Interaction with peritoneal mesothelial cells inhibits the growth of gastric cancer organoids and induces drug resistance

Interaction with peritoneal mesothelial cells inhibits the growth of gastric cancer organoids and induces drug resistance | 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 Short Report Interaction with peritoneal mesothelial cells inhibits the growth of gastric cancer organoids and induces drug resistance Masahiro Inoue, Hiroyuki Uematsu, Shota Shimizu, Kunishige Onuma, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7229795/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Nov, 2025 Read the published version in Human Cell → Version 1 posted 5 You are reading this latest preprint version Abstract The prognosis of gastric cancer with peritoneal dissemination is poor because of its resistance to chemotherapy. To create an in vitro model of peritoneal metastases, cancer organoids were established from ascites fluid of patients with peritoneal metastases of gastric cancer. The histological characteristics of the tumors were preserved in the organoids. A co-culture system was established by overlaying human-derived mesothelial cells on gastric cancer organoids embedded in type I collagen, mimicking peritoneal dissemination foci. When co-cultured with mesothelial cells, the proliferation of ascites-derived gastric cancer organoids and other primary gastric cancer organoids was suppressed. Soluble factors derived from mesothelial cells were involved in suppressing cell proliferation. Organoids in co-culture showed reduced sensitivity to paclitaxel. This co-culture model may provide a useful platform for studying drug resistance mechanisms in the microenvironment of gastric cancer peritoneal metastases. organoid gastric cancer peritoneal dissemination co-culture mesothelial cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Peritoneal dissemination is found in 40–60% of patients with gastric cancer 1 , and in approximately 14% 2 and 18–26% 3 with synchronous peritoneal metastasis. The median survival time of patients with peritoneal dissemination is approximately 4 months, indicating a very poor prognosis 2 . The main treatment option for gastric cancer peritoneal dissemination is systemic chemotherapy; however, the response rate for peritoneal lesions remains low at 14–25% 4 . In contrast, the response rates of primary tumors and lymph node lesions to the same treatment are relatively high at 71% and 79%, respectively, indicating that peritoneal dissemination lesions are resistant to chemotherapy. Recently, intraperitoneal chemotherapy has been attempted for gastric cancer with peritoneal metastasis, with an improved response rate of 27–49% 5–7 . However, as evidenced by the low response rate and rarity of complete remission, the therapeutic effect remains limited, and the development of new treatment strategies is urgently needed. One factor that contributes to treatment resistance is the presence of a unique tumor microenvironment 8 in peritoneal dissemination. To date, many studies have analyzed cancer cell adhesion and invasion using models in which cancer cell lines are layered on the peritoneal mesothelium 9 , 10 . To evaluate the therapeutic efficacy and drug resistance of established peritoneal metastases and develop new treatments, it is necessary to establish an in vitro model that more accurately reproduces the tumor microenvironment and reflects the clinical characteristics of peritoneal dissemination. In recent years, patient-derived organoids have attracted attention as three-dimensional culture models that can be applied in personalized medicine and drug sensitivity evaluation because of their ability to retain the histological/molecular biological characteristics of the original tumor 11 , 12 . In this study, we established organoids from ascites of a patient with peritoneal dissemination of gastric cancer and constructed a novel co-culture model with human mesothelial cells. The effects of interactions with mesothelial cells on cancer organoid proliferation and drug sensitivity were evaluated. Materials and methods Patient samples This study was approved by the Institutional Ethics Committees of Kyoto University (R1575, R1671), Tottori University (21A177), and Osaka International Cancer Institute (1803125402). Tumor and ascites specimens were collected with informed consent from patients at Tottori University and Osaka International Cancer Institute. The clinical data of the four patients with gastric cancer are shown in Tables S1 and S2. Organoid preparation Organoids of MK30 and MK35 were prepared using the CTOS method 13 . Organoids of TUG6 and TUG7 were prepared by mincing without enzymatic digestion. Organoids were cultured in a previously reported medium 14 with some modifications (Table S3 ). Organoid culture The organoids were maintained and passaged according to previously reported methods 15 . For cell viability assays, 50 organoids (40–100 µm) were embedded in type I collagen (Nitta Gelatin, Osaka, Japan) in each well of a 96-well plate (Thermo Fisher Scientific, Waltham, MA, USA). Seven days later, ATP levels were measured using CellTiter-Glo (Promega, Madison, WI, USA) with a GloMax Discover microplate reader (Promega) and normalized to the organoid area on day 0. Culture of HOMC-B1 cells HOMC-B1 cells 16 were obtained from Riken Bio-Resource Research Center (Ibaraki, Japan). HOMC-B1 cells (1 × 10³ cells/well in a 96-well plate) were seeded, cultured for 7 days in RPMI-1640 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% FBS (Thermo Fisher Scientific), and cell survival rates were measured by ATP assay according to the above procedure. Conditioned medium (CM) of HOMC-B1 cells was collected after 24-h culture in the co-culture medium. Adhesion assay Adhesion assays were conducted according to a previously reported method 17 . Control IgG (Thermo Fisher Scientific) or ITGB1-neutralizing antibodies (Sigma-Aldrich, St Louis, MO, USA) (10 µg/mL) were added to the organoids 1 h before the start of co-culture. HOMC-B1 cells were seeded on type IA collagen, and the pre-treated organoids were added 24 h later with 5 µg/mL of the ITGB1-neutralizing antibody. After 48 h, non-adherent organoids were washed out, and the adhesion rate of the organoids to HOMC-B1 cells was calculated. Co-culture of gastric cancer organoids and HOMC-B1 cells Fifty organoids were suspended in type I collagen, and 10 µL of the suspension was seeded into a single well of a 96-well plate on ice. The