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A biomimetic pancreatic cancer on-chip reveals endothelial ablation via ALK7 signaling

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Science Advances  28 Aug 2019:
Vol. 5, no. 8, eaav6789
DOI: 10.1126/sciadv.aav6789
  • Fig. 1 Organotypic model for PDAC-on-a-chip to capture pancreatic tumor vascular invasion.

    (A) Schematic of PDAC-on-a-chip with a biomimetic blood vessel and a pancreatic cancer duct. The microfluidic device is composed of two hollow cylindrical channels embedded within 3D collagen matrix. One channel was seeded with endothelial cells to form a perfusable biomimetic blood vessel, while the other channel was seeded with pancreatic cancer cells to form a pancreatic cancer duct. Phase-contrast image shows seeded cells in the device before PDAC migration. (B) Average invasion distance of a PDAC cell line, YFP PD7591, toward a gradient of FBS with and without human umbilical vein endothelial cells (HUVECs). The presence of HUVECs increased the migration speed of PD7591 (n = 3 individual experiments). Representative phase-contrast images of PD7591 migration at days 0 and 8 demonstrated the collective migration of PDAC invasion. (C) YFP PD7591 (in green) invaded toward the biomimetic blood vessel (in red), migrated along the vessel (i), and wrapped around the blood vessel, as shown in the cross-sectional image of the biomimetic blood vessel (ii). (D) A confocal image of a section of the blood vessel (in red) invaded by YFP PD7591 (in green) showed that part of the blood vessel was ablated by cancer cells in our organotypic model (i and ii). (E) Apoptosis, marked by cleaved caspase-3 staining (in white), was observed in endothelial cells (in red) during invasion of YFP PD7591 (in green) in the blood vessels in our 3D PDAC organotypic model. Endothelial cells in all images were stained with anti-CD31 antibody. YFP PD7591 was restained with FITC-conjugated anti-GFP (green fluorescent protein) antibody. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (in blue). Error bars are SEM.

  • Fig. 2 Endothelial ablation is observed in in vivo mouse tumor models (subcutaneous tumor implantation model and genetically engineered mouse model).

    (A) Examination of endothelial cells in the ectopic mouse tumors in vivo. YFP PD7591 (in green) was subcutaneously injected into mice for 14 days. Apoptotic endothelial cells were also observed in the in vivo tumor microenvironment, similarly to the observation in the 3D organotypic model. Apoptotic endothelial cells (in red) were marked by cleaved caspase-3 signal (in white). (B) Endothelial ablation in subcutaneous tumor implantation model. Resected tumors at day 14 exhibited partial ablation of endothelial cells by pancreatic cancer cells (red arrows) in hybrid blood vessels. Some small vessels, decorated with collagen IV (in white), a basement membrane protein, demonstrated complete endothelial ablation by YFP PD7591 in the luminal side of the vessels (yellow arrows). Blood vessels were stained with anti-mouse endomucin antibody (in red). (C) Endothelial ablation was observed in GEMM of PDAC. Partial ablation of endothelium in the blood vessel was indicated by red arrows, where the blood vessel displayed the occupation of yellow fluorescent protein (YFP) tumor cells in place of the endothelium. Endothelial cells in all images were stained with either anti-mouse CD31 antibody or anti-mouse endomucin. YFP PD7591 was restained with FITC-conjugated anti-GFP antibody. Cell nuclei were stained with DAPI (in blue).

  • Fig. 3 Inhibition of the TGF-β receptor signaling pathway significantly reduced endothelial ablation.

