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Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma

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Science Translational Medicine  13 Sep 2017:
Vol. 9, Issue 407, eaal4712
DOI: 10.1126/scitranslmed.aal4712
  • Fig. 1. Blocking VEGFR-3 signaling in lymphangiogenic melanomas decreases Treg cell infiltration and delays primary tumor growth.

    B16-OVA and B16-OVA/VC tumor–bearing mice were treated with control (Iso) or αR3 starting on the day of inoculation, and tumors were characterized on day 9. (A) Intratumoral VEGF-C concentration (n = 5). N.D., not detected. (B) Immunostained whole-tumor sections [scale bars, 500 μm and 200 μm (in zoomed images)] showing overall lymphatic density [green, Lyve-1; gray, 4′,6-diamidino-2-phenylindole (DAPI)]. (C) Representative flow cytometry plots of tumor cell suspensions for LECs (CD45gp38+CD31+), BECs (CD45gp38CD31+), and Macs (CD45+F4/80+). (D) Quantification of LECs, BECs, and Macs in tumors (n = 5). (E) Growth and survival curves (n ≥ 5). (F) Total infiltrating leukocytes (CD45+). (G) Treg cells (CD4+FoxP3+) and effector CD8+ T (Teff) cells (CD62LCD44+). (H) Gating strategy for myeloid subsets. (I) Quantification of myeloid subsets (mature, m-Myeloid, CD11cCD11b+MHCII+ and immature, imm-Myeloid, CD11cCD11b+MHCII) and MDSCs (granulocytic, G-MDSCs, CD11cCD11b+MHCIILy6G+Ly6Clow, and monocytic, Mo-MDSCs, CD11cCD11b+MHCIILy6G+Ly6Clow). (J) Gating strategy for DC subsets. (K) Quantification of DC subsets: conventional (cDCs; CD11c+CD11b), cross-presenting (CD8+ DCs; CD11c+CD11bCD8+), and myeloid (Myel. DCs; CD11c+CD11b+) (n = 5). All data represent two independent experiments. *P < 0.05, **P < 0.01 by two-tailed Student’s t test.

  • Fig. 2. VEGF-C/VEGFR-3 signaling increases responsiveness of melanoma to immunotherapy.

    Tumor growth and survival of three different melanoma models treated with control (Iso) or αR3-blocking antibodies receiving different immunotherapies (arrows indicate times of administration). (A and B) B16-OVA/VC tumors treated with ATT in (A) WT (n ≥ 15) and (B) K14-VEGFR-3-Ig mice that lack dermal lymphatics (n = 4). ns, not significant. (C to F) B16-OVA/VC tumors in WT mice treated with (C) ex vivo activated DCs (DC vax; n = 6), (D) 50 μg of CpG (n = 6), (E) 10 μg of OVA + 50 μg of CpG (n ≥ 8), and (F) 2 μg of Trp2 peptide–conjugated nanoparticles (NP-Trp2) + 50 μg of CpG (n = 7). (G) B16/VC tumors treated with NP-Trp2 + 50 μg of CpG (n = 6). (H) Tamoxifen-induced tumors in BrafV600E/Pten−/− mice treated with CpG + gp100 peptide (days 8 and 12) and anti–PD-1 antibody (day 12 and every 4 days thereafter). Each panel shows data from one (B to D, F, and G), two (E), or three (A) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test for growth curves and log-rank (Mantel-Cox) test for comparing survival curves.

  • Fig. 3. VEGFR-3 signaling increases infiltration of naïve T cells in a CCR7-dependent manner.

