Bioengineered Spatially Heterogeneous Tumor Microenvironment Model Mediated by Tumor-Stromal Interactions for Multi-Drug Synergy Evaluation

Bioengineered Spatially Heterogeneous Tumor Microenvironment Model Mediated by Tumor-Stromal Interactions for Multi-Drug Synergy Evaluation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bioengineered Spatially Heterogeneous Tumor Microenvironment Model Mediated by Tumor-Stromal Interactions for Multi-Drug Synergy Evaluation jie liu, You Chen, Yifan Xue, Jiahuan Yang, Jing Li, Junfeng Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8488007/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Stromal cell-involved heterogeneous tumor microenvironments (TMEs) critically regulate tumor metastasis, nutrient transport, metabolism, and proliferation. However, existing in vitro models are predominantly tumor cell-centric, lacking adequate cell-cell contact to recapitulate tumor-stromal crosstalk, and current 3D matrices fail to sustain multiple cell viability uniformly, restricting multicellular TME model utility. To address these limitations, this study presents a strategy for constructing a spatially heterogeneous TME model using two matrix materials with distinct properties: a modulus-tunable hybrid bioink (adEMA) with excellent printability for MCF-7 tumor cell culture, and high-bioactivity, low-modulus acellular adipose extracellular matrix (adECM) for stromal cells (MRC-5 fibroblasts and HUVECs), thus matching the growth demands of different cell types. Combining microfluidic chips and 3D bioprinting, a "chocolate sandwich ball"-mimetic spatially heterogeneous TME model mediated by three cell types was established. Herein, adECM acted as the primary matrix for HUVECs and MRC-5, while adEMA gel microspheres loaded with MCF-7 cells were uniformly distributed within the 3D model, maintaining an independent growth space from stromal cells. Results demonstrated that HUVEC- and MRC-5-mediated heterogeneous TME significantly upregulated the expression of malignant tumor markers, while these stromal cells enhanced tumor drug resistance and in vivo tumorigenicity. Using this model, the synergistic therapeutic effects of Cur, DOX, and OXA were confirmed, highlighting the utility of the three-cell-mediated spatially heterogeneous TME model for multi-drug screening. Physical sciences/Engineering Physical sciences/Materials science Physical sciences/Engineering Physical sciences/Materials science Full Text Additional Declarations There is no conflict of interest Supplementary Files chenetalSupportinformation.docx Bioengineered Spatially Heterogeneous Tumor Microenvironment Model Mediated by Tumor-Stromal Interactions for Multi-Drug Synergy Evaluation Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 29 Mar, 2026 Review # 4 received at journal 26 Mar, 2026 Review # 2 received at journal 15 Mar, 2026 Reviewer # 4 agreed at journal 09 Mar, 2026 Review # 3 received at journal 11 Feb, 2026 Review # 1 received at journal 27 Jan, 2026 Reviewer # 3 agreed at journal 23 Jan, 2026 Reviewer # 2 agreed at journal 18 Jan, 2026 Reviewer # 1 agreed at journal 15 Jan, 2026 Reviewers invited by journal 15 Jan, 2026 Submission checks completed at journal 07 Jan, 2026 First submitted to journal 05 Jan, 2026 Unknown event 05 Jan, 2026 Editor assigned by journal 31 Dec, 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-8488007","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":574930666,"identity":"7dcb2885-2bd1-4b72-9a80-443e00e0f061","order_by":0,"name":"jie liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYBACPmYGBoMEhn9y/EDOgQcwYR48WtggWg4YSzYAtSQQpQVCHUg0OACkiNPCzmNQ8KDiToLxtcMPgbbUJc6fkcD44G0bg7w5TofxGBgknHmWZ3Y7zQCo5XDihhsJzIZz2xgMdzbg0ZLYxlxsdjsBpOVA4gaJBDZp3jaGBLBTcWr5x5y4eXb6B5jD2H8T1tIAdI90DsgW5sSGGwlszPi1sBUYJBxLM5a4nVNwIMHgsPGGMw+bJeeckzDcgEMLP//hbYY/amzk+Genb/7woaJOdn578sEPb8ps5HHZArLIAME2YHBsYGBsALIkcKoHAuYHyDx7fEpHwSgYBaNgZAIA3DJcBspGq+gAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6035-0131","institution":"Sun Yat-Sen University","correspondingAuthor":true,"prefix":"","firstName":"jie","middleName":"","lastName":"liu","suffix":""},{"id":574930661,"identity":"256ad24d-4c82-4bc9-b188-ac5e87bf76c7","order_by":1,"name":"You Chen","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"You","middleName":"","lastName":"Chen","suffix":""},{"id":574930662,"identity":"7f806df9-9949-47fc-ab09-118948911373","order_by":2,"name":"Yifan