Fig 2: Tumor proliferation is suppressed in INHBA conditional knock out mice.a Schematic of the experimental design for tamoxifen-inducible Inhba knockout mice with an orthotopic lung cancer model. b Inhba RT-PCR analysis of AMs isolated from Inhbafl/fl CreERT2 - and Inhbafl/fl CreERT2 + mice treated with tamoxifen (n = 4 mice per group). c Representative images of left lung tumors in tumor-bearing mice. d Gross appearance of tumors from Inhbafl/fl CreERT2 - (upper) and Inhbafl/fl CreERT2 + (lower) mice. Scale bars; 5 mm. e Tumor volume of Inhbafl/fl CreERT2 - and Inhbafl/fl CreERT2 + mice (n = 4 per group). f Representative images of CD163 (purple) and INHBA (brown) immunostaining of the normal lung area (left) and lung cancer area (right) from patients with lung cancer. Scale bars; 50 µm. g Proportion of CD163 + and INHBA + macrophages in total cells within the visual field in normal lung and lung cancer tissues, calculated from the mean value of four different visual fields (n = 10 patients). h Schematic diagram of the activin A-producing AM subtype in the lung tumor microenvironment. Means ± s.e.m. for each group are shown. Symbols represent individual mice (b, e) and patients (g). Statistical significance was determined using unpaired (b, e) and paired (g) two-tailed t-tests.

Fig 3: INHBA upregulation in lung alveolar macrophages (AMs) enhances proliferation of lung cancer cells in vivo.a Definitions of R1, R2, and R3 cells in the lungs of the control and tumor-bearing mice. Left images show schematic diagram of control (Ctrl) mice with PBS (upper) or tumor-bearing mice with LLC cells fluorescently labeled with tdTomato (lower). Dot plots on the right indicate the analysis result of CD45+ autofluorescence+ cells (black rectangular regions in the middle dot plots). The red rectangular region and blue pentagonal region indicate AMs and TAMs, respectively. The data are representative of three independent experiments with similar results. b Principal component analysis of R1, R2, and R3 cells by RNA-Seq (n = 3 mice for R1, R2, and R3 populations). c Heatmaps of tumor-associated macrophage marker genes and AM marker genes (left), and the top 15 upregulated genes from R1 to R2 cells (right). d RT-PCR analysis of Inhba expression in AMs and CD45+ (autofluorescence-) cells isolated from control mice, and AMs, CD45+ (autofluorescence-) cells, and tumor cells isolated from tumor-bearing mice (n = 3 mice per group). e Measurement of activin A concentration in AMs sorted from control or tumor-bearing mice using ELISA (n = 6 per group). f WST-1 cell proliferation assay of LLC cells after administration of each dose of recombinant activin A. Viability of LLC cells in each well with or without recombinant activin A was measured on days 0, 1, and 4. Each value indicates the mean ± s.e.m. of the three wells (n = 4 per group for day 0 and day 1, n = 3 per group for day 4). g Comparison of LLC cell number after 2-day culture with or without conditioned media (Sup.) from shRNA-expressing AM cells (AMJ2-C11) (n = 3 per group). h Representative images of the left lung of tumor-bearing mice treated with PBS (left; control) and follistatin (right). Arrowheads indicate the tumor (surrounded by a dashed line). i Tumor volume of tumor-bearing mice treated with PBS (control) or follistatin (n = 3 mice for control, n = 4 mice for follistatin). Means ± s.e.m. for each group are shown. Symbols represent individual mice (d, e, i) or wells (g). Statistical significance was determined using one-way ANOVA with Bonferroni’s post hoc test (d, g) or unpaired two-tailed t-test (e, i).

Fig 4: Single-cell RNA-seq identifies a subtype of tumor-supporting alveolar macrophages (AMs) with high expression of INHBA.a–c UMAP plot of single-cell RNA-seq data of AMs from control and tumor-bearing mice. a A total of 13,413 AM cell transcriptomes were analyzed and hierarchically clustered into 14 subgroups using uniform manifold approximation and projection (UMAP) analysis. b, c Ratio of AMs isolated from control mice (blue) to those isolated from tumor-bearing mice (orange). d Schematic image of RNA-velocity analysis results with scVelo. e, f Distribution of Inhba gene expression levels in AMs isolated from tumor-bearing mice (e) and those isolated from control mice (f), shown using binarization by fitting the expression distribution with Gaussian mixture distribution. g, h In vitro culture of mouse primary AMs from bronchoalveolar lavage fluid with or without damage-associated molecular pattern molecules (DAMPs) from LLC cells. g Flow cytometry histogram of AMs with MARCO. Shaded regions indicate staining with only the second antibody (i.e., without the anti-MARCO antibody). h RT-PCR analysis of Inhba expression in AMs after in vitro culture for 1 day with or without DAMPs (n = 3 per group). Means ± s.e.m. for each group are shown. Symbols represent individual wells (h). Statistical significance was determined using one-way ANOVA with Bonferroni’s post hoc test (h).