Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity

Journal name:
Nature
Volume:
523,
Pages:
231–235
Date published:
DOI:
doi:10.1038/nature14404
Received
Accepted
Published online

Melanoma treatment is being revolutionized by the development of effective immunotherapeutic approaches1, 2. These strategies include blockade of immune-inhibitory receptors on activated T cells; for example, using monoclonal antibodies against CTLA-4, PD-1, and PD-L1 (refs 3, 4, 5). However, only a subset of patients responds to these treatments, and data suggest that therapeutic benefit is preferentially achieved in patients with a pre-existing T-cell response against their tumour, as evidenced by a baseline CD8+ T-cell infiltration within the tumour microenvironment6, 7. Understanding the molecular mechanisms that underlie the presence or absence of a spontaneous anti-tumour T-cell response in subsets of cases, therefore, should enable the development of therapeutic solutions for patients lacking a T-cell infiltrate. Here we identify a melanoma-cell-intrinsic oncogenic pathway that contributes to a lack of T-cell infiltration in melanoma. Molecular analysis of human metastatic melanoma samples revealed a correlation between activation of the WNT/β-catenin signalling pathway and absence of a T-cell gene expression signature. Using autochthonous mouse melanoma models8, 9 we identified the mechanism by which tumour-intrinsic active β-catenin signalling results in T-cell exclusion and resistance to anti-PD-L1/anti-CTLA-4 monoclonal antibody therapy. Specific oncogenic signals, therefore, can mediate cancer immune evasion and resistance to immunotherapies, pointing to new candidate targets for immune potentiation.

At a glance

Figures

  1. Melanoma-intrinsic [bgr]-catenin pathway activation correlates with T-cell exclusion.
    Figure 1: Melanoma-intrinsic β-catenin pathway activation correlates with T-cell exclusion.

    a, b, Heat maps of 266 metastatic melanomas clustered in low versus high T-cell signature gene groups (a), and β-catenin target genes within the T-cell-signature high and low cohorts (b). c, Pearson correlation of CD8A expression with c-MYC, TCF1 and WNT7B (red indicates T-cell-signature high, blue indicates T-cell-signature low). d, Correlation between β-catenin and CD8 in melanoma biopsies. Fisher’s exact test with n = 49. e, Tumour incidence rates of GEMs (median time to tumour event): BrafV600E/Pten−/−: 100%, 21 days (n = 14); BrafV600E/CAT-STA: 85%, 55.5 days (n = 8); BrafV600E/Pten−/−/CAT-STA: 100%, 26 days (n = 14). f, CD3+ T cells depicted as percentage living cells and absolute numbers per gram tumour. n = 20, mean ± standard error of the mean (s.e.m.), Mann–Whitney U test. g, Representative example out of five for fluorescent immunohistochemistry staining against CD3+ T cells. Scale bars, 100 μm. See Extended Data Fig. 3 for overview. ***P ≤ 0.001, ****P ≤ 0.0001, *****P ≤ 0.00001.

  2. BrafV600E/Pten-/-/CAT-STA mice show impaired priming of anti-tumour T cells and reduced numbers of CD103+ dermal dendritic cells.
    Figure 2: BrafV600E/Pten−/−/CAT-STA mice show impaired priming of anti-tumour T cells and reduced numbers of CD103+ dermal dendritic cells.

    a, Abundance and proliferation of TCR-transgenic 2C T cells. Depicted are representative examples pre-gated on live, CD45+CD3+CD8+ cells. b, Statistical analysis of a (n = 8). c, Percentages of dendritic cell subsets within BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA tumours (n = 8). d, Representative example of CD103/CD8α staining (gated CD45+MHCIIhiCD11c+). e, Quantification of CD103+ dendritic cells (n = 12). f, Amount of CD3+ T cell and CD103+ dendritic cell (DC) infiltration in BrafV600E/Pten−/− tumours reconstituted with control or Batf3−/− bone marrow (n = 4 and n = 11, respectively). g, Intra-tumoural injection of Flt3 ligand-derived dendritic cells into BrafV600E/Pten−/−/CAT-STA tumours (n = 6 control mice and 8 mice, PBS control). All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001; NS, not significant.

