CAR T-cell design dependent remodeling of the brain tumor immune microenvironment identify macrophages as key players that inhibit or promote anti-tumor activity

CAR T-cell design dependent remodeling of the brain tumor immune microenvironment identify macrophages as key players that inhibit or promote anti-tumor activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CAR T-cell design dependent remodeling of the brain tumor immune microenvironment identify macrophages as key players that inhibit or promote anti-tumor activity Dalia Haydar, Jorge Ibañez-Vega, Jeremy Crawford, Ching-Heng Chou, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2972427/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Nov, 2023 Read the published version in Cancer Research Communications → Version 1 posted You are reading this latest preprint version Abstract Understanding interactions between adoptively transferred immune cells and the tumor immune microenvironment (TIME) is critical for developing successful T-cell based immunotherapies. Here we investigated the impact of the TIME and chimeric antigen receptor (CAR) design on anti-glioma activity of B7-H3-specific CAR T-cells. We show that five out of six B7-H3 CARs with varying transmembrane, co-stimulatory, and activation domains, exhibit robust functionality in vitro . However, in an immunocompetent glioma model, these CAR T-cells demonstrated significantly varied levels of anti-tumor activity. We used single-cell RNA sequencing to examine the brain TIME after CAR T-cell therapy. We show that the TIME composition was influenced by CAR T-cell treatment. We also found that successful anti-tumor responses were supported by the presence and activity of macrophages and endogenous T-cells. Together, our study demonstrates that efficacy of CAR T-cell therapy in high-grade glioma is dependent on CAR structural design and its capacity to modulate the TIME. Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Biological sciences/Cancer/CNS cancer Health sciences/Medical research/Translational research chimeric antigen receptor CAR T-cell therapy brain tumors immunocompetent mouse models scRNAseq brain tumor immune microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Supplementalfigures.pdf Supplemental Figures Cite Share Download PDF Status: Published Journal Publication published 15 Nov, 2023 Read the published version in Cancer Research Communications → Version 1 posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2972427","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":207027296,"identity":"0faf9b18-4b50-4174-b679-47a7d327c705","order_by":0,"name":"Dalia Haydar","email":"","orcid":"https://orcid.org/0000-0002-7959-2699","institution":"Children's National Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dalia","middleName":"","lastName":"Haydar","suffix":""},{"id":207027297,"identity":"a5226437-49f7-4d32-b5f8-3e07f17a6113","order_by":1,"name":"Jorge Ibañez-Vega","email":"","orcid":"","institution":"St. Jude Children’s Research Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"","lastName":"Ibañez-Vega","suffix":""},{"id":207027298,"identity":"6d611868-2182-478f-ba9b-c036b51f95ee","order_by":2,"name":"Jeremy Crawford","email":"","orcid":"","institution":"St. Jude Children’s Research Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"","lastName":"Crawford","suffix":""},{"id":207027299,"identity":"33488cab-ebcd-4471-bbeb-11edfbe8b591","order_by":3,"name":"Ching-Heng Chou","email":"","orcid":"","institution":"St. Jude Children’s Research Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ching-Heng","middleName":"","lastName":"Chou","suffix":""},{"id":207027300,"identity":"92c2048a-b335-4017-837d-52ad25668a56","order_by":4,"name":"Cliff Guy","email":"","orcid":"","institution":"St. Jude Children's Research 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Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Thomas","suffix":""},{"id":207027309,"identity":"39abe4d7-1ac6-45a3-8e12-85ea7d4f5321","order_by":13,"name":"Giedre Krenciute","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-4335-0644","institution":"St Jude Children's Research Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Giedre","middleName":"","lastName":"Krenciute","suffix":""}],"badges":[],"createdAt":"2023-05-23 15:51:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2972427/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2972427/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1158/2767-9764.CRC-23-0424","type":"published","date":"2023-11-16T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":38117455,"identity":"3c89ad32-a7f4-4fe7-a18f-81ad57955acb","added_by":"auto","created_at":"2023-06-06 17:59:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1291998,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration and functional characterization of syngeneic B7-H3 CAR T-cells with different CAR structures. (A) Scheme of murine (m) B7-H3-CAR constructs. (B) Summary plot of %F(ab′)2-positive T-cells at 3-5 days post transduction. (C) Summary plot of %F(ab′)2-positive T-cells titrated to 40% CAR expression with NT T-cells (n = 7, mean ± SD, 1-way ANOVA with Tukey’s test for multiple comparisons). (D) Summary plot of CD4 and CD8 composition of mB7-H3-CAR products at day 5 post-transduction (n = 4, mean ± SD, 2-way ANOVA with Tukey’s test for multiple comparisons). (E) Summary plot of memory phenotypes (Effector memory (EM): CD44+/CD62L−, central memory (CM): CD44+/CD62L+, naïve (N): CD44−/CD62L) of mB7-H3-CAR products as determined CD44 and CD62L expression (n = 4, mean ± SD, 2-way ANOVA with Tukey’s test for multiple comparisons).