Notch1 regulates angio-supportive bone marrow-derived cells in mice: relevance to chemoresistance

Abstract

Host responses to chemotherapy can induce resistance mechanisms that facilitate tumor regrowth. To determine the contribution of bone marrow-derived cells (BMDCs), we exposed tumor-bearing mice to chemotherapeutic agents and evaluated the influx and contribution of a genetically traceable subpopulation of BMDCs (vascular endothelial-cadherin-Cre-enhanced yellow fluorescent protein [VE-Cad-Cre-EYFP]). Treatment of tumor-bearing mice with different chemotherapeutics resulted in a three- to 10-fold increase in the influx of VE-Cad-Cre-EYFP. This enhanced influx was accompanied by a significant increase in angiogenesis. Expression profile analysis revealed a progressive change in the EYFP population with loss of endothelial markers and an increase in mononuclear markers. In the tumor, 2 specific populations of VE-Cad-Cre-EYFP BMDCs were identified: Gr1⁺/CD11b⁺ and Tie2high/platelet endothelial cell adhesion moleculelow cells, both located in perivascular areas. A common signature of the EYFP population that exits the bone marrow is an increase in Notch. Inducible inactivation of Notch in the EYFP⁺ BMDCs impaired homing of these BMDCs to the tumor. Importantly, Notch deletion reduced therapy-enhanced angiogenesis, and was associated with an increased antitumor effect of the chemotherapy. These findings revealed the functional significance of a specific population of supportive BMDCs in response to chemotherapeutics and uncovered a new potential strategy to enhance anticancer therapy.

Figure 1

Contribution of the different VE-cadherin–expressing cells to the tumor vasculature of unperturbed tumors. (A) Immunohistochemical analysis of β-gal expression in bone marrow sections and sections of subcutaneous growing c-neu transformed mammary epithelial tumors in 5 different mouse models is shown: (Aa-b) constitutive VE-cadherin-Cre/R26R mice (VECAD), (Ac-d) bone marrow transplantation of constitutive VECAD mice in lethally irradiated wild-type (WT) C57BL/6, (Ae-f) inducible CIVE mice, (Ag-h) bone marrow transplantation of CIVE mice in lethally irradiated WT C57BL/6 mice, and (Ai-j) bone marrow transplantation of WT C57BL/6 mice into lethally irradiated CIVE mice. (B) The fluorescent versions of the above models with EYFP instead of lacZ expression were used for FACS analysis of subcutaneous growing c-neu transformed mammary epithelial tumors. The graph depicts the percentage of EYFP⁺ cells in the tumors. (C-F) Shown is the immunohistochemical analysis of β-gal (and CD31 expression for [E] and [F]) in the tumors of the constitutive VE-cadherin transplantation into (C-E) WT C57BL/6 mice and (F) the WT C57BL/6 transplantation into CIVE mice. All images were 20× except for (D) (63×) and (F) (40×).

Figure 2

Chemotherapy-enhanced angiogenesis is associated with an influx of BMDCs. (A) A schematic overview of the mouse model is shown. sc, subcutaneous. (B) The graph shows the tumor growth of LLC cells in C57BL/6 mice either untreated or treated with cisplatin or with paclitaxel. (C-D) The graphs show the percentage of PECAM⁺/CD45⁻ cells of the total cells in subcutaneous growing LLC cells in BL/6 mice transplanted with CIVE bone marrow, (C) 1 day or (D) 8 days after the start of treatment. (E-F) The graphs show the percentage of endothelial cells in subcutaneous growing C26 cells in BALB/c mice (E) 1 day or (F) 8 days after the start of treatment. (G-H) The graphs show the percentage of EYFP⁺ cells in LLC tumors in BL/6 mice transplanted with CIVE bone marrow (G) 1 or (H) 8 days after treatment. *P < .05; **P < .01; ***P < .001 compared with the vehicle control.

