Article Figures & Data
Figures
- Figure 1.
CMTM6 is overexpressed and predicts a poor prognosis for patients with HNSCC. A, Representative IHC staining images of CMTM6 in normal mucosa and HNSCC tissues with different clinical pathologic grades (scale bar, 50 μm). B, Expression of CMTM6 protein in human HNSCC (n = 210) was higher than in oral mucosa (n = 42) and dysplasia (n = 69) by IHC analysis (**, P < 0.01; ***, P < 0.001). C, Quantification analysis of CMTM6 staining in HNSCC with different grades (I, n = 53; II, n = 121; III, n = 36; *, P < 0.05). D, Boxplot indicating CMTM6 expression in different grades of HNSCC from the UALCAN database (***, P < 0.001 for I vs. III, I vs. IV, and II vs. IV; **, P < 0.01 for II vs. III and III vs. IV). Representative IHC staining of CMTM6 in metastasis-free (top, n = 138) and metastatic HNSCC tissue (E; bottom, n = 72; scale bar, 50 μm) and quantification analysis (F), N(−) = N0, N(+) = N1 + N2; **, P < 0.01. G, Kaplan–Meier survival analysis showed that patients with HNSCC with higher CMTM6 expression had a shortened overall survival (cutoff = 95.05). Error bar, SD.
- Figure 2.
Elevated CMTM6 expression in HNSCC cells promotes tumor growth. A, Western blot analysis of CMTM6 expression in five HNSCC cell lines and a normal OKC line. B, Flow cytometry analysis of cell surface PD-L1 in these cell lines. C, Western blot analysis of total CMTM6 and PD-L1 expression in CMTM6 knockdown or control CAL27 and SCC4 cells. D, Cell surface PD-L1 expression was reduced in CMTM6 knockout cells by flow cytometry analysis. E, Growth curves of CMTM6-depleted or control SCC4 cells as measured by a CCK-8 assay (***, P < 0.001). F, Representative images and quantitative analysis of anchorage-dependent colony formation assays in SCC4 cells (***, P < 0.001); error bar, SD. No., number. The data shown are representative of three experiments (A–F).
- Figure 3.
CMTM6 expression has a positive correlation with the Wnt/β-catenin pathway in HNSCC. A, Volcano plot of CMTM6-associated genes by Spearman correlation coefficient test in the LinkedOmics database. The gray dashed line of P = 0.05 was plotted. B, Expression correlation between CMTM6 and CTNNB1 RNA expression in the TCGA HNSCC database. Correlation was analyzed using Spearman correlation coefficient test, n = 520. C, Representative IHC staining of CMTM6 and β-catenin in HNSCC specimens (scale bar, 50 μm). D, Pearson correlation analysis of CMTM6 and β-catenin protein expression in HNSCC tissue microarray (n = 210). E, The distribution of β-catenin (green) in CMTM6 knockdown and control CAL27 cells was analyzed by immunofluorescence (scale bar, 20 μm), and the red arrow indicates the location of the nucleus. F, Western blot analysis showed β-catenin expression levels in total or nuclear proteins of the control and CMTM6-silenced groups. The data shown are representative of three experiments (E and F).
- Figure 4.
CMTM6 is expressed in cancer stem cells and promotes stemness properties. A, Representative coimmunofluorescent staining images of CMTM6 (green) and CSC markers: ALDH1 (red), BMI1 (red), and CD44 (green) in tumorspheres (scale bar, 50 μm). B, The expression of CMTM6 and CSC-related proteins in spheres and adherent cells was detected by Western blotting. C, An ALDEFLUOR assay was conducted in CMTM6-deficient and control cells, and the percentage of ALDHhigh cells was quantified by flow cytometry (**, P < 0.01; ***, P < 0.001); error bar, SD. D, Representative images and quantitative analysis of a sphere formation assay (**, P < 0.01); error bar, SD. E, Immunofluorescence images show the expression of CSC markers BMI1 (green) and CD44 (red) after CMTM6 knockout (scale bar, 20 μm). F, Western blot results confirmed that CMTM6 knockout reduced the expression of CSC-related proteins. G, CMTM6 was positively correlated with CSC genes in the TCGA HNSCC database using Spearman correlation coefficient test, n = 520. The data shown are representative of three experiments (A–F).
- Figure 5.
Aberrant expression of CMTM6 is associated with EMT in HNSCC. A, Spearman correlation analysis of the public TCGA HNSCC database for the expression of CMTM6 and mesenchymal (VIM and FN1) or EMT-TF genes (ZEB1 and ZEB2), n = 520. B, Western blot analysis of CMTM6 in CAL27 cells undergoing TGFβ-induced EMT (8 ng/mL TGFβ1). C, Representative immunofluorescent staining images of E-cadherin (green) and Vimentin (red) in CMTM6 knockdown and control CAL27 cells treated with vehicle or 8 ng/mL TGFβ1 (36 hours; scale bar, 20 μm). D, Western blot analysis confirmed that CMTM6 depletion inhibited TGFβ-induced EMT in HNSCC (8 ng/mL TGFβ1, 36 hours). The data shown are representative of three experiments (B–D).
- Figure 6.
Increased CMTM6 is correlated with immune checkpoint members in HNSCC. A, Expression correlation between CMTM6 and immune checkpoint gene RNA amounts in the TCGA HNSCC database, n = 520, CMTM6 (CMTM6), PD-L1 (CD274), PD-1 (PDCD1), VISTA (VSIR), B7-H3 (CD276), B7-H4 (VTCN1), LAG-3 (LAG3), CTLA-4 (CTLA4), and TIM-3 (HAVCR2). B, Pearson correlation analysis of CMTM6 and several immune checkpoint expressions in human HNSCC tissue microarray based on the IHC results, n = 116.
- Figure 7.
Targeting CMTM6 inhibits tumor growth in an immunocompetent HNSCC allograft mouse model. A, SCC7 tumor tissue sections were stained with PD-L1 antibody and IgG control. Scale bars, 50 μm. B, shCMTM6 and shCtrl SCC7 cells were cultured in the presence or absence of IFNγ (10 ng/mL) for 24 hours. The cells were stained with PD-L1 antibody and analyzed by flow cytometry. C, Cell surface PD-L1 expression on tumor cells after the allograft assay was detected by flow cytometry (tumor cells were gated by CD45−; **, P < 0.01). D, Growth curve of SCC7 tumors (shCtrl, n = 10; WT, n = 6; shCMTM6, n = 9; ***, P < 0.001). E, CD4+ and CD8+ T cells in CD45+ TILs were analyzed by flow cytometry (*, P < 0.05). F, Effects of CD8+ T-cell depletion on the growth of CMTM6 knockdown or control SCC7 tumors (n = 6 mice per group). ns, not significant. G, Representative flow cytometry plots of PD-1+, TIM-3+, and VISTA+ T-cell populations in control and CMTM6 knockdown SCC7 TILs. Quantification and statistical analysis results of PD-1+, TIM-3+, VISTA+, LAG-3+, and B7-H3+ proportion in CD4+ (H) and CD8+ (I) T cells in two groups (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). Error bar, SD. The data shown are representative of three (A–C) and two experiments (D).
Additional Files
Supplementary Data
- Supplementary Legends - Supplementary Figure Legends
- Supplementary Figures - Supplementary Figure 1-6
- Original Western Blot Data - Original Western Blot Data
- Supplementary Table 1 - Association of CMTM6 expression and clinicopathologic parameters in HNSCC
- Supplementary Table 2 - Pearson's correlation coefficients (r) of CMTM6 with immune checkpoints correlation analysis
Volume 8, Issue 2
