Mouse pulmonary interstitial macrophages mediate the pro-tumorigenic effects of IL-9

Although IL-9 has potent anti-tumor activity in adoptive cell transfer therapy, some models suggest that it can promote tumor growth. Here, we show that IL-9 signaling is associated with poor outcomes in patients with various forms of lung cancer, and is required for lung tumor growth in multiple mouse models. CD4+ T cell-derived IL-9 promotes the expansion of both CD11c+ and CD11c− interstitial macrophage populations in lung tumor models. Mechanistically, the IL-9/macrophage axis requires arginase 1 (Arg1) to mediate tumor growth. Indeed, adoptive transfer of Arg1+ but not Arg1- lung macrophages to Il9r−/− mice promotes tumor growth. Moreover, targeting IL-9 signaling using macrophage-specific nanoparticles restricts lung tumor growth in mice. Lastly, elevated expression of IL-9R and Arg1 in tumor lesions is associated with poor prognosis in lung cancer patients. Thus, our study suggests the IL-9/macrophage/Arg1 axis is a potential therapeutic target for lung cancer therapy.

. IMs are the TAMs that respond to IL-9 and promote tumor growth (a-b), LLC cells were injected to the lung, Tumor-infiltrating immune cells were analyzed by flow cytometry (a) (n = 9 mice for WT group, n = 2 mice for Il9r -/group). (b), Cytokine production was analyzed (n = 9 mice for WT GranzB, Perforin, IL-17, and GM-CSF, n = 10 mice for WT IL-10, IL-13, IL-9, IFNg and TNFa group, n = 2 mice Il9r -/group). (c), Monocytes from bone marrow, blood and lung were analyzed 21days after B16 tumor injection (n = 6 mice for groups in left and middle panel, n = 5 mice for groups in right panel). (d), Ki67 expression in monocytes from bone marrow, blood and lung were analyzed 21 days after B16 tumor injection (n = 5 mice for groups in left panel, n = 6 mice for groups in middle panel, n = 6 for WT group and n = 8 mice for Il9r -/group in right panel). (e), Ki67 expression of lung AMs were analyzed 21 days after B16 tumor injection (n = 4 mice for WT PBS group, n = 2 mice for for Il9r -/-PBS group, n = 6 for WT-B-16 group and n = 8 mice for Il9r -/--B16 group). (f-g), IL-9R expression in TAMs (f) and macrophage percentage (g) from s.c. B16 tumor bearing mice (n = 4 mice for WT and Il9r -/group, n = 2 mice for Il9 -/group). Data are the mean ± SEM. Unpaired two-tailed Student t-test was used for comparison in d-e. Supplementary Fig. 3. IL-9 promotes macrophage mediated tumor migration (a), IL-9R expression on LLC cancer cell line was analyzed. (b), LLC cancer cells were treated with IL-9 for 24hs. Ki67 expression was analyzed (n = 3 independent wells). (c), B16 cells were treated with IL-9 for 48hs. Cell death and proliferation was analyzed. (d), Total lung macrophages were isolated from LLC tumor bearing mice and cocultured with LLC cells for 72 hours. LLC cell proliferation and cell death were analyzed by flow cytometry (n = 4 mice).
(e-f), Total lung macrophages were isolated from tumor bearing mice and plated in the lower chamber of the transwell with or without IL-9, tumor cells were plated in the upper chamber (e). (f), Migrated cells were visualized with crystal violet staining (n=3 mice). (g-h), Human monocytes were cultured with GM-CSF or M-CSF for polarizing into M1 or M2 macrophage for 5 days before IFN-γ or IL-4 were respectively added to the cultures for 48hs. IL-9 was added for an additional 24 hours. The migration assay was performed (g) and human 838 cells were stained with crystal violet (h), Scale bar = 100 µm.
Data are the mean ± SEM. Unpaired two-tailed Student t-test was used for comparison in b and f. One-way ANOVA with a Dunnett's multiple comparison test was used for multiple comparisons in d.

