Cells from Scirrhous Gastric Carcinoma. Cancer Res. 2003;63:8848–52. Yashiro M, Kuroda K, Masuda G, Okuno T, Miki Y, Yamamoto Y, Sera T, Sugimoto A, Kushiyama S, Nishimura S, et al. Clinical difference between fibroblast growth factor receptor 2 subclass, type IIIb and type IIIc, in gastric cancer. Sci Rep. 2021;11:4698. Kato Y, Yashiro M, Noda S, Tendo M, Kashiwagi S, Doi Y, Nishii T, Matsuoka J, Fuyuhiro Y, Shinto O, et al. Establishment and characterization of a new hypoxia-resistant cancer cell line, OCUM-12/Hypo, derived from a scirrhous gastric carcinoma. Br J Cancer. 2010;102:898–907. Pimentel H, Bray NL, Puente S, Melsted P, Pachter L. Differential analysis of RNA-seq incorporating quantification uncertainty. Nat Methods. 2017;14:687–90. Bray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016;34:525–7. Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, Benner C, Chanda SK. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun. 2019;10:1523. Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 2001;29:e45. Mookerjee S, Whitley G, Banerjee D. Stanniocalcin-1: A Novel Mediator in Diabetic Kidney Disease and Cardiovascular Disease. Kidney International Reports. 2024;10:321–7. Zhao F, Yang G, Feng M, Cao Z, Liu Y, Qiu J, You L, Zheng L, Zhang T, Zhao Y. Expression, function and clinical application of stanniocalcin-1 in cancer. Journal of Cellular and Molecular Medicine. 2020;24:7686–96. Fang Z, Tian Z, Luo K, Song H, Yi J. Clinical significance of stanniocalcin expression in tissue and serum of gastric cancer patients. Chinese Journal of Cancer Research. 2014;26:602–10. Bai S, Zhao Y, Chen W, Peng W, Wang Y, Xiong S, Aruna, Li Y, Yang Y, Chen S, et al. The stromal-tumor amplifying STC1-Notch1 feedforward signal promotes the stemness of hepatocellular carcinoma. J Transl Med. 2023;21:236. Avalle L, Raggi L, Monteleone E, Savino A, Viavattene D, Statello L, Camperi A, Stabile SA, Salemme V, De Marzo N, et al. STAT3 induces breast cancer growth via ANGPTL4, MMP13 and STC1 secretion by cancer associated fibroblasts. Oncogene. 2022;41:1456–67. Yang Y, Yin S, Li S, Chen Y, Yang L. Stanniocalcin 1 in tumor microenvironment promotes metastasis of ovarian cancer. Onco Targets Ther. 2019;12:2789–98. Peña C, Céspedes MV, Lindh MB, Kiflemariam S, Mezheyeuski A, Edqvist PH, Hägglöf C, Birgisson H, Bojmar L, Jirstrom K, et al. STC1 expression by cancer-associated fibroblasts drives metastasis of colorectal cancer. Cancer Res. 2013;73:1287–97. He LF, Wang TT, Gao QY, Zhao GF, Huang YH, Yu LK, Hou YY. Stanniocalcin-1 promotes tumor angiogenesis through up-regulation of VEGF in gastric cancer cells. J Biomed Sci. 2011;18:39. Huang J, Wang X, Wen J, Zhao X, Wu C, Wang L, Cao X, Dong H, Xu X, Huang F, et al. Gastric cancer cell-originated small extracellular vesicle induces metabolic reprogramming of BM-MSCs through ERK-PPARγ-CPT1A signaling to potentiate lymphatic metastasis. Cancer Cell Int. 2023;23:87. Yu H, Lee H, Herrmann A, Buettner R, Jove R. Revisiting STAT3 signalling in cancer: New and unexpected biological functions. Nature Reviews Cancer. 2014;14:736–46. Park MK, Cho MK, Kang SA, Park HK, Kim YS, Kim KU, Ahn SC, Kim DH, Yu HS. Protease-activated receptor 2 is involved in Th2 responses against Trichinella spiralis infection. Korean Journal of Parasitology. 2011;49:235–43. Wang F, Hu YL, Feng Y, Guo YB, Liu YF, Mao QS, Xue WJ. High-level expression of PRSS3 correlates with metastasis and poor prognosis in patients with gastric cancer. J Surg Oncol. 2019;119:1108–21. Hockla A, Miller E, Salameh MA, Copland JA, Radisky DC, Radisky ES. PRSS3/mesotrypsin is a therapeutic target for metastatic prostate cancer. Molecular Cancer Research. 2012;10:1555–66. Han S, Lee CW, Trevino JG, Hughes SJ, Sarosi GA. Autocrine Extra-Pancreatic Trypsin 3 Secretion Promotes Cell Proliferation and Survival in Esophageal Adenocarcinoma. PLoS One. 2013;8:e76667. Rolland-Fourcade C, Denadai-Souza A, Cirillo C, Lopez C, Jaramillo JO, Desormeaux C, Cenac N, Motta JP, Larauche M, Taché Y, et al. Epithelial expression and function of trypsin-3 in irritable bowel syndrome. Gut. 2017;66:1767–78. Grishina Z, Ostrowska E, Halangk W, Sahin-Tóth M, Reiser G. Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): Mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1. Br J Pharmacol. 2005;146:990–9. Arshad S, Naveed M, Ullia M, Javed K, Butt A, Khawar M, Amjad F. Targeting STAT-3 signaling pathway in cancer for development of novel drugs: Advancements and challenges. Genetics and Molecular Biology. 2020;43:e20180160. Yang Y Lou, Liu P, Li D, Yang Q, Li B, Jiang XJ. Stat-3 signaling promotes cell proliferation and metastasis of gastric cancer through PDCD4 downregulation. Kaohsiung Journal of Medical Sciences. 2020;36:244–9. Zhao D, Zhang J, Zhang L, Wu Q, Wang Y, Zhang W, Xiao Y, Chen J, Zhan Q. PAFR/Stat3 axis maintains the symbiotic ecosystem between tumor and stroma to facilitate tumor malignancy. Acta Pharm Sin B. 2023;13:694–708. Liu A, Li Y, Lu S, Cai C, Zou F, Meng X. Stanniocalcin 1 promotes lung metastasis of breast cancer by enhancing EGFR–ERK–S100A4 signaling. Cell Death Dis. 2023;14:395. Patel CK, Mukherjee TK. Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca2+-Mediated Activation of Esterase Activity. Biomacromolecules. 2024;25:6082–92. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.docx SupplementaryTable2.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 10 May, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers invited by journal 30 Apr, 2026 Editor assigned by journal 30 Apr, 2026 Editor invited by journal 27 Apr, 2026 Submission checks completed at journal 24 Apr, 2026 First submitted to journal 24 Apr, 2026 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-9424453","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":632225315,"identity":"f72b346e-1d40-417b-ab4d-cffe10c9e954","order_by":0,"name":"Takashi Sakuma","email":"","orcid":"","institution":"Osaka Metropolitan University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Sakuma","suffix":""},{"id":632225316,"identity":"51c1b81d-7fe3-4eb2-8525-cb4a4458403a","order_by":1,"name":"Masakazu 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09:36:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9424453/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9424453/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108959956,"identity":"438e6629-0389-45c7-925f-f9764e694976","added_by":"auto","created_at":"2026-05-11 08:34:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":778403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA sequencing and enrichment analysis with CAFs and NFs 128–130, and identification of STC-1. A:\u003c/strong\u003e Volcano plot \u003cem\u003ein silico\u003c/em\u003e analysis with CAFs and NFs 128–130. It showed 223 genes satisfied the criteria of p\u0026lt;0.05 (\u003cem\u003eblack line\u003c/em\u003e), and in 55 genes (\u003cem\u003eRed plots\u003c/em\u003e), the log\u003csub\u003e2\u003c/sub\u003e fold change in the expression level (i.e., the number of CAFs divided by that of NFs) was ≥1.5-fold. \u003cstrong\u003eB:\u003c/strong\u003e GO process enrichment analysis of 55 DEGs. \"GO:0002064: epithelial cell development\" pathway was significantly enriched (q=0.045). \u003cstrong\u003eC:\u003c/strong\u003e Comparison of mRNA expression levels in CAF174 and NF174, with six genes as candidate CAF functional markers. \u003cem\u003eSTC-1\u003c/em\u003e mRNA expression level in CAF174 was higher than that in NF174 (\u003cem\u003earrow\u003c/em\u003e). \u003cstrong\u003eD:\u003c/strong\u003e The effect of CM from SGC cells on \u003cem\u003eSTC-1\u003c/em\u003e mRNA expression of CAF174 and NF174. \u003cem\u003eSTC-1\u003c/em\u003e mRNA expression in CAF174 was significantly upregulated by CM from OCUM-12 cells (p\u0026lt;0.01), but not by CM from MKN-74 cells. The \u003cem\u003eSTC-1\u003c/em\u003e mRNA expression obtained with CM from OCUM-12 cells was also significantly higher than that obtained with CM from MKN-74 cells (p=0.016). No significant difference was revealed in NF174 with CM from each GC cell line. The graph depicts expression levels relative to control NF174 (P3) at day 3. *p\u0026lt;0.05, **p\u0026lt;0.01, \u003cem\u003eN.S.\u003c/em\u003e: not significant.