plates were inverted and left on ice for 5 min. The plates were returned to their original position, incubated at 37°C for 10 min, and then 4 × 10⁴ HOMC-B1 cells suspended in 100 µL of co-culture medium were seeded on top. Organoid growth was evaluated using the area ratio from day 4 to day 0. For the cell death assay, the organoids were stained with 10 µg/mL propidium iodide (PI) (Thermo Fisher Scientific) at 37°C for 10 min, washed twice, and imaged. PI-positive areas were quantified using the ImageJ/Fiji software (NIH, Bethesda, MD, USA). Chemosensitivity assay Organoid drug sensitivity assays were conducted according to previously reported methods 18 . Paclitaxel (Nippon Kayaku, Tokyo, Japan) was added 24 h after seeding the HOMC-B1 cells, and the cells were cultured in the co-culture medium for 4 days. Organoid growth was evaluated using the area ratio from day 4 to day 0. Animal experiment All animal studies were approved by the Institutional Animal Care and Use Committee of Kyoto University (18564). In the peritoneal dissemination model, organoids (diameter 40–100 µm) equivalent to 5 × 10⁶ cells were suspended in 500 µL of HBSS (Fujifilm Wako Pure Chemical Corporation) and injected into the peritoneal cavity of 13-week-old male non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (CLEA Japan, Tokyo, Japan) using a 27-gauge needle (Terumo, Tokyo, Japan). Morphological analyses Paraffin block preparation of the organoids was performed according to previously described methods with some modifications 17 . For the analysis of co-culture, organoids and HOMC-B1 cells were cultured in culture inserts (Corning, Corning, NY, USA). Four days later, the cells were fixed with 10% formaldehyde (Fujifilm Wako Pure Chemical Corporation), the membranes were removed, and the cells were embedded in paraffin. Hematoxylin and eosin (HE) staining, Alcian blue staining, and immunohistochemistry (IHC) were performed according to previously reported methods 13 . The following primary antibodies were used for IHC: anti-E-cadherin (BD Biosciences, San Jose, CA, USA, Cat#610181, 1:500), anti-cytokeratin 8 (DSHB, Iowa City, IA, USA, 1:100), anti-Ki67 (Cell Signaling Technology, Beverly, MA, USA, Cat#9027, 1:400), and anti-CEA (Cell Signaling Technology, Cat#2383, 1:200). The nuclei were stained with DAPI, 4',6-diamidino-2-phenylindole (Thermo Fisher Scientific). Images were obtained using an Olympus BX50 fluorescence microscope and CellSens standard image analysis software (Olympus). Statistical analysis All statistical analyses were performed using GraphPad Prism 10.5.0. Student's t-test was used for single comparisons, and Tukey's multiple comparison test was performed for multiple comparisons after one-way analysis of variance (ANOVA). When the data did not follow a normal distribution, the Mann-Whitney U test was used. A p-value < 0.05 was considered statistically significant. Result Organoids derived from ascites of a patient with peritoneal dissemination of gastric cancer reproduced clinical histological features Organoids (TUG7) were established from the ascites obtained from a patient with peritoneal metastases of gastric cancer (Table S1 , Fig. 1A). Disseminated lesions were observed when the organoids were transplanted into the peritoneal cavity of immunodeficient mice. Histological features were compared among the patient's peritoneal metastases, cancer organoids, and mouse peritoneal metastases (Fig. 1B). HE staining revealed a mixture of undifferentiated cancer cells and signet ring cells in all samples. Alcian blue staining confirmed mucus production. The staining patterns of CEA (a tumor marker), E-cadherin, CK8 (an epithelial marker), and Ki67 (a proliferation marker) were generally preserved in the organoids and xenografts. These results suggest that these models are clinically relevant. Optimization of co-culture conditions for peritoneal mesothelial cells and cancer organoids Culture conditions for the co-culture model were examined. The co-culture medium was prepared by adding additives from the organoid culture medium (excluding A83-01) to the mesothelial cell medium (RPMI-1640, 10% FBS) of the human mesothelial cell line HOMC-B1 (Table S3 ). In the co-culture medium, the growth of HOMC-B1 cells was promoted, and cell morphology was maintained (Fig. 2A). On the other hand, no changes in organoid proliferation or morphology were observed between the organoid culture and co-culture media (Fig. 2B). Next, we evaluated the co-culture conditions using adhesion assays 19 . Integrin β1 (ITGB1) plays an important role in the adhesion of gastric cancer cells to mesothelial cells 9 , 20 . The ITGB1-neutralizing antibody significantly inhibited the adhesion of TUG7 organoids to mesothelial cells (Fig. 2C). Therefore, the optimized co-culture medium may enable the functional evaluation of the interactions between gastric cancer organoids and mesothelial cells. Inhibition of cancer organoid growth by co-culture with mesothelial cells The peritoneal dissemination model was constructed by embedding the organoids in type I collagen and layering HOMC-B1 cells (Fig. 3A, 3B). Using this model, the proliferation of organoids derived from gastric cancer ascites (TUG7) and organoids derived from primary gastric cancer tumors (TUG6, MK30, and MK35) was investigated (Fig. 3C). Organoid proliferation and cell death were examined using Ki67 and PI staining, respectively. Co-culture resulted in a decrease in Ki67-positive cells and an increase in PI-positive cells (Fig. 3D-3F, Fig. S1 A, S1B). Thus, in this co-culture model, the proliferation of gastric cancer organoids was suppressed, and cell death was induced by co-culture with mesothelial cells. To elucidate the cause of growth inhibition in the co-culture, TUG7 cells were cultured in various media, as shown in Fig. 3G. TUG7 growth in Medium A was similar to that in the co-culture medium (control) (Fig. 3H). In contrast, TUG7 expression was significantly inhibited in Medium B containing CM of HOMC-B1 cells (Fig. 3H). These results suggest that the inhibition of cancer organoid growth by co-culture with peritoneal mesothelial cells was mediated by soluble factors secreted from mesothelial cells. Paclitaxel sensitivity was reduced in the peritoneal dissemination model Paclitaxel sensitivity assays were performed using a peritoneal dissemination model in the four gastric cancer organoid lines. All organoids co-cultured with HOMC-B1 cells showed reduced sensitivity to paclitaxel compared with those cultured