Notch pathway inhibition targets chemoresistant insulinoma cancer stem cells

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   Figure 1

   Isolation and characterisation of CM and canINS cancer stem cells (CSC). (A and B) CM in adherent (A) and in tumoursphere (B) culturing conditions (scale bar: 100 µm). (C and D) canINS in adherent (C) and tumoursphere (D) culturing conditions (scale bar: 100 µm). (E and F) Western blot analysis of CM (E) and canINS (F) stem cell markers OCT4 and SOX9 and beta actin as loading control. (G) qRT-PCR of stem cell and self-renewal pathway related genes comparing CM and canINS in both adherent and sphere culturing conditions. The mRNA expression of embryonic stem cell genes (SOX9, OCT4, SOX2) and stem cell-associated surface markers (CD133, CD34) were upregulated in sphere culturing conditions. The expression of NOTCH receptor (NOTCH2) and downstream target genes (HES1, HEY1) was upregulated, whereas no significant differences were recorded in NOTCH1, NOTCH3 and NOTCH4 expression in human and canine INS spheres. Values are mean of triplicates ± s.d. The P-values represent the comparison with a stated hypothesis (values >1) using one samples t-test. *P-values <0.05 were considered statistically significant.
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   Figure 2

   Invasive properties of INS CSCs in vitro. (A) Representative images of invasive capacity of human (top row) and canine (bottom row) CSC-enriched spheres and adherent cells using a collagen-based cell invasion assay kit (scale bar: 20 µm) (B and C) Invading cells were stained and quantified by colourimetric measurement at 560 nm. Values are mean of 3 ± s.e.m. *P-value <0.05.
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   Figure 3

   Putative canine and human INS CSCs show an increased in vivo tumourigenic potential (A) Representative photographs of the chorioallantoic membrane (CAM) 11 days after inoculation with either canINS adherent cells or CSC-enriched spheres following red fluorescent membrane labelling. Pictures on the top row show the merging of the brightfield channel; pictures on the bottom row show the red channel. A3 represents a magnified picture of the circles shown in A2. Magnification is specified on top of each picture. (B) Representative photographs of the chorioallantoic membrane (CAM) 11 days after inoculation with either CM adherent cells or CSC-enriched spheres following red membrane labelling. C3 represents magnified pictures of the circles shown in C2. (C and D) Graphs show the differences in fluorescence between the two populations after quantification using ImageJ. Values are mean of 3 ± s.e.m. *P-value <0.05.
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   Figure 4

   Invasive properties of INS CSCs in vivo. (A and F) Representative images of immunohistochemistry of CAM sections embedded in agar and stained with anti-cytokeratin that stains only human and canine cells (brown). The structure of CAM layers is comprised by ectoderm (ET), mesoderm (M) and endoderm (ED). Cancer cell matrigel grafts (CG) were seeded on the CAM. Pictures show the migration of CM adherent (A) and canINS adherent (B) and CM CSC-enriched sphere cells (C) and canINS CSC-enriched sphere cells (D) in the inner part of the CAM 11 days after being seeded. Results show that the CM adherent (A) and the canINS adherent (B) migrate less through the different layers of the CAM compared with the CM CSC-enriched sphere cells (C) and the canINS CSC-enriched sphere cells (D). High magnifications (20× and 60×) shows in details how the CM (E) and canINS (F) CSC-enriched sphere cells disrupt the CAM membrane and invade through the CAM layers. Magnification is specified on top of each picture (scale bar: 200 µm).
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   Figure 5

   Chemosensitivity and colony formation assays of CM and canINS. (A and B) Chemosensitivity assay in CM (A) and canINS (B): cells were treated with increasing concentrations of 5-FU (from 0.5 to 5 μM) comparing the adherent population (dashed line) and the CSC-enriched sphere population (continuous line). (C and D) Colony formation assay CM (C) and canINS (D) Human and canine cells were treated with increasing concentrations of 5-FU (from 0.5 to 5 μM) comparing the adherent population (dashed) and the CSC-enriched sphere population (solid). Values represent mean of triplicates ± s.d. The P-values represent the comparison using 2 sample t-test within the adherent and the CSC-enriched spheres. *P-value <0.05 was considered statistically significant.
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   Figure 6

   Analysis of Notch pathway protein expression and activation in human and canine insulinoma (INS) cells. (A and B) Graph showing the percentage of cells positive to NOTCH2 antibody using flow cytometry in human (A) and canine (B) INS cell lines. (C and D) Western blot analysis of NOTCH2 in its inactive transmembrane form (NOTCH2-TM) and its active intracellular form (NOTCH2-IC), and HES1 with beta actin as a loading control in human (C) and canine (D) INS cell lines, treated with increasing doses of 5-Fluorouracil (5-FU).
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   Figure 7

   Function of the Notch pathway in canine and human insulinoma (INS) cancer stem cells (CSC). (A and B): Cell viability assay of human (A) and canine (B) INS cell lines using increasing concentrations of DAPT comparing adherent cells (dashed line) against CSC-enriched spheres (solid line). (C and D) Western blot analysis of NOTCH2 in its inactive transmembrane form (NOTCH2-TM) and in its active intracellular form (NOTCH2-IC), and HES1, with beta actin as a loading control in human (C) and canine (D) INS cell lines treated with increasing doses of DAPT. (E and F) Colony formation assay of human (E) and canine (F) INS cell lines using a combination of DAPT and 5-fluorouracil (5-FU). Values represent mean of triplicates ± s.d. The P-values represent the comparison using 2 sample t-tests within the adherent and the CSC-enriched spheres. *P-value <0.05. (G and H) Calculation of the synergistic effect of the DAPT and 5-FU using e-bliss calculation in CM (G) and canINS (H). The method compares the observed combined response with the predicted combined response. The combined effect is synergistic as it is greater than the predicted one.
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   Figure 8

   Combined 5-FU and DAPT treatment decreases human and canine INS CSC-like cells tumourigenic potential in the in vivo chorioallantoic membrane (CAM) model. (A) Representative photographs of the CAM 11 days after inoculation with CSC-enriched CM spheres following red membrane labelling. Cells have been treated with 5-FU (5 μM) and DAPT (20 μg/mL). Pictures on the top row show the merging of the brightfield channel; pictures on the bottom row show the red channel (scale bar: 100 μm). (B) Representative photographs of the CAM 11 days after inoculation with CSC-enriched canINS spheres following red membrane labelling. Cells have been treated with 5-FU (0.5 μM) and DAPT (20 μg/mL). Pictures on the top row show the merging of the brightfield channel; pictures on the bottom row show the red channel (scale bar: 100 μm). (C and D) Graphs show the differences in fluorescence between the different conditions after quantification using ImageJ. Values are the mean of 3 ± s.e.m. *P-value <0.05.

• © 2018 Society for Endocrinology