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9424453/v1/877cc5cfa849339abc9a5266.jpg"},{"id":108978061,"identity":"d6dacdde-e69a-4a06-9ef6-0e01b8936434","added_by":"auto","created_at":"2026-05-11 11:33:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1569942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of STC-1 on the invasion activity and EMT of SGC cells. A:\u003c/strong\u003e The effect of STC-1 from CAFs on invasion activity of GC cells. The number of cells invading the pore membrane filter was significantly increased by STC-1 recombinant protein (p=0.014) or CM from CAF174 (p=0.016). However, no significant difference was observed when both CM from CAF174 and anti-STC-1 antibody were added as neutralization, compared to control (Ctrl) cells. *p\u0026lt;0.05. \u003cstrong\u003eB:\u003c/strong\u003eRepresentative images of invading OCUM-12 cells in two-chamber Matrigel invasion assay. STC-1 neutralization significantly decreased the number of invading OCUM-12 cells (\u003cem\u003earrowhead\u003c/em\u003e). Bar: 200 µm. \u003cstrong\u003eC:\u003c/strong\u003e The mRNA expression level of EMT markers of GC cells with STC-1. In OCUM-12 cells, STC-1 stimulated the downregulation of \u003cem\u003eE-cad\u003c/em\u003e and the upregulations of \u003cem\u003eN-cad\u003c/em\u003e, \u003cem\u003eVim\u003c/em\u003e, and \u003cem\u003eslug\u003c/em\u003e. In NUGC-3 cells, only \u003cem\u003eE-cad\u003c/em\u003e downregulation was observed. In MKN-74 cells, no EMT markers were changed. \u003cstrong\u003eD:\u003c/strong\u003eRepresentative images of morphological changes in OCUM-12 cells. CM from CAF174 transformed OCUM-12 cells into spindle-shaped morphology after 48 hours, whereas no change was obtained with addition of both the same CM and anti-STC-1 antibody (\u003cem\u003earrow\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9424453/v1/328a7d2cdb45d55bdbf43a05.jpg"},{"id":108959954,"identity":"7e331b9a-1b9e-47d7-b90c-f4672470a075","added_by":"auto","created_at":"2026-05-11 08:34:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":480114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe identification of molecular mechanism for producing STC-1 (trypsin isoform from SGC cells and receptor on CAFs).\u003c/strong\u003e \u003cstrong\u003eA:\u003c/strong\u003e The comparison of expression levels of three mRNA isoforms of trypsin (\u003cem\u003ePRSS1\u003c/em\u003e, \u003cem\u003ePRSS2\u003c/em\u003e, \u003cem\u003ePRSS3\u003c/em\u003e) in the three GC cell lines. \u003cem\u003ePRSS3\u003c/em\u003ewas expressed at a higher level compared to the other two isoforms. \u003cem\u003ePRSS3 \u003c/em\u003ewas expressed in OCUM-12 cells and NUGC-3 cells, but not expressed in MKN-74 cells. \u003cstrong\u003eB:\u003c/strong\u003e \u003cem\u003ePAR-2\u003c/em\u003e mRNA expression level between CAF174 and NF174. \u003cem\u003ePAR-2\u003c/em\u003e(\u003cem\u003eF2RL1\u003c/em\u003e) expression in CAF174 was significantly higher than in NF174 (p=0.028).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9424453/v1/7679794d976227495b674cfc.jpg"},{"id":108977801,"identity":"b6569071-8002-4818-aa42-78f961f47d6d","added_by":"auto","created_at":"2026-05-11 11:32:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":696542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe evaluation of the interaction between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRSS3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e/mesotrypsin from SGC cells and STC-1 from CAFs. A:\u003c/strong\u003e The effect of mesotrypsin from SGC cells on \u003cem\u003eSTC-1\u003c/em\u003e and \u003cem\u003ePAR-2\u003c/em\u003e mRNA expressions of CAF174. Stimulation with 100 ng/mL of mesotrypsin or CM from OCUM-12 cells resulted in significant increases in the expression of both \u003cem\u003ePAR-2\u003c/em\u003eand \u003cem\u003eSTC-1\u003c/em\u003e mRNA, but not significant with 30 or 300 ng/mL of mesotrypsin. The mRNA expression level of \u003cem\u003ePAR-2\u003c/em\u003e was significantly decreased by anti-mesotrypsin neutralization antibody. The mRNA expression of CAF174 (P5) with no stimulation at day 3 was used as the control. *p\u0026lt;0.05, **p\u0026lt;0.01. \u003cstrong\u003eB:\u003c/strong\u003e Western blot analysis for CAF174. After addition of mesotrypsin recombinant protein, concentration-dependent upregulations of p-STAT3, PAR-2, and STC-1 were observed. The application of AZ-3451 (PAR-2 inhibitor) suppressed those signaling. The protein expression level of CAF174 (P5) with no stimulation at day 3 was used as the control. \u003cstrong\u003eC:\u003c/strong\u003e \u003cem\u003ePRSS3\u003c/em\u003e mRNA expression in each GC cell line with STC-1. In OCUM-12 cells, \u003cem\u003ePRSS3\u003c/em\u003e was upregulated by STC-1. \u003cstrong\u003eD:\u003c/strong\u003e Western blot analysis of OCUM-12 cells. After stimulation with STC-1, the upregulation of mesotrypsin was induced. The expression level of OCUM-12 cells with no stimulation at day 3 was examined as the control.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9424453/v1/83d60f6e307d03e7ff8f311b.jpg"},{"id":108959958,"identity":"a5d0bc90-fb31-4ae6-b4f4-90c96a79278e","added_by":"auto","created_at":"2026-05-11 08:34:01","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1053142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relationship between the mesotrypsin expression in GC tissues and the overall survival (OS) and recurrence-free survival (RFS) of patients with GC. A:\u003c/strong\u003e Representative images of mesotrypsin staining in GC cells. Mesotrypsin was expressed on the cytoplasm staining of GC cells. Mesotrypsin expression was considered positive when the sum score of intensity and positivity was ≥2. Bar: 200 µm. \u003cstrong\u003eB:\u003c/strong\u003e The survival curves of total 1,205 GC patients, and 112 patients with Borrmann type 4 in OS and RFS. Kaplan–Meier curve indicated that mesotrypsin-positive GC group (n=378) contributed to significantly poorer OS and RFS (both p\u0026lt;0.01). Among the 112 patients with Borrmann type 4, OS of mesotrypsin-positive GC patients (n=52) was also significantly poorer than of negative patients (p=0.045).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9424453/v1/cdb5ad56e14126ef31f52d97.jpg"},{"id":108959961,"identity":"54ec2268-032b-455e-b54e-47b926d95cb3","added_by":"auto","created_at":"2026-05-11 08:34:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":218108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchema of the interaction between SGC cells and CAFs in GC microenvironment. A:\u003c/strong\u003eSTC-1 derived from CAFs is involved in inducing invasion activity and EMT. \u003cem\u003eSTC-1\u003c/em\u003emRNA expression level was significantly increased with CM from SGC cells. \u003cstrong\u003eB:\u003c/strong\u003eMesotrypsin from SGC cells, induces PAR-2 activation and upregulates STC-1 via STAT-3 signaling. \u003cstrong\u003eC:\u003c/strong\u003e \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin was also upregulated by STC-1.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9424453/v1/9602436c6f8316217dd179f6.jpg"},{"id":108979934,"identity":"9c3468a3-53b2-4e60-a167-050db2ca50e4","added_by":"auto","created_at":"2026-05-11 12:02:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5245379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9424453/v1/12ab6f1d-02a8-49de-b44d-7230220a97fc.pdf"},{"id":108978203,"identity":"aae03083-e521-4113-903a-55cb01c22b70","added_by":"auto","created_at":"2026-05-11 11:34:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":169505,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9424453/v1/6e866af8e173943e2737a982.docx"},{"id":108977802,"identity":"e0261be2-13e0-4394-aa46-3881578a746e","added_by":"auto","created_at":"2026-05-11 11:32:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":168769,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9424453/v1/d8f9955c47ae9ac784582ac5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"PRSS3/Mesotrypsin from Scirrhous Gastric Cancer Cells and Stanniocalcin-1 from Cancer-associated Fibroblasts Mutually Promote Invasion Activity","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eScirrhous-type gastric cancer (SGC), defined as Borrmann type 4, is characterized by a rapid infiltration of cancer cells with extensive stromal fibrosis. Clinically, SGC induces a high incidence of peritoneal metastasis and has a quite poor prognosis, with a 5-year overall survival rate\u0026thinsp;\u0026lt;\u0026thinsp;20%.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] It has been speculated that the crosstalk between SGC cells and stromal cells in the cancer microenvironment might play an important role in the rapid progression of this type of cancer.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] One of the main stromal cell types in cancer microenvironments is cancer-associated fibroblasts (CAFs), which are a crucial and abundant cellular component in SGC microenvironment. Our research group's studies have revealed that (\u003cem\u003ei\u003c/em\u003e) CAFs stimulate the proliferation activity of SGC cells,[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and (\u003cem\u003eii\u003c/em\u003e) factors from SGC cells stimulate the proliferation of CAFs.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Clarifying the molecular interactions between SGC cells and CAFs will help improve the therapeutic management of SGC.