alone (Fig. 4A-4D). These results indicated that gastric cancer peritoneal metastasis was clinically more resistant to chemotherapy, suggesting that this model may be useful as an in vitro model for evaluating the efficacy of chemotherapy in gastric cancer peritoneal metastasis. Discussion In this study, we established organoids from the ascites of a patient with gastric cancer and peritoneal metastasis. Organoids and their xenografts generally maintained their morphological characteristics despite some differences in the expression levels of differentiation markers. In cases of morphologically mixed types of cancer, such as in the present case, some cancer cells may be selected during the establishment of the organoids. The organoids were used in a co-culture model with a human mesothelial cell line. In conventional co-culture models, gastric cancer cell lines are seeded on mesothelial cells, and adhesion assays 9 , 10 or transwell chamber-based invasion assays 21 , 22 are performed. However, these conventional models are based on two-dimensional culture and represent the early stages of metastasis. Furthermore, most studies have used established cell lines, which have limitations in modelling three-dimensional cancer tissues 13 . The model used in this study is unique in that it uses organoids that retain the three-dimensional (3D) structure and diversity of a patient's tumor. Furthermore, this model may represent the three-dimensional structure of established peritoneal metastases and reflect the interactions between cancer and mesenchymal cells. Using this model, we have demonstrated for the first time, to the best of our knowledge, that interactions between gastric cancer cells and mesothelial cells increase drug resistance. As paclitaxel is a proliferation-dependent drug, resistance may be due to reduced cell proliferation. In this study, we demonstrated that the CM from HOMC-B1 cells inhibited organoid growth. Soluble factors secreted from co-cultured organoids or mesothelial cells may affect tumor cell growth, although the possibility of nutrient depletion owing to co-culture cannot be ruled out. In this study, we did not attempt to identify soluble factors that inhibit organoid proliferation. In the future, these liquid factors (e.g., TGF-β 23 , 24 secreted from mesothelial cells) are expected to be identified, and elucidation of the expression dynamics of the humoral factors involved in these interactions will contribute to the development of new therapeutic approaches. In conclusion, this 3D co-culture model may be useful for analyzing the influence of the tumor microenvironment on established peritoneal metastasis through interactions with mesothelial cells, and is expected to contribute to elucidating drug resistance mechanisms and evaluating new treatment strategies in clinical practice. Declarations Acknowledgements We thank the members of Inoue Laboratory for their helpful discussions. Author contributions HU, TM, YF and MI designed the study. HU and YS performed the experiments. MI supervised the experiments. SS, TM, and YF acquired the clinical samples and data. HU and MI conducted the data analysis. HU and MI wrote the manuscript. HU, KO, RC, YS and YF reviewed and revised the manuscript. All authors read and approved the final manuscript. • Funding This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan, 22K08893 (T.M., Y.F., M.I.); by a collaboration grant from Kyoto University-KBBM (M.I,). • Conflicts of interest/Competing interests: K.O., R.C, and M.I. are members of the Department of Clinical Bio-resource Research and Development at Kyoto University, which is sponsored by KBBM, Inc. H.U. is an employee of KBBM Inc. The other authors declare no conflict of interest. This work was supported in part by the collaboration grant from KBBM-Kyoto University. • Ethics approval This study was approved by the institutional ethics committees of Kyoto University (R1575, R1671), Tottori University (21A177), and Osaka International Cancer Institute (1803125402). • Informed consent Written informed consent was obtained from participants. This study was performed in accordance with the Declaration of Helsinki. References Manzanedo I, Pereira F, Perez-Viejo E, Serrano A. Gastric Cancer with Peritoneal Metastases: Current Status and Prospects for Treatment. Cancers (Basel). 2023; 15. Thomassen I, van Gestel YR, van Ramshorst B, et al. Peritoneal carcinomatosis of gastric origin: a population-based study on incidence, survival and risk factors. Int J Cancer. 2014; 134:622-8. Koemans WJ, Lurvink RJ, Grootscholten C, Verhoeven RHA, de Hingh IH, van Sandick JW. Synchronous peritoneal metastases of gastric cancer origin: incidence, treatment and survival of a nationwide Dutch cohort. Gastric Cancer. 2021; 24:800-09. Yonemura Y, Endou Y, Sasaki T, et al. Surgical treatment for peritoneal carcinomatosis from gastric cancer. Eur J Surg Oncol. 2010; 36:1131-8. Kobayashi S, Kamiya K, Miki T, et al. Association Between Changes in Skeletal Muscle Quality and Prognosis in Postoperative Patients with Early Gastric Cancer. Ann Surg Oncol. 2024; 31:7722-29. Tu L, Zhang W, Ni L, et al. Study of SOX combined with intraperitoneal high-dose paclitaxel in gastric cancer with synchronous peritoneal metastasis: A phase II single-arm clinical trial. Cancer Med. 2023; 12:4161-69. Vatandoust S, Bright T, Roy AC, et al. Phase 1 trial of intraperitoneal paclitaxel in combination with intravenous cisplatin and oral capecitabine in patients with advanced gastric cancer and peritoneal metastases (IPGP study). Asia Pac J Clin Oncol. 2022; 18:404-09. 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Am J Pathol. 2016; 186:899-911. Ito Y, Kondo J, Masuda M, et al. Ex vivo chemosensitivity assay using primary ovarian cancer organoids for predicting clinical response and screening effective drugs. Human Cell. 2022; 36:752-61. Kawata M, Kondo J, Onuma K, et al. Polarity switching of ovarian cancer cell clusters via SRC family kinase is involved in the peritoneal dissemination. Cancer Sci. 2022; 113:3437-48. Takatsuki H, Komatsu S, Sano R, Takada Y, Tsuji T. Adhesion of gastric carcinoma cells to peritoneum mediated by alpha3beta1 integrin (VLA-3). Cancer Res. 2004; 64:6065-70. Kang X, Li W, Liu W, et al. LIMK1 promotes peritoneal metastasis of gastric cancer and is a therapeutic target. Oncogene. 2021; 40:3422-33. Wang L, Xu Z, Hu C, et al. Peritoneal metastatic gastric carcinoma cells exhibit more malignant behavior when co-cultured with HMrSV5 cells. Aging (Albany NY). 