Endothelial cells promote the colorectal cancer stem cell phenotype through a soluble form of Jagged-1

Abstract

We report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive CRC cells colocalized in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC-conditioned medium or blockade of ADAM17 activity. Collectively, ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1.

Figure 1. Endothelial Cells Promote the CSC Phenotype in CRC Cells in vitro

(A) Freshly isolated xenografted human CRC (xhCRC) cells were co-cultured with freshly isolated human liver parenchyma endothelial cells (LPECs) and the xhCRC Aldefluor-positive cell population was determined. (B) Freshly isolated xhCRC cells were co-cultured with freshly isolated LPECs and the xhCRC CD133-positive cell population was determined. (C) Freshly isolated xhCRC cells were co-cultured with freshly isolated LPECs and the xhCRC sphere forming assay was performed. (D) Freshly isolated human CRC (hCRC) cells were treated with LPEC-derived conditioned medium (CM), and the Aldefluor-positive cell population was determined. (E) Freshly isolated human CRC (hCRC) cells were treated with LPEC-derived conditioned medium (CM), and the CD133-positive cell population was determined. (F) Sphere-forming capability in 1°, 2°, and 3° cultures of freshly isolated hCRC cells treated with LPEC CM. (G) xhCRC cells were FAC-sorted by the Aldefluor assay into high, middle, and low cell populations (upper panel). The Aldefluor-high, -middle, and -low cell populations were treated with LPEC CM in parallel (middle panel); the Aldefluor assay was then repeated to determine which ALDH fraction could be enriched for CSCs by EC CM (lower panel). (H) xhCRC cells were FAC-sorted for high, middle, and low CD133 expression (upper panel). The CD133-high, -middle, and -low fractions were treated with LPEC CM in parallel (middle panel); the flow cytometry analysis on CD133 was then repeated to determine which CD133 fraction could be enriched for CSCs by EC CM (lower panel). *p<0.05, mean ± SEM. See also Figure S1.

Figure 2. Endothelial Cells Promote the CSC Phenotype of CRC Cells in vivo

(A) In vivo tumorigenicity assay with limited dilution; HT29 cells pretreated with control CM or LPEC CM. (B) In vivo tumorigenicity assay with limited dilution combined with serial transplantation using freshly isolated xhCRC cells pretreated with control CM or LPEC CM. (C/D) Hepatic metastatic incidence and burden of xhCRC cells pre-treated with control CM or LPEC CM in a splenic injection model (*p<0.05, mean ± SEM). (E) Representative immunofluorescent staining of CD133 and CD31 in human primary CRC surgical specimens (left panel), and human CRC liver metastases (right panel). The highlighted region in the upper panel is enlarged in the lower panel. See also Figure S2.

Figure 3. Endothelial Cell Conditioned Medium Promotes Chemoresistance of Colorectal Cancer Cells

(A) HCT116 cells treated with oxaliplatin and 5-FU simultaneous to the addition of control CM or RF24 CM (MTT assay). (B) Western blot analysis of the expression of pro-apoptotic markers in HCT116 cells treated with oxaliplatin and 5-FU simultaneous to the addition of control CM or RF24 CM. (C) xhCRC cells treated with oxaliplatin and 5-FU simultaneous to the addition of control CM or LPEC CM (MTT assay). (D) Western blot analysis of the expression of pro-apoptotic markers in xhCRC cells treated with 5-FU simultaneous to the addition of control CM or LPEC CM. *p<0.05, mean ± SEM.

Figure 4. Endothelial Cells Activate Notch Signaling in Neighboring Colorectal Cancer Cells

(A) Promoter activity of Hes-1, Gli, and TCF in HT29 cells after treatment with control CM or LPEC CM. (B) Hes-1 promoter activity in freshly isolated xhCRC cells treated with control CM or LPEC CM (*p<0.05, mean ± SEM). (C) NICD and Hes-1 expression of freshly isolated hCRC cells treated with control CM or LPEC CM. (D) Representative immunofluorescent staining for NICD and CD31 in human primary CRCs (left panel) and CRC liver metastases (right panel). The highlighted region in the upper panel is enlarged in the lower panel. (E) Representative immunofluorescent staining of NICD and CD133 in primary CRCs (left panel) and CRC liver metastases (right panel). See also Figure S3.