  3. Active [bgr]-catenin signalling within tumour cells suppresses the recruitment of CD103+ dendritic cells.
    Figure 3: Active β-catenin signalling within tumour cells suppresses the recruitment of CD103+ dendritic cells.

    a, Chemokine expression in GEM tumours assessed via gene array analysis (n = 4). b, Confirmatory quantitative polymerase chain reaction with reverse transcription (qRT–PCR) (n = 8) with fold change (FC) indicated at the top. c, Transcript levels of Ccl3, Ccl4, Cxcl1 and Cxcl2 assessed from YFP+ and CD45 YFP cells from BrafV600E/Pten−/−/YFP+ tumours (n = 5), sorted on day 7 after tamoxifen administration. ND, not detected. d, Expression level of CCR5 in sorted CD45+CD11c+ dendritic cells (n = 8). e, Migration assay of dendritic cell subsets towards recombinant mouse CCL4 or conditioned medium (SF) (two independent experiments, duplicates per experiment). f, Atf3 transcripts in tumour tissues (n = 8). g, ATF3-specific ChIP assay in BP and BPC cell lines (two independent experiments, duplicates per experiment). h, Amount of secreted CCL4 in 48-h-conditioned siRNA-treated tumour-cell BP and BPC supernatants, assessed by enzyme-linked immunosorbent assay (ELISA) and Atf3 expression at the endpoint detected by qRT–PCR (two independent experiments, duplicates per experiment). All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001; NS, not significant.

  4. Reconstitution with Flt3 ligand dendritic cells reverses resistance to immunotherapy.
    Figure 4: Reconstitution with Flt3 ligand dendritic cells reverses resistance to immunotherapy.

    a, b, Tumour growth in BrafV600E/Pten−/− (a) and BrafV600E/Pten−/−/CAT-STA (b) mice untreated or treated with anti-CTLA-4 and anti-PD-L1 therapy (n = 10). c, Tumour growth of BrafV600E/Pten−/−/CAT-STA tumour-bearing mice that were untreated, treated with anti-CTLA-4 and anti-PD-L1 therapy, intra-tumoural Flt3 ligand (Flt3-L) dendritic cell injections, or combination therapy (n = 5). BM-DC, bone-marrow dendritic cell; mAb, monoclonal antibody. All data are mean ± s.e.m., two-way analysis of variance (ANOVA) test. **P ≤ 0.01, ****P ≤ 0.0001; NS, not significant.

  5. Correlation between active [bgr]-catenin and CD8 T-cell infiltrate in human patients.
    Extended Data Fig. 1: Correlation between active β-catenin and CD8 T-cell infiltrate in human patients.

    a, A continuous numerical score was generated using six β-catenin target genes (CTNNB1 score). Using this score, patients from the TCGA data set were grouped in high or low CTNNB1 score (centred on the average score) (low, 91 patients; high, 108 patients). Subsequent correlation analysis was performed using a Fisher’s exact test. b, Representative examples for CD8 and β-catenin staining in human needle biopsies used for analysis shown in Fig. 1d.

  6. Tumour growth of genetically engineered mice.
    Extended Data Fig. 2: Tumour growth of genetically engineered mice.

    a, Overall survival of all three models: BrafV600E/Pten−/− with 100% lethality and mean time to death of 31 days (n = 14), BrafV600E/CAT-STA with 85% lethality and mean time to tumour event of 93 days (n = 8), and BrafV600E/Pten−/−/CAT-STA with 100% lethality and mean time to tumour event of 36 days (n = 14). b, Tumour outgrowth of BrafV600E/Pten−/− (red) and BrafV600E/Pten−/−/CAT-STA (blue) tumours shown as mm3 at days after tamoxifen application (n = 10). c, Representative macroscopic pictures for tumour growth over time when tamoxifen was applied on the lower back of the mouse (see illustration). d, Gene array analysis of tumours isolated from GEMs (n = 4, Mann–Whitney U test). e, Histology slides showing representative examples for haematoxylin and eosin stain in all three mouse models (left, ×20, scale bars indicate 100 μm; right, ×100, scale bars indicate 20 μm). *P ≤ 0.05; NS, not significant.