2020; 12:3238-48. Liu H, Zhu Y, Zhu H, et al. Role of transforming growth factor beta1 in the inhibition of gastric cancer cell proliferation by melatonin in vitro and in vivo. Oncol Rep. 2019; 42:753-62. Mutsaers SE. Mesothelial cells: their structure, function and role in serosal repair. Respirology. 2002; 7:171-91. Supplementary Files FigureS1.tif Figure S1. Suppression of proliferation and induction of cell death in cancer organoids by co-culture with HOMC-B1 cells. (A, B) Quantitative analysis of the Ki67-positive rates (A) and PI-positive areas (B) in the indicated gastric cancer organoid lines after 4-day co-culture with HOMC-B1 cells. Each dot represents an individual organoid. In the box-and-whisker diagram, the boxes represent the 25th-75th percentiles, the central line represents the median, and the whiskers represent the 10th-90th percentiles. *, p <0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Table.S1.xlsx Table.S2.xlsx Table.S3.xlsx Cite Share Download PDF Status: Published Journal Publication published 13 Nov, 2025 Read the published version in Human Cell → Version 1 posted Editorial decision: Major Revisions Needed 20 Aug, 2025 Reviewers agreed at journal 01 Aug, 2025 Reviewers invited by journal 01 Aug, 2025 Editor assigned by journal 30 Jul, 2025 First submitted to journal 27 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7229795","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":494175096,"identity":"61b46b46-c3f3-4cbb-8ecd-fb4b89dcb7e8","order_by":0,"name":"Masahiro Inoue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYDACZjApwcPG3nwAxJAhVouNDB/PsQSwXmLtSrORk8gxALEIa9Ft5z34mKfiMA8bQ87nVzdqLHgY2A8f3YBPi9lhvmRjnjMgLWe3WeccAzqMJy3tBn4tPGbSvG1ALYy924xz2IBaJHjMiNDyD6iFmeeZcc4/orU0pPGwsfEwP85tI06LseGcYzY8bDxsZsy5fcAIIuiX82cMH7ypkbCXn//48eecb3Vy/OyHj+HVggzYJMAkscpBgPkDKapHwSgYBaNg5AAAWA89ax+s2I4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7315-026X","institution":"Kyoto University Graduate School of Medicine Faculty of Medicine: Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu","correspondingAuthor":true,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Inoue","suffix":""},{"id":494175097,"identity":"0dfdde8d-41ae-43f6-ae8a-9d951267ad0c","order_by":1,"name":"Hiroyuki Uematsu","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine Faculty of Medicine: Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Uematsu","suffix":""},{"id":494175098,"identity":"beeb6388-c3a3-4d19-886d-2413605d271f","order_by":2,"name":"Shota Shimizu","email":"","orcid":"","institution":"Tottori University Faculty of Medicine Graduate School of Medicine: Tottori Daigaku Igakubu Daigakuin Igakukei Kenkyuka","correspondingAuthor":false,"prefix":"","firstName":"Shota","middleName":"","lastName":"Shimizu","suffix":""},{"id":494175099,"identity":"fd11dadd-76a6-4f6f-b474-1e36826aa8b1","order_by":3,"name":"Kunishige Onuma","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine Faculty of Medicine: Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu","correspondingAuthor":false,"prefix":"","firstName":"Kunishige","middleName":"","lastName":"Onuma","suffix":""},{"id":494175100,"identity":"e9cb20a0-e3c0-4f62-8c4f-a1d32310f06d","order_by":4,"name":"Roberto Coppo","email":"","orcid":"","institution":"Kyoto University Graduate School of Medicine Faculty of Medicine: Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu","correspondingAuthor":false,"prefix":"","firstName":"Roberto","middleName":"","lastName":"Coppo","suffix":""},{"id":494175101,"identity":"6c74e3fe-90b0-45ba-a30a-5d869c2a7ccc","order_by":5,"name":"Yumi Sato","email":"","orcid":"","institution":"Fukushima Medical University School of Medicine: Fukushima Kenritsu Ika Daigaku Igakubu Daigakuin Igaku Senko","correspondingAuthor":false,"prefix":"","firstName":"Yumi","middleName":"","lastName":"Sato","suffix":""},{"id":494175102,"identity":"397ffe09-4ce4-4f7a-8191-87cd14b45db2","order_by":6,"name":"Tomoyuki Matsunaga","email":"","orcid":"","institution":"Tottori University Faculty of Medicine Graduate School of Medicine: Tottori Daigaku Igakubu Daigakuin Igakukei Kenkyuka","correspondingAuthor":false,"prefix":"","firstName":"Tomoyuki","middleName":"","lastName":"Matsunaga","suffix":""},{"id":494175103,"identity":"b03cdec7-2bbf-4fa6-93cf-db0da6cb17a7","order_by":7,"name":"Yoshiyuki Fujiwara","email":"","orcid":"","institution":"Tottori University Faculty of Medicine Graduate School of Medicine: Tottori Daigaku Igakubu Daigakuin Igakukei Kenkyuka","correspondingAuthor":false,"prefix":"","firstName":"Yoshiyuki","middleName":"","lastName":"Fujiwara","suffix":""}],"badges":[],"createdAt":"2025-07-28 04:59:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7229795/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7229795/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13577-025-01311-x","type":"published","date":"2025-11-13T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88412002,"identity":"1adb8e95-fe4c-4ccd-9bb9-9697a515fb6b","added_by":"auto","created_at":"2025-08-06 08:30:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10634674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological features of gastric cancer organoids (TUG7) derived from ascites.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phase-contrast images of the TUG7 organoids. (B) Comparison of microscopic features: Patient-derived gastric cancer peritoneal metastasis tissue (top), ascites-derived organoids from the same patient (middle), and peritoneal dissemination tissue in mouse xenografts (bottom). HE staining, Alcian blue staining, and immunohistochemical staining for CEA, E-cadherin, CK8, and Ki-67 are shown. Scale bars: 100 μm (A); 50 μm (B).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7229795/v1/9772979ce5cc35c6efa6c3c1.png"},{"id":88410351,"identity":"e265b110-2261-4e01-9272-812730005c17","added_by":"auto","created_at":"2025-08-06 08:22:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1917471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the co-culture medium in HOMC-B1 cells and TUG7 organoids.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Growth (upper panel) and morphology (lower panel) of cells cultured for 7 days in the indicated media: (A) HOMC-B1 cell, (B) TUG7 organoids. (C) Quantitative analysis of the adhesion of TUG7 organoids to HOMC-B1 cells treated with a neutralizing antibody against integrin β1 (Anti-ITGB1).