Selective and effective killing of angiogenic vascular endothelial cells and cancer cells by targeting tissue factor using a factor VII-targeted photodynamic therapy for breast cancer

Abstract

The cell surface receptor tissue factor (TF) is regarded as a common but specific target on angiogenic tumor vascular endothelial cells (VECs) and tumor cells in many types of cancer including breast cancer. The purpose of this study is to develop a selective and effective TF-targeting photodynamic therapy (PDT) by using its natural ligand factor VII (fVII)-conjugated Sn(IV) chlorin e6 (SnCe6) for the treatment of breast cancer. A cross linker EDC was used to covalently conjugate fVII protein to SnCe6, and the binding activity and phototoxicity was confirmed by ELISA and in vitro PDT. The efficacy of fVII-tPDT was assessed in vitro by crystal violet staining assay and in vivo by measuring tumor size in mice carrying murine or human breast cancer xenografts. We show that active site-mutated (K341A) fVII protein can be internalized into breast cancer cells and vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs) as angiogenic VECs. fVII-tPDT not only enhances 12-fold the in vitro efficacy but also selectively and effectively kills angiogenic HUVECs and breast cancer cells via specifically binding of fVII to TF and inducing apoptosis and necrosis as the underlying mechanism. Furthermore, fVII-tPDT can significantly inhibit the tumor growth of murine and human breast cancer without obvious toxicities in mice. We conclude that fVII-tPDT using fVII-SnCe6 conjugate can selectively and effectively kill angiogenic VECs and breast cancer cells in vitro and significantly inhibit the tumor growth of murine and human breast cancer in mice.

Fig. 1

The fVII–SnCe6 conjugate retains the same absorption spectrum as free SnCe6 and the same binding activity as unconjugated fVII protein. a Chemical structure of SnCe6. b Absorption spectra of SnCe6 and fVII–SnCe6. The samples in all three panels were diluted 1:50 in ddH₂O to a concentration of 0.6 µM SnCe6. c Binding activity of fVII–SnCe6 and fVII to MDA-MB-231 as determined by cell ELISA. Results in b and c are representative of eleven and two experiments, respectively

Fig. 2

fVII is internalized by TF-expressing VEGF-stimulated angiogenic HUVECs and MDA-MB-231 breast cancer cells. fVII protein primarily bound to the cell plasma membrane (green, arrows in a and d) of the fixed cells (0 min), while endocytosis of fVII, shown as co-localization intracellularly with Alexa Fluor 633-phalloidin-stained cytoskeletal F-actin (red, arrows in b and e), was observed at 37°C for 30 min after incubation with VEGF-stimulated HUVECs (b) and human breast cancer MDA-MB-231 cells (e). c and f Secondary antibody FITC controls for non-specific FITC background. Scale bars, 20 µm

Fig. 3

fVII-targeting enhances the ability of SnCe6 PDT to kill human MD-MBA-231 breast cancer cells in vitro more than 12-fold. The half maximal effective doses (EC₅₀, represented by dashed line) of SnCe6 for MDA-MB-231 cancer cells were shown (a, c for fVII-tPDT; b, d for ntPDT) using crystal violet staining. Equations and R² values were generated using semilog line for a–c and one phase decay for d with Prism 5.0c software. a–d Representative of two experiments

Fig. 4

fVII-tPDT selectively kills breast cancer cells (a, b) and angiogenic HUVECs (d) but has no side effects in normal cells such as 293 cells (c) and unstimulated HUVECs (d). a–c TF expression and selective effect of fVII-tPDT on human breast cancer MDA-MB-231 (a) and MCF-7 (b) cells and normal 293 cells (c). The level of TF expression was determined by flow cytometry using goat anti-HTF antibody (solid line). The control (dashed line) was incubated only with the secondary antibody FITC. The fVII-tPDT (2 µM SnCe6) treatment of these cell lines was performed with various laser fluence values, and efficacy was determined by crystal violet staining. d fVII-tPDT and ntPDT (2 µM SnCe6) treatment of angiogenic (VEGF-stimulated) and quiescent (unstimulated) HUVECs. a–d Representative of two experiments

Fig. 5

The mechanism of action of fVII-tPDT is to induce necrosis and apoptosis, and the effect of fVII-tPDT is specifically mediated by fVII protein. MDA-MB-231 cancer cells were treated separately with fVII-tPDT, ntPDT (2 µM, 36 J/cm²), photosensitizer alone (2 µM) or laser light alone (36 J/cm²). After treatment, the cells were separately assessed for necrosis (a) and for apoptosis (b). After these assays, the MDA-MB-231 cancer cells were stained with crystal violet for the therapeutic effect (c). d 2 µM fVII–SnCe6 was pre-mixed with mfVII protein in various molar ratios prior to incubation with MD-MB-231 cells, and the cells were then treated with fVII-tPDT (36 J/cm²). e MDA-MB-231 cells were treated with BSA-targeted SnCe6 PDT (2 µM, 36 J/cm²)

Fig. 6

fVII-targeted SnCe6 PDT is efficacious for treatment of mouse (a, b) and human (c, d) breast cancer in mouse models. a Mouse EMT6 tumors in Balb/c mice were treated by fVII-tPDT (2 or 4 µM SnCe6, 72 J/cm²) or ntPDT (4 µM SnCe6, 72 J/cm²) on days 0 and 7. b Mouse EMT6 tumor weights from the Balb/c mice (a) on day 12. c Human MDA-MB-231 tumors in SCID mice were treated with fVII-tPDT or ntPDT (2 µM SnCe6, 72 J/cm²) on days 0, 7, 14, 21, and 28. d MDA-MB-231 tumor weights from the SCID mice on day 45. Each group contained five mice, and the data are presented as mean ± S.D. in all panels and analyzed using ANOVA single factor