Supplementary Fig. 4. IL-9 impacts lung macrophage function by regulating Arg1 expression.
(a) iNOS expression from IMs were analyzed by flow (n= 5 mice for WT group, n = 3 for Il9r -/group). (b-e), YARG mice were injected with B16 cells, and lung Arg1 +/YFP macrophages were sorted on day 14. Cells were intravenously injected into Il9r -/mice 4 days after tumor injection (b). Lung weights were measured on day 23 (c) (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). Arg1 expression was analyzed in macrophages by flow cytometry (d). Il9r expression was analyzed from FACS sorted donor macrophages (e). (f), CD206 expression in IMs from Arg1 fl/fl LysM-Cre + mice or littermate control mice (n = 6 mice for Arg1 fl/fl LysM-Cregroup, n = 8 mice for Arg1 fl/fl LysM-Cre + group). (g), Percentages of lung CD4 and CD8 T cells, Ki67 expression in T cells and cytokine expression from CD8 cells were analyzed by flow cytometry (n = 7 mice for Arg1 fl/fl LysM-Cregroup and n = 10 mice for Arg1 fl/fl LysM-Cre + group). (h), Percentages of lung CD4 and CD8 T cells, CD44 + and CD62L + T cells were analyzed by flow (n= 10 mice for WT group, n = 7 mice for Il9r -/group). (i), Macrophage adoptive transfer experiment was performed as shown in Fig. 5d. Lung CD62L+ CD8 T cells were analyzed (n =8 mice for WT group, n = 3 mice for PBS group, n = 5 mice for AM and CD11c + IM groups, n = 6 mice for CD11c -IM group). Data are the mean ± SEM. One-way ANOVA with a Dunnett's multiple comparison test was used for multiple comparisons in c and i. Unpaired two-tailed Student t-test was used for comparison in h. Supplementary Fig. 5. IL-9 induces IL-6 expression in Arg1 expressing IMs. (a-b), WT tumor bearing mice were treated with isotype antibody or anti-IL-6 (a) and tumor growth was analyzed (b) (n = 7 mice). (c), B16 cells were stimulated for 60 minutes and pSTAT3 expression was analyzed.
(d-f), DC numbers (d) (n = 5 mice for WT group, n = 6 for Il9r -/group), pSTAT3 expression (e) (n = 19 mice for WT group, n = 11 mice for Il9r -/group), and activation markers (f) (n = 3 mice for WT group, n = 4 mice for Il9r -/group) were analyzed from B16 tumor bearing mice. (g), Mice were treated as shown in b, MHCII expression from lung DCs was analyzed by flow cytometry (n = 5 mice for isotype group, n = 7 mice for anti-IL-6 group). Data are the mean ± SEM. Unpaired two-tailed Student t-test was used for comparison. Supplementary Fig. 6. Therapeutic targeting of the IL-9-macrophage axis prevents lung cancer growth. (a), Immunohistochemistry staining of CD163 and IL-9R in lung cancer patient tissue, Scale bar = 100 µm.
(b-f), WT mice were intravenously injected with B16 tumor cell line. Seven days after tumor inoculation, tumor-bearing mice were intravenously injected with nanoparticle-siRNA complexes every 72 hours. Scr/Il9r-siRNA was conjugated with Alexa Flour 555. Nanoparticles were tagged with SIRPα peptide. (b-c) Lung cells that had nanoparticle-siRNA complex uptake were analyzed by flow cytometry ( n = 5 mice for groups in b, n = 6 mice for Scr-siRNA group and n = 7 for Il9r-siRNA group in c). (d), IL-9R expression in siRNA + (Alexa Flour 555) lung macrophages were analyzed by flow (n = 4 mice). (e), siRNA + (Alexa Flour 555) lung macrophages were sorted by gating on Alexa Flour 555 + MerTK + CD64 + live cells. Gene expression was analyzed (n = 3 mice). (f) Lung macrophages were analyzed by flow cytometry. (n = 6 mice for Scr-siRNA group and n = 7 for Il9r-siRNA group in the middle and right panels). Data are the mean ± SEM. Unpaired two-tailed Student t-test was used for comparison. Supplementary Fig. 7. Gating strategies used for identifying different cell types Gating strategies for identifying lung macrophages (a), T cells (b), mast cell and basophil (c), monocytes and DC (d). Strategy for macrophages was used in Figures 2c-f, h, i, k, Figure 4d Supplementary Figures 1a-c, e-f, h, i-l, q-r, 2e-g, 4a, d, f, and 6c-f. T cell gating was used in Supplementary 4g-i. Monocyte gating was used in Supplementary 2c-d. DC gating was used in Supplementary 5c-g. All strategies were used for Figures 2c and f, 7l, and Supplementary figures 1m-o, 2a-b, and 6b.

Supplementary Tables
Supplementary Table 1. Lung cancer patient sample information related to Fig. 9c