\u003c/p\u003e \u003cp\u003eSeveral molecules have been reported to be a CAFs-specific marker for gastric cancer (GC).[\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Our group has demonstrated that secretion of fibroblast growth factor 7 (FGF7) by gastric CAFs is associated with the remarkable proliferation of SGC cells via FGF receptor type 2 (FGFR2).[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] The expression frequency of FGF7/FGFR2 in SGC is only 20%,[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and molecular mechanism of SGC remains unknown. It is also not clear how these molecules affect the characteristics of SGC, or how a potential chain reaction underlying the interaction between cancer cells and CAFs affects the pathogenesis of SGC. We conducted the present study to clarify the molecules that are associated with the mutual stimulating interaction between SGC cells and CAFs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003ePatients and cell culture.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThree GC cell lines (OCUM-12,[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] NUGC-3, MKN74), and four pairs of CAFs (CAF128, CAF129, CAF130, CAF174) and normal fibroblasts (NFs) (NF128, NF129, NF130, NF174), were used. OCUM-12 and NUGC-3 were derived from diffuse-type (SGC cell lines), and MKN-74 from intestinal-type (non-SGC cell lines). Four CAFs were from tumoral gastric serosa, and NFs from non-tumoral. CAF128, CAF130, CAF174 were established from each three patient with poorly differentiated adenocarcinoma of GC, and CAF129 from signet ring cell. GC cell lines were incubated in a culture medium consisting of Dulbecco's modified Eagle medium (DMEM; Wako, Osaka, Japan) with 5 % ftal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan), 100 IU/mL penicillin (Wako), 100 mg/mL streptomycin (Wako), and 0.5 mM sodium pyruvate (Wako). CAFs and NFs were cultured under similar conditions except that 10 % FS was used. 11 cells were cultured in 21 % O at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNext-generation sequencing.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTotal RNA was isolated from three pairs of CAFs and NFs 128\u0026ndash;130 with RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany), and was subjected to RNA sequencing using Ion GeneStudio S5 Sequencer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollection of conditioned medium.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eConditioned medium (CM) was collected from semi-confluent GC cell lines or CAFs after incubation in serum-free DMEM for 72 hours. The supernatant was stored at -20\u0026deg;C until use.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effects of various molecules or CM on each cell.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGC cells were cultured with culture medium as control, or 50% of CM from CAF174 with/without 100 ng/mL of anti-stanniocalcin-1 (STC-1) antibody (Proteintech, Rosemont, Illinois, USA) as neutralization, or 100 ng/mL of recombinant human STC-1 (CLOUD-CLONE CORP., Houston, Texas, USA). Each fibroblast was also educated with culture medium as control, or 50% of CM from GC cell lines with/without 150 ng/mL of anti-mesotrypsin neutralizing antibody (MAB3710, R\u0026amp;D Systems, Minneapolis, Minnesota, USA), or various concentration (30, 100, 300 ng/mL) of recombinant human mesotrypsin (CLOUD-CLONE CORP.) with/without 20 nM of AZ-3451 (MedChemexpress, Monmouth, NJ, USA) for protease-activated receptor 2 (PAR-2) inhibitor. After 72 hours incubation, mRNA or protein was extracted for subsequent analyses. Concentration of STC-1 recombinant protein was determined by proliferation assay for GC cell lines, and each antibody and inhibitor was by IC\u003csub\u003e50\u003c/sub\u003e values.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBioinformatic analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll raw FASTQ files for RNA sequencing were analyzed against ENSEMBL cDNA transcriptomes [Human assembly hg38 (GRCh38), release 94][\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] to quantify transcript expression for each sample for indexing with the number of bootstraps set to 100 using kallisto (v0.46.2).[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] Gene expression between CAFs and NFs was compared using sleuth.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Differentially expressed genes (DEGs) satisfied p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Volcano plots were generated to display DEGs within the overall gene expression levels. All DEGs were input into Metascape database [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org\u003c/span\u003e\u003cspan address=\"https://metascape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] for Gene Ontology (GO) process enrichment analysis, with q\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReverse transcription-polymerase chain reaction (RT-PCR).\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTotal cellular RNA was extracted with RNeasy Plus Mini Kit (QIAGEN). The quality of RNA was analyzed using NanoDrop 2000c (Thermo Fisher Scientific). Subsequently, complementary DNA (cDNA) was synthesized from 1 \u0026micro;g of RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) and DNA was subjected to 28 PCR cycles with each primer (Sigma-Aldrich, Burlington, Massachusetts, USA) \u003cb\u003e(Supplementary Table\u0026nbsp;1)\u003c/b\u003e using AmpliTaq Gold 360 DNA polymerase (Thermo Fisher Scientific) under gel electrophoresis. The PCR conditions were as follows: annealing for 30 sec at 60\u0026deg;C, extension for 1 min at 72\u0026deg;C, and final incubation for 7 min at 72\u0026deg;C. \u003cem\u003eGlyceraldehyde-3-phosphate dehydrogenase\u003c/em\u003e (\u003cem\u003eGAPDH\u003c/em\u003e) was used to normalize mRNA levels for differences.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGel electrophoresis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTAE buffer in 50\u0026times;TAE (NIPPON GENE, Tokyo, Japan) and agarose S (NIPPON GENE) was allowed to melt and solidify, and GelRed\u0026trade; (Biotium, Fremont, California, USA) were added for staining. 5 \u0026micro;l of each amplicon and 50bp DNA Ladder RTU (GeneDireX, Inc., Zuhan, Taiwan) were loaded. The gel was run in a gel electrophoresis apparatus at 85 Volts for 30 min, and then observed under a UV illuminator.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative real-time RT-PCR.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eReverse transcription to cDNA was performed as above. Real-time RT-PCR was performed on ABI Prism 7000 (Applied Biosystems, Waltham, Massachusetts, USA) according to manufacturer\u0026rsquo;s protocol using TaqMan Fast Advanced Master Mix (2\u0026times;) (Thermo Fisher Scientific). Total reaction volume was 20 \u0026micro;l. The amplification parameters were set at 95\u0026deg;C for 3 sec and 60\u0026deg;C for 30 sec (40 cycles total). \u003cem\u003eHypoxanthine phosphoribosyltransferase 1\u003c/em\u003e (\u003cem\u003eHPRT1\u003c/em\u003e) was used to normalize mRNA levels for each gene. The threshold cycle (Ct) values were used for calculation of the relative expression ratios between control and treated cells using the formula described by Pfaffl.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] All quantitative PCR reactions were performed three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMorphology.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOCUM-12 cells under CM from CAF174, or under CM from CAF174 with/without 100 ng/mL of anti-STC-1 antibody as neutralization, were cultured for 48 hours. Cell morphological changes were observed microscopically and continuously. Epithelial-mesenchymal transition (EMT) was determined when spindle-shaped cancer cells were found compared to OCUM-12 cells under normal condition, by phase-contrast microscopy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInvasion assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e invasiveness was measured by two-chamber Matrigel invasion assay as previously reported.