Figure 3

Influx of 2 subpopulations of VE-cadherin BMDCs. (A) Representative confocal pictures show EYFP⁺ cells in the LLC tumors (blue: TO-PRO, red: PECAM, green: EYFP). (B) Shown are adult bone marrow cells expressing VE-cadherin from 2 distinct populations: an endotheliallike population expressing PECAM and VE-cadherin and a monocytic population expressing Gr1 and CD11b. (C) Cytospins of FACS sorted BMD-EYFP⁺ cells stained for Gr1, PECAM, and VE-cadherin are shown. (D-E) The graphs show the percentage of (D) EYFP⁺/PECAM⁺ or (E) EYFP⁺/PECAM⁺/CD45⁻ cells in subcutaneous growing LLC cells in BL/6 mice transplanted with CIVE bone marrow 8 days after the start of treatment. (F-G) The graphs show the percentage of (F) EYFP⁺/Gr1⁺/CD11b⁺ cells or (G) Gr1⁺/CD11b⁺ cells in subcutaneous growing LLC cells in BL/6 mice transplanted with CIVE bone morrow 1 day after the start of treatment. *P < .05 compared with the vehicle control. *, single green EYFP cells; arrowheads, double-positive cells (EYFP⁺/PECAM⁺). All images were obtained at 20× except for the zoom images; scale bar sizes are indicated.

Figure 4

Notch is upregulated in VE-cadherin–expressing cells that egressed the bone marrow. (A-C) FACS sorting results are shown for 3 populations of cells from (A) the bone marrow, (B) the blood, and (C) the tumor of the CIVE mice after tamoxifen induction. (D) Shown is a schematic overview of the different stages of the EYFP⁺ cells. (E-H) From the bone marrow, a population of EYFP⁻/PECAM⁺ and a population of EYFP⁺/PECAM⁺ were sorted. From the blood, the EYFP⁺/PECAM⁺ population was sorted. RT-PCR was performed for levels of different proteins in the 4 populations: (E) BM EYFP⁻/PECAM⁺, (F) BM EYFP⁺/PECAM⁺, (G) blood EYFP⁺/PECAM⁺, and (H) tumor PECAM+/EYFP+, showing the endothelial markers (gray), Notch1/Dll4 (red), and the macrophage markers (black). eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; ICAM, intercellular adhesion molecule; mRNA, messenger RNA; VCAM, vascular cell adhesion molecule.

Figure 5

The influx of BMD-VE-cadherin/Notch^high cells confers chemoresistance and enhanced angiogenesis. (A) Shown is a schematic overview of the transplantation model. Tumor growth occurred in LLC cells in C57BL/6 mice transplanted with either CIVE bone marrow or CIVE–Notch KO bone marrow; mice were either untreated or treated with cisplatin. (B-C) The graphs show a comparison of the EYFP⁺ cells in (B) the bone marrow (BM) and (C) the blood between the mice transplanted with CIVE or the CIVE–Notch KO bone marrow. (D) The graphs shows a comparison of the tumor growth of LLC cells in C57BL/6 mice transplanted with the CIVE bone marrow vs the CIVE–Notch KO bone marrow, either untreated or treated with cisplatin. (E) Representative confocal pictures show EYFP⁺ cells in the LLC tumors (blue: DAPI, red: PECAM, green: EYFP) in the mice with the CIVE–Notch KO bone marrow (upper panels) and the mice with the CIVE bone marrow (lower panels). (F) Shown is the contribution of EYFP⁺ cells to the LLC tumors in BL/6 mice transplanted with CIVE or CIVE–Notch KO bone marrow 8 days after start treatment. (G) The graph shows the percentage of endothelial cells (PECAM⁺ cells) of the total cells in subcutaneous growing LLC cells in BL/6 mice transplanted with CIVE or CIVE–Notch KO bone marrow 8 days after the start of treatment. *P < .05; **P < .01; ***P < .001 compared with the CIVE vehicle control. The scale bar applies to all images.

Figure 6

Late deletion of Notch, 2 days after tumor cell inoculation, renders the same effect. (A) The schematic overview shows the transplantation model and experimental procedure. (B) Representative confocal pictures show EYFP⁺ cells in the LLC tumors (red: PECAM, green: EYFP) in the mice with the Notch-Cre^−/− bone marrow (left panel) and the mice with the Notch-Cre^+/+ bone marrow (right panel) (both treated with the vehicle control, cisplatin, or paclitaxel). In the table (below), mean (standard deviation) PECAM⁺/CD45⁻ cells per treatment group are shown. (C) The graph shows the contribution of EYFP⁺ cells to the LLC tumors in BL/6 mice, transplanted, respectively, with the Notch-Cre^−/− or the Notch-Cre^+/+ bone marrow, shown by FACS analysis and confocal microscopy. (D) The graph shows a comparison of the tumor growth of LLC cells in C57BL/6 mice transplanted with the CIVE bone marrow vs the CIVE–Notch KO bone marrow, either untreated or treated with cisplatin. *P < .05; **P < .01; ***P < .001. Panel B images were obtained with a 20× objective.