Xue","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Xue","suffix":""},{"id":574930669,"identity":"77dc3fac-efd3-4ee2-8538-37cee9b80ee6","order_by":3,"name":"Jiahuan Yang","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Jiahuan","middleName":"","lastName":"Yang","suffix":""},{"id":574930670,"identity":"5cb04c30-4d72-4587-96c6-57da65a1df31","order_by":4,"name":"Jing Li","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""},{"id":574930671,"identity":"dcb1605d-b61d-49b1-9c76-339763fe8880","order_by":5,"name":"Junfeng Hu","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Junfeng","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2025-12-31 08:55:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8488007/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8488007/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100747263,"identity":"8e853423-abb5-4d83-8eaf-b47059e49dd8","added_by":"auto","created_at":"2026-01-21 03:52:47","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10345958,"visible":true,"origin":"","legend":"","description":"","filename":"Chenetalmanuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/a11a834c4a32355df4858d4e.docx"},{"id":100747243,"identity":"59577485-b4f1-4093-929c-b0e1b466b0ed","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7282,"visible":true,"origin":"","legend":"","description":"","filename":"MICRONANO05361.json","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/71a4c0db412f5e655fa5ff01.json"},{"id":100747251,"identity":"d9fe9b44-f983-47c8-8423-c4b3e10d8467","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3627457,"visible":true,"origin":"","legend":"","description":"","filename":"chenetalSupportinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/98931b56a8eb0305e0baa655.docx"},{"id":100747250,"identity":"e80acf66-642c-4241-8196-32459e465d6c","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132381,"visible":true,"origin":"","legend":"","description":"","filename":"MICRONANO053610enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/5449094a7422e0c3bd7d6c51.xml"},{"id":100747248,"identity":"129e51df-6fa7-410d-bcc8-5f4543b840c8","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":402630,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/eed295bfcf9190f2160596b0.png"},{"id":100747255,"identity":"6fb5ac35-1d8e-4ea7-b060-537af08ba0ec","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153511,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/e5a4a2ad01ae7fb7ca69831c.png"},{"id":100747258,"identity":"32841578-c7d0-4fae-9934-efbc5f3d096e","added_by":"auto","created_at":"2026-01-21 03:52:47","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":810820,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/93005ae45bbc546d4eb4a743.png"},{"id":100796685,"identity":"e5d758ea-a93e-49d6-806c-f890af0f410b","added_by":"auto","created_at":"2026-01-21 13:45:09","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":543806,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/3513bacc9d223c1ce66d45f0.png"},{"id":100796565,"identity":"f6fe2051-8b14-4e8a-93ab-62d772dd672d","added_by":"auto","created_at":"2026-01-21 13:44:14","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1560487,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/f246abca0c55215988bfe243.png"},{"id":100747257,"identity":"83ff3639-2123-4ca2-8669-627a0f5241b6","added_by":"auto","created_at":"2026-01-21 03:52:47","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":744649,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/9735c77d5d3f17cf681bc2c6.png"},{"id":100747245,"identity":"6df3a85d-a2b8-4172-98ff-0187d74255fd","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1364474,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/60b93252b2d044421f41b752.jpeg"},{"id":100747249,"identity":"0db605be-c5bb-46b4-92cc-b3d2e46c1f0d","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2482026,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/d445ddb7e4488f1b1b0a1724.jpeg"},{"id":100747247,"identity":"39c79576-9ad5-417c-9e2f-ef603b7a1be1","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1360278,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/95eae8c00405271f16058687.png"},{"id":100747256,"identity":"63d7554e-4848-419d-bf4c-df6daa579fe4","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":952303,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/064198319bdbbff3f5200c2c.png"},{"id":100804064,"identity":"965a697c-8e12-4908-a68f-af817b064f1d","added_by":"auto","created_at":"2026-01-21 