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] GC cell lines (2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per chamber) were seeded in the upper chamber, with 100 ng/mL of STC-1 recombinant protein, or CM from CAF174, or CM from CAF174 and 100 ng/mL of anti-STC-1 antibody as neutralization. After 72 hours, cancer cells invading the lower surface of the membrane through a filter were stained with Diff-Quik (Sysmex, Hyogo, Japan), and were manually counted at \u0026times;200 magnification. Six randomly chosen fields and the mean of six fields were calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blot analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCell lysates from each cell was collected and 2 \u0026micro;g of total protein were subjected on 4\u0026ndash;20% Mini Protean TGX\u0026trade; Precast Gel (#4561096, Bio-Rad, Hercules, California, USA). The protein bands were transferred to Trans-Blot Turbo Transfer Pack (#1704156, Bio-Rad). Primary antibodies were as follows: β-actin (ACTB) (1:1000; Sigma-Aldrich), signal transducer and activator of transcription 3 (STAT-3) (1:1000; Proteintech), phosphorylation of STAT-3 (p-STAT3) (1:1000; Abcepta, San Diego, California, USA), PAR-2 (1:1000; Santa Cruz Biotechnology, Dallas, Texas, USA), STC-1 (1:1000), mesotrypsin (1:1000; Abcepta).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePatients with GC.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e A total of 1,205 patients, who underwent gastrectomy with regional lymph node dissection for GC at Osaka Metropolitan University Hospital from 2001 and 2019, were retrospectively enrolled. None of patients had received radiotherapy or chemotherapy before the surgery. This study was approved by Osaka Metropolitan University Ethics Committee (approval number 0924, 2022-077). The study protocol conformed to the ethical guidelines of the Declaration of Helsinki, and informed consent was obtained from all patients.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunohistochemical staining of mesotrypsin on GC tissue.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe tissue samples were deparaffinized, and slides were heated. After endogenous peroxidase activity was blocked, the samples were incubated with anti-mesotrypsin antibody (1:100; Abcepta) for 1 hour at room temperature. The samples were incubated with biotinylated secondary antibody (Nichirei Bioscience Inc.) for 1 hour at room temperature, then treated with streptavidin-peroxidase reagent and counterstained with Mayer\u0026rsquo;s hematoxylin.\u003c/p\u003e \u003cp\u003eThe expression level of mesotrypsin in cancer cells was analyzed by evaluating the staining intensity and the percentage of positively stained areas. Intensity was rated 0\u0026ndash;2 as follows (0\u0026thinsp;+\u0026thinsp;=\u0026thinsp;no staining, 1\u0026thinsp;+\u0026thinsp;=\u0026thinsp;weak staining, 2\u0026thinsp;+\u0026thinsp;=\u0026thinsp;strong staining), and positivity was also graded on a scale 0\u0026ndash;3 (0\u0026thinsp;=\u0026thinsp;0\u0026ndash;19%, 1\u0026thinsp;=\u0026thinsp;20\u0026ndash;39%, 2\u0026thinsp;=\u0026thinsp;40\u0026ndash;59%, and 3\u0026thinsp;=\u0026thinsp;60\u0026ndash;100%). Two scores were added to gain the final result of 0\u0026ndash;6 points. Expression was considered positive when the mesotrypsin score was \u0026ge;\u0026thinsp;2. The evaluations were conducted by two double-blinded independent observers who were unaware of patients\u0026rsquo; clinical data and outcomes. When a different evaluation between the two independent observers was found, the evaluation was rechecked and discussed.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis.\u003c/h2\u003e \u003cp\u003eStatistical analysis for RNA sequencing and GO analysis for DEGs was performed as above. As for gene expression data and invasion assay, the unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to determine the significance of differences between groups. Survival rates of overall survival (OS) and recurrence free survival (RFS) were calculated by Kaplan-Meier method, and survival curves were compared with the log-rank test. Univariate and multivariate analyses were also performed using the Cox proportional hazards model. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant. SPSS software (SPSS Japan) was applied for all analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eRNA sequencing data and enrichment analysis with three pairs of CAFs and NFs derived from GC tissue, and identification of STC-1\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRNA sequencing with CAFs and NFs 128\u0026ndash;130 revealed that 223 of the total 40,453 genes satisfied the criteria of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. In 55 of the 223 genes, the log\u003csub\u003e2\u003c/sub\u003e fold change of the expression level (i.e., the number of CAFs divided by the number of NFs) was \u0026ge;\u0026thinsp;1.5-fold \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. We considered these 55 genes differentially expressed genes (DEGs). GO process enrichment analysis of 55 DEGs indicated that the ratio of genes with the annotation \"epithelial cell development\" was significantly higher than that of the background gene population \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further extracted ten DEGs with annotations related to \"epithelial cell,\" i.e., cancer cells, from the database \u003cb\u003e(Supplementary Table\u0026nbsp;2)\u003c/b\u003e. Four of these genes were subsequently excluded because of the type of receptor or the absence of expression of NFs in the RNA sequencing data. We examined the remaining six genes as candidate CAFs-specific functional markers: a \u003cem\u003edisintegrin and metalloprotease 17\u003c/em\u003e (\u003cem\u003eADAM17\u003c/em\u003e), a\u003cem\u003ennexin A3\u003c/em\u003e (\u003cem\u003eANXA3\u003c/em\u003e), \u003cem\u003eFraser extracellular matrix complex subunit 1\u003c/em\u003e (\u003cem\u003eFRAS1\u003c/em\u003e), \u003cem\u003enetrin-1\u003c/em\u003e (\u003cem\u003eNTN-1\u003c/em\u003e), \u003cem\u003eSRY-box transcription factor 9\u003c/em\u003e (\u003cem\u003eSOX9\u003c/em\u003e), and \u003cem\u003eSTC-1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo determine the final single molecule of CAFs, we compared mRNA expression levels of these six genes in another pair of fibroblast lines, CAF174 and NF174 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, and we eventually identified STC-1 as the molecule of CAFs because \u003cem\u003eSTC-1\u003c/em\u003e expression was also higher in CAF174 than in NF174. The results of the quantitative RT-PCR revealed that the expression level of \u003cem\u003eSTC-1\u003c/em\u003e was significantly higher in CAF174 at passage 3 (P3) compared to NF174 at P3 (p\u0026thinsp;=\u0026thinsp;0.030). Moreover, after 72 hours with CM from OCUM-12 cells (an SGC cell line), \u003cem\u003eSTC-1\u003c/em\u003e mRNA expression in CAF174 was significantly increased, and it was also significantly higher than that obtained with CM from MKN-74 cells (a non-SGC cell line) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effects of STC-1 from CAFs on the invasion activity and EMT of GC cell lines\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe evaluated the effects of STC-1 from CAFs on SGC cell lines. Matrigel invasion assay revealed that in OCUM-12 cells, both STC-1 recombinant protein and CM from CAF174 significantly increased the number of invading cells, whereas the addition of both CM from CAF174 and anti-STC-1 antibody significantly decreased the number of invading cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Representative images of OCUM-12 cells that passed through 12-\u0026micro;m-pore membrane filter in invasion assay are provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. Regarding EMT markers, we observed that stimulation by STC-1 decreased \u003cem\u003eE-cadherin\u003c/em\u003e (\u003cem\u003eE-cad\u003c/em\u003e) mRNA expression and increased the expressions of \u003cem\u003eN-cadherin\u003c/em\u003e (\u003cem\u003eN-cad\u003c/em\u003e), \u003cem\u003eVimentin\u003c/em\u003e (\u003cem\u003eVim\u003c/em\u003e), and \u003cem\u003eSnail Family Transcriptional Repressor 2\u003c/em\u003e (\u003cem\u003eslug\u003c/em\u003e) mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD is a representative by phase-contrast microscopy image of OCUM-12 cells. After 48 hours of culturing with CM from CAF174, OCUM-12 cells showed a morphology that was more spindle-shaped than that of control cells, but when both the same CM and anti-STC-1 antibody were applied, no morphological changes occurred. Together these results indicated that STC-1 from CAFs stimulated invasion activity and EMT of SGC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe factors derived from SGC cells and the receptors on CAFs that upregulated STC-1 from CAFs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe identified the factors derived from SGC cells that upregulate STC-1 from CAFs. We first observed that (\u003cem\u003ei\u003c/em\u003e) \u003cem\u003eProtease serine 3\u003c/em\u003e (\u003cem\u003ePRSS3\u003c/em\u003e) was expressed more than the other two isoforms, i.e., \u003cem\u003eProtease serine 1\u003c/em\u003e (\u003cem\u003ePRSS1\u003c/em\u003e) and \u003cem\u003eProtease serine 2\u003c/em\u003e (\u003cem\u003ePRSS2\u003c/em\u003e) in the GC cell lines, and (\u003cem\u003eii\u003c/em\u003e) \u003cem\u003ePRSS3\u003c/em\u003e