\u003cstrong\u003e \u003c/strong\u003eScale bars: 100 μm. ns, not significant; **, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7229795/v1/b13a30bbd53db735db3d4a8e.png"},{"id":88412003,"identity":"bf13ed34-701d-4393-85c0-5eb37b804f15","added_by":"auto","created_at":"2025-08-06 08:30:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6120602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppression of growth in gastric cancer organoids co-cultured with mesothelial cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Schematic illustration (A) and HE staining (B) of the co-culture model of gastric cancer organoids and HOMC-B1 cells. Scale bars: 50 μm. (C) Growth of gastric cancer organoid lines (TUG6, TUG7, MK30, and MK35) cultured alone (mono) or co-cultured with HOMC-B1 cells (co). (D) Representative images of TUG7 organoids cultured alone (Monoculture) or co-cultured with HOMC-B1 cells (Co-culture). Ki-67 immunohistochemistry (left panels) and PI staining (right panels). Scale bars: 100 μm (left panel), 50 μm (right panel). (E, F) Quantitative analysis of the Ki67-positive rates (E) and PI-positive areas (F) in TUG7 organoids after 4-day co-culture with HOMC-B1 cells. Each dot represents an individual organoid. In the box-and-whisker diagram, the boxes represent the 25th-75th percentiles, the central line represents the median, and the whiskers represent the 10th-90th percentiles. (G) Components of the indicated media. CM, conditioned medium. (H) Evaluation of TUG7 organoid growth in the medium indicated in Fig. 3G. ns, nonsignificant; **, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001; ****, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7229795/v1/48f05d8cfd2683878aa820d3.png"},{"id":88410349,"identity":"0d4e2d74-2092-4c4e-a2d1-c2302cc35764","added_by":"auto","created_at":"2025-08-06 08:22:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1254927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDecreased sensitivity of gastric cancer organoids to paclitaxel under co-culture conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D) Paclitaxel sensitivity of the indicated gastric cancer organoid lines in monoculture (black lines) and co-culture with HOMC-B1 cells (red lines). Average and SD are also shown.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7229795/v1/4c6ee0198cbfbbd09f6c731c.png"},{"id":96105237,"identity":"36040ddb-a0f7-4ce8-a87d-6fdb3da0befe","added_by":"auto","created_at":"2025-11-17 16:10:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19120941,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7229795/v1/3c1c29ce-cf92-4dc9-8eba-e770e868b9c0.pdf"},{"id":88410348,"identity":"59174773-5e24-41eb-8a5e-333c29f56ea5","added_by":"auto","created_at":"2025-08-06 08:22:55","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1919272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1. Suppression of proliferation and induction of cell death in cancer organoids by co-culture with HOMC-B1 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Quantitative analysis of the Ki67-positive rates (A) and PI-positive areas (B) in the indicated gastric cancer organoid lines after 4-day co-culture with HOMC-B1 cells. Each dot represents an individual organoid. In the box-and-whisker diagram, the boxes represent the 25th-75th percentiles, the central line represents the median, and the whiskers represent the 10th-90th percentiles. *, \u003cem\u003ep \u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001; ****, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7229795/v1/09695f49e0a7d63860523133.tif"},{"id":88410345,"identity":"3a69a21b-3d31-4155-974b-409597533b95","added_by":"auto","created_at":"2025-08-06 08:22:55","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10075,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7229795/v1/c668b51ec6211e2a0c851621.xlsx"},{"id":88410346,"identity":"7c99ba67-7006-4c67-aff2-12c464978842","added_by":"auto","created_at":"2025-08-06 08:22:55","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10042,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7229795/v1/73fe34b2e486c53caf531933.xlsx"},{"id":88410354,"identity":"49d1e8f6-8eda-48b3-ac46-5d425e48ee78","added_by":"auto","created_at":"2025-08-06 08:22:56","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10368,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7229795/v1/0e614c20e5486fdab205d921.xlsx"}],"financialInterests":"","formattedTitle":"Interaction with peritoneal mesothelial cells inhibits the growth of gastric cancer organoids and induces drug resistance","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePeritoneal dissemination is found in 40\u0026ndash;60% of patients with gastric cancer\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and in approximately 14%\u003csup\u003e2\u003c/sup\u003e and 18\u0026ndash;26%\u003csup\u003e3\u003c/sup\u003e with synchronous peritoneal metastasis. The median survival time of patients with peritoneal dissemination is approximately 4 months, indicating a very poor prognosis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The main treatment option for gastric cancer peritoneal dissemination is systemic chemotherapy; however, the response rate for peritoneal lesions remains low at 14\u0026ndash;25%\u003csup\u003e4\u003c/sup\u003e. In contrast, the response rates of primary tumors and lymph node lesions to the same treatment are relatively high at 71% and 79%, respectively, indicating that peritoneal dissemination lesions are resistant to chemotherapy. Recently, intraperitoneal chemotherapy has been attempted for gastric cancer with peritoneal metastasis, with an improved response rate of 27\u0026ndash;49%\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e. However, as evidenced by the low response rate and rarity of complete remission, the therapeutic effect remains limited, and the development of new treatment strategies is urgently needed.\u003c/p\u003e\u003cp\u003eOne factor that contributes to treatment resistance is the presence of a unique tumor microenvironment \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e in peritoneal dissemination. To date, many studies have analyzed cancer cell adhesion and invasion using models in which cancer cell lines are layered on the peritoneal mesothelium\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To evaluate the therapeutic efficacy and drug resistance of established peritoneal metastases and develop new treatments, it is necessary to establish an in vitro model that more accurately reproduces the tumor microenvironment and reflects the clinical characteristics of peritoneal dissemination.