Fig 1: IL-9 impacts lung macrophage function by regulating Arg1 expression.a Heatmap showing Arg1 expression from RNA-Seq experiment described in Fig. 5. b, c Arg1 expression in macrophages from B16 tumor model (b) (n = 9 mice for WT group, n = 7 mice for Il9r−/− group) and orthotopic LLC lung tumor model (c) (n = 6 mice for WT group, n = 5 mice for Il9r−/− group). d Arg1 expression in mixed bone marrow chimeric mice described in Fig. 4f (n = 9 mice). e CD11c−IM and CD11c+IM were isolated from WT tumor bearing mice and stimulated with PBS or 40 ng/ml IL-9 for 48 h. Arg1 production was analyzed by flow. f Arginase activity were analyzed in total lung macrophages and tumor foci (n = 5 mice for WT and Il9r−/− - B16-Lung Mac group, n = 5 mice for WT-LLC group, n = 12 mice for Il9r−/− -LLC−Lung Mac, n = 7 mice for WT- orthotopic LLC tumor group, n = 2 mice for Il9r−/−- orthotopic LLC tumor group). g–i YARG mice were injected with B16 cells, and total lung Arg1+/− macrophages were sorted from intact lungs of tumor bearing mice on day 14. Cells were intravenously injected into Il9r−/− mice 4 days after tumor injection. f, g Tumor development was analyzed on day 23. h Donor macrophages were analyzed by flow cytometry (n = 2 mice for WT group, n = 8 mice for Il9r−/− + PBS group, n = 12 mice for Il9r−/− + YFP+ Mac group, n = 14 mice for Il9r−/− +YFP- Mac group). i Lung Arg1+ macrophages were analyzed by flow cytometry (n = 2 mice for WT group, n = 8 mice for Il9r−/− + PBS group, n = 10 mice for Il9r−/− + YFP+ Mac group, n = 13 mice for Il9r−/− +YFP− Mac group). Mac: macrophage. j Arg1fl/fl LysM-Cre+/- mice were injected with B16 tumor, tumor development was analyzed on day 14 and day 21 (n = 13 mice for D14 Arg1fl/fl LysM-Cre+ group, n = 6 mice for D21 Arg1fl/fl LysM-Cre+ group, n = 9 mice for D14 and D21 Arg1fl/fl LysM-Cre- groups). k IMs from tumor-bearing Arg1fl/fl LysM-Cre+ mice or littermate control mice were sorted from intact lungs of tumor bearing mice 14 days after tumor injection and transferred to Il9r−/− mice which have been injected with tumor 4 days before. Tumor development was analyzed on day 17 (n = 6 mice for Arg1fl/fl LysM-Cre- group, n = 5 mice for Arg1fl/fl LysM-Cre+ group). l Arg1+ cells were analyzed from WT tumor bearing mice by flow cytometry (n = 8 mice per group). m Dot plot showing ARG1 expression in different clusters from human lung cancer patient scRNA-Seq. Data are the mean ± SEM. Unpaired two-tailed Student t-test was used for comparison in b, c, f and k. Two-tailed paired t test was used for generating p value in d. One-way ANOVA with a Dunnett’s multiple comparison test was used for multiple comparisons in h and i. Two-way ANOVA with Sidak’s multiple comparisons was used for comparisons in j.
Fig 2: TAMs express IL9R in lung cancer patient tissue.a Kaplan-Meier plots showing differences in survival among lung cancer patients (n = 982) using data derived from published transcriptomic data and online tools described in the Methods for IL9 and IL9R. HR: hazard ratio. b Comparison of gene expression in normal lung tissue and metastatic lung tissues by using data derived from published transcriptomic data obtained by gene-array and online tools described in the “Methods”. FC: fold change. K.W.p Kruskal–Wallis p value. The bars represent the proportions of metastatic tumor samples that show higher expression of the selected gene compared to normal samples at each of the quantile cutoff values (minimum, 1st quartile, median, 3rd quartile, maximum). c UMAP showing clusters from normal and lung tumor tissue from human lung cancer patients. DC, dendritic cell. d UMAP showing gene expression. e Dot plot showing IL9R expression in different clusters.
Fig 3: IMs are the TAMs that respond to IL-9.a–d LLC cells were directly injected to the lung. Tumor appearance (a) and tumor incidence (b) (n = 2 experiments) were assessed after resection. Tumor weight (c) (n = 8 mice for WT group, n = 10 mice for Il9r−/− group) was calculated from two independent experiments. d Lung cells from tumor bearing mice and cells from tumor foci were isolated for flow analysis (n = 5 mice for lung Mac group, n = 6 mice for TAM group). TAM: tumor associated macrophage. e Venn graph showing the overlap genes from human TAMs and mouse IMs. f–h WT (CD45.1) and Il9r−/− mice (CD45.2) bone marrow cells were mixed in 1:1 ratio and transferred to lethally irradiated recipient mice (CD45.1+ CD45.2+ mice). After reconstitution, chimeric mice were intravenously injected with B16 tumor cells, donor lung macrophages were analyzed by flow cytometry (n = 9 mice). i–k Il9r−/− mice were injected with B16 tumor on day 0. Fluorescent bead-labeled monocytes were transferred to recipient mice on day 10 (i). Tumor growth (j) and lung macrophages (k) were analyzed on day 18, dot plots were gated on MerTK+ CD64+ SiglecF− live cells (n = 4 mice per group). Data are the mean ± SEM. Unpaired two-tailed Student t-test was used for comparison in c, d and j. Two-way ANOVA with Sidak’s multiple comparisons was used for comparisons in h.