was expressed at a considerably higher level in the SGC cell lines (OCUM-12 cells and NUGC-3 cells) than in the non-SGC cell line (MKN-74 cells) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Regarding the receptor on the fibroblasts, \u003cem\u003ePAR-2\u003c/em\u003e, encoded by \u003cem\u003eF2R-like trypsin receptor 1\u003c/em\u003e (\u003cem\u003eF2RL1\u003c/em\u003e), was expressed at a significantly higher level in CAF174 than in NF174, as also shown by the RNA sequencing data of CAFs and NFs 128\u0026ndash;130 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe interaction between mesotrypsin from SGC cells and the PAR-2/STAT-3/STC-1 axis in CAFs\u003c/h3\u003e\n\u003cp\u003eMesotrypsin affected the expression levels of PAR-2 and STC-1 in CAF174. CAF174 cells were cultured with various mesotrypsin concentrations (30, 100, 300 ng/mL). With 100 ng/mL of mesotrypsin recombinant protein, the expressions of both \u003cem\u003ePAR-2\u003c/em\u003e and \u003cem\u003eSTC-1\u003c/em\u003e mRNA were significantly higher than the control values. Furthermore, the expression of \u003cem\u003ePAR-2\u003c/em\u003e mRNA was significantly increased by CM from OCUM-12 cells, but anti-mesotrypsin antibody significantly suppressed the increase by the same CM \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn western blot analysis, the expression levels of p-STAT-3, PAR-2, and STC-1 were all upregulated by mesotrypsin stimulation in a concentration-dependent manner. However, the addition of both 100 ng/mL of recombinant human mesotrypsin and 20 nM PAR-2 inhibitor (AZ-3451) decreased these expression levels to nearly the same levels as the control values \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. This result demonstrated that in CAF174, mesotrypsin stimulation induced STAT-3 signaling through activated PAR-2, and no other receptors. The results on the side of GC cells are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D. The mRNA expression level of \u003cem\u003ePRSS3\u003c/em\u003e obtained with STC-1 for each GC cell line is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. The results of western blot analysis clarified that the mesotrypsin protein level in OCUM-12 cells was upregulated by STC-1 stimulation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. These results (both the mRNA and protein expression levels) suggested that the level of STC-1 from CAFs was upregulated by mesotrypsin from SGC cells through activated PAR-2 via STAT-3 signaling, and conversely, \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin from SGC cells was upregulated by STC-1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe relationship between the expression of mesotrypsin in GC and the clinicopathological characteristics of patients with GC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe observed the expression of mesotrypsin in the cytoplasm of GC cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The correlation between mesotrypsin expression in GC cells and clinicopathological characteristics of the patients with GC are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Among the 1,205 cases of GC patients examined in this study, 378 (31.4%) expressed a high level of mesotrypsin. Mesotrypsin expression in GC cells was significantly correlated with the following characteristics: Borrmann type 4 GC, tumor size (\u0026gt;\u0026thinsp;50 mm), tumor depth (T2\u0026ndash;T4), lymph node metastases, and pathological stage (2\u0026ndash;4). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB provides OS and RFS curves classified by mesotrypsin expression in the total series of 1,205 GC patients and the 112 patients with Borrmann type 4 GC. The 5-year OS rate was 59.5% in the patients with high mesotrypsin expression in GC cells and 73.8% in the patients with low mesotrypsin expression in GC cells. The 5-year RFS rates in the same groups of patients were 72.0% and 80.4%, respectively. Both OS and RFS of mesotrypsin-positive patients in the total series (n\u0026thinsp;=\u0026thinsp;378) were significantly poorer than those of the mesotrypsin-negative patients. Among the 112 patients with Borrmann type 4 cancer, mesotrypsin-positive patients (n\u0026thinsp;=\u0026thinsp;52) had significantly poorer OS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between Mesotrypsin expression in gastric cancer cells and clinicopathological features in 1,205 gastric cancer patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eMesotrypsin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;378)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;827)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u0026thinsp;\u0026ge;\u0026thinsp;60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(78.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(75.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.242\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(65.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e568\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(68.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.215\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(34.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(31.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMacroscopic type\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBorrmann type 4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(13.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(7.26%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOther types\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e326\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(86.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(92.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMicroscopic type\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUndifferentiated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(52.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(51.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.355\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDifferentiated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(47.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(48.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor size (\u0026ge;\u0026thinsp;50 mm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(52.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(37.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor depth (T\u0026thinsp;\u0026ge;\u0026thinsp;2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(73.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(58.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLymph node metastases (N\u0026thinsp;\u0026ge;\u0026thinsp;1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(58.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(45.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStage\u0026thinsp;\u0026ge;\u0026thinsp;2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(66.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(49.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the results of the univariate and multivariate analyses for OS after surgery in the 1,205 patients with GC. The univariate analysis revealed that OS was significantly correlated with age (\u0026ge;\u0026thinsp;60 yrs), Borrmann type 4, undifferentiated microscopic type, tumor size (\u0026gt;\u0026thinsp;50 mm), tumor depth (T2\u0026ndash;T4), lymph node metastases, the pathological stage (2\u0026ndash;4), and mesotrypsin expression in GC cells. The multivariate analysis with the Cox proportional hazards model identified the following as independent predictive parameters for the patients' OS: age (\u0026ge;\u0026thinsp;60 yrs), tumor size (\u0026gt;\u0026thinsp;50 mm), lymph node metastases, stage (2\u0026ndash;4), and mesotrypsin expression in GC cells.