Csf2rb-Dependent Development of Yolk Sac-Derived Macrophages Drives Persistent Pro-Tumorigenic Activity

Csf2rb-Dependent Development of Yolk Sac-Derived Macrophages Drives Persistent Pro-Tumorigenic 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 Csf2rb-Dependent Development of Yolk Sac-Derived Macrophages Drives Persistent Pro-Tumorigenic Activity Yong Zhao, Tong Lei, Qian Zhang, Zhaoqi Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7182707/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tissue-resident macrophages (RTMs), predominantly originating from embryonic progenitors, play a pivotal role in maintaining tissue homeostasis and immune regulation. Their functional diversity is shaped by both their developmental origins—including the yolk sac, fetal liver, and bone marrow—and the local tissue microenvironment. In this study, we identified a unique subset of RTMs in Lyz2cre-RosaYFP reporter mice that lack Lyz2 expression and persist long-term in peripheral tissues. These Lyz2-YFP- RTMs follow a distinct developmental trajectory, transitioning from Lyz2-YFP-CD117+CD45-AA4.1- early erythro-myeloid progenitors (EMPs) to Lyz2-YFP-CD117+CD45+AA4.1- intermediate EMPs in a Csf2rb-dependent manner. Transcriptional profiling reveals that yolk sac-derived Kupffer cells (KCs) lacking Lyz2 expression are enriched in genes associated with T cell chemotaxis and tumor immunity. Functionally, these Lyz2-never expressed (Lyz2-YFP-) KCs are preferentially localized within intratumoral regions, where they promote hepatocellular carcinoma progression by recruiting CD4+CD25+Foxp3+ regulatory T cells (Tregs) and inducing T cell exhaustion. Conversely, Lyz2-ever expressed (Lyz2-YFP+) KCs exhibit anti-tumor activity by enhancing effector CD8+ T cell function. Our findings unveil a long-lived subset of RTMs with sustained pro-tumorigenic potential, underscoring the critical role of their yolk sac-derived developmental trajectory in shaping their functional properties. This study provides novel insights into the ontogeny-dependent heterogeneity of RTMs and their dual roles in tumor immunity. Biological sciences/Immunology/Haematopoiesis/Leukopoiesis Biological sciences/Immunology/Chemokines Health sciences/Diseases/Cancer/Tumour immunology Biological sciences/Cell biology/Cell migration/Chemotaxis Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages/Kupffer cells tissue-resident macrophages yolk sac erythro-myeloid progenitors Kupffer cells pro-tumor potential Csf2rb hepatocellular carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Supplementtable.xlsx Table 1 SupplementFigure.pdf SupplementMaterial.pdf Supplementary Materials GA.png Cite Share Download PDF Status: Posted 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. 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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-7182707","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":493419478,"identity":"ab62f436-c7ce-4e3c-8a20-75e860206b89","order_by":0,"name":"Yong Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYHACNiCWkGNgJlGLhTFQC2MDKVoqEoHKidRicID92YMfFRLp89uZnz9gqLFj4J9NQKfBAYZ0w54zErkbDrMZNjAcS2aQuHOAoJZjErxtQC3MDEAtbAcYDCQSCGlhbJP82yaRLt/M/rGB4R9RWpjZpIG2JDAc5jFsYGwjQovkYTY2aZkzEoYbDvMUzkjsS+aRuEFAC9/x9meSbyrq5OX7j2/48OGbnRz/DAJaFA4j84CKefCrBwL5BoJKRsEoGAWjYMQDAEDFPGuXXBmQAAAAAElFTkSuQmCC","orcid":"","institution":"State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.","correspondingAuthor":true,"prefix":"","firstName":"Yong","middleName":"","lastName":"Zhao","suffix":""},{"id":493419479,"identity":"d24529cd-045e-47a5-90eb-c2d654b2c27e","order_by":1,"name":"Tong Lei","email":"","orcid":"","institution":"State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Lei","suffix":""},{"id":493419480,"identity":"da09f526-406d-4e23-867a-1e1285842282","order_by":2,"name":"Qian Zhang","email":"","orcid":"https://orcid.org/0000-0001-5469-4878","institution":"State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Zhang","suffix":""},{"id":493419481,"identity":"ba47c235-5496-449f-ad4f-a75f7f9dcaf8","order_by":3,"name":"Zhaoqi Zhang","email":"","orcid":"https://orcid.org/0000-0003-1568-0857","institution":"Faculty of Synthetic Biology, Shenzhen University of Advanced Technology, 1068 Xueyuan Boulevand, University Town of Shenzhen, Xili Nanshan, Shenzhen 518055, China;","correspondingAuthor":false,"prefix":"","firstName":"Zhaoqi","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-07-22 