  7. T-cell infiltration of genetically engineered mice.
    Extended Data Fig. 3: T-cell infiltration of genetically engineered mice.

    a, Representative images of immmunofluorescent staining against CD3 (red, left panel) and TRP1 (green, right panel) in all three tumour tissues (scale bar, 100 μm; ×4, ×10, ×20 with ×4 differential interference contrast (DIC) on top; nuclei Hoechst ×20 CD3 stain as shown in Fig. 1). b, Representative immmunofluorescent staining against CD3 (red, left panel) and TRP1 (green, right panel) in a highly pigmented area of BrafV600E/Pten−/− tumour tissues (scale bar, 100 μm; ×10, ×20 with ×10 DIC left) excluding that the lack of T cells is associated with increased pigmentation (nuclei Hoechst). c, Numbers of CD3+ T cells were counted within 13 different fields (0.5 mm × 1 mm) from two tumour samples. Mean of 12 T cells or 3.2 T cells per 0.5 mm2 in BrafV600E/CAT-STA or BrafV600E/Pten−/−/CAT-STA tumours, respectively, versus 100 T cells per 0.5 mm2 in BrafV600E/Pten−/− tumours. Data are given as mean with minimum and maximum, as well as individual values. Statistical analysis was performed using Mann–Whitney U test. ****P ≤ 0.0001.

  8. Characterization of the T-cell infiltrate in BrafV600E/Pten-/-/CAT-STA mice.
    Extended Data Fig. 4: Characterization of the T-cell infiltrate in BrafV600E/Pten−/−/CAT-STA mice.

    a, Distribution of T-cell subsets in BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA tumours (n = 6). b, c, Representative flow cytometry plots to discriminate αβ-TCR T cells and γδ-TCR T cells (b), naive (CD62L+CD44) and effector (CD62LCD44+) T cells (pre-gated on CD3+CD8+ T cells), and one representative example of CD44/CD45RA staining (c). Quantification of naive (CD62L+CD44CD45RA+), effector (CD62LCD44+CD45RA) and memory (CD62L+CD44+CD45RA) T cells is indicated on the right (n = 6). d, Representative flow cytometry plots of FoxP3+ T regulatory cells (n = 6). e, Quantification and comparison of PD-1/Lag3 double-positive T cells in BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA tumours (n = 12). f, Representative flow cytometry of PD-1- and Lag3-positive T cells (pre-gated on CD3+CD8+ T cells) in BrafV600E/Pten−/− tumours. g, Il2 transcripts present in sorted CD3+ T cells from BrafV600E/Pten−/− tumours and spleen (n = 10). h, Ifng transcripts present in sorted CD3+ T cells from BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA mice (n = 10). i, Expression level of PD-L1 in whole tumour tissue from both mouse models assessed by qRT–PCR (n = 8). j, Flow cytometric analysis of PD-L1 expression of non-haematopoietic tumour cells (CD45), CD45+CD11c+ dendritic cells (DC) and CD45+CD3+ T cells. Shown is a representative example as histogram (grey isotype, red BrafV600E/Pten−/−; blue, BrafV600E/Pten−/−/CAT-STA) with mean fluorescent intensity of n = 3 given each histogram (red, BrafV600E/Pten−/−; blue, BrafV600E/Pten−/−/CAT-STA). k, Percentage of Gr1+ cells within the CD11b+ fraction of the tumour immune cell infiltrate (n = 8; absolute numbers BrafV600E/Pten−/−: 1,047 ± 418 cells per gram tumour to BrafV600E/Pten−/−/CAT-STA: 739 ± 185 cells per gram tumour; P = 0.7429). All data are mean ± s.e.m., Mann–Whitney U test.*P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001; NS, not significant.

  9. Injection of Flt3 ligand-derived dendritic cells into tumours of BrafV600E/Pten-/-/CAT-STA mice is sufficient to overcome the lack of CD103+ dermal dendritic cells.
    Extended Data Fig. 5: Injection of Flt3 ligand-derived dendritic cells into tumours of BrafV600E/Pten−/−/CAT-STA mice is sufficient to overcome the lack of CD103+ dermal dendritic cells.

    a, Expression level of Ifnb in CD45+CD11c+ sorted dendritic cells from tumours from BrafV600E/Pten−/− (open bars) and BrafV600E/Pten−/−/CAT-STA (filled bars) mice. FC, fold change. b, Expression level of Batf3, Irf8 and Itgae in sorted dendritic cells. Fold change is indicated in each graph (n = 8). c, Mean (± s.e.m.) tumour weight of BrafV600E/Pten−/−/CAT-STA assessed at the endpoint of the experiment depicted in Fig. 3e, after intra-tumoural injection of dendritic cells. d, Per cent of GFP+CD11c+ dendritic cells (DC) present at the tumour site after injections of Flt3 ligand-derived dendritic cells from actin–GFP mice. Depicted are the percentages detected in the tumour of both genotypes injected with either wild-type or actin–GFP dendritic cells as well as in the TdLNs for the actin–GFP injected mice (n = 4). All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05.