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUnivariate and Multivariable analysis for overall survival after surgery in 1,205 gastric cancer patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eUnivariate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eMultivariate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHazard ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHazard ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge (\u0026ge;\u0026thinsp;60)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.018\u0026ndash;1.679\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.789\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.039\u0026ndash;2.880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender (male)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.798\u0026ndash;1.554\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMacroscopic type (Borrmann type 4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.096\u0026ndash;5.078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.787\u0026ndash;2.292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.279\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMicroscopic type (Undifferentiated)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.171\u0026ndash;1.770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.743\u0026ndash;1.418\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.874\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor size (\u0026ge;\u0026thinsp;50 mm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.893\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.138\u0026ndash;4.830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.624\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.143\u0026ndash;2.307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor depth (T\u0026thinsp;\u0026ge;\u0026thinsp;2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.785\u0026ndash;6.975\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.637\u0026ndash;2.409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.528\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLymph node metastases (N\u0026thinsp;\u0026ge;\u0026thinsp;1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.622\u0026ndash;5.933\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.806\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.126\u0026ndash;2.897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStage\u0026thinsp;\u0026ge;\u0026thinsp;2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.488\u0026ndash;7.888\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.659\u0026ndash;2.843\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.400\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMesotrypsin positive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.495\u0026ndash;2.262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.039\u0026ndash;2.082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.030\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eCI: confidence interval.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur findings demonstrated that the level of \u003cem\u003ePRSS3\u003c/em\u003e from SGC cells was upregulated by stimulation with STC-1 from CAFs. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e is a schema of the interaction between SGC cells and CAFs via a mutual activation of mesotrypsin and STC-1. Our analyses revealed that (\u003cem\u003ei\u003c/em\u003e) STC-1 from CAFs stimulated cell-invasion activity and promoted EMT, especially on SGC cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e; (\u003cem\u003eii\u003c/em\u003e) mesotrypsin from SGC cells stimulated PAR-2 activation and an upregulation of STC-1 via STAT-3 signaling in CAFs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e; and (\u003cem\u003eiii\u003c/em\u003e) \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin was upregulated by STC-1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Our search of the relevant literature identified no report regarding the function of STC-1 derived from CAFs for SGC cells; our present study is thus the first to clarify the interaction between \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin from SGC cells and PAR-2/STAT-3/STC-1 axis from CAFs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStanniolcalcin-1 (STC-1) moderates serum calcium and phosphate homeostasis.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] STC-1 has been reported to be overexpressed in tissues of various types of cancer including GC[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and to promote malignant phenotypes.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] The expression of STC-1 in CAFs was observed to be higher than that in NFs in several types of cancers.[\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] To the best of our knowledge, the significance of \u003cem\u003eSTC-1\u003c/em\u003e in CAF tissues in GC has not been reported. The results of the present study demonstrated that \u003cem\u003eSTC-1\u003c/em\u003e expression in CAFs was significantly higher than that in NFs in GC tissues, and STC-1 derived from CAFs promoted invasion activity and was involved in EMT progression of SGC cells by the downregulation of \u003cem\u003eE-cadherin\u003c/em\u003e and the upregulation of \u003cem\u003eN-cadherin\u003c/em\u003e, \u003cem\u003eVimentin\u003c/em\u003e, and \u003cem\u003eslug\u003c/em\u003e. SGC exhibits a rapid infiltration of cancer cells, and the changes in the expression levels of EMT markers suggest both the suppression of cellular adhesion ability and the acquisition of mesenchymal characteristics. We suspect that the change in SGC cell morphology to spindle-shaped might be associated with invasion activity of SGC cells. STC-1 from GC cells (not CAFs) was observed to help promote metastasis via tumor angiogenesis.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] We speculated that in light of the effects of STC-1 from CAFs on SGC cells observed in the present study, STC-1 might affect not only SGC cells but also the cancer microenvironment.\u003c/p\u003e \u003cp\u003eOur findings revealed that the expression of \u003cem\u003eSTC-1\u003c/em\u003e mRNA tended to increase with CM from SGC cells more than with CM from non-SGC cells. These results suggest that the factors from OCUM-12 cells might mediate the conversion of NFs to CAFs. It has been reported that activated STAT-3 signaling induced STC-1 secretion,[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] contributing to cancer progression.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] In contrast, our present study's RNA sequencing data revealed that \u003cem\u003eF2RL1\u003c/em\u003e was significantly expressed in CAFs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cb\u003eSupplementary Table\u0026nbsp;2)\u003c/b\u003e, and this gene encodes PAR-2, which is one type of G protein-coupled receptor (GPCR). In addition, endogenous serine proteases such as trypsin and tryptase specifically activate PAR-2 by cleaving the extracellular N-terminus to expose its tethered ligand domain.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] These findings might indicate that STC-1 from CAFs is regulated by PAR-2 on CAFs via activated STAT-3 signaling, and that serine protease derived from SGC cells might upregulate STC-1 from CAFs through PAR-2.\u003c/p\u003e \u003cp\u003eThere are several serine proteases, and we focused on \u003cem\u003eProtease serine 3\u003c/em\u003e (\u003cem\u003ePRSS3\u003c/em\u003e), a component of a trypsin mRNA isoform. Unlike cationic trypsinogen encoded by \u003cem\u003ePRSS1\u003c/em\u003e and anionic trypsinogen encoded by \u003cem\u003ePRSS2\u003c/em\u003e, mesotrypsin encoded by \u003cem\u003ePRSS3\u003c/em\u003e contributes only 3%\u0026ndash;10% of the trypsin activity in human pancreatic fluid.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] Mesotrypsin has been recognized as a molecule that is associated with tumor progression in several types of cancer,[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] but little is known about the regulation of mesotrypsin expression, downstream signaling, and receptor selectivity. Especially, trypsin activates PAR-2 by cleaving,[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] but the question of whether mesotrypsin acts on PAR-2 remains a matter of discussion.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] This contradiction might be due to multiple factors such as the differences in PAR-2 expression, cell types used, and the effects of the extracellular matrix.\u003c/p\u003e \u003cp\u003eThe results of our present investigation indicate that STC-1 from CAFs is upregulated by mesotrypsin from SGC cells through activated PAR-2 on CAFs via STAT-3 signaling, and conversely, \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin from SGC cells is upregulated by STC-1. \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin and PAR-2/STAT-3/STC-1 might interact with each other. It has been reported that STAT-3 is related to the proliferation and invasion of tumor cells through cyclin D1[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] or mircoRNA-499-5p activation.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] STAT-3 is frequently activated in CAFs in various types of cancer including GC.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] A positive activating interaction between cancer cells and CAFs via STAT-3 signaling might be essential to the invasion of cancer cells.\u003c/p\u003e \u003cp\u003eOur immunohistochemical examination of 1,205 cases of GC showed that high mesotrypsin expression contributed to a significantly poor prognosis (both OS and RFS). Corresponding to the report by Wang \u003cem\u003eet al.