Article Figures & Data
Figures
- Figure 1.
Ectopic expression of BRAF(V600E) upregulates IL-1α/β expression in melanocytes and melanoma cells. A and B, flow cytometric analysis of green fluorescent protein (GFP) and BRAF expression in dermal melanocytes following transduction with lentiviral expression vectors BRAF(wt)-IRES-GFP, BRAF(V600E)-IRES-GFP, or empty-IRES-GFP. Gated GFP(dim) cells were flow sorted for use in subsequent studies. C, Affymetrix gene expression profiling of selected genes classically implicated in immune modulation of the tumor microenvironment. Transduced and sorted dermal melanocytes (36 hours following transduction) or HS294T cells (24 hours post-transduction) were analyzed, and the heatmap shown represents color-coded expression levels for each sample compared to GFP-transduced controls. D, Luminex assay showing cytokine profiles in supernatants of transduced dermal melanocyte preparations cultured for 5 days. Results are representative of 4 independent experiments. ND, not detected.
- Figure 2.
Inhibition of BRAF(V600E) abrogates IL-1 expression in melanoma cell lines and patient tumors. A, RT-PCR analysis of IL-1α, IL-1β, and GAPDH transcripts in BRAF(V600E)-positive WM793p2 cells at different time points following treatment with 1 μmol/L vemurafenib. B, flow cytometric analysis showing intracellular IL-1β protein expression in live cell-gated WM793p2 cells 48 hours following treatment with titrated doses of PLX4032. C, RT-PCR analysis showing transcript levels of IL1α, IL1β, and CNX in 5 vemurafenib-treated melanoma cell lines expressing either wt BRAF (HS294T) or V600E-mutated BRAF (A375, EB16-MEL, KUL84-MEL, and WM793p2). Transcript levels were normalized to GAPDH expression and adjusted to corresponding baseline samples. D, immunohistochemical (IHC) analysis of IL-1α protein expression in tumor biopsies resected from 2 representative metastatic melanoma patients harboring the BRAF(V600E) mutation, both before and on vemurafenib treatment. E, summary of changes to IL-1α expression in response to vemurafenib treatment, as assessed by IHC analysis of 12 total melanoma patient tumor biopsy pairs analyzed.
- Figure 3.
IL–1α-treated tumor-associated fibroblasts induce suppression of melanoma-specific CD8+ T-cells. A, tissue sections from 2 representative melanoma metastases labeled with anti-αSMA antibody and visualized with peroxidase immunostaining. Reddish brown color shows staining of αSMA-positive tumor-associated fibroblasts (TAF), asterisks denote tumor cells, arrows indicate tumor-infiltrating lymphocytes (TIL), and ‘V’ denotes tumor vasculature. B, interferon-gamma release by MART-1 reactive TIL stimulated with MART-1 peptide-pulsed T2 cells in the presence or absence of untreated or IL–1α-treated melanoma TAFs, with or without the addition of IL–1-neutralizing antibodies. Data are representative of 6 different TAF lines analyzed and 3 experimental replicates. C, frequency of CD107a-positive TIL following co-culture with MART-1 peptide-pulsed dermal fibroblasts pretreated with or without IL-1α, as determined by flow cytometry. Data from 2 different melanoma patient TILs are shown, and are representative of 2 independent experiments. Asterisks indicate statistical significance (P < 0.05); ns, not significant.
- Figure 4.
IL-1α upregulates the expression of immunosuppressive genes in melanoma-derived TAFs. A, phase-contrast images of 3 short-term cultured TAFs derived from melanoma patient biopsies (10×). B, normalized relative transcriptional expression levels of PTGS2 (COX-2), PD-L1, and PD-L2 in 24-hour IL–1α-treated or untreated TAFs, as analyzed by Affymetrix gene expression array. C, Western blot analysis showing COX-2 and β-actin protein expression in 4 additional patient-derived TAF lines, before and 24 hours after treatment with IL-1α. D, surface expression of PD-1 ligands PD-L1 and PD-L2 on TAFs 24 hours after treatment with IL-1β or IFN-γ, as determined by flow cytometry. Data from 9 different melanoma-derived TAF lines are shown. Asterisks indicate statistical significance (P < 0.05); ns, not significant.
- Figure 5.
BRAF(V600E) can induce T-cell suppression through IL–1-mediated upregulation of PD-1 ligands and COX-2 on TAFs. A, flow cytometric analysis of surface PD-L1 expression on TAFs cultured overnight with IL-1α, IL-1β, IL-6, TNF-α, or conditioned medium from cultured primary melanocytes transduced with lentiviral vectors expressing BRAF(wt)-GFP, BRAF(V600E)-GFP, or GFP alone. As indicated, conditioned media experiments were also carried out in the presence of isotype control or anti-IL1α- and anti-IL1β-blocking antibodies. B, interferon-gamma release by T2-stimulated MART-1-reactive TIL in the presence of melanoma patient-derived TAFs previously exposed to conditioned media from BRAF(V600E) mutant-expressing melanoma cell lines that were either untreated or treated with the BRAF(V600E) inhibitor vemurafenib. Results from 5 different TAF lines are shown. C, to assess the relative contributions of IL-1α/β, COX-2, and PD-1 ligands in the induction of T-cell suppression, 3 melanoma TAF lines were pretreated with conditioned media from untreated or vemurafenib-treated melanoma cell lines (WM793p2 and EB16-MEL), in the presence of either IL–1α/β-blocking antibodies or the COX-2 inhibitor NS398. Preconditioned TAFs were then incubated with MART-1-reactive TIL and MART-1 peptide-pulsed T2 cells in the presence of isotype control antibody or antibodies specific for PD-L1 and PD-L2. Asterisks indicate statistical significance (P < 0.05); ns, not significant.