Figure 5. Endothelial Cells Secrete a Soluble Form of Jagged-1 to Promote the CSC Phenotype in Colorectal Cancer Cells

(A) Western blot analysis of HT29 and LPEC cell lysates and conditioned medium utilizing antibodies to the N-terminus and C-terminus regions of Jagged-1 (JAG1). Western blotting for DLL4 was also performed. (B) Detection of soluble Jagged-1 in the conditioned medium from HT29 cells and LPECs after siRNA-mediated Jagged-1 knockdown. (C) NICD and Hes-1 expression in HT29 cells after treatment with control CM or LPEC CM with decreased Jagged-1 levels by siRNA knockdown. (D) Sphere forming assay of HT29 cells exposed to control CM or LPEC CM with decreased Jagged-1 levels by siRNA knockdown. (E) Sphere forming assay of freshly isolated xhCRC cells exposed to control CM or LPEC CM with decreased Jagged-1 levels by siRNA knockdown. (F) Sphere forming assay of freshly isolated xhCRC cells exposed to the LPEC CM that is Jagged-1 depleted by immunoprecipitation. See also Figure S4.

Figure 6. Proteomics Analysis Demonstrate that the EC-secreted Soluble Form of Jagged-1 is C-terminally Truncated by ADAM17

(A) Concentrated LPEC CM was fractionated by FPLC gel filtration. The fractions were applied to xhCRC cells containing the Hes-1 promoter - luciferase construct, and Hes-1 promoter activity was assessed (upper panel). Western blot detection of soluble Jagged-1 in combined adjacent fractions is shown in the lower panel. (B) Jagged-1 from LPEC CM was immunoprecipitated using an N-terminal antibody and subjected them to deglycosylation followed by digestion. Mass spectrometric analysis of Jagged-1 proteins was performed with multiple peptides that identified consistent with the N-terminal region of Jagged-1 with a C-terminus at amino acid 1054. (C) Mass spectrometric analysis of the ADAM17 cleavage site of Jagged-1. A synthetic peptide corresponding to aa1047–1061 of Jagged-1 was incubated with buffer only (left panel). ADAM17 (middle panel), or ADAM17 with its inhibitor TAPI-2 (right panel), and the reaction products were subjected to mass spectrometry. Peak 1 represents the intact substrate SLIAAVAEVRVQRRP, and Peak 2 represents the ADAM17 cleavage product, a 906.56 Da peptide that was determined to be VRVQRRP. See also S5.

Figure 7. Inhibition of ADAM17 in Endothelial Cells Blocks the Conditioned Medium Promotion of the CSC Phenotype in Human CRC Cells

(A) Western blot detection of ADAM17 in the cell lysate of LPECs or LPECs with decreased ADAM17 by siRNA knockdown (left panel). The soluble form of Jagged-1 in the CM from the cells shown in the left panel, are shown in the right panel. (B) Secretion of the soluble form of Jagged-1 into the CM of HT29 cells and LPECs treated with the ADAM17 inhibitor TAPI-2. (C/D) Sphere forming assays on HT29 cells (Panel C) and freshly isolated xhCRC cells (Panel D) after treatment with CM from LPECs, with or without the ADAM17 inhibitor TAPI-2 (*p<0.05, mean ± SEM). (E) In vivo tumorigenicity assay (day 10) using freshly isolated xhCRC cells co-injected with LPECs with/without daily TAPI-2 treatment (*p<0.05, LPEC/no TAPI-2 vs all other groups). See also S6.

Figure 8. Proposed Model for EC-mediated Paracrine Activation of Notch Signaling in Colorectal Cancer Cells

(A) The conical model for Notch pathway activation, where membrane bound ligands such as Jagged-1 activates Notch signaling in contacting cells. (B) Schematic summarizing our proposed model for paracrine activation of the Notch pathway in CRC cells. ADAM17 cleaves membrane bound Jagged-1 on ECs, releasing an N-terminal soluble fragment that binds, and activates, Notch on CRC cells.