Research ArticleTUMOR IMMUNOLOGY

Myeloid cell–synthesized coagulation factor X dampens antitumor immunity

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Science Immunology  20 Sep 2019:
Vol. 4, Issue 39, eaaw8405
DOI: 10.1126/sciimmunol.aaw8405
  • Fig. 1 Direct FXa inhibitors improve anti-tumor immunity.

    (A) Schematic illustration of the treatment scheme. WT mice injected with 5 × 104 T241 cells were randomized at day 14 to treatment with saline injection (mock), LMWH injection (dalteparin, 200 mg/kg body weight), or oral anticoagulation with FXa inhibitor (rivaroxaban, 0.4 mg/g chow). (B) Tumor volumes of treatment groups (n = 9) on the day of randomization. (C) Macroscopic lung metastasis in treatment groups (day 22; n = 9/8/9). (D) Tumor weights at the end of the experiment (day 22; n = 8/7/8). (E) Quantification of total and Mrc1+CD204+ macrophages in the TME (n = 4). (F) Regulatory and cytotoxic T cell frequencies in the TME (n = 7). (G) Quantification of CD169+CD11c+ MHC II+ macrophages and CD8+CD11c+ DCs in draining lymph nodes of T241 tumors (n = 7); for all: one-way analysis of variance (ANOVA), mean ± SD; *P < 0.05; **P < 0.005; ***P < 0.001; ns, not significant. (H) Illustration of predicted distribution of rivaroxaban and LMWH/antithrombin in the TME.

  • Fig. 2 Expression of FX by tumor-associated immune cells.

    (A) Peripheral blood monocytes and neutrophils from PyMT or tumor-free mice were isolated by density gradient centrifugation and additional CD115+ or CD11b+ bead selection. Shown are representative cytospins and F10 mRNA expression relative to r18s (n = 6/5/7/5/6); one-way ANOVA, mean ± SEM; **P < 0.005; ***P < 0.001. (B) Flow cytometric analysis of FX (intracellular), Mrc1, and CD11c expression in CD11b+/F4/80 (neutrophils) and CD11b+/F4/80+ (TAMs) of PyMT mice. (C) Clustering of Gel bead-in-EMulsion (GEM) single-cell sequenced CD11c-selected cells from early-stage PyMT WT tumors by t-SNE analysis. (D) Violin and tSNE plots revealing F4/80 (Adgre1) and Mrc1 expressing myeloid cell clusters. (E) Expression of F7 and F10 in CD68+ and F4/80+ myeloid cells. (F) Differential transcript abundance in F10+ clusters 5 versus 14 (top 40 log fold change). (G) FX expression by CD68+ TAMs on serial sections from human clear cell (representative of 3/15 tumors) and papillary (representative of 8/10 tumors) RCC.

  • Fig. 3 Monocyte/macrophage-autonomous FX synthesis promotes immune evasion.

    (A) FXa activity and FX protein in serum-free supernatant of F10fl/flLysMcre peritoneal macrophages after stimulation as indicated for 18 hours (n = 3/4/8/8); mean ± SEM, two-way ANOVA, Sidak’s multiple comparisons test. (B) Expression of F10 mRNA of peritoneal macrophages exposed to DMEM + IFN-γ or PyMT cell culture supernatant supplemented with IFN-γ (n = 4/5/4/5). (C) Ccl17 and Ccl22 mRNA expression of peritoneal macrophages exposed to PyMT cell culture supernatant with or without purified human FX (n = 4); mean ± SEM, two-way ANOVA, Sidak’s multiple comparisons test. (D) Effect of FX deletion in F10fl/flLysMcre peritoneal macrophages on polarization marker expression after 18 hours of stimulation in PyMT tumor cell–conditioned medium with IFN-γ; Z score and individual replicates (n = 4/5) are shown, unpaired, two-tailed t test. (E) FX plasma levels in 19-week-old female mice with or without PyMT tumors (n = 12/12, 6/8); mean ± SEM, two-way ANOVA, Sidak’s multiple comparisons test. (F) Tumor-free survival (n = 30) and tumor growth (n = 33) of PyMT F10fl/flLysMcre and PyMT F10fl/fl littermate control (n = 36 and 37) mice; log-rank (Mantel-Cox) test (tumor-free survival), mean ± SEM, two-way ANOVA, Sidak’s multiple comparisons test (tumor growth). Quantification of (G) GrB+CD8+ T cell (n = 6/8) and FoxP3+CD4+ T cells (n = 6/7) in the TME and of (H) CD169+ macrophages and CD8+ DCs in the draining lymph nodes of late-stage tumors of 20-week-old PyMT F10fl/flLysMcre or PyMT F10fl/fl littermate control mice (n = 9/9). Tumor weight at sacrifice (day 22) of (I) F10fl/flLysMcre (n = 5/6) and (J) F10fl/flCX3CR1cre (n = 5/5) mice injected with 5 × 104 T241 fibrosarcoma cells; mean ± SEM, unpaired, two-tailed t test. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