\u003c/em\u003e,[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] our study demonstrated high mesotrypsin expression and lymph node metastases were common independent prognostic factors for OS. It has been observed that \u003cem\u003ePRSS3\u003c/em\u003e/Mesotrypsin promoted the proliferation, invasion, and migration activities of GC cells, displaying properties of an oncogene.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] We observed that mesotrypsin contributes to not only the maintenance of stemness of GC cells and the promotion of autonomic tumor progression such as lymph node metastases; it also indirectly enhances invasion activity of SGC cells via an upregulation of STC-1. These findings suggested that mesotrypsin might be a crucial factor for the invasion of cancer cells by affecting the whole SGC microenvironment along with stromal tissues. STC-1 in CAFs and/or mesotrypsin in SGC cells might be a promising therapeutic target for SGC.\u003c/p\u003e \u003cp\u003eWe should acknowledge some study limitations. First, STC-1 has been reported to act on receptors such as VEGF-R2 and Notch-1.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] However, VEGF-A2 did not affect the expression level of OCUM-12 cells in our study (data not shown). Trypsin effectively prevents autolysis and preserves esterase activity for a prolonged time under Ca\u003csup\u003e2+\u003c/sup\u003e binding conditions.[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] There might be a molecular interaction between STC-1 and \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin through calcium-associated receptors. Second, we did not perform \u003cem\u003ein vivo\u003c/em\u003e experiments to investigate the interaction between \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin from SGC cells and STC-1 from CAFs. To clarify the therapeutic effects of mesotrypsin or an STC-1 inhibitor on SGC cells, \u003cem\u003ein vivo\u003c/em\u003e experiments using a mouse SGC model might be informative.\u003c/p\u003e \u003cp\u003eIn conclusion, the results of this study suggest that \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin from SGC cells and a PAR-2/STAT-3/STC-1 axis in CAFs might interact and promote invasion activity of SGC cells. Our findings might provide the foundation for a promising therapeutic target for SGC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAFs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecancer associated fibroblasts\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econditioned medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eF2RL1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eF2R Like Trypsin Receptor 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egastric cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNFs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enormal fibroblasts\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAR-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprotease-activated receptor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ePRSS3\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eprotease serine 3\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTAT-3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eactivator of transcription 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSGC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003escirrhous-type gastric cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTC-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estanniocalcin-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA sequencing datasets generated during the current study are available in the in the DDBJ Sequenced Read Archive under the accession numbers from DRR963239 to DRR963244. The other data can be obtained from the corresponding author or the Ethics Committee ([email protected]) on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate Rachel Feldman of KN International, Inc. for assistance with editing the manuscript for English grammar and usage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTS (Takashi Sakuma) and MY designed and performed the experiments, and co-wrote the manuscript. HG, DM, CF, HN, YY, and HK accumulated the data. TS (Tomoya Sano) and DI prepared and collected GC tissue specimens. KM suggested and co-designed the study. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethics Committee of Osaka Metropolitan University (approval number 0924, 2022-077). Patients provided written informed consent. This retrospective study was conducted in accordance with the principles of the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegistry and the Registration No. of the study/trial: 0924, 2022-077.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnimal Studies: N/A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare regarding this study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYook JH, Oh ST, Kim BS. Clinicopathological Analysis of Borrmann Type IV Gastric Cancer. Cancer Res Treat. 2005;37:87\u0026ndash;91.\u003c/li\u003e\n \u003cli\u003eNashimoto A, Akazawa K, Isobe Y, Miyashiro I, Katai H, Kodera Y, Tsujitani S, Seto Y, Furukawa H, Oda I, et al. Gastric cancer treated in 2002 in Japan: 2009 annual report of the JGCA nationwide registry. Gastric Cancer. 2013;16:1\u0026ndash;27.\u003c/li\u003e\n \u003cli\u003eMiki Y, Yashiro M, Okuno T, Kitayama K, Masuda G, Hirakawa K, Ohira M. CD9-positive exosomes from cancer-associated fibroblasts stimulate the migration ability of scirrhous-type gastric cancer cells. Br J Cancer. 2018;118:867\u0026ndash;77.\u003c/li\u003e\n \u003cli\u003eKinoshita H, Yashiro M, Fukuoka T, Hasegawa T, Morisaki T, Kasashima H, Masuda G, Noda S, Hirakawa K. Diffuse-type gastric cancer cells switch their driver pathways from FGFR2 signaling to SDF1/CXCR4 axis in hypoxic tumor microenvironments. Carcinogenesis. 2015;36:1511\u0026ndash;20.\u003c/li\u003e\n \u003cli\u003eKushiyama S, Yashiro M, Yamamoto Y, Sera T, Sugimoto A, Nishimura S, Togano S, Kuroda K, Okuno T, Miki Y, et al. Dipeptidyl Peptidase-4 from Cancer-associated Fibroblasts Stimulates the Proliferation of Scirrhous-type Gastric Cancer Cells. Anticancer Res. 2022;42:501\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eFuyuhiro Y, Yashiro M, Noda S, Kashiwagi S, Matsuoka J, Doi Y, Kato Y, Hasegawa T, Sawada T, Hirakawa K. Upregulation of cancer-associated myofibroblasts by TGF-\u0026beta; from scirrhous gastric carcinoma cells. Br J Cancer. 2011;105:996\u0026ndash;1001.\u003c/li\u003e\n \u003cli\u003eKim KT, Lee MH, Shin SJ, Cho I, Kuk JC, Yun J, Choi YY. Decorin as a key marker of desmoplastic cancer-associated fibroblasts mediating first-line immune checkpoint blockade resistance in metastatic gastric cancer. Gastric Cancer. 2025;28:12\u0026ndash;26.\u003c/li\u003e\n \u003cli\u003eOzmen E, Demir TD, Ozcan G. Cancer-associated fibroblasts: protagonists of the tumor microenvironment in gastric cancer. Frontiers in Molecular Biosciences. 2024;11:1340124.\u003c/li\u003e\n \u003cli\u003eZhang Y, Chen H, Zhang W, Zhou H. Identification of cancer-associated fibroblast-related Ectodysplasin-A as a novel indicator for prognosis and immune response in gastric cancer. Heliyon. 2024;10:e34005.\u003c/li\u003e\n \u003cli\u003eNurmik M, Ullmann P, Rodriguez F, Haan S, Letellier E. In search of definitions: Cancer-associated fibroblasts and their markers. International Journal of Cancer. 2020;146:895\u0026ndash;905.\u003c/li\u003e\n \u003cli\u003eNakazawa K, Yashiro M, Hirakawa K. Keratinocyte Growth Factor Produced by Gastric Fibroblasts Specifically Stimulates Proliferation of Cancer Cells from Scirrhous Gastric Carcinoma. Cancer Res. 2003;63:8848\u0026ndash;52.\u003c/li\u003e\n \u003cli\u003eYashiro M, Kuroda K, Masuda G, Okuno T, Miki Y, Yamamoto Y, Sera T, Sugimoto A, Kushiyama S, Nishimura S, et al. Clinical difference between fibroblast growth factor receptor 2 subclass, type IIIb and type IIIc, in gastric cancer. Sci Rep. 2021;11:4698.\u003c/li\u003e\n \u003cli\u003eKato Y, Yashiro M, Noda S, Tendo M, Kashiwagi S, Doi Y, Nishii T, Matsuoka J, Fuyuhiro Y, Shinto O, et al. Establishment and characterization of a new hypoxia-resistant cancer cell line, OCUM-12/Hypo, derived from a scirrhous gastric carcinoma. Br J Cancer. 2010;102:898\u0026ndash;907.\u003c/li\u003e\n \u003cli\u003ePimentel H, Bray NL, Puente S, Melsted P, Pachter L. Differential analysis of RNA-seq incorporating quantification uncertainty. Nat Methods. 2017;14:687\u0026ndash;90.\u003c/li\u003e\n \u003cli\u003eBray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016;34:525\u0026ndash;7.\u003c/li\u003e\n \u003cli\u003eZhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, Benner C, Chanda SK. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun. 2019;10:1523.