05:10:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7182707/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7182707/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88295767,"identity":"23fbc3f0-adc6-4649-ac96-6e20a99eb551","added_by":"auto","created_at":"2025-08-05 03:24:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":325748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLyz2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-YFP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e RTMs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLyz2\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003ecre\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-Rosa\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eYFP\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e reporter mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Gating strategy and YFP labeling of tissue resident macrophages of lung, liver, spleen, and kidney in newborn \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice (n = 5). (b) YFP labeling of AMs, KCs, Rp-Mac, RN-RTM in newborn \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice (n = 5). Each scatterplot point represents one mouse, data show means with SD. (c) Percent stacked column chart analysis of YFP\u003csup\u003e-\u003c/sup\u003e RTM (red) and YFP\u003csup\u003e+\u003c/sup\u003e RTM (blue) in AM, KC, Rp-Mac, and RN-RTM in embryonic 17.5 days (E17.5), newborn (NB), postnatal 40 days (P40), and postnatal 300 days (P300) \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice (n = 5 for each group). Data show means with SD. (d) Experimental procedure of YFP\u003csup\u003e-\u003c/sup\u003e KCs and YFP\u003csup\u003e+\u003c/sup\u003e KCs adoptive transfer model. 5×10\u003csup\u003e5\u003c/sup\u003e YFP\u003csup\u003e-\u003c/sup\u003e KCs and YFP\u003csup\u003e+\u003c/sup\u003e KCs were sorted from 6~8\u003c/p\u003e\n\u003cp\u003eweeks old CD45.2\u003csup\u003e+\u003c/sup\u003e\u003cem\u003e Lyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and immediately transferred into CD45.1\u003csup\u003e+\u003c/sup\u003e 6 Gy irradiated recipient mice. Examined the YFP protein expression of CD45.2\u003csup\u003e+\u003c/sup\u003e donor-derived KCs in liver 2, 4 and 8 weeks after transplantation. (e) Flow cytometry analysis of YFP expression of CD45.2\u003csup\u003e+\u003c/sup\u003e grafted Tim4\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003elow\u003c/sup\u003e KCs. The flow cytometry analysis showed means. (f) Frequency analysis of proportion of CD45.2\u003csup\u003e+\u003c/sup\u003e donor-KCs in total KCs (above) and numbers of CD45.2\u003csup\u003e+\u003c/sup\u003e donor-KCs (below) (n = 3 in 2 weeks, n = 5 in 4 weeks and n = 4 in 8 weeks). The frequency analysis showed means with SD. Two-way ANOVA. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/35af92f4a469d33bb7088e1f.png"},{"id":88295768,"identity":"d8b0819f-fb18-46bc-a13e-cccfc9f2e87a","added_by":"auto","created_at":"2025-08-05 03:24:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":355101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLyz2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-YFP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e KCs and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLyz2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-YFP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e KCs have distinct transcriptional and functional signatures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Experimental procedures of sample preparation for bulk RNA-seq analysis of YFP\u003csup\u003e-\u003c/sup\u003eKCs and YFP\u003csup\u003e+\u003c/sup\u003e KCs. YFP\u003csup\u003e-\u003c/sup\u003e Tim4\u003csup\u003e+\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003eTim4\u003csup\u003e+\u003c/sup\u003e KCs were sorted from 6~8 weeks old \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice (n = 2). (b) Volcano plot showed the differential gene expression (DEGs) between YFP\u003csup\u003e-\u003c/sup\u003e KCs and YFP\u003csup\u003e+\u003c/sup\u003e KCs. The blue dots indicate genes downregulated in YFP\u003csup\u003e-\u003c/sup\u003e KCs, and the red dots indicate upregulated genes in YFP\u003csup\u003e-\u003c/sup\u003e KCs. DEGs are obtained by standard as foldchange (FC) greater than 1.5 and P-value less than 0.05. (c) Radar