14:35:45","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70827,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/bf7d369d86c5e0e5f1edb1de.png"},{"id":100747259,"identity":"b5e3618f-353c-4353-b8d0-f891aa628af4","added_by":"auto","created_at":"2026-01-21 03:52:47","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":38235,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/8df7d44d7393cdf5d3ed4814.png"},{"id":100747265,"identity":"9139aea5-cf03-47af-976f-275387e19e2c","added_by":"auto","created_at":"2026-01-21 03:52:47","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135258,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/c1b0e25b603ae8240585e944.png"},{"id":100857744,"identity":"6ae0f9cf-d490-42c4-8299-311eacb7c6d8","added_by":"auto","created_at":"2026-01-22 07:21:30","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68298,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/86fdfac44b10c89aa956d9a2.png"},{"id":100747268,"identity":"e7649864-b81b-4ced-b82c-27dc2e22cf9d","added_by":"auto","created_at":"2026-01-21 03:52:47","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":205842,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/85b9ab370d2c57b3793d1f23.png"},{"id":100747253,"identity":"b191da89-f10c-49d1-a156-04ebfd7de6df","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139180,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/597d0df6415dc3b0f3506aac.png"},{"id":100747266,"identity":"3ffb792b-a1d5-4dc9-8e0f-e2bdf605245b","added_by":"auto","created_at":"2026-01-21 03:52:47","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":211976,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/afec3e733a4db121f97c9555.png"},{"id":100796690,"identity":"0f806799-a92e-45d7-ab7f-7e163bc4176c","added_by":"auto","created_at":"2026-01-21 13:45:11","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":400291,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/ed8f42560aa9611202409fda.png"},{"id":100796498,"identity":"4a44b144-f5f9-4724-ade7-3b8d03080755","added_by":"auto","created_at":"2026-01-21 13:43:41","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":177915,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/ecb874fe73efa65031ee033f.png"},{"id":100747267,"identity":"ef95458d-9392-42ec-8050-08ff85c4c520","added_by":"auto","created_at":"2026-01-21 03:52:47","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169237,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/d214a6cad3a9e9abf462a15d.png"},{"id":100747269,"identity":"243301e1-cb2f-45b9-bf06-a68149919359","added_by":"auto","created_at":"2026-01-21 03:52:48","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131013,"visible":true,"origin":"","legend":"","description":"","filename":"MICRONANO053610structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/1c7e7a64b7b28c3b54b759b0.xml"},{"id":100747270,"identity":"bc2be1bb-70c4-43d4-9bbc-2adb52b77409","added_by":"auto","created_at":"2026-01-21 03:52:48","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142879,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/ba6ed993988dc2687e1ba1c1.html"},{"id":100859735,"identity":"e40a65b4-f5bb-429a-9d31-f5b0e1d8958a","added_by":"auto","created_at":"2026-01-22 07:32:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2012179,"visible":true,"origin":"","legend":"","description":"","filename":"Chenetalmanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1_covered_9baf2d12-3916-4ba0-a7ed-4a2d86e1c972.pdf"},{"id":100747244,"identity":"7579e149-84e7-4145-b5bc-fda49ddc1ad0","added_by":"auto","created_at":"2026-01-21 03:52:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3627457,"visible":true,"origin":"","legend":"Bioengineered Spatially Heterogeneous Tumor Microenvironment Model Mediated by Tumor-Stromal Interactions for Multi-Drug Synergy Evaluation","description":"","filename":"chenetalSupportinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8488007/v1/6e9b758da2210d661d3bcd88.