Research ArticleCancer

Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

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Science Translational Medicine  24 Oct 2018:
Vol. 10, Issue 464, eaat3487
DOI: 10.1126/scitranslmed.aat3487
  • Fig. 1 DKK3 is expressed by HPSCs in PDAC.

    Dkk3 expression was measured in HPSCs and PDAC cell lines by RT-PCR (A) and qPCR (B) in monoculture and coculture. Striped bars indicate expression in HPSCs after coculture with PDAC cells. (C) Dkk3 expression in human PDAC and normal pancreatic tissue was determined by Affymetrix array. (D) IHC of DKK3 in a tissue microarray of human PDAC. Shown are representative fields from PDAC expressing low and high amounts of DKK3 with normal pancreas and negative controls. (E) DKK3 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in plasma samples from patients with PDAC, CP, or no pancreatic disease and in conditioned medium (CM) from HPSCs (HPSC-CM). (F) In a GEMM of PDAC, DKK3 is expressed early in development with CP and PanIN lesions and increases in PDAC. Scale bars, 200 μm. (G) Relative expression of Dkk3 in the GEMM of PDAC and in cancer cells isolated from GEMM tumors was quantified by Affymetrix. *P < 0.05, ***P < 0.001. Data are means ± SEM.

  • Fig. 2 DKK3 stimulates HPSC and PDAC activity and increases chemoresistance.

    (A) HPSC proliferation was measured by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] after treatment with PBS or rhDKK3 (10 μg/ml). OD490, optical density at 490 nm. DKK3 was silenced in HPSCs by shDKK3 (fig. S1B), cell proliferation was measured by MTT assay (B), and migration was determined at 24 hours (C). Control cells were transfected with scrambled short hairpin RNA (shRNA). (D) Panc1 cells were treated with rhDKK3 (10 μg/ml) or serum-free medium control, and cell migration and invasion were measured after 24 hours. (E) Panc1 cells were stably silenced for DKK3, and (E) cell proliferation and (F) colony formation in soft agar were measured. (G) BxPC3 cell migration was measured after treatment with CM from HPSCs or HPSCs silenced for DKK3. HPF, high-power field. (H) Soft agar colony formation in gemcitabine and (I) apoptosis were determined in chemosensitive L3.6pl cells expressing DKK3 compared with transfection controls. (J) Gemcitabine-induced apoptosis was measured in chemoresistant HS766T cells silenced for DKK3. *P < 0.01, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Scale bars, 200 μm.

  • Fig. 3 NF-κB is activated by DKK3 in PSCs and PDAC cells and is necessary for DKK3-mediated stimulation of cell activity.

    (A) Phosphorylation of p65 and IκBα induced by DKK3 treatment (10 μg/ml) was determined by Western blotting. Relative protein loading was shown by using anti–β-actin Ab. (B) Western blot showing the time course of p65 activation in HPSC and Panc1 cells. Cells were treated with recombinant DKK3 (10 μg/ml) for 0 to 24 hours, and changes in band density relative to baseline were quantified. (C) DKK3 stimulates NF-κB luciferase reporter in HPSC and PDAC cells, compared to the mutant luciferase reporter (MT). NF-κB activity induced by DKK3 was measured in Panc28 with phosphorylation-defective IκBαM by (D) luciferase reporter and (E) Western blotting. (F) Proliferation of Panc28 and Panc28/IκBαM was measured by MTT assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus PBS control.

  • Fig. 4 Neutralization of DKK3 inhibits tumor growth and prolongs survival.

    BxPC3 tumor cells labeled with firefly luciferase were orthotopically implanted into nude mice, with or without either control HPSCs or HPSCs stably silenced for DKK3, in a 1:3 tumor/stroma ratio. (A) Average pancreas tumor volume and percentage of animals with metastases are shown at 35 days after injection. (B) IVIS imaging showing the growth of Panc02 tumor cells implanted subcutaneously in syngeneic C57/BL6 or DKK3-null mice, with Ki67 expression assessed by IHC. (C and D) Dkk3-deficient mice were crossed with KPC mice to produce P48-Cre; KrasLSL-G12D;Trp53fl/fl; Dkk3−/− progeny. Kaplan-Meier survival curve (C) and survival table (D) for mice with WT DKK3 (black), DKK3-null (red), or heterozygous DKK3 (blue). (E) Representative images of tumors from (C) from DKK3-WT mice (moribund, day 47), DKK3-heterozygous mice (moribund, day 63), or DKK3-null mice (early time point at day 48, or when moribund at day 68). (F) DKK3, (G) Ki67 proliferation index by IHC, and (H) collagen type I expression by qPCR. Data are means ± SEM [n = 8 to 15 mice per group in (A), 5 mice per group in (B), 10 to 16 mice per group in (C) to (D) and (F) to (H)]. *P < 0.05. Scale bars, 200 μm.