    (A) Inhibition of TGF-β receptor signaling pathway with SB431542 in 3D organotypic model. PDAC tumor cells from the pancreatic cancer duct were allowed to invade into the biomimetic vessels. Once the PDAC tumor cells reached the blood vessels, 5 μM SB431542 or dimethyl sulfoxide (DMSO) was administered for 7 days. Representative confocal images of vessels treated with SB431542 (i) and DMSO (ii) in 3D organotypic model. The percentage of endothelium-ablated area showed that SB431542 significantly reduced the endothelial ablation in 3D organotypic model (n = 4 individual experiments). Western blot for phosphorylated Smad2 in endothelial cells and PD7591 in 2D monoculture confirmed the effectiveness of SB431542 to inhibit the TGF-β signaling pathway in endothelial cells and in pancreatic cancer cells PD7591. (B) Inhibition of TGF-β receptor signaling pathway with SB431542 in a mouse tumor model for either 1 or 2 weeks. SB431542 was peritoneally administered into the mice daily for 1 and 2 weeks. Mice were sacrificed at days 16 and 23. Quantification of endothelial cell density revealed a significantly higher endothelial cell density within the tumors treated with SB431542 as compared to vehicle control (n = 5 mice per experimental group) for both 1 and 2 weeks. Cleaved caspase-3 activity in endothelial cells was also less in SB431542-treated tumors versus control tumors in both 1 and 2 weeks. Cleaved caspase-3 activity was significantly increased in tumors treated with SB431542 for 2 weeks versus 1 week. Right: Representative images of tumor samples in vehicle control DMSO and SB431542 conditions for 1-week condition. Endothelial cells were stained with anti-mouse CD31 antibody. YFP PD7591 was restained with FITC-conjugated anti-GFP antibody. Cell nuclei were stained with DAPI (in blue). *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistical significance. NS, not significant. Two-tailed Student’s t test. Error bars are SEM.

  • Fig. 4 Endothelial ablation required activin-ALK7 signaling in the invasive PDAC.

    (A) A schematic demonstrates a setup for 2D patterned coculture of PDAC and endothelial cells. Tumor cells were plated inside an annulus, and endothelial cells were plated outside the annulus. Once the annulus was removed, tumor invaded into the endothelial cells, resulting in an expansion of the tumor area. (B) SB431542 reduced endothelial ablation in 2D patterned coculture (n = 3 individual experiments). (C) ALK7 was knocked out in YFP PD7591 by CRISPR-Cas9 and plated in 2D patterned cocultures with wild-type (WT) HUVECs. Invasion area of ALK7 knockout (KO) YFP PD7591 was significantly less than scramble PD7591, suggesting the role of ALK7 in PD7591 to mediate endothelial ablation (n = 3 individual experiments). (D) Inhibin βA and inhibin βB, major subunits of activins, were up-regulated in pancreatic cancer cells as compared to normal mouse pancreatic ductal epithelial cells (mPDE) (n = 3 individual experiments). (E) Inhibition of activin with the endogenous inhibitor follistatin significantly diminished endothelial ablation of PDAC in 2D patterned cocultures (n = 3 individual experiments). (F) Inhibition of activin by follistatin (200 ng/ml) in 3D organotypic model revealed a significant reduction in tumor replaced blood vessel (n = 3 individual experiments). (G) In vivo mouse tumor model to confirm the role of ALK7 in endothelial ablation. ALK7 knockout PD7591 versus scramble cells were subcutaneously implanted into mice for 2 weeks. Cleaved caspase-3 signal indicated a substantial reduction in apoptotic endothelial cells in ALK7 knockout condition versus scrambled PD7591 condition (n = 5 mice per experimental group). A significant increase in endothelial cell density was also observed with ALK7 knockout PD7591 tumors versus scramble PD7591 tumors (n = 5 mice per experimental group). (i) Representative images of tumors implanted with scramble PD7591 depicted less endothelial cell density and high apoptotic signal. (ii) Representative images of tumor implanted with ALK7 knockout PD7591 depicted high endothelial cell density and less apoptotic signal. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistical significance. Two-tailed Student’s t test. Error bars are SEM.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaav6789/DC1

    Fig. S1. Screening for chemoattractants for pancreatic cancer cell invasions and characterization of gradient in the 3D organotypic model.

    Fig. S2. PD7591 in the biomimetic duct channel proliferated in response to the FBS gradient introduced from the biomimetic blood vessel.

    Fig. S3. Vascular invasion and endothelial ablation were observed in multiple primary murine PDAC cell lines and a human pancreatic cancer cell line in our 3D biomimetic PDAC-on-a-chip model.

