Targeting tissue factor as a novel therapeutic oncotarget for eradication of cancer stem cells isolated from tumor cell lines, tumor xenografts and patients of breast, lung and ovarian cancer

Abstract

Targeting cancer stem cell (CSC) represents a promising therapeutic approach as it can potentially fight cancer at its root. The challenge is to identify a surface therapeutic oncotarget on CSC. Tissue factor (TF) is known as a common yet specific surface target for cancer cells and tumor neovasculature in several solid cancers. However, it is unknown if TF is expressed by CSCs. Here we demonstrate that TF is constitutively expressed on CD133 positive (CD133+) or CD24-CD44+ CSCs isolated from human cancer cell lines, tumor xenografts from mice and breast tumor tissues from patients. TF-targeted agents, i.e., a factor VII (fVII)-conjugated photosensitizer (fVII-PS for targeted photodynamic therapy) and fVII-IgG1Fc (Immunoconjugate or ICON for immunotherapy), can eradicate CSC via the induction of apoptosis and necrosis and via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, respectively. In conclusion, these results demonstrate that TF is a novel surface therapeutic oncotarget for CSC, in addition to cancer cell TF and tumor angiogenic vascular endothelial TF. Moreover, this research highlights that TF-targeting therapeutics can effectively eradicate CSCs, without drug resistance, isolated from breast, lung and ovarian cancer with potential to translate into other most commonly diagnosed solid cancer, in which TF is also highly expressed.

Keywords: cancer stem cells; solid cancer; targeted immunotherapy; targeted photodynamic therapy; tissue factor.

Figure 1. TF expression in human lung cancer H460 CD133+ CSCs isolated from in vitro cultured cancer cell line

a-d. CD133+ CSCs (a, c) and CD133- non-CSCs (b, d) were immunofluorescently stained for expression of CD133 (red) and TF (green). Their nuclei were stained by DAPI (blue) and the cells were photographed under confocal microscopy (Zeiss). Scale bar: 20 μm.

Figure 2. TF expression on lung cancer H460 CD133+ CSCs isolated from subcutaneous human lung tumor xenografts in mice

a-b. Representative confocal microscopy imaging of co-expression of TF and CD133 on CD133+ CSCs (a) and only TF expression on CD133- non-CSCs (b) isolated from H460 tumor xenografts. c-d. Representative imaging of co-expression of TF and CD44 on CD133+ CSCs (c) and only TF expression on CD133- non-CSCs (d). Scale bar: 20 μm.

Figure 3. TF expression on CD133+ CSCs isolated from OVCAR-5 line, MDA-MB-231 line and from patients' breast tumor tissues

a. Representative imaging of TF expression on CD133+ CSC OVCAR-5 cells. Original magnification: 200 × b. Immunoblotting for TF expression on CD133+ CSCs and CD133- non-CSC MDA-MB-231 cells. CD133 expression was confirmed on CD133+ CSCs and GAPDH was for assessing sample loading. c. Representative imaging of TF expression on breast cancer patients' CD133+ CSCs and CD133- non-CSCs, CD133 expression was confirmed only on CD133+ CSCs, not on CD133- cells (Original magnification: 25 μm under ZEO Fluorescent Cell Imager, Bio-Rad).

Figure 4. TF-targeted PDT with fVII-SnCe6 is effective in eradicating CD133+ CSCs and CD133- non-CSC cancer cells by inducing apoptosis and necrosis

a-b. % survival of CD133+ CSCs, CD133- non-CSC and parental cancer cells H460 (a) and A549 (b) after being treated by fVII-tPDT for 36 J/cm² 635nm laser light, as determined by clonogenic assay. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001 by linear mixed model with t-test. c-d. After fVII-tPDT, the CD133+ H460 CSC cells (c) were stained with Annexin V-FITC and then stained with propidium iodide (PI). Untreated CD133+ H460 CSCs were the control cells (d). The cells were photographed under a fluorescent microscope using green (FITC), red (PI), and phase channels. Original magnification: 200 ×. e-f. Representative imaging of CD133+ CSCs from OVCAR5 line were stained with crystal violet dye after fVII-tPDT (e), and the control CSCs (f) were not treated. Original magnification: 400 ×. Data presented as Mean ± SEM %. The results in a-b were repeated 2-4 times per cancer line for a total of 10 independent experiments. The mechanism studies in c-d were representative from two independent experiments with H460 line and repeated one time in OVCAR5 line. Observations on cellular destruction by fVII-tPDT in e-f were representative from two independent experiments with OVCAR5 and were reproducible in other cancer lines (Supplementary Figure S6).

Figure 5. TF-targeted ICON is effective in mediating ADCC and CDC to kill CSCs and non-CSC cancer cells

a. ICON-dependent NK-mediated ADCC was effective in killing CD133+ CSCs and CD133- non-CSC H460 cells; b. ICON-mediated CDC to CD133+ CSCs and non-CSC H460 cells. c-d. ICON can kill CD24-CD44+ CSCs (c) and CD133+ CSCs (d), as well as non-CSCs (CD24-CD44- in c and CD133- in d), isolated from MDA-MB-231 line separately using CD24- and CD44-based breast cancer stem cell kit and CD133-PE positive selection protocol. e. ICON-mediated ADCC to CSCs (CD133+) and non-CSCs (CD133-) breast cancer cells isolated from breast cancer patients. p values were analyzed by linear mixed model (a, c, d, e) or ANOVA model (b) with t-test. Data presented as Mean ± SEM. Each experiment was repeated 2-3 times.