Fig 4: Lung macrophages promote IL-9 mediated lung tumor growth.a, b Boy/J mice were injected with B16 melanoma cells. Macrophages were sorted from the entire lung of tumor bearing mice on d17 and intravenously injected to Il9r−/− mice 4 days after tumor injection (a). b, c Donor macrophages were detected on day 20 (n = 4 mice for AM group, n = 5 mice for CD11c+ IM group, n = 6 for CD11c− IM group in panel b) (n = 3 mice for PBS group, n = 5 mice for other groups in panel c). d Tumor development was assessed (n = 8 mice for WT group, n = 3 mice for PBS group, n = 5 mice for other groups). e, f WT and Il9r−/− macrophages were sorted from the entire lung of mixed bone marrow chimeric mice described in Fig. 4f and transferred into Il9r−/− tumor bearing mice (e). Tumor growth was analyzed on day 17 (f) (n = 3 mice). Mac: macrophage. g–j Total lung macrophages were isolated from intact lungs of B16 (g, h) (n = 1 for left two groups, n = 4 mice for middle group, n = 3 mice for right two groups) or LLC (i) (n = 1 well of cell for left group, n = 2 wells of cell for the second left group, n = 4 mice for other groups) tumor-bearing mice and plated in the lower chamber of a transwell with or without IL-9; B16 or LLC cells were plated in the upper chamber. B16 cells were allowed to migrate for 16 h, and LLC cells for 3 h before counting. Unmigrated cells were removed and migrated cells were calculated from the average of two views under 20× microscopy, Scale bar = 250 µm. j Human monocytes were isolated from human PBMC and differentiated into M1 or M2 macrophage for 7 days. Cells were treated with IL-9 overnight, and human 838 lung cancer cells were plated in the upper chamber of the transwell. Migrated cells were counted after 3 h (n = 4 donors per group). Data are the mean ± SEM. One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p values for multiple comparisons in b, c, d. Unpaired two-tailed Student t-test was used for comparison in f, g, i and j.
Fig 5: Therapeutic targeting of IL-9-macrophage axis prevents lung cancer growth.a Kaplan–Meier plots showing differences in survival among lung cancer patients (n = 982 donors) by using data and online tools described in Methods for ARG1 and IL6. b Comparison of gene expression in normal lung tissue and metastatic lung tissues by using data and online tool described in the method. The bars represent the proportions of metastatic tumor samples that show higher expression of the selected gene compared to normal samples at each of the quantile cutoff values (minimum, 1st quartile, median, 3rd quartile, maximum). c IL9R and IL6 gene expression were analyzed in cells from normal lung tissue and cells in the lung tumor (n = 8 donors for non-tumor group and n = 9 donors for lung cancer group in left panel, n = 6 donors for non-tumor group and n = 4 donors for lung cancer group in right panel). d Serum IL-9 level, IL-6 level and arginase activity were analyzed from healthy donors and lung cancer patient samples. (n = 5 donors for left panel, n = 7 donors for non-tumor group and n = 4 donors for lung cancer group in middle panel, n = 7 donors for non-tumor group and n = 5 donors for lung cancer group in right panel). e Dot plot showing gene expression in macrophages between normal lung tissue and lung tumor. f, g Immunohistochemistry staining of CD68 and IL-9R. Protein expression quantification was performed by using Image J software (n = 7 donors), Scale bar = 100 µm. h–l WT mice were intravenously injected with B16 tumor cell line, 7 days after tumor inoculation, tumor bearing mice were intravenously injected with nanoparticle-siRNA complexes every 72 h. Scr/Il9r-siRNA was conjugated with Alexa Flour 555. Nanoparticles were tagged with SIRPα peptide (h). i Lung tumor growth were analyzed on day 21 (n = 6 mice for Scr-siRNA group, n = 7 mice for Il9r-siRNA group). j IL-9R expression in siRNA+ (Alexa Flour 555) lung macrophages were analyzed by flow (n = 6 mice for Scr-siRNA group, n = 7 mice for Il9r-siRNA group). k Arg1 production from siRNA+ (Alexa Flour 555) macrophages were analyzed by flow (n = 6 mice for Scr-siRNA group, n = 7 mice for Il9r-siRNA group). l siRNA+ (Alexa Flour 555) lung macrophages were sorted by gating on Alexa Flour 555+ MerTK+ CD64+ live cells. Gene expression was analyzed (n = 6 mice for groups in left panel, n = 6 mice for Scr-siRNA group and n = 7 mice for Il9r-siRNA group in right panel). Data are the mean ± SEM. Unpaired two-tailed Student t-test was used for comparison.
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