    Fig. S4. Staining for cleaved caspase-3 and collagen IV (Col IV) in vivo and in 3D organotypic model.

    Fig. S5. Tumor growth rate and caspase signal in endothelial cells in subcutaneous tumor implantation model.

    Fig. S6. Inhibition of the TGF-β receptor signaling pathway reduced the endothelial ablation in human pancreatic cancer cell lines in 2D patterned coculture invasion assay and caspase staining in 2D patterned coculture.

    Fig. S7. Examination of ALK4, ALK5, and ALK7 in endothelial cells and PD7591 in endothelial cell ablation.

    Fig. S8. Representative images of vessel area replaced by tumor cells in 3D organotypic model.

    Fig. S9. Representative images of tumor cell pattern in 2D coculture pattern assays.

    Fig. S10. Schematics describing how CD31 signal intensity and cleaved caspase-3 signal intensity were measured for quantification.

    Movie S1. 3D rendering of confocal image z-stack showed that YFP PD7591 (in green) invaded toward the biomimetic blood vessel (stained with CD31 in red) and wrapped around the blood vessel.

    Movie S2. YFP PD7591 (in green) invaded toward the biomimetic blood vessel (stained with CD31 in red).

    Movie S3. YFP PD7591 (in green) and HUVECs (in phase) were plated in 2D patterned coculture invasion assay in the presence of vehicle control (dimethyl sulfoxide).

    Movie S4. YFP PD7591 (in green) and HUVECs (in phase) were plated in 2D patterned coculture invasion assay in the presence of 5 μM SB431542.

    Table S1. Oligo sequences for CRISPR.

    Table S2. Primer sequences for qPCR.

    Supplementary Methods

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Screening for chemoattractants for pancreatic cancer cell invasions and characterization of gradient in the 3D organotypic model.
    • Fig. S2. PD7591 in the biomimetic duct channel proliferated in response to the FBS gradient introduced from the biomimetic blood vessel.
    • Fig. S3. Vascular invasion and endothelial ablation were observed in multiple primary murine PDAC cell lines and a human pancreatic cancer cell line in our 3D biomimetic PDAC-on-a-chip model.
    • Fig. S4. Staining for cleaved caspase-3 and collagen IV (Col IV) in vivo and in 3D organotypic model.
    • Fig. S5. Tumor growth rate and caspase signal in endothelial cells in subcutaneous tumor implantation model.
    • Fig. S6. Inhibition of the TGF-β receptor signaling pathway reduced the endothelial ablation in human pancreatic cancer cell lines in 2D patterned coculture invasion assay and caspase staining in 2D patterned coculture.
    • Fig. S7. Examination of ALK4, ALK5, and ALK7 in endothelial cells and PD7591 in endothelial cell ablation.
    • Fig. S8. Representative images of vessel area replaced by tumor cells in 3D organotypic model.
    • Fig. S9. Representative images of tumor cell pattern in 2D coculture pattern assays.
    • Fig. S10. Schematics describing how CD31 signal intensity and cleaved caspase-3 signal intensity were measured for quantification.
    • Legends for movies S1 to S4
    • Table S1. Oligo sequences for CRISPR.
    • Table S2. Primer sequences for qPCR.
    • Supplementary Methods

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). 3D rendering of confocal image z-stack showed that YFP PD7591 (in green) invaded toward the biomimetic blood vessel (stained with CD31 in red) and wrapped around the blood vessel.
    • Movie S2 (.avi format). YFP PD7591 (in green) invaded toward the biomimetic blood vessel (stained with CD31 in red).
    • Movie S3 (.avi format). YFP PD7591 (in green) and HUVECs (in phase) were plated in 2D patterned coculture invasion assay in the presence of vehicle control (dimethyl sulfoxide).
    • Movie S4 (.avi format). YFP PD7591 (in green) and HUVECs (in phase) were plated in 2D patterned coculture invasion assay in the presence of 5 μM SB431542.

    Files in this Data Supplement:

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