Additional file 1 of Lyve-1 deficiency enhances the hepatic immune microenvironment entailing altered susceptibility to melanoma liver metastasis
Creators: name: Jauch, Anna Sophia
name: Wohlfeil, Sebastian A.
name: Weller, Céline
name: Dietsch, Bianca
name: Häfele, Verena
name: Stojanovic, Ana
name: Kittel, Maximilian
name: Nolte, Hendrik
name: Cerwenka, Adelheid
name: Neumaier, Michael
name: Schledzewski, Kai
name: Sticht, Carsten
name: Reiners-Koch, Philipp-Sebastian
name: Goerdt, Sergij
name: Géraud, Cyrill
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Supplementary Figures. Figure S1. Additional phenotypic characterization of Ctrl and Lyve-1-/- mice. Analysis of the iron homeostasis. Further analyses of hyaluronan levels by different ELISAs and by age. A. Body length (female, P = 0.5438, n = 5-6/group; male, P = 0.7832, n = 5/group) measured of Ctrl and Lyve-1-/- mice. B. Levels of ALT (female, P = 0.3176, n = 8-9/group), AST (female, P = 0.1270, Mann-Whitney U-test, n = 6-7/group), GLDH (female, P = 0.2095, n = 7-9/group) in blood plasma of Ctrl and Lyve-1-/- mice are displayed. C. Iron concentration in liver (female, P = 0.4110, n = 3/group), plasma (female, P = 0.0387, n = 6/group), and Hepcidin concentration in plasma (female, P = 0.3747, n = 6-8/group) of Ctrl and Lyve-1-/- mice. D. Hyaluronan concentration in plasma of Ctrl and Lyve-1-/- mice in the age of a 6 weeks (female, P &lt; 0.0001, n = 6/group), b 8 weeks (female, P &lt; 0.0001, n = 6/group) and c 10 weeks (female, P &lt; 0.0001, n = 6/group) measured with Hyaluronan DuoSet ELISA (DY3614-05, R&amp;D Systems, Minneapolis, MN, USA). E. Hyaluronan concentration in plasma of Ctrl and Lyve-1-/- mice in the age of a 12 weeks (female, P = 0.0022, Mann-Whitney U-test, n = 6/group) and b 14 weeks (female, P = 0.0079, Mann-Whitney U-test, n = 5/group) measured with Hyaluronan Enzyme-Linked Immunosorbent Assay (K-1200, Echelon Biosciences, Salt Lake City, UT, USA). Data information: * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001; **** P &lt; 0.0001; ns = not significant. Figure S2. Analysis of platelet counts, functions and hemostasis in Ctrl or Lyve-1-/- mice. A. Platelet count (female, P = 0.7831, n = 5-9/group), thromboelastography (TEG) R [min] (male, P = 0.0536, n = 5/group), TEG K [min] (male, P &gt;0.9999, Mann-Whitney U-test, n = 5/group), TEG Angle [degree] (male, P = 0.3413, Mann-Whitney U-test, n = 5/group) and TEG MA [mm] (male, P = 0.1211, n = 5/group) quantified in blood of Ctrl and Lyve-1-/- mice. B. Activated partial thromboplastin time (aPTT) (female, P = 0.1040, n = 6/group), INR (female, P = 0.5647, n = 4-5/group) and thrombin time (female, P = 0.8084, n = 6/group) measured in blood of Ctrl and Lyve-1-/- mice. Data information: * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001; **** P &lt; 0.0001; ns = not significant. Figure S3. Quantification of hepatic metastases after intrasplenic of WT31 cells as well as quantification of lung metastases after intravenous injection of WT31 cells. Measurement of microscopic and macroscopic metastatic area. A. After spleen injection of WT31 melanoma cells the numbers of macroscopic visible hepatic metastases (57.6 vs. 7.6, P = 0.0164) were quantified in Ctrl (n = 10) and Lyve-1-/- (n = 5) mice at day 21. The ROUT method was used to detect outliers. Two outliers were excluded in the Lyve-1-/- group. Representative photographs of livers with melanoma metastases are shown. Scale bars: 1 cm. B. WT31 cells were