    (A to D) B16-OVA and B16-OVA/VC tumor–bearing mice were treated with control (Iso) or VEGFR-3–blocking antibodies (αR3) starting on the day of inoculation, and tumors were characterized on day 9 by flow cytometry (data represent two independent experiments, n = 5 each). (A) Quantification of tumor-infiltrating conventional CD4+ T cells (conv CD4+; FoxP3) and CD8+ T cells. (B) Activation status of CD4+ (top) and CD8+ (bottom) T cells. Naïve, CD44CD62L+; effector/effector memory (EM), CD44+CD62L; central memory (CM), CD44+CD62L+. (C) Ratio of naïve/EM in infiltrating CD4+ and CD8+ T cells. (D) CCL21 concentration as assessed by enzyme-linked immunosorbent assay (ELISA) in the tumor, dLNs, and ndLNs. (E) Representative image of a lymphangiogenic B16/VC tumor section immunostained for CD4+ T cells (red), CCL21 (green), LECs (Lyve-1, white), and DAPI (blue). Scale bar, 100 μm. (F) Quantification of CCR7+ T cell subsets in untreated B16-OVA and B16-OVA/VC tumors after 14 days (n ≥ 4). (G to J) B16-OVA/VC tumor–bearing mice were treated with Iso or anti-CCR7 (αCCR7)–blocking antibodies on days 0, 3, and 6, and (G to I) tumors were characterized on day 9 by flow cytometry or (J) mice were given adoptive transfer of 106 naïve OT-I CD8+ T cells (n ≥ 6). (G) Representative flow cytometry plots of T cell activation status. (H) Quantification of naïve and CM fractions of conventional CD4+ (left) and CD8+ (right) T cells. (I) Ratio of naïve/EM subsets. (J) Quantification of intratumoral OT-I cells as percentage of overall CD8+ T cells on day 10 after inoculation. *P < 0.05, **P < 0.01, ***P < 0.001 performed with two-tailed Student’s t test or one-way analysis of variance (ANOVA).

  • Fig. 4. Primary human metastatic melanomas contain CCL21-expressing LECs, and expression of VEGFC positively correlates with hallmarks of tumor inflammation.

    (A) Representative image of a human primary melanoma immunostained for LVs (green, podoplanin; blue, DAPI). Scale bars, 500 μm (left) and 200 μm (right). (B) Quantification of LV density in tumor (n = 14) and, when present, neighboring skin (n = 7) of primary melanoma tumor sections stratifying patients with elevated intratumor LV density (closed circles), indicating tumor lymphangiogenesis, from those without (open circles). (C) Representative image of a lymphangiogenic melanoma immunostained for VEGF-C (brown). Scale bar, 100 μm. (D) Representative image of an intratumoral LV (podoplanin, green) expressing CCL21 (red). Blue, nuclei (DAPI). Scale bar, 10 μm. (E and F) Correlations of gene expression data of human primary cutaneous metastatic melanoma patients from TCGA. (E) Heat map showing correlation between the expression of 30 genes indicative of T cell inflammation versus VEGFC, FIGF (VEGFD), and VEGFA. Colors indicate minimum and maximum r values using nonparametric Spearman’s test. (F) Dot plots of genes of interest (n = 103) shown with linear regression correlations using nonparametric Spearman’s test.

  • Fig. 5. Serum VEGF-C correlates with antitumor immune responses and PFS after immunotherapy in human metastatic melanoma patients.

    (A to C) Correlations of magnitude and quality of T cell responses with serum VEGF-C concentrations (n = 20) in patients enrolled in a phase 1 clinical study (NCT00112229) evaluating an antitumor Melan-A/MART-1 peptide vaccine. T cell responses reflect peak values across four weekly blood samples in Melan-A tetramer+ CD8+ T cells, and serum VEGF-C was measured before therapy. (A) Antigen-specific T cells as % of circulating CD8+ T cells versus serum VEGF-C. Left: Absolute values for each patient (dotted line indicates mean VEGF-C). Right: Comparison of T cell numbers in patients with low (<mean) versus high (>mean) VEGF-C. (B) IFN-α expression and (C) polyfunctionality in terms of IFN-α, TNF-α (tumor necrosis factor–α), IL-2 (interleukin-2), and CD107 expression in tetramer+ CD8+ T cells. (D) PFS of human melanoma patients (n = 76) enrolled in a phase 2 clinical study (NCT01927419) receiving combined αPD-1 and αCTLA-4 checkpoint blockade. Patients were stratified into three groups (high, mid, low) according to serum VEGF-C, VEGF-D, and VEGF-A concentrations measured before immunotherapy. Groups were compared using a nonparametric Spearman’s test for correlations, two-tailed Student’s t test for dot plots (*P < 0.05), and log-rank (Mantel-Cox) test for survival curves.

  • Fig. 6. Increased efficacy of immunotherapy in lymphangiogenic B16 melanomas depends on CCR7 signaling before therapy and local activation and expansion of TILs after therapy.

