Additional file 1 of Combinational blockade of MET and PD-L1 improves pancreatic cancer immunotherapeutic efficacy
Creators: name: Li, Enliang
name: Huang, Xing
name: Zhang, Gang
name: Liang, Tingbo
Publisher: name: figshare
Year: 2021
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Figure S1. MET overexpression in PDAC tissue samples. (A-B) MET protein expressions in 18 paired PDAC and adjacent noncancerous tissue specimens assessed immunohistochemically. T, tumor; N, normal. Figure S2. MET promotes transcriptional upregulation of PD-L1 in PDAC cells. (A) MET (up) and PD-L1 (down) mRNA expressions in BXPC3 cells. The cells were transfected with shMET or CTRL shRNA as assessed by qPCR. (B) MET, STAT1, pSTAT1 and PDL1 protein levels are assessed by immunoblotting in BXPC3 and KPC cells were transfected with MET silenced (shMET) and/or were treated with IFNγ. Figure S3. MET inhibition hampers IFNγ-induced PD-L1 upregulation in PDAC cells. (A) BXPC3 cells underwent transfection with shMET or CTRL shRNA, and were treated with IFNγ for 48 h, followed by PD-L1 level assessment by flow cytometry. Representative images (right) and quantification (left) are shown. (B) KPC cells underwent transfection with shMET or CTRL shRNA, and were treated with IFNγ for 48 h, followed by PD-L1 level assessment by flow cytometry. Representative images (right) and quantification (left) are shown. (C) PD-L1 cell membrane protein expression in KPC cells after 48 h of treatment with IFNγ alone or IFNγ combined with capmatinib. Representative flow-cytograms (right) and quantification (left) are shown. Figure S4. MET inhibits protein degradation of PD-L1 in PDAC cells. (A-B) Vector control and shMET BXPC3 (A) and KPC cells (B) were treated with CHX (100 µg/mL) and analyzed by Western blotting to determine the stability of PD-L1 protein. (C) Endogenous co-immunoprecipitation of BXPC3 cells by MET and CMTM6 antibodies. MET and CMTM6 expression levels are analyzed by Western blotting. (D) MET and CMTM6 protein levels are assessed by immunoblotting in BXPC3 cells transfected with MET silenced (shMET) or negative control (CTRL) vectors. (E) MET, CMTM6, and PD-L1 protein levels are assessed by immunoblotting in BXPC3 cells transfected with MET silenced (shMET) and/or CMTM6 overexpressed (CMTM6-OE). Figure S5. Immuno-profiling of combinational blockade of MET and PD-L1 in orthotopic and subcutaneous mouse models. (A-B) Quantification of CD4+ T cells from purified tumor-infiltrating lymphocytes in an orthotopic (A) and a subcutaneous (B) mouse model. (C-D) The myeloid cell density in tumors was quantitated based on the area of positive IHC staining of the CD11B marker in an orthotopic (C) and a subcutaneous (D) mouse model. Figure S6. Synergistic effects of PD-1 monoclonal antibody and MET inhibition in a subcutaneous mouse model. (A) Drug treatment protocol for capmatinib and PD-1 antibody in C57/BL6 mice. At the endpoint, tumors were extracted and assessed via flow cytometry. (B-E) Sizes of subcutaneous KPC tumors in C57/BL6 mice treated with capmatinib and/or PD-1 antibody. Mouse body weights (B), representative tumor images (C), tumor volumes (D), and tumor weights (E) are shown. (F-G) Quantification of CD3+ T cells (F) and CD8+ T cells (G) from purified tumor-infiltrating lymphocytes.