  • Fig. 4 Pharmacological targeting of macrophage FXa-PAR2 signaling by direct FXa inhibitors.

    (A) PD-L1 and EPCR expression in TAMs from late-stage tumors of PyMT F10fl/flLysMcre mice compared with PyMT F10fl/fl littermate controls. SSC-A, side scatter area. (B) Effects of rivaroxaban on TAM polarization marker expression in WT peritoneal macrophages after 18 hours in PyMT tumor cell–conditioned medium with hirudin/vitamin K and IFN-γ; heatmap (control, n = 6; 62 nM rivaroxaban, n = 7; 500 nM rivaroxaban, n = 7); log2 scale; values relative to mean of control; Z score, unpaired, two-tailed Student’s t test. (C) Rivaroxaban plasma levels of untreated tumor-free WT mice and PyMT F10fl/flLysMcre and PyMT F10fl/fl littermate controls receiving rivaroxaban in the diet (n = 6/11/11); means ± SEM, one-way ANOVA with Dunnett's multiple comparisons test. (D) Tumor growth of PyMT F10fl/flLysMcre (n = 28) and PyMT F10fl/fl littermate control (n = 36) mice with oral rivaroxaban administration; means ± SEM; two-way ANOVA, Sidak's multiple comparisons test. (E) Quantification of CD11b+CD11c+F4/80+ TAMs (n = 10) and Mrc1+ (n = 4/4/6/6) and PD-L1+ (n = 8/6/6/7) subpopulations in late-stage tumor TME of 20-week-old PyMT mice with or without oral rivaroxaban therapy; means ± SEM; two-way ANOVA, Dunnett's multiple comparisons test. (F) Expression profiles of CD11c-selected TAMs from the TME of late-stage tumors in 20-week-old PyMT mice (n = 7/6/7/7); mean ± SEM, unpaired, two-tailed Student’s t test. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

  • Fig. 5 FXa inhibitor synergizes with checkpoint inhibitor therapy.

    (A) Treatment scheme. i.p., intraperitoneal. (B) Tumor growth of 5 × 104 T241 cells injected into WT mice with or without treatment of rivaroxaban and/or α-PD-L1 (n = 9/9/10/8); mean ± SEM, two-way ANOVA, Tukey’s multiple comparisons test. (C) Quantification of CD103+ CCR7+ MHCIIlo DCs, GrB+ CTL, activated CD137+ CD8+ T cells, and Treg (n = 4) in the TME at sacrifice 27 days after tumor cell injection (n = 4); mean ± SEM, one-way ANOVA, Tukey’s multiple comparison test. (D) Tumor growth of 5 × 104 MC38 cells injected into WT mice with or without treatment of rivaroxaban and/or α-PD-L1 (n = 9/11/11/11); mean ± SEM, two-way ANOVA, Tukey’s multiple comparisons test. Quantification of (E) CD103+ CCR7+ MHCIIlo DCs, GrB+ CTL, and Treg (n = 4) in the TME and of (F) CD8+ DCs, CD103+ DCs and CD169+ macrophages (n = 4) at sacrifice 21 days after tumor injection; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

  • Fig. 6 Attenuated tumor growth in FXa-resistant PAR2 mutant mice.