plot of different pathway of macrophage function and each dimension of the radar plot represents the mean of the fold change of transcripts per kilobase million (TPM) of genes of each pathway, in YFP\u003csup\u003e+\u003c/sup\u003e KCs (blue) and YFP\u003csup\u003e-\u003c/sup\u003e KCs (red). Fold change ranges from 0% to 200%. (d) Experimental procedure of endocytosis expression of YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs. YFP\u003csup\u003e-\u003c/sup\u003e Tim4\u003csup\u003e+\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003eTim4\u003csup\u003e+\u003c/sup\u003e KCs were sorted from\u003c/p\u003e\n\u003cp\u003e6~8 weeks old \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice, then mixed with late or early endosome dye as recommended by the manual. Analysis of RFP protein expression of late or early endosome dye after cultured in vitro for 24 hours. (e) Histograms show the RFP expression of each group (left) and frequency analysis of RFP\u003csup\u003e+\u003c/sup\u003e cells (right). The flow cytometry data are presented as means; each point represented in frequency analysis represents an independent experiment (n = 3). (f) Flow cytometry analysis visualization of \u003cem\u003eEsam \u003c/em\u003eexpression of YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs in 6~8 weeks old \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and frequency analysis of proportion of Esam\u003csup\u003e+\u003c/sup\u003e cells in YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs. Each scatterplot point in frequency analysis represents one mouse (n = 3). (g) Flow cytometry analysis visualization of \u003cem\u003eCcr7 \u003c/em\u003eexpression of YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs in 6~8 weeks old \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and frequency analysis of proportion of Ccr7\u003csup\u003e+\u003c/sup\u003e cells in YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs. Each scatterplot point in frequency analysis represents one mouse (n = 5). (h) Sankey diagram analysis visualization of chemokine expressions in YFP\u003csup\u003e-\u003c/sup\u003eand YFP\u003csup\u003e+\u003c/sup\u003e KCs (red) and target chemokine receptor genes (blue). (i) Frequency analysis of ratio of CD45.1\u003csup\u003e+\u003c/sup\u003e grafted CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e T cells and proportion of CD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e cells in total CD4\u003csup\u003e+\u003c/sup\u003e T cells. Each scatterplot point in frequency analysis represents one mouse (n = 3). (j) Heatmap analysis of Hif1-α pathway, cell adhesion pathway, and ECM pathway, and scaled relative expression of pathway core genes between YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs in bulk RNA-seq data. The histogram data showed means with SD. (k) Histogram analysis of relative expression of pro-tumor genes \u003cem\u003eTrem2\u003c/em\u003e, \u003cem\u003ePdcd1 \u003c/em\u003eand \u003cem\u003eCxcl9 \u003c/em\u003ebetween YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs in bulk RNA-seq data. The histogram data showed means with SD. (l) Histogram analysis of relative expression of anti-tumor genes \u003cem\u003eFolr2\u003c/em\u003e, \u003cem\u003eApoe \u003c/em\u003eand \u003cem\u003eId3\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ebetween YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs in bulk RNA-seq data. The histogram data showed means with SD. Two-way ANOVA. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/a447a9ded0b55fa4b12548d5.png"},{"id":88295913,"identity":"7ca98c25-b1f7-44c2-9f2b-e1b90bab164f","added_by":"auto","created_at":"2025-08-05 03:32:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1709695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLyz2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-YFP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e KCs display pro-tumor potentiality in HCC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Experimental procedure of orthotopic HCC mouse model. 