Fig 2: Taqman qPCR validation of differentially regulated genes in human IMA compared to normal lung tissues Mucin genes (MUC5AC, MUC5B, and MUC3A/B) were significantly induced in human IMA (n = 7) compared to normal lung tissues (n = 6).Transcription factors SPDEF, FOXA3, and HNF4A but not NKX2-1 were significantly induced in human IMA (n = 7) compared to normal lung tissues (n = 6).Immune checkpoint gene VTCN1 but not PD-L1 was significantly induced in human IMA (n = 7) compared to normal lung tissues (n = 6).Data information: See patient information in Dataset EV1. Taqman gene expression qPCR analysis was performed as described in Materials and Methods. Each gene expression was normalized by comparison with the constitutive expression of ACTB (GAPDH was not used for normalization since GAPDH was induced in human IMA compared to normal lung tissue; see Dataset EV2). Results are expressed as mean ± SEM of biological replicates for each group. P < 0.05 versus normal was considered significant (Mann–Whitney test).

Fig 3: VTCN1 but not PD-L1 is expressed in human IMA Shown is immunohistochemistry (IHC) detecting VTCN1 but not PD-L1 in human IMA. Alcian blue detects mucus. Scale bar: 50 µm. Insets show higher magnification of regions indicated by arrows.64% of the human IMA expressed VTCN1 but most of the human IMA did not express PD-L1 as determined by IHC.Left panel, genes highly correlated with VTCN1 or PD-L1 in NSCLC cell lines (n = 105; Klijn et al, 2015) were assessed as to whether they are expressed in human IMA. 38 genes highly correlated with VTCN1 were expressed in human IMA. However, only seven genes highly correlated with PD-L1 were expressed in human IMA. Right panel, likewise, 28 genes highly correlated with VTCN1 in the TCGA LUAD cases (n = 230; Cancer Genome Atlas Research Network, 2014) were expressed in human IMA but only 1 gene highly correlated with PD-L1 was expressed in human IMA. Red indicates genes in the Mucinous Lung Tumor Signature (the 143 genes).Hierarchical clustering of the expression of IMA-related genes and cell type markers in the NSCLC cell lines (n = 105; Klijn et al, 2015). Genes in red color: pro-mucous genes. Genes in green color: anti-mucous genes. VTCN1 but not PD-L1 positively correlates with a mucous gene marker MUC5B in the NSCLC cell lines (n = 105; Klijn et al, 2015). Of note, the anti-mucous transcription factor NKX2-1 was correlated with the pro-mucous transcription factor SPDEF in the 105 human NSCLC cell lines; however, NKX2-1 was not correlated with SPDEF when 41 cell lines that lack the expression of both NKX2-1 and SPDEF (RPKM = 1) were excluded from the calculation, suggesting that the positive correlation was seen due to the large number of the cell lines that lack the expression of both NKX2-1 and SPDEF. Genes in red color: pro-mucous genes. Genes in green color: anti-mucous genes.Hierarchical clustering of the expression of IMA-related genes and cell type markers in the TCGA LUAD cases (n = 230; Cancer Genome Atlas Research Network, 2014). Red indicates specimens with IMA pathology. Genes in red color: pro-mucous genes. Genes in green color: anti-mucous genes. VTCN1 but not PD-L1 positively correlates with a mucinous tumor marker HNF4A in the TCGA LUAD cases (n = 230; Cancer Genome Atlas Research Network, 2014). Overall, VTCN1 but not PD-L1 associates with mucous gene markers. Genes in red color: pro-mucous genes. Genes in green color: anti-mucous genes.

Fig 4: Expression of the B7 family ligands/receptors on immune cells of CPs. (A) PD-L1, B7-H3, B7-H4 and VISTA expression on immune cells in ACP and PCP. The red arrow refers to the immune cells with positive VISTA. (B) Representative images of VISTA, CD3 and IBA-1 codetection are shown. (C, D) QIF scores show IBA-1+ tumor-associated macrophage expressed more VISTA than CD3+ T lymphocytes in both ACP and PCP. VISTA expression is negatively correlated with the number of CD3+ cells (E) in the tumor and positively correlated with the number of IBA-1+ cells (F). Red bar: 0.5 mm, black bar: 50 µm, white bar: 50 µm. ***P<0.001, ****P<0.0001. ACP, adamantinomatous-type craniopharyngioma; PCP, papillary-type CP; QIF, quantitative immunofluorescence.

Fig 5: VISTA is positively correlated with PD-L1 in CPs. Analysis of the correlation between the expression of VISTA and the expression of PD-L1, B7-H3 and B7-H4 in CPs (A). Immunofluorescence colocalization revealed the difference that expressions of PD-L1 and VISTA were different in tumor cells (B), but the two molecules were colocalized in tumor associated immune cells (C). Red bar: 0.5 mm, white bar: 50 µm. CPs, craniopharyngioma; PD-L1, programmed cell death ligand 1.