  10. Chemokine expression patterns indicate that CCL4 expression from tumour cells is directly inhibited by active [bgr]-catenin-signalling.
    Extended Data Fig. 6: Chemokine expression patterns indicate that CCL4 expression from tumour cells is directly inhibited by active β-catenin-signalling.

    a, Expression of Ccl4 mRNA in established tumour cell lines BP and BPC (8 independent experiments). b, Amount of secreted CCL4 in 48 h conditioned BP and BPC tumour cell supernatants, assessed by ELISA (4 independent experiments). c, d, Control qRT–PCR for the experiment shown in Fig. 4e with Ifnb expression (c) and Ifng expression (d) (n = 6). ND, not detected. All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05.

  11. Active [bgr]-catenin signalling blocks CCL4 production in human melanoma cell lines.
    Extended Data Fig. 7: Active β-catenin signalling blocks CCL4 production in human melanoma cell lines.

    a, Western blot on mel537 and mel888 showing stabilized β-catenin expression. b, Expression level of human ATF3 and human CCL4 in mel537 and mel888 (three independent experiments, duplicates per experiment). c, Expression level of β-catenin target genes in mel537 and mel888. d, ATF3-specific ChIP assay in mel537 and mel888 cell lines for the CCL4 gene locus (two independent experiments, duplicates per experiment). e, CCL4 secretion (left) and ATF3 transcription levels (right) after siRNA-mediated knockdown of CTNNB1 and ATF3 in mel537 and mel888 assessed by ELISA or qRT–PCR, respectively (two independent experiments, duplicates per experiment). All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05.

  12. [bgr]-Catenin target gene expression correlates inversely with markers for human BATF3-lineage dendritic cells and T cells.
    Extended Data Fig. 8: β-Catenin target gene expression correlates inversely with markers for human BATF3-lineage dendritic cells and T cells.

    Pearson correlation of CTNNB1 score with CD8Α (R2 = 0.214), THBD (R2 = 0.109) and IRF8 (R2 = 0.2374) (red indicates T-cell-signature high, blue indicates T-cell-signature low).

  13. Graphical summary.
    Extended Data Fig. 9: Graphical summary.

    Left, tumour without active β-catenin signalling in which ATF3 transcription is not induced and thus CCL4 (red circles) is transcribed and secreted. Downstream CD103+ dendritic cells (DC) (blue) are attracted and subsequent activation of CD8+ T cells (green) is enabled. Right, tumour with active β-catenin signalling (green), which leads to induction of ATF3 transcription (red), which in turn leads, among others effects, to suppression of CCL4 transcription. This leads to an active escape from the anti-tumour immune response since dendritic cell recruitment is insufficient.

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Author information

Affiliations

  1. Department of Pathology, The University of Chicago, Chicago, Illinois 60637, USA

    • Stefani Spranger &
    • Thomas F. Gajewski
  2. Center for Research Informatics, The University of Chicago, Chicago, Illinois 60637, USA

    • Riyue Bao
  3. Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA

    • Thomas F. Gajewski

Contributions

S.S. contributed to the overall project design, planned and performed experiments, and performed data analysis. R.B. performed analysis of the TCGA data set. T.F.G. designed the overall project. S.S. and T.F.G. wrote the manuscript.

Corresponding author

Correspondence to:

Gene array data have been deposited in the Gene Expression Omnibus under accession number GSE63543.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Correlation between active β-catenin and CD8 T-cell infiltrate in human patients. (1,537 KB)

    a, A continuous numerical score was generated using six β-catenin target genes (CTNNB1 score). Using this score, patients from the TCGA data set were grouped in high or low CTNNB1 score (centred on the average score) (low, 91 patients; high, 108 patients). Subsequent correlation analysis was performed using a Fisher’s exact test. b, Representative examples for CD8 and β-catenin staining in human needle biopsies used for analysis shown in Fig. 1d.

