Estrogen prevents anti-tumor effects of Zoledronic acid in breast cancer through the NK and CD8+T cell PD-1/PD-L1 axes

Estrogen prevents anti-tumor effects of Zoledronic acid in breast cancer through the NK and CD8+T cell PD-1/PD-L1 axes | 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 Estrogen prevents anti-tumor effects of Zoledronic acid in breast cancer through the NK and CD8+T cell PD-1/PD-L1 axes Christopher George, Jiabao Zhou, Victor Canuas-Landero, Diane Lefley, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9426058/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Clinical trials in breast cancer have demonstrated reduced recurrence in bone and soft tissue in postmenopausal women following adjuvant treatment with Zoledronic acid (Zol). In contrast however, the same treatment resulted in an increased risk of extra-skeletal recurrence in premenopausal women. The reason for these stark differences has remained unclear, until now preventing thousands of young breast cancer patients from being able to benefit from the life prolonging effects of Zol. Here we demonstrate that Zol induces an anti-tumor response by increasing infiltration of CD8+T, dendritic and NK cells into soft tissue metastases, but only when estrogen concentrations are low. Anti-tumor response to Zol appears to be CD8+T and NK cell-dependent in soft tissue, but only NK cell-dependent in bone. Conversely, estrogen significantly reduces systemic and tumor infiltrating CD8+ and CD4+T cells, increases PD-1 expression on T and NK cells and increases PD-L1 on tumor cells. We propose that estradiol reduces anti-cancer lymphoid cell infiltration into tumors and increases their exhaustion status, preventing Zol-induced activation of T and NK cells. Our data suggests that under premenopausal concentrations of estradiol, anti-tumor response to Zol can be restored by blocking PD-1/PD-L1 interactions, thereby overcoming estrogen driven immune suppression. Biological sciences/Cancer Biological sciences/Immunology Health sciences/Oncology Breast Cancer Zoledronic acid T cells NK cells PD-1 PD-L1 Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresandTablesfinal.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 16 May, 2026 Reviews received at journal 15 May, 2026 Reviews received at journal 11 May, 2026 Reviews received at journal 01 May, 2026 Reviewers agreed at journal 28 Apr, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers agreed at journal 26 Apr, 2026 Reviewers invited by journal 26 Apr, 2026 Editor assigned by journal 25 Apr, 2026 Submission checks completed at journal 20 Apr, 2026 First submitted to journal 15 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. 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-9426058","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":623577582,"identity":"c69bfa23-78c8-41c8-945e-0ed837217483","order_by":0,"name":"Christopher George","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"George","suffix":""},{"id":623577583,"identity":"aac0c0ba-3794-480f-b419-2bc4ffb8838d","order_by":1,"name":"Jiabao Zhou","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Jiabao","middleName":"","lastName":"Zhou","suffix":""},{"id":623577584,"identity":"266d2aa8-e400-43fe-bf12-3ae7fb3434c1","order_by":2,"name":"Victor Canuas-Landero","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"","lastName":"Canuas-Landero","suffix":""},{"id":623577585,"identity":"40bec682-ae97-44a8-b463-3e9651e0aa92","order_by":3,"name":"Diane Lefley","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Diane","middleName":"","lastName":"Lefley","suffix":""},{"id":623577586,"identity":"a77755f6-f471-41d8-8e51-ce7e0cb62160","order_by":4,"name":"Russell Hughes","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Russell","middleName":"","lastName":"Hughes","suffix":""},{"id":623577587,"identity":"25153729-d4b5-4fa2-b8ef-b7c3d7adec0c","order_by":5,"name":"Fiona Thistletwaite","email":"","orcid":"","institution":"The Christie NHS Foundation Trust","correspondingAuthor":false,"prefix":"","firstName":"Fiona","middleName":"","lastName":"Thistletwaite","suffix":""},{"id":623577588,"identity":"0d48ce9d-95d9-4347-900d-8a0d31d551c4","order_by":6,"name":"Caroline Wilson","email":"","orcid":"","institution":"The Christie NHS Foundation Trust","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Wilson","suffix":""},{"id":623577589,"identity":"f97442b4-749d-4571-bbdd-e5cd4cdce937","order_by":7,"name":"Ingunn Holen","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Ingunn","middleName":"","lastName":"Holen","suffix":""},{"id":623577590,"identity":"fb99260b-d8d2-4185-9662-a2592e379d40","order_by":8,"name":"Janet Brown","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Janet","middleName":"","lastName":"Brown","suffix":""},{"id":623577591,"identity":"e6f4e584-903f-4714-af30-0a3349cad49a","order_by":9,"name":"Mark Dunning","email":"","orcid":"","institution":"Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Dunning","suffix":""},{"id":623577592,"identity":"ee06aa37-6dcd-4122-ac09-87830e26e33f","order_by":10,"name":"Sarah Danson","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Danson","suffix":""},{"id":623577593,"identity":"cb5b290b-a46b-4f6b-8963-b07b39b2d09a","order_by":11,"name":"Matthew Winter","email":"","orcid":"","institution":"Weston Park Cancer Centre","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Winter","suffix":""},{"id":623577594,"identity":"10d3cb29-391c-4fca-b340-40b5115ba6bb","order_by":12,"name":"Lewis Quayle","email":"","orcid":"","institution":"Sheffield Hallam University","correspondingAuthor":false,"prefix":"","firstName":"Lewis","middleName":"","lastName":"Quayle","suffix":""},{"id":623577595,"identity":"04d9e9d4-5390-458b-bc4c-8d945ff52715","order_by":13,"name":"Munitta