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The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy

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Science Translational Medicine  11 Apr 2018:
Vol. 10, Issue 436, eaan3464
DOI: 10.1126/scitranslmed.aan3464
  • Fig. 1 The outgrowth of D2A1-GFP tumors in Balb/c mice is restricted by a GFP-specific CD8+ T cell response.

    (A) Endpoint tumor incidence, 32 days after the orthotopic injection of 2.5 × 104, 5 × 104, or 1 × 105 D2A1–green fluorescent protein (GFP) cells into syngeneic Balb/c mice; 1 × 105 D2A1-GFP cells injected into immunodeficient nonobese diabetic/severe combined immunodeficient mice; and 1 × 105 unlabeled D2A1 cells injected into Balb/c mice (n = 10 per group). Tumor incidence is reported as the fraction of mice bearing tumors of diameter ≥ 2 mm (see Supplementary Materials and Methods). (B) Fraction of tumor-bearing mice as a function of time after the orthotopic injection of D2A1-GFP cells into Balb/c mice at doses of 2.5 × 104, 5 × 104, or 1 × 105 per injection (n = 8 to 10 per group). (C) Secretion of interferon-γ (IFN-γ) into culture medium during ex vivo coculture of irradiated D2A1-GFP cells with lymph node (LN) cells isolated from the inguinal LNs of tumor-free or D2A1-GFP–bearing Balb/c mice. Data for cells isolated from the ipsilateral (tumor-draining) and contralateral LNs are shown for tumor-bearing mice (n = 5 per group). (D) Tumor diameter after the orthotopic injection of 1 × 105 D2A1-GFP cells into Balb/c mice 1 day after initiating injections of either anti-CD8 antibodies or control immunoglobulin G (IgG) (n = 9 to 10 per group). (E) Tumor diameter after the orthotopic injection of 1 × 105 D2A1-GFP cells into wild-type (WT) or GFP-transgenic (Tg) mice, both on a Balb/c:C57BL/6 F1 background (n = 11 per group). (F) Number of tumor nodules in the lungs after the intravenous injection via the tail vein of 1 × 106 D2A1-GFP cells into Balb/c mice that had previously rejected D2A1-GFP tumors or into naïve mice (n = 3 to 5 per group). (G) Number of tumor nodules in the lungs after the intravenous injection via the tail vein of D2A1-GFP 5 × 105 cells into Balb/c mice bearing D2A1-GFP cells in the mammary fat pad (MFP) at different stages of growth or rejection (n = 3 to 6 per group). For all panels, data are plotted as means ± SEM. P values were calculated using the Mann-Whitney test (*P < 0.05, **P < 0.005, ***P < 0.0005).

  • Fig. 2 The postsurgical wound-healing response protects local immunogenic tumors from immune-mediated destruction.

    (A) Identification of sponge-infiltrating myeloid cells using flow cytometry after staining for the presence of neutrophils (CD45+CD11b+Ly6G+) and macrophages (CD45+CD11b+F4/80+), 7 days after sponge implantation. (B) Immunofluorescence staining of tissue sections containing stroma-infiltrated sponges. Sections were stained with anti-F4/80 (macrophages, top) or anti–α–smooth muscle actin (αSMA) (myofibroblasts and blood vessel–lining pericytes, bottom). Representative images at ×10 magnification are shown for sections isolated 14 days after wounding by subcutaneous sponge implantation. DAPI, 4′,6-diamidino-2-phenylindole. (C to E) Injection of immunogenic D2A1-GFP cells into control or wounded Balb/c mice. (C) Schematic illustrating the experimental design in which 1 × 105 tumor cells were injected into the MFP of control mice (left) or mice bearing a local wound (center) or were injected directly into a site of wound healing (right). The incidence (D) and mass (E) of the resulting tumors were determined 30 days after the injection of tumor cells (n = 10 per group). For all panels, data are plotted as means ± SEM. P values were calculated using the Mann-Whitney test (*P < 0.05).

  • Fig. 3 The systemic response to surgery triggers the outgrowth of immunogenic tumor cells at distant anatomical sites.

    (A to C) Injection of immunogenic D2A1-GFP cells into syngeneic Balb/c mice wounded at distant sites. (A) Schematic illustrating the experimental design in which 1 × 105 D2A1-GFP cells were injected into the MFP of unwounded mice or into mice that had been previously wounded by sponge implantation at one or two distant sites (1× and 2×, respectively). (B) Tumor diameter at the conclusion of the experiment and (C) tumor incidence as a function of time are shown (n = 9 to 10 per group). The dashed red line indicates a tumor diameter of 2 mm, the threshold for tumor incidence (see Materials and Methods). inj, injection. (D to F) Tumor incidence as a function of time for three experiments in which tumors and surgical wounds were inflicted at contralateral sites: (D) mice were wounded by sponge implantation 7 days before the subcutaneous (SC) injection of 1 × 105 tumor cells; (E) mice were wounded by sponge implantation 7 days after the subcutaneous injection of 1 × 105 tumor cells; and (F) mice were wounded by a cutaneous incision 7 days after the orthotopic injection of 2 × 105 tumor cells into the MFP (n = 12 to 14 per group in each experiment). (G) Meta-analysis of tumor diameter and tumor incidence for experiments in which D2A1-GFP cells were injected into unwounded Balb/c mice or into mice surgically wounded at distant anatomical sites. Data are shown for all experiments with D2A1-GFP cells (left) and for experiments in which 1 × 105 D2A1-GFP cells were injected orthotopically into an MFP (right). (H) Linkage plots of tumor incidence for experiments in which D2A1-GFP cells were injected into unwounded mice or into mice surgically wounded at distant anatomical sites. Data sets are the same as in (G). For all panels, data are plotted as means ± SEM. P values were calculated using the Mann-Whitney test (B) (*P < 0.05) or Fisher’s exact test (G).