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Bioengineered Spatially Heterogeneous Tumor Microenvironment Model Mediated by Tumor-Stromal Interactions for Multi-Drug Synergy Evaluation","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microsystems-and-nanoengineering","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"micronano","sideBox":"Learn more about [Microsystems \u0026 Nanoengineering](http://www.nature.com/micronano/)","snPcode":"41378","submissionUrl":"https://mts-micronano.nature.com/","title":"Microsystems \u0026 Nanoengineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8488007/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8488007/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStromal cell-involved heterogeneous tumor microenvironments (TMEs) critically regulate tumor metastasis, nutrient transport, metabolism, and proliferation. However, existing in vitro models are predominantly tumor cell-centric, lacking adequate cell-cell contact to recapitulate tumor-stromal crosstalk, and current 3D matrices fail to sustain multiple cell viability uniformly, restricting multicellular TME model utility. To address these limitations, this study presents a strategy for constructing a spatially heterogeneous TME model using two matrix materials with distinct properties: a modulus-tunable hybrid bioink (adEMA) with excellent printability for MCF-7 tumor cell culture, and high-bioactivity, low-modulus acellular adipose extracellular matrix (adECM) for stromal cells (MRC-5 fibroblasts and HUVECs), thus matching the growth demands of different cell types. Combining microfluidic chips and 3D bioprinting, a \"chocolate sandwich ball\"-mimetic spatially heterogeneous TME model mediated by three cell types was established. Herein, adECM acted as the primary matrix for HUVECs and MRC-5, while adEMA gel microspheres loaded with MCF-7 cells were uniformly distributed within the 3D model, maintaining an independent growth space from stromal cells. Results demonstrated that HUVEC- and MRC-5-mediated heterogeneous TME significantly upregulated the expression of malignant tumor markers, while these stromal cells enhanced tumor drug resistance and \u003cem\u003ein vivo\u003c/em\u003e tumorigenicity. Using this model, the synergistic therapeutic effects of Cur, DOX, and OXA were confirmed, highlighting the utility of the three-cell-mediated spatially heterogeneous TME model for multi-drug screening.\u003c/p\u003e","manuscriptTitle":"Bioengineered Spatially Heterogeneous Tumor Microenvironment Model Mediated by Tumor-Stromal Interactions for Multi-Drug Synergy Evaluation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 03:52:41","doi":"10.21203/rs.3.rs-8488007/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-30T00:42:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-26T16:43:28+00:00","index":4,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-16T00:25:30+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-10T01:59:56+00:00","index":4,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-11T08:58:03+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-27T06:49:24+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-24T02:10:54+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-18T11:49:09+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-15T07:29:12+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-15T07:16:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-07T06:50:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microsystems \u0026 Nanoengineering","date":"2026-01-05T08:46:58+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-01-05T06:55:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-31T08:53:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microsystems-and-nanoengineering","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"micronano","sideBox":"Learn more about [Microsystems \u0026 Nanoengineering](http://www.nature.com/micronano/)","snPcode":"41378","submissionUrl":"https://mts-micronano.nature.com/","title":"Microsystems \u0026 Nanoengineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56b1ef80-4997-4f67-942f-ed7896ca20b7","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61176728,"name":"Physical sciences/Engineering"},{"id":61176729,"name":"Physical sciences/Materials science"},{"id":61176730,"name":"Physical sciences/Engineering"},{"id":61176731,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2026-05-04T00:50:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 03:52:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8488007","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8488007","identity":"rs-8488007","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}