\u003c/li\u003e\n \u003cli\u003ePfaffl MW. A new mathematical model for relative quantification in real-time RT\u0026ndash;PCR. Nucleic Acids Res. 2001;29:e45.\u003c/li\u003e\n \u003cli\u003eMookerjee S, Whitley G, Banerjee D. Stanniocalcin-1: A Novel Mediator in Diabetic Kidney Disease and Cardiovascular Disease. Kidney International Reports. 2024;10:321\u0026ndash;7.\u003c/li\u003e\n \u003cli\u003eZhao F, Yang G, Feng M, Cao Z, Liu Y, Qiu J, You L, Zheng L, Zhang T, Zhao Y. Expression, function and clinical application of stanniocalcin-1 in cancer. Journal of Cellular and Molecular Medicine. 2020;24:7686\u0026ndash;96.\u003c/li\u003e\n \u003cli\u003eFang Z, Tian Z, Luo K, Song H, Yi J. Clinical significance of stanniocalcin expression in tissue and serum of gastric cancer patients. Chinese Journal of Cancer Research. 2014;26:602\u0026ndash;10.\u003c/li\u003e\n \u003cli\u003eBai S, Zhao Y, Chen W, Peng W, Wang Y, Xiong S, Aruna, Li Y, Yang Y, Chen S, et al. The stromal-tumor amplifying STC1-Notch1 feedforward signal promotes the stemness of hepatocellular carcinoma. J Transl Med. 2023;21:236.\u003c/li\u003e\n \u003cli\u003eAvalle L, Raggi L, Monteleone E, Savino A, Viavattene D, Statello L, Camperi A, Stabile SA, Salemme V, De Marzo N, et al. STAT3 induces breast cancer growth via ANGPTL4, MMP13 and STC1 secretion by cancer associated fibroblasts. Oncogene. 2022;41:1456\u0026ndash;67.\u003c/li\u003e\n \u003cli\u003eYang Y, Yin S, Li S, Chen Y, Yang L. Stanniocalcin 1 in tumor microenvironment promotes metastasis of ovarian cancer. Onco Targets Ther. 2019;12:2789\u0026ndash;98.\u003c/li\u003e\n \u003cli\u003ePe\u0026ntilde;a C, C\u0026eacute;spedes MV, Lindh MB, Kiflemariam S, Mezheyeuski A, Edqvist PH, H\u0026auml;ggl\u0026ouml;f C, Birgisson H, Bojmar L, Jirstrom K, et al. STC1 expression by cancer-associated fibroblasts drives metastasis of colorectal cancer. Cancer Res. 2013;73:1287\u0026ndash;97.\u003c/li\u003e\n \u003cli\u003eHe LF, Wang TT, Gao QY, Zhao GF, Huang YH, Yu LK, Hou YY. Stanniocalcin-1 promotes tumor angiogenesis through up-regulation of VEGF in gastric cancer cells. J Biomed Sci. 2011;18:39.\u003c/li\u003e\n \u003cli\u003eHuang J, Wang X, Wen J, Zhao X, Wu C, Wang L, Cao X, Dong H, Xu X, Huang F, et al. Gastric cancer cell-originated small extracellular vesicle induces metabolic reprogramming of BM-MSCs through ERK-PPAR\u0026gamma;-CPT1A signaling to potentiate lymphatic metastasis. Cancer Cell Int. 2023;23:87.\u003c/li\u003e\n \u003cli\u003eYu H, Lee H, Herrmann A, Buettner R, Jove R. Revisiting STAT3 signalling in cancer: New and unexpected biological functions. Nature Reviews Cancer. 2014;14:736\u0026ndash;46.\u003c/li\u003e\n \u003cli\u003ePark MK, Cho MK, Kang SA, Park HK, Kim YS, Kim KU, Ahn SC, Kim DH, Yu HS. Protease-activated receptor 2 is involved in Th2 responses against Trichinella spiralis infection. Korean Journal of Parasitology. 2011;49:235\u0026ndash;43.\u003c/li\u003e\n \u003cli\u003eWang F, Hu YL, Feng Y, Guo YB, Liu YF, Mao QS, Xue WJ. High-level expression of PRSS3 correlates with metastasis and poor prognosis in patients with gastric cancer. J Surg Oncol. 2019;119:1108\u0026ndash;21.\u003c/li\u003e\n \u003cli\u003eHockla A, Miller E, Salameh MA, Copland JA, Radisky DC, Radisky ES. PRSS3/mesotrypsin is a therapeutic target for metastatic prostate cancer. Molecular Cancer Research. 2012;10:1555\u0026ndash;66.\u003c/li\u003e\n \u003cli\u003eHan S, Lee CW, Trevino JG, Hughes SJ, Sarosi GA. Autocrine Extra-Pancreatic Trypsin 3 Secretion Promotes Cell Proliferation and Survival in Esophageal Adenocarcinoma. PLoS One. 2013;8:e76667.\u003c/li\u003e\n \u003cli\u003eRolland-Fourcade C, Denadai-Souza A, Cirillo C, Lopez C, Jaramillo JO, Desormeaux C, Cenac N, Motta JP, Larauche M, Tach\u0026eacute; Y, et al. Epithelial expression and function of trypsin-3 in irritable bowel syndrome. Gut. 2017;66:1767\u0026ndash;78.\u003c/li\u003e\n \u003cli\u003eGrishina Z, Ostrowska E, Halangk W, Sahin-T\u0026oacute;th M, Reiser G. Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): Mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1. Br J Pharmacol. 2005;146:990\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eArshad S, Naveed M, Ullia M, Javed K, Butt A, Khawar M, Amjad F. Targeting STAT-3 signaling pathway in cancer for development of novel drugs: Advancements and challenges. Genetics and Molecular Biology. 2020;43:e20180160.\u003c/li\u003e\n \u003cli\u003eYang Y Lou, Liu P, Li D, Yang Q, Li B, Jiang XJ. Stat-3 signaling promotes cell proliferation and metastasis of gastric cancer through PDCD4 downregulation. Kaohsiung Journal of Medical Sciences. 2020;36:244\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eZhao D, Zhang J, Zhang L, Wu Q, Wang Y, Zhang W, Xiao Y, Chen J, Zhan Q. PAFR/Stat3 axis maintains the symbiotic ecosystem between tumor and stroma to facilitate tumor malignancy. Acta Pharm Sin B. 2023;13:694\u0026ndash;708.\u003c/li\u003e\n \u003cli\u003eLiu A, Li Y, Lu S, Cai C, Zou F, Meng X. Stanniocalcin 1 promotes lung metastasis of breast cancer by enhancing EGFR\u0026ndash;ERK\u0026ndash;S100A4 signaling. Cell Death Dis. 2023;14:395.\u003c/li\u003e\n \u003cli\u003ePatel CK, Mukherjee TK. Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca2+-Mediated Activation of Esterase Activity. Biomacromolecules. 2024;25:6082\u0026ndash;92.\u003c/li\u003e\n\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":"","lastPublishedDoi":"10.21203/rs.3.rs-9424453/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9424453/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eScirrhous gastric cancer (SGC) is characterized by rapid cancer-cell infiltration with extensive stromal fibrosis and has a poor prognosis compared to the other types of gastric cancer (GC). SGC cells and cancer-associated fibroblasts (CAFs) might interact with each other in the tumor microenvironment and contribute to progressive invasion. We investigated these cells' interaction with functional CAFs markers in SGC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eFour pairs of CAFs and normal fibroblasts (NFs) and three GC cell lines were used. We examined the CAF marker by RNA sequencing and evaluated the effects of CAFs on SGC cells by invasion assay and morphologic analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIn silico analysis indicated that stanniocalcin-1 (STC-1) was a CAF marker. The \u003cem\u003eSTC-1\u003c/em\u003emRNA expression in CAFs was significantly higher than in NFs, and was significantly upregulated by the supernatant of an SGC cell line (OCUM-12 cells). STC-1 from CAFs was associated with invasion activity and epithelial-mesenchymal transition of SGC cells. Mesotrypsin (encoded by \u003cem\u003ePRSS3\u003c/em\u003e) from OCUM-12 cells activated protease-activated receptor 2 (PAR-2) and upregulated STC-1 expression in CAFs via signal transducer and activator of transcription 3 (STAT-3) pathway. In turn, \u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin was upregulated by STC-1. High mesotrypsin expression contributed to significantly poorer survival in GC patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003e\u003cem\u003ePRSS3\u003c/em\u003e/mesotrypsin from SGC cells and the PAR-2/STAT-3/STC-1 axis in CAFs might contribute to the mutual interaction between SGC cells and CAFs that promotes SGC invasion.\u003c/p\u003e","manuscriptTitle":"PRSS3/Mesotrypsin from Scirrhous Gastric Cancer Cells and Stanniocalcin-1 from Cancer-associated Fibroblasts Mutually Promote Invasion Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 08:33:46","doi":"10.21203/rs.3.rs-9424453/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-10T04:12:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262249170034010271107329914190795845613","date":"2026-04-30T07:05:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-30T06:45:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T06:43:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-27T05:27:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-24T08:16:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2026-04-24T07:29:32+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":"d0d617b5-d642-471c-91cf-d8b1ac983f7f","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-10T04:12:55+00:00","index":36,"fulltext":""},{"type":"reviewerAgreed","content":"262249170034010271107329914190795845613","date":"2026-04-30T07:05:36+00:00","index":35,"fulltext":""},{"type":"reviewersInvited","content":"20","date":"2026-04-30T06:45:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T06:43:06+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T08:33:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 08:33:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9424453","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9424453","identity":"rs-9424453","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}