\u003c/p\u003e\u003cp\u003eIn recent years, patient-derived organoids have attracted attention as three-dimensional culture models that can be applied in personalized medicine and drug sensitivity evaluation because of their ability to retain the histological/molecular biological characteristics of the original tumor\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In this study, we established organoids from ascites of a patient with peritoneal dissemination of gastric cancer and constructed a novel co-culture model with human mesothelial cells. The effects of interactions with mesothelial cells on cancer organoid proliferation and drug sensitivity were evaluated.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003ePatient samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003e This study was approved by the Institutional Ethics Committees of Kyoto University (R1575, R1671), Tottori University (21A177), and Osaka International Cancer Institute (1803125402). Tumor and ascites specimens were collected with informed consent from patients at Tottori University and Osaka International Cancer Institute. The clinical data of the four patients with gastric cancer are shown in Tables S1 and S2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOrganoid preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOrganoids of MK30 and MK35 were prepared using the CTOS method\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Organoids of TUG6 and TUG7 were prepared by mincing without enzymatic digestion. Organoids were cultured in a previously reported medium\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e with some modifications (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eOrganoid culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe organoids were maintained and passaged according to previously reported methods\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For cell viability assays, 50 organoids (40–100 µm) were embedded in type I collagen (Nitta Gelatin, Osaka, Japan) in each well of a 96-well plate (Thermo Fisher Scientific, Waltham, MA, USA). Seven days later, ATP levels were measured using CellTiter-Glo (Promega, Madison, WI, USA) with a GloMax Discover microplate reader (Promega) and normalized to the organoid area on day 0.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCulture of HOMC-B1 cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHOMC-B1 cells\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e were obtained from Riken Bio-Resource Research Center (Ibaraki, Japan). HOMC-B1 cells (1 × 10³ cells/well in a 96-well plate) were seeded, cultured for 7 days in RPMI-1640 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% FBS (Thermo Fisher Scientific), and cell survival rates were measured by ATP assay according to the above procedure. Conditioned medium (CM) of HOMC-B1 cells was collected after 24-h culture in the co-culture medium.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdhesion assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAdhesion assays were conducted according to a previously reported method\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Control IgG (Thermo Fisher Scientific) or ITGB1-neutralizing antibodies (Sigma-Aldrich, St Louis, MO, USA) (10 µg/mL) were added to the organoids 1 h before the start of co-culture. HOMC-B1 cells were seeded on type IA collagen, and the pre-treated organoids were added 24 h later with 5 µg/mL of the ITGB1-neutralizing antibody. After 48 h, non-adherent organoids were washed out, and the adhesion rate of the organoids to HOMC-B1 cells was calculated.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCo-culture of gastric cancer organoids and HOMC-B1 cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFifty organoids were suspended in type I collagen, and 10 µL of the suspension was seeded into a single well of a 96-well plate on ice. The plates were inverted and left on ice for 5 min. The plates were returned to their original position, incubated at 37°C for 10 min, and then 4 × 10⁴ HOMC-B1 cells suspended in 100 µL of co-culture medium were seeded on top. Organoid growth was evaluated using the area ratio from day 4 to day 0. For the cell death assay, the organoids were stained with 10 µg/mL propidium iodide (PI) (Thermo Fisher Scientific) at 37°C for 10 min, washed twice, and imaged. PI-positive areas were quantified using the ImageJ/Fiji software (NIH, Bethesda, MD, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemosensitivity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOrganoid drug sensitivity assays were conducted according to previously reported methods\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Paclitaxel (Nippon Kayaku, Tokyo, Japan) was added 24 h after seeding the HOMC-B1 cells, and the cells were cultured in the co-culture medium for 4 days. Organoid growth was evaluated using the area ratio from day 4 to day 0.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal experiment\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All animal studies were approved by the Institutional Animal Care and Use Committee of Kyoto University (18564). In the peritoneal dissemination model, organoids (diameter 40–100 µm) equivalent to 5 × 10⁶ cells were suspended in 500 µL of HBSS (Fujifilm Wako Pure Chemical Corporation) and injected into the peritoneal cavity of 13-week-old male non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (CLEA Japan, Tokyo, Japan) using a 27-gauge needle (Terumo, Tokyo, Japan).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMorphological analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eParaffin block preparation of the organoids was performed according to previously described methods with some modifications\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. For the analysis of co-culture, organoids and HOMC-B1 cells were cultured in culture inserts (Corning, Corning, NY, USA). Four days later, the cells were fixed with 10% formaldehyde (Fujifilm Wako Pure Chemical Corporation), the membranes were removed, and the cells were embedded in paraffin. Hematoxylin and eosin (HE) staining, Alcian blue staining, and immunohistochemistry (IHC) were performed according to previously reported methods\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The following primary antibodies were used for IHC: anti-E-cadherin (BD Biosciences, San Jose, CA, USA, Cat#610181, 1:500), anti-cytokeratin 8 (DSHB, Iowa City, IA, USA, 1:100), anti-Ki67 (Cell Signaling Technology, Beverly, MA, USA, Cat#9027, 1:400), and anti-CEA (Cell Signaling Technology, Cat#2383, 1:200). The nuclei were stained with DAPI, 4',6-diamidino-2-phenylindole (Thermo Fisher Scientific). Images were obtained using an Olympus BX50 fluorescence microscope and CellSens standard image analysis software (Olympus).