injected into the tail vein and melanoma lung metastases (41.92 vs. 59.71, P = 0.1343) were counted in Ctrl (n = 13) and Lyve-1-/- (n = 14) mice at day 19. Photographs of lungs are displayed. Scale bars: 1 cm. C. The hepatic macroscopic metastatic area in relation to the total liver area was analyzed per mouse. MC38 hepatic metastatic area (P = 0.6336) in Ctrl (n = 5) and Lyve-1-/- (n = 5). B16F10 luc2 hepatic metastatic area (P = 0.0175, Mann-Whitney U-test) in Ctrl (n = 12) and Lyve-1-/- (n = 14). Hepatic metastatic area after intrasplenic injection of WT31 melanoma (P = 0.0256) in Ctrl (n = 10) and Lyve-1-/- (n = 5). Hepatic metastatic area after i.v. injection of WT31 melanoma (P = 0.4502, Mann-Whitney U-test) in Ctrl (n = 13) and Lyve-1-/- (n = 14). D. The hepatic microscopic metastatic area in relation to the total liver area was analyzed per mouse. MC38 hepatic metastatic area (P = 0.9675) in Ctrl (n = 5) and Lyve-1-/- (n = 5). B16F10 luc2 hepatic metastatic area (P = 0.0068, Mann-Whitney U-test) in Ctrl (n = 6) and Lyve-1-/- (n = 9). Hepatic metastatic area after intrasplenic injection of WT31 melanoma (P = 0.0186, Mann-Whitney U-test) in Ctrl (n = 8) and Lyve-1-/- (n = 5). Hepatic metastatic area after i.v. injection of WT31 melanoma (P = 0.8443, Mann-Whitney U-test) in Ctrl (n = 12) and Lyve-1-/- (n = 14). Data information: * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001; **** P &lt; 0.0001; ns = not significant. Figure S4. Analysis of cell proliferation or apoptosis in hepatic melanoma metastases of Ctrl and Lyve-1-/- mice. A. Immunofluorescence stainings of hepatic B16F10 luc2 melanoma metastases for Ki67 and Emcn in Ctrl (nmet = 14, n = 4) and Lyve-1-/- (nmet = 12, n = 5) mice as well as for cleaved Caspase-3 (cCasp-3) and Emcn in Ctrl (nmet = 14, n = 4) and Lyve-1-/- (nmet = 12, n = 5) mice. Scale bars: 100 µm. B. Immunofluorescence stainings of hepatic WT31 melanoma metastases for Ki67 and Emcn in Ctrl (nmet = 16, n = 7) and Lyve-1-/- (nmet = 11, n = 6) mice as well as for cCasp-3 and Emcn in Ctrl (nmet = 9, n = 5) and Lyve-1-/- (nmet = 6, n = 5) mice. Scale bars: 100 µm. C. Percentage of Ki67+DAPI+ cells (P = 0.2972, Mann-Whitney U-test) in Ctrl (nmet = 14, n = 4) and Lyve-1-/- (nmet = 12, n = 5) mice or cCasp-3+DAPI+ cells (P = 0.7808, Mann-Whitney U-test) in Ctrl (nmet = 14, n= 4) and Lyve-1-/- (nmet = 12, n = 5) mice in relation to total DAPI+ melanoma cells counted in hepatic B16F10 luc2 melanoma metastases. D. Percentage of Ki67+DAPI+ cells (P = 0.9594) in Ctrl (nmet = 16, n = 7) and Lyve-1-/- (nmet = 11, n = 6) mice or cCasp-3+DAPI+ cells (P = 0.8637, Mann-Whitney U-test) in Ctrl (nmet = 9, n = 5) and Lyve-1-/- (nmet = 6, n = 5) mice in relation to total DAPI+ melanoma cells counted in hepatic WT31 melanoma metastases. Data information: * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001; **** P &lt; 0.0001; ns = not significant. Figure S5. Analysis of F4/80+Lyve-1+ cells in tumor-free and metastasized Ctrl livers. A. Immunofluorescence stainings for F4/80 and Lyve-1 in 3-day-old skin wound healing tissue of wild type mice (n = 2). Wounds were previously induced by 4mm punch biopsy under anaesthesia with Isoflurane. B. Immunofluorescence stainings of F4/80 and Lyve-1 in untreated, tumor-free Ctrl and Lyve-1 -/- liver tissue (female, n = 6/group). Scale bars: 100 µm. C. Immunofluorescence stainings of F4/80 and Lyve-1 in livers with metastases of B16F10 luc2 melanoma cells in Ctrl (nmet = 10, n = 6) and