Muthana","email":"","orcid":"","institution":"University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Munitta","middleName":"","lastName":"Muthana","suffix":""},{"id":623577596,"identity":"ea2f9efb-a3e7-4a61-bfb4-57103a121f75","order_by":14,"name":"Penelope Ottewell","email":"data:image/png;base64,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","orcid":"","institution":"University of Sheffield","correspondingAuthor":true,"prefix":"","firstName":"Penelope","middleName":"","lastName":"Ottewell","suffix":""}],"badges":[],"createdAt":"2026-04-15 11:10:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9426058/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9426058/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107062312,"identity":"78b23d08-2074-45c1-922f-a26ba983044b","added_by":"auto","created_at":"2026-04-16 10:28:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":846252,"visible":true,"origin":"","legend":"","description":"","filename":"OttewellnpjBresatCancerv9.1final.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9426058/v1_covered_0dd9d8d3-4fe1-4681-9f81-851964281955.pdf"},{"id":107062262,"identity":"c9ee32c7-7ea6-484e-ae43-f037d50602a3","added_by":"auto","created_at":"2026-04-16 10:27:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2276044,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresandTablesfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9426058/v1/b09cb6c89e21459ffad5dce5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Estrogen prevents anti-tumor effects of Zoledronic acid in breast cancer through the NK and CD8+T cell PD-1/PD-L1 axes","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"npj-breast-cancer","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjbcancer","sideBox":"Learn more about [npj Breast Cancer](http://www.nature.com/npjbcancer/)","snPcode":"41523","submissionUrl":"https://mts-npjbcancer.nature.com/","title":"npj Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast Cancer, Zoledronic acid, T cells, NK cells, PD-1, PD-L1","lastPublishedDoi":"10.21203/rs.3.rs-9426058/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9426058/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Clinical trials in breast cancer have demonstrated reduced recurrence in bone and soft tissue in postmenopausal women following adjuvant treatment with Zoledronic acid (Zol). In contrast however, the same treatment resulted in an increased risk of extra-skeletal recurrence in premenopausal women. The reason for these stark differences has remained unclear, until now preventing thousands of young breast cancer patients from being able to benefit from the life prolonging effects of Zol. Here we demonstrate that Zol induces an anti-tumor response by increasing infiltration of CD8+T, dendritic and NK cells into soft tissue metastases, but only when estrogen concentrations are low. Anti-tumor response to Zol appears to be CD8+T and NK cell-dependent in soft tissue, but only NK cell-dependent in bone. Conversely, estrogen significantly reduces systemic and tumor infiltrating CD8+ and CD4+T cells, increases PD-1 expression on T and NK cells and increases PD-L1 on tumor cells. We propose that estradiol reduces anti-cancer lymphoid cell infiltration into tumors and increases their exhaustion status, preventing Zol-induced activation of T and NK cells. Our data suggests that under premenopausal concentrations of estradiol, anti-tumor response to Zol can be restored by blocking PD-1/PD-L1 interactions, thereby overcoming estrogen driven immune suppression.","manuscriptTitle":"Estrogen prevents anti-tumor effects of Zoledronic acid in breast cancer through the NK and CD8+T cell PD-1/PD-L1 axes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-16 10:26:15","doi":"10.21203/rs.3.rs-9426058/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-16T17:16:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T23:26:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T13:32:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T17:38:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126357281434250864872509531892928768081","date":"2026-04-29T00:49:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170710550187015916688727794068401285559","date":"2026-04-27T13:02:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121239185018877954729171964394636412334","date":"2026-04-26T18:52:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-26T16:04:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-25T23:58:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-20T05:32:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Breast Cancer","date":"2026-04-15T10:57:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-breast-cancer","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjbcancer","sideBox":"Learn more about [npj Breast Cancer](http://www.nature.com/npjbcancer/)","snPcode":"41523","submissionUrl":"https://mts-npjbcancer.nature.com/","title":"npj Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b675cc7-ad25-47b9-893a-ff72866ebd52","owner":[],"postedDate":"April 16th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-16T17:16:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T23:26:04+00:00","index":21,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T13:32:03+00:00","index":20,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T17:38:48+00:00","index":19,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":66365504,"name":"Biological sciences/Cancer"},{"id":66365505,"name":"Biological sciences/Immunology"},{"id":66365506,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-16T17:24:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-16 10:26:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9426058","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9426058","identity":"rs-9426058","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}