  2. Extended Data Figure 2: Tumour growth of genetically engineered mice. (424 KB)

    a, Overall survival of all three models: BrafV600E/Pten−/− with 100% lethality and mean time to death of 31 days (n = 14), BrafV600E/CAT-STA with 85% lethality and mean time to tumour event of 93 days (n = 8), and BrafV600E/Pten−/−/CAT-STA with 100% lethality and mean time to tumour event of 36 days (n = 14). b, Tumour outgrowth of BrafV600E/Pten−/− (red) and BrafV600E/Pten−/−/CAT-STA (blue) tumours shown as mm3 at days after tamoxifen application (n = 10). c, Representative macroscopic pictures for tumour growth over time when tamoxifen was applied on the lower back of the mouse (see illustration). d, Gene array analysis of tumours isolated from GEMs (n = 4, Mann–Whitney U test). e, Histology slides showing representative examples for haematoxylin and eosin stain in all three mouse models (left, ×20, scale bars indicate 100 μm; right, ×100, scale bars indicate 20 μm). *P ≤ 0.05; NS, not significant.

  3. Extended Data Figure 3: T-cell infiltration of genetically engineered mice. (692 KB)

    a, Representative images of immmunofluorescent staining against CD3 (red, left panel) and TRP1 (green, right panel) in all three tumour tissues (scale bar, 100 μm; ×4, ×10, ×20 with ×4 differential interference contrast (DIC) on top; nuclei Hoechst ×20 CD3 stain as shown in Fig. 1). b, Representative immmunofluorescent staining against CD3 (red, left panel) and TRP1 (green, right panel) in a highly pigmented area of BrafV600E/Pten−/− tumour tissues (scale bar, 100 μm; ×10, ×20 with ×10 DIC left) excluding that the lack of T cells is associated with increased pigmentation (nuclei Hoechst). c, Numbers of CD3+ T cells were counted within 13 different fields (0.5 mm × 1 mm) from two tumour samples. Mean of 12 T cells or 3.2 T cells per 0.5 mm2 in BrafV600E/CAT-STA or BrafV600E/Pten−/−/CAT-STA tumours, respectively, versus 100 T cells per 0.5 mm2 in BrafV600E/Pten−/− tumours. Data are given as mean with minimum and maximum, as well as individual values. Statistical analysis was performed using Mann–Whitney U test. ****P ≤ 0.0001.

  4. Extended Data Figure 4: Characterization of the T-cell infiltrate in BrafV600E/Pten−/−/CAT-STA mice. (314 KB)

    a, Distribution of T-cell subsets in BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA tumours (n = 6). b, c, Representative flow cytometry plots to discriminate αβ-TCR T cells and γδ-TCR T cells (b), naive (CD62L+CD44) and effector (CD62LCD44+) T cells (pre-gated on CD3+CD8+ T cells), and one representative example of CD44/CD45RA staining (c). Quantification of naive (CD62L+CD44CD45RA+), effector (CD62LCD44+CD45RA) and memory (CD62L+CD44+CD45RA) T cells is indicated on the right (n = 6). d, Representative flow cytometry plots of FoxP3+ T regulatory cells (n = 6). e, Quantification and comparison of PD-1/Lag3 double-positive T cells in BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA tumours (n = 12). f, Representative flow cytometry of PD-1- and Lag3-positive T cells (pre-gated on CD3+CD8+ T cells) in BrafV600E/Pten−/− tumours. g, Il2 transcripts present in sorted CD3+ T cells from BrafV600E/Pten−/− tumours and spleen (n = 10). h, Ifng transcripts present in sorted CD3+ T cells from BrafV600E/Pten−/− and BrafV600E/Pten−/−/CAT-STA mice (n = 10). i, Expression level of PD-L1 in whole tumour tissue from both mouse models assessed by qRT–PCR (n = 8). j, Flow cytometric analysis of PD-L1 expression of non-haematopoietic tumour cells (CD45), CD45+CD11c+ dendritic cells (DC) and CD45+CD3+ T cells. Shown is a representative example as histogram (grey isotype, red BrafV600E/Pten−/−; blue, BrafV600E/Pten−/−/CAT-STA) with mean fluorescent intensity of n = 3 given each histogram (red, BrafV600E/Pten−/−; blue, BrafV600E/Pten−/−/CAT-STA). k, Percentage of Gr1+ cells within the CD11b+ fraction of the tumour immune cell infiltrate (n = 8; absolute numbers BrafV600E/Pten−/−: 1,047 ± 418 cells per gram tumour to BrafV600E/Pten−/−/CAT-STA: 739 ± 185 cells per gram tumour; P = 0.7429). All data are mean ± s.e.m., Mann–Whitney U test.*P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001; NS, not significant.