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1/a184e6cbace86e714641fb9d.jpg"},{"id":38117995,"identity":"14c48689-cf75-4f61-9097-5205bf73cb44","added_by":"auto","created_at":"2023-06-06 18:15:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1191168,"visible":true,"origin":"","legend":"\u003cp\u003eCD28-based mB7-H3 CAR T-cells with mutated activation domains outperform other constructs in in vitro functional assays. (A) MTS cytotoxicity assay against GL261 tumor cells at an effector to target (E:T) ratio of 4:1 and (B) 0.25:1 (n = 7, mean ± SD, 2-way ANOVA with Tukey’s test for multiple comparisons). (C-E) T-cells expressing different mB7-H3 CAR constructs were cocultured with GL261 tumor cells at a 2:1 ratio with restimulation every 3-days against fresh tumor cells until they no longer killed and/or expanded. (C) Fold expansion of different T-cell donors upon successive stimulations (x-axis: each stimulation is a 3-day co-culture with fresh GL261 tumor cells, n = 7). (D) Summary of the maximum fold expansion of mB7-H3 CAR T-cells from individual donors upon repeat stimulation with GL261 tumor cells (n = 7, minimum to maximum range, 1-way ANOVA with Tukey’s test for multiple comparisons). (E) Maximum number of times CAR T-cells were able to kill Gl261 tumor cells (n = 7, minimum to maximum range, 1-way ANOVA with Tukey’s test for multiple comparisons).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1/615821a9fc16f1380186bfe5.jpg"},{"id":38117456,"identity":"f923a162-243f-4175-b51a-6bf65aae0926","added_by":"auto","created_at":"2023-06-06 17:59:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1121686,"visible":true,"origin":"","legend":"\u003cp\u003eSurface expression of 4-1BBL on CD28-based mB7-H3-CAR T-cells enhances effector cytokines release in repeat stimulation assay. Culture supernatants were collected at 24-hour post repeated-stimulation with GL261 tumor cells at 2:1 ratio and analyzed using Multiplex assay. (A) Summary plots of cytokines and chemokines produced by CAR T-cells post first stimulation and (B) fourth stimulation against GL261 tumor cells (n = 4, mean ± SEM, 2-way ANOVA with Tukey’s test for multiple comparisons). (C-E) CAR T-cell production of IFNγ, IL-2, and GM-CSF at 24 hours’ post-stimulations one and four (n = 4, mean ± SEM, 2-way ANOVA with Tukey’s test for multiple comparisons).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1/0f33b619249e526f16879cdf.jpg"},{"id":38117777,"identity":"9a2379ee-0971-44d2-95f8-6cdeb82f19b6","added_by":"auto","created_at":"2023-06-06 18:07:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1885236,"visible":true,"origin":"","legend":"\u003cp\u003eCAR structural design significantly impacts anti-glioma efficacy of mB7-H3 CAR T-cells in the GL261 immunocompetent model. Albino C57BL/6 mice were transplanted with 1×10\u003csup\u003e5\u003c/sup\u003e GL261 cells orthotopically, followed 7-days later by intra-tumoral injection of 3×10\u003csup\u003e6\u003c/sup\u003e mB7-H3-CAR T-cells transduced with different constructs and adjusted to 40% CAR expression. (A) Axial brain MRI images from 3 representative mice per treatment group at days 16 and 29 post-tumor implantation. (B) Summary plots for change in tumor volumes as measured by MRI at days 16 and 29 post-tumor implantation with lines connecting individual mice (n denotes the number of mice surviving at the day of imaging in each group). (C) Bar graph showing percentage of survival and deceased mice within each treatment group at days 16, 29, and 45 post-tumor implantation (D) Kaplan-Meier survival curve (n = 11, log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons, *P\u0026lt;0.05; ***P\u0026lt;0.001). Experiments were repeated twice with CAR T-cells generated from 2 different T-cell donors. (E) Summary table for performance of different mB7-H3 CAR designs from in vitro and in vivo data ((-) means no response, increasing number of (+) signs mean better response).