- Figure 6.
Model of BRAF(V600E)-mediated immune suppression by TAFs in the melanoma tumor microenvironment. Illustration of proposed mechanistic model showing how constitutively activated BRAF(V600E) in melanoma tumor cells may initiate and sustain T-cell suppression in vivo. In this model, T-cell suppression is manifested by tumor-associated fibroblasts (TAF) that upregulate COX-2 and PD-1 ligands PD-L1 and PD-L2 in response to BRAF(V600E)-induced IL-1α/β production by melanoma cells. Targeted therapies that inhibit BRAF(V600E) could abrogate IL-1α/β production by tumor cells, thus interfering with the cellular crosstalk leading to TAF-mediated immune suppression.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF file, 94K.
- Supplementary Figure 1 - PDF file, 473K, (A) Frequencies of IL-1α and IL-1β staining at different disease stages. Sections of a melanoma progression tissue array stained with antibodies specific for IL-1α or IL-1β and visualized by Vector-red immunostaining. Red color indicates positive staining for (C-E) IL-1α or (G-I) IL-1β in representative primary and metastatic tumors. (J) IL-1α or (K) IL-1β from stage 3 lymph node metastasis of tumors with (red) or without (black) the BRAF V600E mutation. Scores indicates the product of the intensity of signal (0-3) and frequency (0-100) of immunoreactive cells. (L) Presence or absence of IL-1α or IL-1β in melanoma cell lines with known BRAF mutational status. Asterisks, in house, or references (ref.) indicate the source of mutational analysis, (first) and the source of the IL-1 analysis (first and last respectively). Methods of BRAF mutational analysis are indicated by P, pyrosequencing; S, Sanger sequencing; Q, sequenome. Bold text indicates cell lines used in this study.
- Supplementary Figure 2 - PDF file, 303K, IL-1α production is reduced by BRAF knockdown shRNA expression in melanoma cells. IL-1α staining in WM793 melanoma cells transduced with scrambled shRNA vector, BRAF targeting shRNA vectors, or untranduced as indicated. Expression of shRNA is indicated by coexpression of RFP (x-axis) and is induced by treatment with 2 ug/mL Doxycycline (DOX) (Lower). Cells were treated with DOX for one week prior to flow analysis by intracellular staining for IL-1α.
- Supplementary Figure 3 - PDF file, 212K, (A) H&E staining of representative, A375 human xenograft tumor sections excised from NOD/SCID mice. Prior to excision, mice with established tumors were treated for 3 days with either PLX4720 or DMSO vehicle control. Magnification: 4x. (B) RT-PCR analysis of human IL1α, IL1β, IL-8, GRO-α, PD-L1, and PD-L2 transcripts derived from A375 xenografts shown in (A). Data show the average of 3 mice per group, and are representative of 3 separate experiments.
- Supplementary Figure 4 - PDF file, 188K, Tumor-associated fibroblasts (TAF) cultures were derived from three separate metastatic melanoma biopsies from three distinct anatomical sites: lymph node, lung and soft tissue. Cultured TAFs were exposed overnight to IL-1α, and treated and untreated cells were subjected to Affymetrix gene expression analysis. (A) Heatmap displaying the 197 most differentially expressed genes selected at FDR 0.01. Relative expression values are indicated by the color bar. (B) Gene set enrichment analysis of IL-1α-treated TAFs demonstrates a strong correlation with gene sets previously identified to be associated with NF-kappaB upregulation and the interferon response. (C) Heatmap showing IL-1α induced expression of nine genes previously reported to be associated with T-cell suppression.
- Supplementary Figure 5 - PDF file, 78K, (A) Dose titration curve showing surface expression of PD-L1 on TAFs as measured by flow cytometry 18 hours following exposure to titrated doses of IL-1α or IL-1β. (B) Time course experiment assessing surface expression of PD-L1 by flow cytometry following exposure of TAFs to 1 ng/ml IL-1β.
- Supplementary Figure 6 - PDF file, 99K, Four separate TAF lines were derived from human melanoma tumor biopsies and exposed overnight to recombinant human IL-1α (1 ng/ml) or IFN-γ (500 U/ml). TAFs were then stained using antibodies specific for PD-L1 and PD-L2, and analyzed for surface expression by flow cytometry.
Volume 18, Issue 19