1×10\u003csup\u003e6\u003c/sup\u003e Hepa1-6 tumor cells were injected into liver lobe in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice for 2 weeks with no gender restrict (n = 5). (b) Flow cytometry analysis visualization of YFP protein expression in KCs of adjacent tissues and tumor tissues (n = 5). The flow cytometry data are presented as means. (c) Frequency analysis of ratio of YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs in adjacent tissues and tumor tissues respectively. Each scatterplot point in frequency analysis represents one mouse (n = 5). (d) Immunofluorescence images of intertumoral, tumor margin, and peritumoral region of Hepa1-6-induced HCC (above), and frequency analysis of proportion of YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs in intertumoral, tumor margin, and peritumoral region. Scale bar, 100µm. Each scatterplot point in frequency analysis represents one independent sample (n = 5). (e) Experimental procedures for YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs reconstructed orthotopic HCC mouse model. First, 6~8 weeks old CD45.1\u003csup\u003e+\u003c/sup\u003e recipient mice were treated with intravenous injection of CLL to deplete total KCs. 1 day after CLL injection, 1×10\u003csup\u003e5\u003c/sup\u003e YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs were sorted from 6~8 weeks old \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and mixed with 1×10\u003csup\u003e6\u003c/sup\u003e Hepa1-6/B16 tumor cells respectively and immediately injected into liver lobe of CD45.1\u003csup\u003e+\u003c/sup\u003e KCs deficient recipient mice. Analysis 2 weeks after injection. (f) Macroscopic evaluation of the liver lobe of B16 induced YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs reconstructed orthotopic HCC mouse. (g)\u003c/p\u003e\n\u003cp\u003eRepresentative liver lobe histopathology images of YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs reconstructed B16-induced orthotopic HCC mouse and (h) frequency analysis of percentage of macroscopically visible tumor nodules area in liver lobes after HCC (right). Each scatterplot point in frequency analysis represents one mouse. (n=4, it was independent from the above experiments, KCs reconstructed HCC experiments B). (i) Frequency analysis of tumor weight of each group of Hepa1-6-induced orthotopic HCC mouse. Each scatterplot point in frequency analysis represents one mouse. (n=9 in PBS and CLL, n=8 in YFP\u003csup\u003e+\u003c/sup\u003e KCs, n=7 in YFP\u003csup\u003e-\u003c/sup\u003e KCs, which include two independent KCs reconstructed HCC experiments A, C). (j) Flow cytometry analysis visualization of grafted KCs in tumor tissues of orthotopic HCC mouse (left) and frequency analysis of percentage of grafted KCs in CD45\u003csup\u003e+\u003c/sup\u003e cells in tumors. The flow cytometry data are presented as means, each scatterplot point in frequency analysis represents one mouse. (n=9 in PBS and CLL, n=8 in YFP\u003csup\u003e+\u003c/sup\u003e KCs, n=7 in YFP\u003csup\u003e-\u003c/sup\u003e KCs, which include two independent KCs reconstructed HCC experiments A, C). (k) Analysis of proportion of CD4\u003csup\u003e+\u003c/sup\u003e T cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells in tumor. Each scatterplot point in frequency analysis represents one mouse. (n=9 in PBS and CLL, n=8 in YFP\u003csup\u003e+\u003c/sup\u003e KCs, n=7 in YFP\u003csup\u003e-\u003c/sup\u003e KCs, which include two independent KCs reconstructed HCC experiments A, C). (l) Analysis of numbers of Treg in YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e KCs reconstructed orthotopic HCC mouse. (n = 3 in YFP\u003csup\u003e-\u003c/sup\u003e KCs HCC mice and n = 4 in YFP\u003csup\u003e+\u003c/sup\u003e KCs HCC mice, KCs reconstructed HCC experiments A). (m) Analysis of proportion of TNF-α\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003e cells in CD4\u003csup\u003e+\u003c/sup\u003e T cells (left) and CD8\u003csup\u003e+\u003c/sup\u003e T cells (right). Each scatterplot point in frequency analysis represents one mouse. (n=4, it was independent from the above experiments, KCs reconstructed HCC\u003c/p\u003e\n\u003cp\u003eexperiments C). (m) Analysis of proportion of CD4\u003csup\u003e+\u003c/sup\u003e T cells (left) and CD8\u003csup\u003e+\u003c/sup\u003e T cells (right) in total CD45\u003csup\u003e+\u003c/sup\u003e T cells in B16-induced orthotopic HCC mouse with anti-CD4 treatment. Each scatterplot point in frequency analysis represents one mouse. (n = 3 in CLL, n = 5 in PBS + YFP\u003csup\u003e-\u003c/sup\u003e KCs and n = 4 in anti-CD4 + YFP\u003csup\u003e-\u003c/sup\u003e KCs, KCs reconstructed HCC experiments D). (n) Frequency analysis of tumor weight of each group of B16-induced orthotopic HCC mouse (n = 3 in CLL, n = 5 in PBS + YFP\u003csup\u003e-\u003c/sup\u003e KCs and n = 4 in anti-CD4 + YFP\u003csup\u003e-\u003c/sup\u003e KCs, KCs reconstructed HCC experiments D). Each scatterplot point in frequency analysis represents one mouse. Here included four independent KCs reconstructed HCC experiments A, B, C and D. Two-way ANOVA. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/6b2c0d0bbfc41ef726ae2564.png"},{"id":88297137,"identity":"d4e4c7d0-20d9-4228-8139-eefea306e9c0","added_by":"auto","created_at":"2025-08-05 03:40:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1354560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLyz2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-YFP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e RTMs originate from YS-derived EMPs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Flow cytometry analysis visualization of EMPs populations from E8.5 to E11.5 yolk sac. The flow cytometry data are presented as means. (b) Frequency analysis of proportion of YFP\u003csup\u003e-\u003c/sup\u003e cells in each population from E8.5 to E11.5 yolk sac. Each scatterplot point in frequency analysis represents one independent sample (n = 5). (c) Cloud graph analysis of single cell data of yolk sac precursor cells. (d) Violin graph analysis of gene expression of erythro-myeloid progenitors. (e) Pseudo-time analysis of erythro-myeloid progenitors in five distinct stages. (f) Pseudo-time analysis of each erythro-myeloid progenitor clusters in developmental stage. (g) Lyz2 expression in five distinct developmental stages. (h) Cloud graph analysis of gene expression of cluster C1 specific genes. (i) Experimental procedures of mRNA expression analysis between\u003c/p\u003e\n\u003cp\u003esorted early-EMPs and late-EMPs, and relative expression of erythro-myeloid progenitor cluster C1 specific genes in sorted early-EMPs and late-EMPs. (j) Heatmap analysis of erythro-myeloid progenitor cluster C1 specific genes in bulk-seq data of sorted YFP\u003csup\u003e-\u003c/sup\u003e KCs and YFP\u003csup\u003e+\u003c/sup\u003e KCs. (k) Experimental procedures of early-EMPs and late-EMPs development experiment in vivo. CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e-\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003eYFP\u003csup\u003e-\u003c/sup\u003e early-EMPs and CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e+\u003c/sup\u003eYFP\u003csup\u003e+\u003c/sup\u003e late-EMPs were sorted from E9.5 CD45.2\u003csup\u003e+\u003c/sup\u003e\u003cem\u003e Lyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and then injected into CD45.1\u003csup\u003e+\u003c/sup\u003e 6 Gy irradiated recipient mice (n = 3). Analysis of YFP protein expression of donor-RTM in lung, liver, spleen, and kidney. (l) Histograms show YFP expression of YFP\u003csup\u003e-\u003c/sup\u003e early-EMPs-derived RTMs and YFP\u003csup\u003e+\u003c/sup\u003e late-EMPs-derived RTMs (n = 3). The flow cytometry data are presented as means (n = 3). Two-way ANOVA. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/fcd83ff164bfcd695708d776.png"},{"id":88295774,"identity":"3bcc483d-b63b-49be-ad2c-0c935c8521e9","added_by":"auto","created_at":"2025-08-05 03:24:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":681003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLyz2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-YFP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e RTMs derived from early-EMPs to intermediate-EMPs in M-CSF-independent manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Cloud graph analysis of macrophage maturation-related receptor genes in single cell data of yolk sac precursors. (b) Experimental procedures of mRNA expression analysis of early-EMPs and late-EMPs. CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e-\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003e early-EMPs (red) and CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e+\u003c/sup\u003e late-EMPs (blue) were sorted from E9.5 \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice. (c) Relative expression of Lyz2 between CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e-\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003e early-EMPs (red) and CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e+\u003c/sup\u003e late-EMPs (blue). Each scatterplot point in analysis represents one independent sample (n = 3). (d) Relative