    (A and B) B16-OVA/VC tumor–bearing mice treated with control IgG (Iso) or anti–VEGFR-3 (αR3)–blocking antibodies were euthanized 3 days after ATT, and tumor single-cell suspensions were analyzed by flow cytometry (n = 5). Quantification of overall naïve CD8+ (CD45+CD8+CD44CD62L+), effector CD8+ (CD45+CD8+CD44+CD62L), and OT-I (CD45+CD8+CD45.1+) T cells (A) in the tumor and (B) in the dLNs. (C) Tumor growth and survival curves of B16-OVA/VC tumor–bearing mice treated with anti-CCR7 (αCCR7), control IgG (Iso), or αR3 antibodies combined with ATT on day 9. CCR7 blockade was performed only before ATT (days 0, 3, and 6) (data pooled from two or more independent experiments, n ≥ 15 total). (D) Tumor growth curves of B16-OVA/VC tumor–bearing mice treated with control IgG (Iso) or αR3 antibodies received daily injections of the small molecular S1P inhibitor FTY720 starting on the same day as ATT was performed (day 9) (n ≥ 5). Statistics show differences between Iso + FTY720 and αR3 + FTY720 by one-way ANOVA. (E) Representative flow cytometry plots and (F) quantification of circulating CD4+ and CD8+ T cells (after B220 exclusion) in blood 26 days after tumor inoculation. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test or one-way ANOVA and log-rank (Mantel-Cox) test for survival curves.

  • Fig. 7. Mice rejecting primary lymphangiogenic B16 melanomas in response to immunotherapy show epitope spreading and long-term protection.

    (A to D) B16-OVA/VC tumor–bearing mice that rejected the primary tumor [primary intradermal (1° i.d.) challenge] after therapeutic vaccination received a metastatic rechallenge with intravenous injections of 2 × 105 B16-WT or B16-OVA/VC cells [secondary intravenous (2° i.v.) challenge] at least 10 days after complete regression. Mice that received either no treatment (naïve) or vaccination only (Vax only) served as controls. (A) Flow cytometry analysis of circulating effector CD4+ (CD45+B220CD4+CD44+CD62L), effector CD8+ (CD45+B220CD8+CD44+CD62L), and tumor antigen–specific CD8+ (CD45+B220CD8+SIINFEKL-pentamer+) T cells 23 days after 1° i.d. challenge but before 2° i.v. rechallenge. (B) Representative images of lung metastases. (C) Quantification of metastatic nodules per lung of mice. (D) Circulating tumor antigen–specific CD8+ T cell responses 9 days after the 2° i.v. challenge (data pooled from two independent experiments, n ≥ 5 total). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/407/eaal4712/DC1

    Materials and Methods

    Fig. S1. VEGF-C/VEGFR-3 signaling does not affect the growth of B16 melanomas, and only potentiates immunotherapy in lymphangiogenic tumors, including those that do not express OVA.

    Fig. S2. In the naturally lymphangiogenic BrafV600E/Pten−/− mouse model, VEGFR-3 blockade reduces intratumoral lymphatics and total CCL21 in the tumor microenvironment.

    Fig. S3. Expression of VEGFC, but not FIGF (VEGFD) or VEGFA, strongly correlates with hallmarks of inflammation within metastatic sites of human melanoma.

    Table S1. Primary data.

  • Supplementary Material for:

    Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma

    Manuel Fankhauser, Maria A. S. Broggi, Lambert Potin, Natacha Bordry, Laura Jeanbart, Amanda W. Lund, Elodie Da Costa, Sylvie Hauert, Marcela Rincon-Restrepo, Christopher Tremblay, Elena Cabello, Krisztian Homicsko, Olivier Michielin, Douglas Hanahan, Daniel E. Speiser, Melody A. Swartz*

    *Corresponding author. Email: melodyswartz{at}uchicago.edu

    Published 13 September 2017, Sci. Transl. Med. 9, eaal4712 (2017)
    DOI: 10.1126/scitranslmed.aal4712

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. VEGF-C/VEGFR-3 signaling does not affect the growth of B16 melanomas, and only potentiates immunotherapy in lymphangiogenic tumors, including those that do not express OVA.
    • Fig. S2. In the naturally lymphangiogenic BrafV600E/Pten−/− mouse model, VEGFR-3 blockade reduces intratumoral lymphatics and total CCL21 in the tumor microenvironment.
    • Fig. S3. Expression of VEGFC, but not FIGF (VEGFD) or VEGFA, strongly correlates with hallmarks of inflammation within metastatic sites of human melanoma.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary data.

    [Download Table S1]

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