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1/5866f8e78fc5bf2a3a9bc15e.jpg"},{"id":38118403,"identity":"84ffa246-8aa8-4886-9473-afb0f7c27da7","added_by":"auto","created_at":"2023-06-06 18:31:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2150984,"visible":true,"origin":"","legend":"\u003cp\u003eTumor immune microenvironment heterogeneity post CAR T-cell treatment. (A) Experimental scheme. Albino C57BL/6 mice were transplanted with 1 × 10\u003csup\u003e5\u003c/sup\u003e GL261 cells orthotopically, followed 25-days later by intra-tumoral injection of 3 × 10\u003csup\u003e6\u003c/sup\u003e mB7-H3-CAR T-cells (28.mζ, BBL-28.mζ, \u003csup\u003eCD8tm\u003c/sup\u003eBB.ζ, or Ctrl). Tumors were collected at 4-days post treatment and processed for single-cell RNA sequencing. (B) UMAP with major cell subsets in all tumor samples. (C) Bar graph showing the percentage of each major cell type per treatment group. (D) UMAP dimensionality reduction of single cell data from all tumors clustered into 21 Seurat clusters annotated by number. (E) UMAP visualization of the 21 Seurat clusters by treatment group. Mac – macrophages, Mono – monocytes, MG – microglia, DC – dendritic cells.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1/2b7fea81cb605c3edce56de2.jpg"},{"id":38117781,"identity":"844d7108-e04d-4483-a6a4-8d21fcd0961f","added_by":"auto","created_at":"2023-06-06 18:07:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1792606,"visible":true,"origin":"","legend":"\u003cp\u003eDiversity of endogenous T-cell responses within the glioma TIME post CAR T-cell treatment. Seurat clusters 2, 8, and 16 were reclustered to further analyze lymphoid responses post CAR T-cell treatment. (A) UMAP plots of the T-cell subclusters visualized by treatment group. (B) Summary plot of T-cell subcluster distribution per treatment. (C) Dot plot depicting T-cell lineage and differentiation markers, T-cell immune inhibitory genes, T-cell cytolysis and immune activation genes, and chemotaxis genes per T-cell subcluster. Dot size represents the percentage of cells expressing each gene and dot color represents mean expression level with a gradient of lowest expression in blue to highest expression in red. (D-E) Dot plots depicting differentially expressed genes associated with T-cell immune activation and inhibition/exhaustion per treatment group. (F) Summary plot showing expression of CAR molecules per T-cell subclusters. (G-I) Volcano plots showing differentially –up and –down regulated genes in T-cell subclusters C3 in (G), C5 in (H), and C8 in (I) as compared to all other T-cell subclusters. Tex – Exhausted T-cells, Treg – Regulatory T-cells, Teff– Effector memory T-cells, Tquin – Quinescent T-cells, Trm – Tissue resident memory T-cells, Prol. – Proliferating.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1/74793f17022490b1e31f117f.jpg"},{"id":38117998,"identity":"ca2b8b34-6983-4725-b8b0-ede763f97d50","added_by":"auto","created_at":"2023-06-06 18:15:29","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2475193,"visible":true,"origin":"","legend":"\u003cp\u003eEffective responses with 28.mζ CAR T-cells are associated with balanced pro-and anti- inflammatory myeloid cell responses. Seurat clusters 0, 7, 10, 14 were reclustered into 10 macrophage/microglia subclusters to further define the diversity of myeloid responses post CAR T-cell treatment. (A) UMAP plots of the macrophage/microglia subclusters visualized by treatment group. (B) Summary plot of Mac/MG subcluster distribution per treatment. (C) Dot plot depicting expression of myeloid lineage markers, genes associated with pro-tumorigenic responses, anti-tumorigenic, and differentiation genes. Dot size represents the percentage of cells expressing each gene and dot color represents mean expression level with a gradient of lowest expression in blue to highest expression in red. (D) Enrichment plots of top six hallmark pathways involved in pro- and anti- inflammatory macrophage functions from GSEA Hallmark analysis comparing macrophage subcluster 2 versus other macrophage subclusters. Ranked genes depicted on the x-axis with a black line with most enriched on the left to least enriched on the right. Normalized enrichment score (NES) depicted as well as adjusted p-value (p. adjusted). (E) Enrichment plots for macrophage hallmark pathways enriched in subcluster 7 as compared to other macrophage subclusters. (F) Volcano plot showing differential gene expression profiles in macrophage subclusters associated with 28.mζ-CAR treatment compared to all other groups along with dot plot for top 10 differentially up- or down- regulated hallmark pathways in macrophage subclusters associated with 28.mζ-CAR treatment compared to other CAR groups. (G) Dot plot depicting differentially expressed genes associated with immunosuppression, invasion, recruitment, M1-like and M2-like macrophage responses per treatment group.