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using GraphPad Prism 10.5.0. Student's t-test was used for single comparisons, and Tukey's multiple comparison test was performed for multiple comparisons after one-way analysis of variance (ANOVA). When the data did not follow a normal distribution, the Mann-Whitney U test was used. A p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cb\u003eOrganoids derived from ascites of a patient with peritoneal dissemination of gastric cancer reproduced clinical histological features\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOrganoids (TUG7) were established from the ascites obtained from a patient with peritoneal metastases of gastric cancer (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;1A). Disseminated lesions were observed when the organoids were transplanted into the peritoneal cavity of immunodeficient mice. Histological features were compared among the patient's peritoneal metastases, cancer organoids, and mouse peritoneal metastases (Fig.\u0026nbsp;1B). HE staining revealed a mixture of undifferentiated cancer cells and signet ring cells in all samples. Alcian blue staining confirmed mucus production. The staining patterns of CEA (a tumor marker), E-cadherin, CK8 (an epithelial marker), and Ki67 (a proliferation marker) were generally preserved in the organoids and xenografts. These results suggest that these models are clinically relevant.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOptimization of co-culture conditions for peritoneal mesothelial cells and cancer organoids\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCulture conditions for the co-culture model were examined. The co-culture medium was prepared by adding additives from the organoid culture medium (excluding A83-01) to the mesothelial cell medium (RPMI-1640, 10% FBS) of the human mesothelial cell line HOMC-B1 (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). In the co-culture medium, the growth of HOMC-B1 cells was promoted, and cell morphology was maintained (Fig.\u0026nbsp;2A). On the other hand, no changes in organoid proliferation or morphology were observed between the organoid culture and co-culture media (Fig.\u0026nbsp;2B). Next, we evaluated the co-culture conditions using adhesion assays\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Integrin β1 (ITGB1) plays an important role in the adhesion of gastric cancer cells to mesothelial cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The ITGB1-neutralizing antibody significantly inhibited the adhesion of TUG7 organoids to mesothelial cells (Fig.\u0026nbsp;2C). Therefore, the optimized co-culture medium may enable the functional evaluation of the interactions between gastric cancer organoids and mesothelial cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInhibition of cancer organoid growth by co-culture with mesothelial cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe peritoneal dissemination model was constructed by embedding the organoids in type I collagen and layering HOMC-B1 cells (Fig.\u0026nbsp;3A, 3B). Using this model, the proliferation of organoids derived from gastric cancer ascites (TUG7) and organoids derived from primary gastric cancer tumors (TUG6, MK30, and MK35) was investigated (Fig.\u0026nbsp;3C). Organoid proliferation and cell death were examined using Ki67 and PI staining, respectively. Co-culture resulted in a decrease in Ki67-positive cells and an increase in PI-positive cells (Fig.\u0026nbsp;3D-3F, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, S1B). Thus, in this co-culture model, the proliferation of gastric cancer organoids was suppressed, and cell death was induced by co-culture with mesothelial cells.\u003c/p\u003e\u003cp\u003eTo elucidate the cause of growth inhibition in the co-culture, TUG7 cells were cultured in various media, as shown in Fig.\u0026nbsp;3G. TUG7 growth in Medium A was similar to that in the co-culture medium (control) (Fig.\u0026nbsp;3H). In contrast, TUG7 expression was significantly inhibited in Medium B containing CM of HOMC-B1 cells (Fig.\u0026nbsp;3H). These results suggest that the inhibition of cancer organoid growth by co-culture with peritoneal mesothelial cells was mediated by soluble factors secreted from mesothelial cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePaclitaxel sensitivity was reduced in the peritoneal dissemination model\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePaclitaxel sensitivity assays were performed using a peritoneal dissemination model in the four gastric cancer organoid lines. All organoids co-cultured with HOMC-B1 cells showed reduced sensitivity to paclitaxel compared with those cultured alone (Fig.\u0026nbsp;4A-4D). These results indicated that gastric cancer peritoneal metastasis was clinically more resistant to chemotherapy, suggesting that this model may be useful as an in vitro model for evaluating the efficacy of chemotherapy in gastric cancer peritoneal metastasis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we established organoids from the ascites of a patient with gastric cancer and peritoneal metastasis. Organoids and their xenografts generally maintained their morphological characteristics despite some differences in the expression levels of differentiation markers. In cases of morphologically mixed types of cancer, such as in the present case, some cancer cells may be selected during the establishment of the organoids.\u003c/p\u003e\u003cp\u003eThe organoids were used in a co-culture model with a human mesothelial cell line. In conventional co-culture models, gastric cancer cell lines are seeded on mesothelial cells, and adhesion assays\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e or transwell chamber-based invasion assays\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e are performed. However, these conventional models are based on two-dimensional culture and represent the early stages of metastasis. Furthermore, most studies have used established cell lines, which have limitations in modelling three-dimensional cancer tissues\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The model used in this study is unique in that it uses organoids that retain the three-dimensional (3D) structure and diversity of a patient's tumor. Furthermore, this model may represent the three-dimensional structure of established peritoneal metastases and reflect the interactions between cancer and mesenchymal cells. Using this model, we have demonstrated for the first time, to the best of our knowledge, that interactions between gastric cancer cells and mesothelial cells increase drug resistance. As paclitaxel is a proliferation-dependent drug, resistance may be due to reduced cell proliferation.