    (A) Mature residue numbering; coding and amino acid sequence of PAR2-WT, G37I, and R38E mice. (B) Confirmation of FXa resistance of mouse PAR2-G37I to cleavage by TF-FVIIa-FXa on CHO cells expressing mouse EPCR (n = 4); mean ± SEM, two-tailed, unpaired Student’s t test. (C) Tumor-free survival (n = 29, 34, and 28); log-rank (Mantel-Cox) test, and (D) total tumor volumes and weights of the largest tumors at sacrifice (n = 30, 34, and 30) of PyMT mice; means ± SEM, two-way ANOVA, Tukey’s multiple comparisons test. (E) Quantification of CD11b+CD11c+CD68+ TAMs (n = 9/9/10) and Mrc1+ (n = 7) and EPCR+ (n = 6/5/4) subpopulations in late-stage TME of 20-week-old PyMT PAR2-WT, G37I, or R38E mice; means ± SEM, one-way ANOVA. (F) CD3+CD8+ T cell (n = 14/14/13) frequencies in late-stage tumors of 20-week-old PyMT mice; means ± SEM, one-way ANOVA. (G) Tumor weights 22 days after subcutaneous injection of B16F10 (n = 12/12/10) or T241 (n = 9) cells into PAR2-WT, G37I, or R38E mice; pooled data from two independent experiments; means ± SEM, one-way ANOVA. (H) Tumor weights 22 days after subcutaneous injection of T241 in PAR2fl/flLysMcre or littermate control mice (n = 5/4); means ± SEM, unpaired, two-tailed t test. Frequencies of granzyme B+ (GrB+) CD8+ T cells in TME of (I) F10fl/flLysMcre mice (n = 4) or (J) PAR2-G37I mice and controls with or without rivaroxaban therapy (n = 4) evaluated 21 days after T241 injection. T cells were cocultured with T241 cells for quantification of antitumor CD8+ T cell responses by ELISpot assay shown by representative spots; means ± SEM, unpaired, two-tailed t test; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

  • Fig. 7 Tumor stage-independent reprogramming of TAM immune-evasive phenotypes in PAR2 FXa-resistant mice.

    (A) Macrophage polarization marker expression in peritoneal macrophages after 18 hours of culture in PyMT tumor cell–conditioned, serum-free DMEM supplemented with hirudin/vitamin K and IFN-γ (n = 4); Z score, unpaired, two-tailed t test. (B) CXCL1 plasma levels of 13-week-old PyMT PAR2-WT, G37I, or R38E mice (n = 6/6/5); means ± SEM, one-way ANOVA. (C to E) Quantification of (C) CD11b+CD11c+CD68+ TAMs (n = 11/10/10) and Mrc1+ (n = 7/6/6) and EPCR+ (n = 5/5/5) subpopulations, (D) neutrophils (n = 14/12/12), and (E) CD8+ and CD4+ T cells in early-stage TME of 20-week-old PyMT PAR2-WT, G37I, or R38E mice (n = 13/12/14); means ± SEM, one-way ANOVA. (F) Expression profiles of CD11c-selected TAMs from late-stage (n = 8/8) and early-stage (n = 5 to 9/7 to 10) tumors of 20-week-old mice; mean ± SEM, unpaired, two-tailed t test; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/39/eaaw8405/DC1

    Fig. S1. Expression of TF signaling components in macrophages and tumor cells.

    Fig. S2. Regulation of metastasis by macrophage FXa-PAR2 signaling.

    Fig. S3. Tumor progression alters myeloid cell composition of the TME.

    Fig. S4. Generation of PAR2 mutant mice.

    Table S1. Raw data file.

    Table S2. Sources of materials used.

    Table S3. Primer list for RT-PCR.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Expression of TF signaling components in macrophages and tumor cells.
    • Fig. S2. Regulation of metastasis by macrophage FXa-PAR2 signaling.
    • Fig. S3. Tumor progression alters myeloid cell composition of the TME.
    • Fig. S4. Generation of PAR2 mutant mice.
    • Table S2. Sources of materials used.
    • Table S3. Primer list for RT-PCR.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

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