expression of \u003cem\u003eCsf1r\u003c/em\u003e, \u003cem\u003eCsf2ra\u003c/em\u003e, \u003cem\u003eCsf2rb\u003c/em\u003e, \u003cem\u003eCsf2rb2 \u003c/em\u003ebetween CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e-\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003e early-EMPs (red) and\u003c/p\u003e\n\u003cp\u003eCD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e+\u003c/sup\u003e late-EMPs (blue). Each scatterplot point in analysis represents one independent sample (n = 3). (e) Experimental procedures of early-EMPs development experiment in vitro. CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e-\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003eYFP\u003csup\u003e-\u003c/sup\u003e early-EMPs were sorted from E9.5 \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and then cultured with 50ng/ml GM-CSF or 50ng/ml M-CSF for 48 hours in vitro (n = 3). (f) Frequency analysis of proportion of EMPs subpopulations in cultured-EMPs under GM-CSF or M-CSF conditions. Data show means with SD. (g) Frequency analysis of proportion of YFP- cells in each cultured-EMP population under GM-CSF or M-CSF conditions. Data show means with SD. (h) Flow cytometry analysis visualization of CD115 and CD11b expression in CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003e intermediate-EMPs from E8.5 to E11.5 yolk sac. The flow cytometry data are presented as means. (i) Histograms show YFP labeling of CD11b\u003csup\u003e+\u003c/sup\u003eCD115\u003csup\u003e+\u003c/sup\u003e (purple), CD11b\u003csup\u003e+\u003c/sup\u003eCD115\u003csup\u003e-\u003c/sup\u003e (black), and CD11b\u003csup\u003e-\u003c/sup\u003eCD115\u003csup\u003e-\u003c/sup\u003e (green) populations of intermediate-EMPs from E8.5 to E11.5 yolk sac. Data show means. (j) Experimental procedures of YFP\u003csup\u003e-\u003c/sup\u003e and YFP\u003csup\u003e+\u003c/sup\u003e intermediate-EMPs development experiment in vitro. CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003eYFP\u003csup\u003e-\u003c/sup\u003e and CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003eYFP\u003csup\u003e+\u003c/sup\u003e intermediate-EMPs were sorted from E9.5 \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and then cultured with 50ng/ml GM-CSF or 50ng/ml M-CSF for 48 hours in vitro (n = 3). (k) Flow cytometry analysis visualization of AA4.1 and YFP expression in cultured intermediate-EMPs. (l) Experimental procedures of YFP\u003csup\u003e-\u003c/sup\u003e int-EMPs and YFP\u003csup\u003e+\u003c/sup\u003e int-EMPs development experiment in vivo. CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003eYFP\u003csup\u003e-\u003c/sup\u003e int-EMPs and CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003eYFP\u003csup\u003e+\u003c/sup\u003e int-EMPs were sorted from E9.5 CD45.2\u003csup\u003e+\u003c/sup\u003e\u003cem\u003e Lyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and then injected into CD45.1\u003csup\u003e+\u003c/sup\u003e 6 Gy irradiated recipient mice (n = 4). (m)\u003c/p\u003e\n\u003cp\u003eFrequent analysis of YFP\u003csup\u003e+\u003c/sup\u003e cells of donor-RTM in lung, liver, spleen, and kidney. Each scatterplot point in analysis represents one mouse (n = 4). Two-way ANOVA. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/dcba25af971e257307c839d2.png"},{"id":88295769,"identity":"5e066a70-6472-4692-a96a-02c179b4e958","added_by":"auto","created_at":"2025-08-05 03:24:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":404147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCsf2rb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-dependent development of YS early-EMPs-derived KCs promotes sustained pro-tumorigenic activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Flow cytometry analysis CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e-\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003e early-EMPs, CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003eint-EMPs, and CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e+\u003c/sup\u003e late-EMPs proportion in E9.5 yolk sac cells of \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice (n = 9 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice, n = 6 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice). (b) Frequent analysis of YFP\u003csup\u003e+\u003c/sup\u003e cells in CD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003e int-EMPs of \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice (n = 9 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice, n = 6 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice). (c) Frequent analysis of YFP\u003csup\u003e+\u003c/sup\u003eCD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003e int-EMPs and YFP\u003csup\u003e-\u003c/sup\u003eCD117\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eAA4.1\u003csup\u003e-\u003c/sup\u003e int-EMPs in total EMPs of \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice (n = 9 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice, n = 6 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice). (d) Flow cytometry analysis of YFP protein expression of Tim4\u003csup\u003e+\u003c/sup\u003e KCs in P40 \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice (n = 5 in each group). (e) Frequent analysis of YFP\u003csup\u003e-\u003c/sup\u003e KCs and YFP\u003csup\u003e+\u003c/sup\u003e KCs proportion in total Tim4\u003csup\u003e+\u003c/sup\u003e KCs in P40 \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice (n = 5 in each group). Data are presented as means ± SD. (f) Percentage of Tim4\u003csup\u003e+\u003c/sup\u003e KCs in total cells in P40 \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice. Each scatterplot point in analysis represents one mouse (n = 5).\u003c/p\u003e\n\u003cp\u003e(g) Percentage of YFP\u003csup\u003e+\u003c/sup\u003eTim4\u003csup\u003e+\u003c/sup\u003eKCs and YFP\u003csup\u003e-\u003c/sup\u003eTim4\u003csup\u003e+\u003c/sup\u003e KCs in total cells in P40 \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice. Each scatterplot point in analysis represents one mouse (n = 5). (h) Schematic diagram of distinct developmental pathway from early-EMPs to YFP\u003csup\u003e-\u003c/sup\u003e int-EMPs and YFP\u003csup\u003e+\u003c/sup\u003e int-EMPs, which finally differentiated into Lyz2-negative RTMs and Lyz2-expressing RTMs respectively. (i) Experimental procedures of Hepa1-6-induced orthotopic hepatocellular carcinoma. 1×10\u003csup\u003e6\u003c/sup\u003e Hepa1-6 tumor cells were injected into liver lobe in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice for 2 weeks. Each scatterplot point in analysis represents one mouse (n = 3 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice, n = 4 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice). (j) Macroscopic evaluation of the liver lobe of Hepa1-6-induced orthotopic HCC in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice and \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice. (k) Frequency analysis of tumor weight of each group of Hepa1-6-induced orthotopic HCC mouse. Each scatterplot point in frequency analysis represents one mouse. (n = 3 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP\u003c/sup\u003e reporter mice, n = 4 in \u003cem\u003eLyz2\u003c/em\u003e\u003csup\u003ecre\u003c/sup\u003e-Rosa\u003csup\u003eYFP-\u003c/sup\u003e\u003cem\u003eCsf2rb\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e'\u003csup\u003e-\u003c/sup\u003e mice). (l) Frequent analysis of Tim4\u003csup\u003e+\u003c/sup\u003e KCs in total cells in Hepa1-6-induced orthotopic HCC. Each scatterplot point in frequency analysis represents one mouse. (m) Percentage analysis of YFP\u003csup\u003e+\u003c/sup\u003e KCs and YFP\u003csup\u003e-\u003c/sup\u003e KCs in total KCs in Hepa1-6-induced orthotopic HCC. Data are presented as means ± SD. (n) Frequent analysis of CD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Treg cells in CD4\u003csup\u003e+\u003c/sup\u003e T cells in Hepa1-6-induced orthotopic HCC. Each scatterplot point in frequency analysis represents one mouse. (o) Frequent analysis of TNF-α\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003e Treg cells in CD8\u003csup\u003e+\u003c/sup\u003e T cells in Hepa1-6-induced orthotopic HCC. Each scatterplot point in frequency analysis represents one mouse. Two-way ANOVA. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/4bc0594970d5a461af565be5.png"},{"id":89956591,"identity":"fbb86bfc-7816-4bad-8188-016bf920bdd4","added_by":"auto","created_at":"2025-08-26 22:18:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3088437,"visible":true,"origin":"","legend":"Article file","description":"","filename":"Manuscriptwithoutauthorinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1_covered_5367cf93-7d31-42e6-944b-321d55cd7dcb.pdf"},{"id":88295771,"identity":"ba790438-5199-40b9-9608-315fbbf83533","added_by":"auto","created_at":"2025-08-05 03:24:13","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1121675,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"Supplementtable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/ceaa51b08b5729bb7470266a.xlsx"},{"id":88295915,"identity":"278f1b66-c1aa-4fd5-9644-edc228f2eba0","added_by":"auto","created_at":"2025-08-05 03:32:13","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4425007,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/d465e8dd421f2d4e02372399.pdf"},{"id":88295770,"identity":"386e884a-6f19-457f-b6c8-c6a85007a7b7","added_by":"auto","created_at":"2025-08-05 03:24:13","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":50530,"visible":true,"origin":"","legend":"Supplementary Materials","description":"","filename":"SupplementMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/919e0135629c7e67d3bf6e60.pdf"},{"id":88295776,"identity":"6497a5d8-30cf-4f92-b838-b125fae8e765","added_by":"auto","created_at":"2025-08-05 03:24:13","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1459773,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-7182707/v1/b42a87d4bb59fbf24916812d.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Csf2rb-Dependent Development of Yolk Sac-Derived Macrophages Drives Persistent Pro-Tumorigenic Activity","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"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":"tissue-resident macrophages, yolk sac erythro-myeloid progenitors, Kupffer cells, pro-tumor potential, Csf2rb, hepatocellular carcinoma","lastPublishedDoi":"10.21203/rs.3.rs-7182707/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7182707/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Tissue-resident macrophages (RTMs), predominantly originating from embryonic progenitors, play a pivotal role in maintaining tissue homeostasis and immune regulation. Their functional diversity is shaped by both their developmental origins—including the yolk sac, fetal liver, and bone marrow—and the local tissue microenvironment. In this study, we identified a unique subset of RTMs in Lyz2cre-RosaYFP reporter mice that lack Lyz2 expression and persist long-term in peripheral tissues. These Lyz2-YFP- RTMs follow a distinct developmental trajectory, transitioning from Lyz2-YFP-CD117+CD45-AA4.1- early erythro-myeloid progenitors (EMPs) to Lyz2-YFP-CD117+CD45+AA4.1- intermediate EMPs in a Csf2rb-dependent manner. Transcriptional profiling reveals that yolk sac-derived Kupffer cells (KCs) lacking Lyz2 expression are enriched in genes associated with T cell chemotaxis and tumor immunity. Functionally, these Lyz2-never expressed (Lyz2-YFP-) KCs are preferentially localized within intratumoral regions, where they promote hepatocellular carcinoma progression by recruiting CD4+CD25+Foxp3+ regulatory T cells (Tregs) and inducing T cell exhaustion. Conversely, Lyz2-ever expressed (Lyz2-YFP+) KCs exhibit anti-tumor activity by enhancing effector CD8+ T cell function. Our findings unveil a long-lived subset of RTMs with sustained pro-tumorigenic potential, underscoring the critical role of their yolk sac-derived developmental trajectory in shaping their functional properties. This study provides novel insights into the ontogeny-dependent heterogeneity of RTMs and their dual roles in tumor immunity.","manuscriptTitle":"Csf2rb-Dependent Development of Yolk Sac-Derived Macrophages Drives Persistent Pro-Tumorigenic Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 03:24:09","doi":"10.21203/rs.3.rs-7182707/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":"721ef836-cdab-4845-aeda-b71eb7d6fd04","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52399309,"name":"Biological sciences/Immunology/Haematopoiesis/Leukopoiesis"},{"id":52399310,"name":"Biological sciences/Immunology/Chemokines"},{"id":52399311,"name":"Health sciences/Diseases/Cancer/Tumour immunology"},{"id":52399312,"name":"Biological sciences/Cell biology/Cell migration/Chemotaxis"},{"id":52399313,"name":"Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages/Kupffer cells"}],"tags":[],"updatedAt":"2025-08-26T22:10:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-05 03:24:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7182707","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7182707","identity":"rs-7182707","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}