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. 2010 May 26:10:235.
doi: 10.1186/1471-2407-10-235.

Targeting tissue factor on tumour cells and angiogenic vascular endothelial cells by factor VII-targeted verteporfin photodynamic therapy for breast cancer in vitro and in vivo in mice

Zhiwei Hu  1 , Benqiang Rao, Shimin Chen, Jinzhong Duanmu
Affiliations

Targeting tissue factor on tumour cells and angiogenic vascular endothelial cells by factor VII-targeted verteporfin photodynamic therapy for breast cancer in vitro and in vivo in mice

Zhiwei Hu et al. BMC Cancer. .

Abstract

Background: The objective of this study was to develop a ligand-targeted photodynamic therapy (tPDT) by conjugating factor VII (fVII) protein with photosensitiser verteporfin in order to overcome the poor selectivity and enhance the effect of non-targeted PDT (ntPDT) for cancer. fVII is a natural ligand for receptor tissue factor (TF) with high affinity and specificity. The reason for targeting receptor TF for the development of tPDT is that TF is a common but specific target on angiogenic tumour vascular endothelial cells (VEC) and many types of tumour cells, including solid tumours and leukaemia.

Methods: Murine factor VII protein (mfVII) containing a mutation (Lys341Ala) was covalently conjugated via a cross linker EDC with Veterporfin (VP) that was extracted from liposomal Visudyne, and then free VP was separated by Sephadex G50 spin columns. fVII-tPDT using mfVII-VP conjugate, compared to ntPDT, was tested in vitro for the killing of breast cancer cells and VEGF-stimulated VEC and in vivo for inhibiting the tumour growth of breast tumours in a mouse xenograft model.

Results: We showed that: (i) fVII protein could be conjugated with VP without affecting its binding activity; (ii) fVII-tPDT could selectively kill TF-expressing breast cancer cells and VEGF-stimulated angiogenic HUVECs but had no side effects on non-TF expressing unstimulated HUVEC, CHO-K1 and 293 cells; (iii) fVII targeting enhanced the effect of VP PDT by three to four fold; (iii) fVII-tPDT induced significantly stronger levels of apoptosis and necrosis than ntPDT; and (iv) fVII-tPDT had a significantly stronger effect on inhibiting breast tumour growth in mice than ntPDT.

Conclusions: We conclude that the fVII-targeted VP PDT that we report here is a novel and effective therapeutic with improved selectivity for the treatment of breast cancer. Since TF is expressed on many types of cancer cells including leukaemic cells and selectively on angiogenic tumour VECs, fVII-tPDT could have broad therapeutic applications for other solid cancers and leukaemia.

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Figures

Figure 1
Figure 1
Veterporfin (VP), mfVII protein, and the conjugate. A. Spectra of VP and Visudyne. B. Diagram and molecular weight of mfVII protein determined in SDS-PAGE. fVII, peptide, and His are murine fVII with a mutation of K341A, and S peptide (WT or D14N mutant) and an 8-histidine tag. M represents standard protein markers. C. Spectra of VP, mfVII protein, and Sephadex G50 spin column-purified mfVII-VP conjugate and PBS-VP control. Arrows indicate the Q-band of VP and arrowheads indicate the protein absorbance peak. D and E. The binding activity of VP-conjugated mfVII and unconjugated mfVII to MDA-MB-231 cancer cells determined by flow cytometric analysis (D) and cell ELISA (E). Results in A-D are representative of two or more experiments.
Figure 2
Figure 2
TF is expressed on tumour cells and VEGF-stimulated HUVECs but not on unstimulated HUVECs. A. TF expression was detected on breast cancer cell lines but not on CHO-K1 cells by flow cytometry using mouse Icon (mfVII/hIgG1 Fc) protein. B. Selective expression of TF on VEGF-stimulated HUVECs but not on unstimulated HUVECs was observed and photographed under confocal microscope. HUVECs were stained for TF expression by mouse Icon or anti-HTF antibody (FITC, green) and then by anti-human CD31 PE (red) to verify that TF-expressing cells were of vascular endothelial origin. Results in A and B are representative of two experiments.
Figure 3
Figure 3
fVII targeting improves the selectivity of VP PDT. A. Incubation time of fVII-VP and free VP affected the effect of fVII-tPDT and ntPDT (2 μM, 689 nm laser at 60 J/cm2) on killing MDA-MB-231 cancer cells. However, VP alone without laser irradiation (VP alone) did not have an effect on killing the cancer cells. B. Comparison of fVII-tPDT and ntPDT (689 nm laser at 60 J/cm2) for side effects on non-TF-expressing CHO-K1 cells as a normal cell line. C. VP PDT (fVII-tPDT or ntPDT with 0.5 or 5 μM VP at 60 J/cm2) for the non-TF expressing 293 line as a normal cell line control. D and E. fVII-tPDT and ntPDT (689 nm laser at 36 J/cm2) for HUVEC with or without induction of TF expression by VEGF, as seen in Figure 2B. Results in A-D are representative of two experiments.
Figure 4
Figure 4
fVII targeting enhances the therapeutic effect of VP PDT for human and mouse breast cancer cells. A and B. fVII targeting decreased the EC50 (crossing with dotted lines) concentration of VP in fVII-tPDT by three to four fold for human (MDA-MB-231) (A) and mouse (EMT6) (B) breast cancer lines, compared to ntPDT (689 nm laser for 60 J/cm2). C. fVII targeting also decreased the EC50 (crossing with dotted line) of the laser energy for VP PDT (2 μM VP). D. The effect of fVII-targeted VP PDT (2 μM VP, 689 nm laser for 60 J/cm2) could be inhibited by unconjugated mfVII. Results in A-C are representative of two experiments.
Figure 5
Figure 5
fVII-tPDT induces stronger levels of apoptosis and cytotoxicity (necrosis) than ntPDT in human breast cancer MDA-MB-231 cells. Both fVII-tPDT and ntPDT were carried out at 2 μM VP and 60 J/cm2. A. Apoptosis assay for Caspase 3/7 activity in the PDT-treated cancer cells. B. Cytotoxicity assay for LDH release from PDT-treated cancer cells into the culture medium. C. PDT therapeutic effect assayed by crystal violet staining.
Figure 6
Figure 6
fVII targeting improved the effect of VP PDT for the treatment of murine breast cancer EMT6 in female Balb/C mice. Mouse breast cancer EMT6 cells were subcutaneously injected into female 4-6-week-old Balb/c mice (Taconic Farms Inc) with 1 × 106 (A) or 2 × 106 (B) cells per mouse. When the tumour size reached about 100 mm3, the mice were treated as follows. A. fVII-VP tPDT and ntPDT with 2 μM VP and a 689 nm laser for 105 J/cm2 were carried out on days 0, 3, 8, and 11 (arrows). B. fVII-tPDT with a final concentration of 2 or 4 μM VP in mfVII-VP was carried out with a 689 nm laser at 105 J/cm2 on days 0, 4, 6, 9, 13, and 16 (arrows). Control mice were i.v. injected with PBS buffer.
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