\u003c/p\u003e\u003cp\u003eIn this study, we demonstrated that the CM from HOMC-B1 cells inhibited organoid growth. Soluble factors secreted from co-cultured organoids or mesothelial cells may affect tumor cell growth, although the possibility of nutrient depletion owing to co-culture cannot be ruled out. In this study, we did not attempt to identify soluble factors that inhibit organoid proliferation. In the future, these liquid factors (e.g., TGF-β \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e secreted from mesothelial cells) are expected to be identified, and elucidation of the expression dynamics of the humoral factors involved in these interactions will contribute to the development of new therapeutic approaches.\u003c/p\u003e\u003cp\u003eIn conclusion, this 3D co-culture model may be useful for analyzing the influence of the tumor microenvironment on established peritoneal metastasis through interactions with mesothelial cells, and is expected to contribute to elucidating drug resistance mechanisms and evaluating new treatment strategies in clinical practice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the members of Inoue Laboratory for their helpful discussions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHU, TM, YF and MI designed the study. HU and YS performed the experiments. MI supervised the experiments. SS, TM, and YF acquired the clinical samples and data. HU and MI conducted the data analysis. HU and MI wrote the manuscript. HU, KO, RC, YS and YF reviewed and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026bull;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan, 22K08893 (T.M., Y.F., M.I.); by a collaboration grant from Kyoto University-KBBM (M.I,).\u003c/p\u003e\n\u003cp\u003e\u0026bull;\u003cstrong\u003eConflicts of interest/Competing interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.O., R.C, and M.I. are members of the Department of Clinical Bio-resource Research and Development at Kyoto University, which is sponsored by KBBM, Inc. H.U. is an employee of KBBM Inc. The other authors declare no conflict of interest. This work was supported in part by the collaboration grant from KBBM-Kyoto University.\u003c/p\u003e\n\u003cp\u003e\u0026bull;\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the institutional ethics committees of Kyoto University (R1575, R1671), Tottori University (21A177), and Osaka International Cancer Institute (1803125402).\u003c/p\u003e\n\u003cp\u003e\u0026bull;\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from participants. This study was performed in accordance with the Declaration of Helsinki.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eManzanedo I, Pereira F, Perez-Viejo E, Serrano A. Gastric Cancer with Peritoneal Metastases: Current Status and Prospects for Treatment. 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Aging (Albany NY). 2020; 12:3238-48.\u003c/li\u003e\n\u003cli\u003eLiu H, Zhu Y, Zhu H, et al. Role of transforming growth factor beta1 in the inhibition of gastric cancer cell proliferation by melatonin in vitro and in vivo. Oncol Rep. 2019; 42:753-62.\u003c/li\u003e\n\u003cli\u003eMutsaers SE. Mesothelial cells: their structure, function and role in serosal repair. Respirology. 2002; 7:171-91.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"organoid, gastric cancer, peritoneal dissemination, co-culture, mesothelial cell","lastPublishedDoi":"10.21203/rs.3.rs-7229795/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7229795/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe prognosis of gastric cancer with peritoneal dissemination is poor because of its resistance to chemotherapy. To create an \u003cem\u003ein vitro\u003c/em\u003e model of peritoneal metastases, cancer organoids were established from ascites fluid of patients with peritoneal metastases of gastric cancer. The histological characteristics of the tumors were preserved in the organoids. A co-culture system was established by overlaying human-derived mesothelial cells on gastric cancer organoids embedded in type I collagen, mimicking peritoneal dissemination foci. When co-cultured with mesothelial cells, the proliferation of ascites-derived gastric cancer organoids and other primary gastric cancer organoids was suppressed. Soluble factors derived from mesothelial cells were involved in suppressing cell proliferation. Organoids in co-culture showed reduced sensitivity to paclitaxel. This co-culture model may provide a useful platform for studying drug resistance mechanisms in the microenvironment of gastric cancer peritoneal metastases.\u003c/p\u003e","manuscriptTitle":"Interaction with peritoneal mesothelial cells inhibits the growth of gastric cancer organoids and induces drug resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 08:22:51","doi":"10.21203/rs.3.rs-7229795/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-08-20T22:17:15+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-01T09:33:28+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-01T08:43:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-30T08:29:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2025-07-28T00:58:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f76cafc3-60f7-4798-9f64-de750e5c5ccf","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:05:14+00:00","versionOfRecord":{"articleIdentity":"rs-7229795","link":"https://doi.org/10.1007/s13577-025-01311-x","journal":{"identity":"human-cell","isVorOnly":false,"title":"Human Cell"},"publishedOn":"2025-11-13 15:57:50","publishedOnDateReadable":"November 13th, 2025"},"versionCreatedAt":"2025-08-06 08:22:51","video":"","vorDoi":"10.1007/s13577-025-01311-x","vorDoiUrl":"https://doi.org/10.1007/s13577-025-01311-x","workflowStages":[]},"version":"v1","identity":"rs-7229795","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7229795","identity":"rs-7229795","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}