Fig 2: IL-9 induces IL-6 expression in Arg1 expressing IMs.a Dot plot showing IL6 expression in different clusters from human lung cancer patients scRNA-Seq. b Naive mice were i.v injected with IL-9 for 4 days, IL-6 expression in IMs was analyzed (n = 4 mice). c IL6 expression in human PBMC monocyte derived M2 macrophage (n = 3 donors). d Serum IL-6 level in B16 tumor bearing mice (n = 9 mice for WT group, n = 8 mice for Il9r−/− mice). e IL-6 expression was analyzed by gating on Arg1+ or Arg1− macrophages from WT B16 tumor bearing mice (n = 9 mice). f Serum IL-6 level from mice described in Fig. 6f were analyzed by ELISA (n = 10 mice for Arg1+ Mac group, n = 12 mice for Arg1− Mac group, n = 5 mice for PBS group). g Arg1+ or Arg1- macrophages were sorted from entire lung of tumor bearing mice and plated in the lower chamber of the transwell; B16 tumor cells were placed in the upper chamber. Cells were allowed to migrate for 12 h (n = 4 mice). h Arg1+ or Arg1− macrophages were sorted from entire lung of tumor bearing mice and cultured for 24 h, IL-6 concentration was analyzed (n = 3 mice). i, j Mixed bone marrow chimeric mice were generated and injected with B16 tumor as described. Donor derived IL-6+ Arg1+ IMs were analyzed by flow cytometry, dot plots were gated on live IMs (n = 9 mice). k–m WT tumor-bearing mice were treated with isotype antibody or anti-IL-6 antibody as shown in k, tumor growth (l) (n = 3 mice for isotype group, n = 5 mice for anti-IL-6 group) and survival (m) (n = 12 mice) were analyzed. Unpaired two-tailed Student t-test was used for comparison in b, c, d, e, h and l. Data are the mean ± SEM. One-way ANOVA with a Dunnett’s multiple comparison test was used for multiple comparisons in f. Paired two-tailed Student t-test was used for comparison in j. Two-way ANOVA with Sidak’s multiple comparisons was used for comparisons in g.

Fig 3: IL-9 promotes tumor growth by altering lung macrophage populations.a–f B16 melanoma cells were intravenously injected into the mice. a Tumor development was assessed on day 21 (n = 11 mice for WT group, n = 7 mice for Il9r−/− group, n = 9 mice for Il9−/− group). b Survival analysis from d0 to d40 after tumor cell injection. c IL-9+ cells were analyzed by flow cytometry. d, e Lung macrophage populations were analyzed by flow (n = 8 mice for WT group, n = 7 mice for Il9r−/− group, n = 4 mice for Il9−/− group). f ΔgMFI of IL-9R were analyzed by flow cytometry, ΔgMFI is the gMFI of each population minus the gMFI of isotype controls in that population (n = 8 mice for MDSC and n = 11 mice for other cell types). gMFI geometric mean fluorescence intensity. MDSC, Myeloid-derived suppressor cells. g, h Mice were i.v. injected with LLC tumor cells, tumor growth (g) (n = 6 mice) and lung macrophages (h) (n = 7 mice) were analyzed on day 20. i.v.: intravenously injection. i B16 melanoma cells were intravenously injected into the mice. Tumor development was assessed on day 21. Lung macrophage numbers were analyzed by FACS (n = 8 mice for Il9fl/fl CD4-Cre− group, n = 7 mice for Il9fl/fl CD4-Cre+). j, k LLC cells were directly injected to the lung. Tumor growth was assessed 14 days after tumor inoculation (j) (n = 3 mice for WT-Matrigel and Il9r−/−-Matrigel group, n = 8 mice for WT-LLC group, n = 7 mice for Il9r−/−-LLC group). Lung macrophages were analyzed by flow (k) (n = 3 mice for WT-Matrigel and Il9r−/−-Matrigel group, n = 4 mice for WT-LLC group and Il9r−/−-LLC group). Data are the mean ± SEM and representative of two independent experiments. One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p values for multiple comparisons in a and e. Log-rank (Mantel–Cox) test was used to generate p value in b. Unpaired two-tailed Student t-test was used for comparison in g–k. Two-way ANOVA with Sidak’s multiple comparisons was used for comparisons in e.

Fig 4: 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 5: 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.