Lyve-1-/- mice (nmet = 6, n = 5). D. Immunofluorescence stainings of F4/80 and Lyve-1 in livers with metastases of WT31 melanoma cells of Ctrl (nmet = 12, n = 5) and Lyve-1-/- mice (nmet = 12, n = 5). Figure S6. Gating strategy applied to identify indicated hepatic immune cell populations in both tumor-free and metastasized livers by flow cytometry. Additional analyses of dendritic cells, NK cells, NKT cells and gamma delta (gd) T cells in tumor-free and metastasized Lyve-1-/- livers by flow cytometry. A. Single-cell suspensions of liver tissue were analyzed by flow cytometry. Cells gated as single/live CD45+ were further defined as: B cells (CD3εnegCD19+), γδT T cells (CD19negCD3ε+TCRγδ+), NKT cells (CD3ε+TCRβ+NK1.1+), CD8+ T cells (CD3ε+TCRβ+NK1.1negCD8a+), CD4+ T cells (CD3ε+TCRβ+NK1.1negCD4+), Treg (CD3ε+TCRβ+NK1.1negCD4+FoxP3+), NK cells (CD3εnegCD19negNKp46+NK1.1+Eomes+); myeloid cells are first gated as Lymphoidneg CD3εnegCD19negNKp46negNK1.1neg), and then further defined as neutrophils (CD11b+Ly6G+), eosinophils (CD11b+Ly6GintSiglecF+), DCs (CD11b+Ly6GnegSiglecFnegCD11chighMHC-IIhigh), macrophages (non-DCs F4/80+Ly6Cneg), and inflammatory monocytes (non-DCs F4/80+Ly6C+). Representative end-gates with simplified ascending gating paths for each population are shown. B. Flow cytometric analysis of liver immune cells including dendritic cells (P = 0.2459), NK cells (P = 0.1165), NKT cells (P = 0.0973) and gdT cells (P = 0.0653) was performed of whole untreated, tumor-free Ctrl and Lyve-1-/- livers (female, n = 5-8/group). C. Flow cytometric analysis of whole liver tissue with B16F10 luc2 melanoma metastases of Ctrl and Lyve-1-/- mice (female, n = 5-8/group) for dendritic cells (P = 0.2624), NK cells (P = 0.6368), NKT cells (P = 0.1412) and gdT cells (P = 0.1668). D. Flow cytometric analysis of whole liver tissue with WT31 melanoma metastases of Ctrl and Lyve-1-/- mice (female, n = 8/group) for dendritic cells (P = 0.3006), NK cells (P = 0.7971), NKT cells (P = 0.3127) and gdT cells (P = 0.8224). Data information: * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001; **** P &lt; 0.0001; ns = not significant. Figure S7. Unaltered numbers of myeloid-derived suppressor cells and Tregs in peritumoral liver tissue of Ctrl and Lyve-1-/- with B16F10 luc2 or WT31 metastases. A. Immunofluorescence stainings of peritumoral hepatic tissue for DAPI, CD45 and Ly6C as well as for DAPI, CD45 and Ly6C/Ly6G in Ctrl (n = 4) and Lyve-1-/- (n = 5) mice with B16F10 luc2 metastases. Scale bars: 100 µm. B. Immunofluorescence stainings of peritumoral hepatic tissue for DAPI, CD45 and Ly6C as well as for DAPI, CD45 and Ly6C/Ly6G in Ctrl (n = 6) and Lyve-1-/- (n = 5) mice with WT31 metastases. Scale bars: 100 µm. C. Quantification of peritumoral CD45+Ly6C+ cells (P = 0.099) or CD45+Ly6C+Ly6G+ cells (P = 0.3896) in livers of Ctrl (n= 4) and Lyve-1-/- (n = 5) mice with metastases of B16F10 luc2. D. Quantification of peritumoral CD45+Ly6C+ cells (P = 0.7117) or CD45+Ly6C+Ly6G+ cells (P = 0.1967) in livers of Ctrl (n= 6) and Lyve-1-/- (n = 5) mice with metastases of WT31. E. Quantification of peritumoral CD4+FoxP3+ cells (P = 0.1852) in livers of Ctrl (n= 8) and Lyve-1-/- (n = 7) mice with B16F10 luc2 metastases. F. Quantification of peritumoral CD4+FoxP3+ cells (P = 0.9984) in livers of Ctrl (n = 9) and Lyve-1-/- (n = 8) mice with WT31 metastases. Data information: * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001; **** P &lt; 0.0001; ns = not significant.