  • Fig. 4 Surgery initiates a systemic inflammatory response that triggers the outgrowth of distant immunogenic tumors and can be inhibited by perioperative anti-inflammatory treatment.

    (A) Relative proportion of circulating neutrophils and inflammatory (Ly6Chi) monocytes in wounded and control Balb/c mice, 1, 3, and 7 days after surgery. The proportion of each cell type in the circulation was determined as a percentage of CD45+ leukocytes, and the values were normalized to those of control mice on each collection day (n = 4 to 6 per group). (B) Concentrations of interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and CCL2 in the circulation of control and wounded Balb/c mice, 24 hours after surgery, as detected by enzyme-linked immunosorbent assay (n = 6 per group). (C) Correlation between the percentage of tumor-infiltrating CD11b+ myeloid cells (left) or the myeloid-to-CD8+ T cell ratio (right) and the percentage of D2A1-GFP cells within orthotopic tumors, 17 days after the injection of tumor cells into unwounded Balb/c mice. (D) Tumor diameter after the subcutaneous injection of 5 × 105 D2A1-GFP cells into Balb/c mice that were subsequently treated with anti-Ly6G or isotype-control antibodies (n = 12 per group). (E) Tumor diameter after the orthotopic injection of 1 × 106 D2A1-GFP–sgLuciferase (sgLuc) or D2A1-GFP–sgCCL2 cells into Balb/c mice (n = 5 per group). (F) Tumor diameter after the orthotopic injection of 1 × 105 D2A1-GFP cells into previously unwounded (left) or wounded (right) Balb/c mice that were subsequently treated with anti-PD1 or isotype-control antibodies (n = 15 mice per group). PD1, programmed cell death protein 1. (G) Tumor diameter after the orthotopic injection of 1 × 105 D2A1-GFP cells into previously unwounded (left) or wounded (right) Balb/c mice treated peri- and postoperatively with saline or meloxicam (n = 15 mice per group). For all panels, data are plotted as means ± SEM. P values were calculated using Student’s t test (A and B) or the Mann-Whitney test (C to G) (*P < 0.05, **P < 0.005, ***P < 0.0005).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/436/eaan3464/DC1

    Materials and Methods

    Fig. S1. The outgrowth of D2A1-GFP tumors in Balb/c mice is restricted by a GFP-specific CD8+ T cell response.

    Fig. S2. Surgical wounding triggers the outgrowth of tumor cells at distant anatomical sites.

    Fig. S3. Meta-analyses demonstrate that surgical wounding promotes the outgrowth of distantly implanted tumor cells.

    Fig. S4. Surgical wounding overcomes the effect of a tumor vaccine to promote the growth of distant B16 tumors.

    Fig. S5. Surgical wounding triggers a systemic inflammatory response characterized by the mobilization of inflammatory myeloid cells into the circulation.

    Fig. S6. Myeloid cells infiltrate D2A1-GFP tumors and promote tumor growth.

    Fig. S7. NSAIDs alter the polarization of tumor-infiltrating macrophages.

    Table S1. Components of flow cytometry antibody cocktails.

    Table S2. Primary data.

    Reference (56)

  • Supplementary Material for:

    The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy

    Jordan A. Krall, Ferenc Reinhardt, Oblaise A. Mercury, Diwakar R. Pattabiraman, Mary W. Brooks, Michael Dougan, Arthur W. Lambert, Brian Bierie, Hidde L. Ploegh, Stephanie K. Dougan, Robert A. Weinberg*

    *Corresponding author. Email: weinberg{at}wi.mit.edu

    Published 11 April 2018, Sci. Transl. Med. 10, eaan3464 (2018)
    DOI: 10.1126/scitranslmed.aan3464

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. The outgrowth of D2A1-GFP tumors in Balb/c mice is restricted by a GFP-specific CD8+ T cell response.
    • Fig. S2. Surgical wounding triggers the outgrowth of tumor cells at distant anatomical sites.
    • Fig. S3. Meta-analyses demonstrate that surgical wounding promotes the outgrowth of distantly implanted tumor cells.
    • Fig. S4. Surgical wounding overcomes the effect of a tumor vaccine to promote the growth of distant B16 tumors.
    • Fig. S5. Surgical wounding triggers a systemic inflammatory response characterized by the mobilization of inflammatory myeloid cells into the circulation.
    • Fig. S6. Myeloid cells infiltrate D2A1-GFP tumors and promote tumor growth.
    • Fig. S7. NSAIDs alter the polarization of tumor-infiltrating macrophages.
    • Table S1. Components of flow cytometry antibody cocktails.
    • Reference (56)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Primary data.

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