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1/1545f5e1979db9efda0a5b4d.jpg"},{"id":38117464,"identity":"52c7ba78-679a-4951-a7d2-41b8c49fbd70","added_by":"auto","created_at":"2023-06-06 17:59:30","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2502305,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal macrophage/microglia depletion abrogates effective CAR T-cell responses. GL261 glioma-bearing mice were treated with BLZ945 at 200mg/Kg starting 5-days post tumor implantation. (A) Experimental scheme of BLZ945 macrophage depletion kinetics experiment. Daily drug dosing via oral gavage was for 2 weeks and tumors were harvested for FACS analysis at days 9, 16, and 20 post tumor implantation. (B) Summary plot showing frequency of TAMs infiltrating tumors as percentage of live CD45+ immune cells. (C) Experimental scheme for combination study. Glioma-bearing mice were treated with BLZ945 at 200mg/Kg starting 5-days post tumor implantation and continued daily for 3 weeks. B7-H3 CAR T-cells with 28.mζ domains were then injected intratumorally at day 16. (D) Kaplan-Meier survival curve (n= 11, log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons, ***P\u0026lt;0.001). (E) Representative images from immunostaining for T-cell and macrophage markers from tumors at endpoint showing CD3 and Iba1 staining in brain samples from each treatment group at 40x magnification (scale bar = 100 μm). (F) H-scores depicting quantitative analysis of CD3 and Iba1 staining in brain tumor samples at endpoint from (E) as evaluated by blinded pathologist.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1/56ef2afd2c78706b6786ad69.jpg"},{"id":46926074,"identity":"ceec2d96-09d0-4177-8bc1-54c77f95f308","added_by":"auto","created_at":"2023-11-22 15:00:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1575968,"visible":true,"origin":"","legend":"","description":"","filename":"Maintext.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1_covered_13156677-53ff-448f-ae64-1c93fe29c466.pdf"},{"id":38117465,"identity":"8fdf2e43-ed87-426a-bb1a-837c76f115e4","added_by":"auto","created_at":"2023-06-06 17:59:30","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8353817,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figures\u003c/p\u003e","description":"","filename":"Supplementalfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2972427/v1/cfd4c1b4bad53cbfa2850498.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"CAR T-cell design dependent remodeling of the brain tumor immune microenvironment identify macrophages as key players that inhibit or promote anti-tumor activity","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"chimeric antigen receptor, CAR T-cell therapy, brain tumors, immunocompetent mouse models, scRNAseq, brain tumor immune microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-2972427/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2972427/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding interactions between adoptively transferred immune cells and the tumor immune microenvironment (TIME) is critical for developing successful T-cell based immunotherapies. Here we investigated the impact of the TIME and chimeric antigen receptor (CAR) design on anti-glioma activity of B7-H3-specific CAR T-cells. We show that five out of six B7-H3 CARs with varying transmembrane, co-stimulatory, and activation domains, exhibit robust functionality \u003cem\u003ein vitro\u003c/em\u003e. However, in an immunocompetent glioma model, these CAR T-cells demonstrated significantly varied levels of anti-tumor activity. We used single-cell RNA sequencing to examine the brain TIME after CAR T-cell therapy. We show that the TIME composition was influenced by CAR T-cell treatment. We also found that successful anti-tumor responses were supported by the presence and activity of macrophages and endogenous T-cells. Together, our study demonstrates that efficacy of CAR T-cell therapy in high-grade glioma is dependent on CAR structural design and its capacity to modulate the TIME.\u003c/p\u003e","manuscriptTitle":"CAR T-cell design dependent remodeling of the brain tumor immune microenvironment identify macrophages as key players that inhibit or promote anti-tumor activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-06 17:59:24","doi":"10.21203/rs.3.rs-2972427/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e16872c1-9113-4bf2-be21-9e7c6fd647e2","owner":[],"postedDate":"June 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":22121255,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":22121256,"name":"Biological sciences/Cancer/CNS cancer"},{"id":22121257,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2023-11-22T14:59:56+00:00","versionOfRecord":{"articleIdentity":"rs-2972427","link":"https://doi.org/10.1158/2767-9764.CRC-23-0424","journal":{"identity":"cancer-research-communications","isVorOnly":true,"title":"Cancer Research Communications"},"publishedOn":"2023-11-16 00:00:00","publishedOnDateReadable":"November 16th, 2023"},"versionCreatedAt":"2023-06-06 17:59:24","video":"","vorDoi":"10.1158/2767-9764.CRC-23-0424","vorDoiUrl":"https://doi.org/10.1158/2767-9764.CRC-23-0424","workflowStages":[]},"version":"v1","identity":"rs-2972427","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2972427","identity":"rs-2972427","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}