  • Fig. 5 DKK3-blocking Abs inhibit PSC and cancer cell activity, chemoresistance, and tumor progression, with improved survival.

    HPSCs and BxPC3 cells were treated with DKK3 mAb clones JM6-6-1 and JM8-12-1 or isotype control mAb or PBS. (A and B) HPSC apoptosis (A) and migration (B) as measured by fluorescence-activated cell sorting (FACS) and Transwell migration assay at 48 hours. (C) BxPC3 migration in response to rhDKK3 (10 μg/ml) as measured by Transwell migration assay at 48 hours. (D) BxPC3 resistance to gemcitabine (100 μM) as measured by MTT proliferation assay at 6 days. HPSC-CM (10 μg/ml). (E) The orthotopic coinjection BxPC3 + HPSC model of PDAC was used to test the efficacy of DKK3 mAb clones JM6-6-1 or JM8-12-1 (5 mg/kg ip, once every 5 days). Overall tumor progression was measured every 3 to 4 days by IVIS imaging. (F) Metastatic tumors in the peritoneal cavity after removal of the primary pancreatic tumor are shown by IVIS imaging. (G) Kaplan-Meier survival curve showing mice treated with DKK3 mAb clone JM8-12 (red), control Ab (blue), or PBS (black). KPC mice (P48-Cre; Kras LSL-G12D;Trp53fl/fl) either with WT DKK3 (solid lines) or deficient in DKK3 (dashed lines) were treated with DKK3 mAb JM6-6-1 (5 mg/kg, ip, once every 5 days), PBS, or control mAb. Kaplan-Meier survival curve (H) is shown with hazard ratios (log-rank test). Data are means ± SEM [n = 7 mice per group in (E) and (F), 6 to 7 mice per group in (G), 3 to 5 mice per group in (H)]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control Ab.

  • Fig. 6 DKK3 blockade is associated with increased tumor immune infiltrates and improves response to checkpoint inhibitor therapy.

    (A) T cells were stimulated and treated with DKK3 (5 to 10 μg/ml), and proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. In a syngeneic orthotopic model with luciferase-labeled KPC cells, (B) tumors were examined for CD3 and CD8 expression by IHC, and (C) additional markers of T cell activity were measured by qPCR. (D) Mice in the syngeneic orthotopic KPC-luciferase model were treated with control IgG, DKK3 mAb JM6-6-1, α-CTLA4, or the combination JM6-6-1 + α-CTLA4, and tumor growth was measured by IVIS imaging up to 190 days. Survival in this orthotopic implantation model is shown in (E). Using a GEMM (F), KPC/DKK3+/+ (black) or KPC/DKK3−/− (blue) mice were treated with α-CTLA4 or control IgG, and the Kaplan-Meier survival curve is shown. Data are means ± SEM [n = 5 mice per group in (B) and (C), 5 to 7 mice per group in (D) and (E), 4 to 7 mice per group in (F)]. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 200 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/464/eaat3487/DC1

    Fig. S1. DKK3 expression and silencing.

    Fig. S2. DKK3 expression and function in HPSCs and PDAC cells.

    Fig. S3. DKK3 expression in syngeneic models of PDAC.

    Fig. S4. DKK3 depletion in the P48-Cre; KrasLSL-G12D;Trp53fl/+ model of PDAC.

    Fig. S5. DKK3 expression in an autochthonous model of PDAC and effects of DKK3 mAb on cell surface binding of DKK3 and an orthotopic model.

    Fig. S6. DKK3 expression in mouse PSCs and effects of treatment with DKK3 mAb on survival.

  • This PDF file includes:

    • Fig. S1. DKK3 expression and silencing.
    • Fig. S2. DKK3 expression and function in HPSCs and PDAC cells.
    • Fig. S3. DKK3 expression in syngeneic models of PDAC.
    • Fig. S4. DKK3 depletion in the P48-Cre; KrasLSL-G12D;Trp53fl/+ model of PDAC.
    • Fig. S5. DKK3 expression in an autochthonous model of PDAC and effects of DKK3 mAb on cell surface binding of DKK3 and an orthotopic model.
    • Fig. S6. DKK3 expression in mouse PSCs and effects of treatment with DKK3 mAb on survival.

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