  5. Extended Data Figure 5: Injection of Flt3 ligand-derived dendritic cells into tumours of BrafV600E/Pten−/−/CAT-STA mice is sufficient to overcome the lack of CD103+ dermal dendritic cells. (229 KB)

    a, Expression level of Ifnb in CD45+CD11c+ sorted dendritic cells from tumours from BrafV600E/Pten−/− (open bars) and BrafV600E/Pten−/−/CAT-STA (filled bars) mice. FC, fold change. b, Expression level of Batf3, Irf8 and Itgae in sorted dendritic cells. Fold change is indicated in each graph (n = 8). c, Mean (± s.e.m.) tumour weight of BrafV600E/Pten−/−/CAT-STA assessed at the endpoint of the experiment depicted in Fig. 3e, after intra-tumoural injection of dendritic cells. d, Per cent of GFP+CD11c+ dendritic cells (DC) present at the tumour site after injections of Flt3 ligand-derived dendritic cells from actin–GFP mice. Depicted are the percentages detected in the tumour of both genotypes injected with either wild-type or actin–GFP dendritic cells as well as in the TdLNs for the actin–GFP injected mice (n = 4). All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05.

  6. Extended Data Figure 6: Chemokine expression patterns indicate that CCL4 expression from tumour cells is directly inhibited by active β-catenin-signalling. (132 KB)

    a, Expression of Ccl4 mRNA in established tumour cell lines BP and BPC (8 independent experiments). b, Amount of secreted CCL4 in 48 h conditioned BP and BPC tumour cell supernatants, assessed by ELISA (4 independent experiments). c, d, Control qRT–PCR for the experiment shown in Fig. 4e with Ifnb expression (c) and Ifng expression (d) (n = 6). ND, not detected. All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05.

  7. Extended Data Figure 7: Active β-catenin signalling blocks CCL4 production in human melanoma cell lines. (165 KB)

    a, Western blot on mel537 and mel888 showing stabilized β-catenin expression. b, Expression level of human ATF3 and human CCL4 in mel537 and mel888 (three independent experiments, duplicates per experiment). c, Expression level of β-catenin target genes in mel537 and mel888. d, ATF3-specific ChIP assay in mel537 and mel888 cell lines for the CCL4 gene locus (two independent experiments, duplicates per experiment). e, CCL4 secretion (left) and ATF3 transcription levels (right) after siRNA-mediated knockdown of CTNNB1 and ATF3 in mel537 and mel888 assessed by ELISA or qRT–PCR, respectively (two independent experiments, duplicates per experiment). All data are mean ± s.e.m., Mann–Whitney U test. *P ≤ 0.05.

  8. Extended Data Figure 8: β-Catenin target gene expression correlates inversely with markers for human BATF3-lineage dendritic cells and T cells. (163 KB)

    Pearson correlation of CTNNB1 score with CD8Α (R2 = 0.214), THBD (R2 = 0.109) and IRF8 (R2 = 0.2374) (red indicates T-cell-signature high, blue indicates T-cell-signature low).

  9. Extended Data Figure 9: Graphical summary. (240 KB)

    Left, tumour without active β-catenin signalling in which ATF3 transcription is not induced and thus CCL4 (red circles) is transcribed and secreted. Downstream CD103+ dendritic cells (DC) (blue) are attracted and subsequent activation of CD8+ T cells (green) is enabled. Right, tumour with active β-catenin signalling (green), which leads to induction of ATF3 transcription (red), which in turn leads, among others effects, to suppression of CCL4 transcription. This leads to an active escape from the anti-tumour immune response since dendritic cell recruitment is insufficient.

Supplementary information

PDF files

  1. Supplementary Information (3.3 MB)

    This file contains Supplementary Tables 1-5 comprising:1 (a) Expression of T cell genes in segregated groups (b) Gene clusters after supervised hierarchical clustering (c) List of differentially expressed genes; 2 - Pearson correlation of β-catenin target genes and CD8a transcripts; 3 - Mutation analysis summary with (a) T cell signature low patients and (b) T cell signature high patients (c) Table summarizing potential pathway activators in patients with an active β-catenin signature; 4 - Mouse gene array data of differentially expressed genes (a) and a summary table focusing on chemokine expression (b); 5 - Detailed primer and antibody information (a) Genotyping primers (b) antibodies (c) qPCR primer/ probes (d) ChIP assay primer and (e) siRNA oligos.

Additional data