Fig. 2: BMP7 modulates MAPK14 in anti-PD1-resistant tumors and immune cells.

a Reverse phase protein array (RPPA) results on expression levels and activation status of 243 proteins in 344SQP (n = 3 biologically independent samples) and 344SQR (n = 3 biologically independent samples) tumors treated with anti-PD1(10 mg/kg). Normalized data were first log2-transformed (log2(x + 1)). Proteins expressed at different levels between groups (downregulated proteins in green and upregulated proteins in red) were identified by a P value of <0.05 obtained from LIMMA’s moderated t statistic (MAPK14, *p = 0.0107). b Representative images of immunohistochemical stains for MAPK14, SMAD1/5/9 phosphorylation, and SMAD1 in formalin-fixed paraffin-embedded tissue sections from 344SQP and 344SQR tumors treated with IgG (10 mg/kg). Scale bar, 100 μm (×40 magnification). Data shown are representative of two reproducible independent experiments of  three biologically independent samples. c, Representative images of immunohistochemical stains for MAPK14, SMAD1/5/9 phosphorylation, and SMAD1 in formalin-fixed paraffin-embedded tissue sections from BMP7-knockdown tumors treated with IgG (10 mg/kg) compared with control (scale bar, 100 μm) (×40 magnification). Data shown are representative of two reproducible independent experiments of three biologically independent samples. d Representative images of immunohistochemical stains for MAPK14, SMAD1/5/9 phosphorylation, and SMAD1 in formalin-fixed paraffin-embedded tissue sections from 344SQP and 344SQR tumors treated with anti-PD1(10 mg/kg). Scale bar, 100 μm (×40 magnification). Data shown are representative of two reproducible independent experiments of three biologically independent samples. e Representative images of immunohistochemical stains for MAPK14, SMAD1/5/9 phosphorylation, and SMAD1 in formalin-fixed paraffin-embedded tissue sections from BMP7-knockdown tumors treated with anti-PD1(10 mg/kg) compared with control (scale bar, 100 μm) (×40 magnification). Data shown are representative of two reproducible independent experiments of three biologically independent samples. f Representative images of immunohistochemical stains for MAPK14 and SMAD1/5/9 phosphorylation in formalin-fixed paraffin-embedded tissue sections from two patients collected before treatment and at the time of disease progression on pembrolizumab or ipimilumab. Scale bar, 100 μm (×40 magnification). Data shown are representative of two reproducible independent experiments. g Nanostring immune panel results for 770 genes in tumor-infiltrating leukocytes (TILs) collected from 344SQP (n = 2 biologically independent samples) and 344SQR (n = 3 biologically independent samples) tumors treated with anti-PD1(10 mg/kg). Genes expressed at different levels between groups (downregulated genes in green and upregulated genes in red) were identified by a P value of <0.05 obtained from LIMMA’s moderated t statistic (MAPK14, p = 0.0047). h Immunofluorescence analysis of MAPK14 and SMAD1/5/9 phosphorylation (green) in the macrophage cell line RAW 264.7 at 24 h after treatment with BMP7 (250 ng) or BMP7 plus follistatin (foll) (250 ng). DAPI (blue) was used to stain cellular nucleus. Data shown are representative of three reproducible independent experiments. Scale bar, 100 μm (×40 magnification).