Oncotarget. 2017 Jan 3;8(1):1481-1494. doi: 10.18632/oncotarget.13644.

Angiogenesis. 2017 Feb;20(1):85-96. doi: 10.1007/s10456-016-9530-9.

Tissue factor is an angiogenic specific receptor, a novel oncotarget in cancer stem cells and the target for factor VII-targeted immunotherapy and photodynamic therapy 

Zhiwei Hu

The Ohio State University College of Medicine, The James Comprehensive Cancer Center, Department of Surgery and Division of Surgical Oncology, Columbus OH 43210.

* Correspondence should be addressed to: Dr. Zhiwei Hu, The Ohio State University College of Medicine, Department of Surgery, OSUCCC, Columbus, OH 43210. USA. Phone: 614-685-4606; Fax: 614-685-4606; Email: zhiwei.hu@osumc.edu

 

Tumor microenvironment is composed of several tumor compartments, importantly the cancer cells, tumor neovascular endothelial cells and cancer stem cells. The cancer cells represent the majority of a tumor mass. Tumor neovascular endothelial cells are the inner layer of tumor neovasculature that provides not only nutrients and oxygen for cancer cells to proliferate, but also serve as conduct for cancer cells to metastasize into distant organs and consequently form metastases. Cancer stem cells (CSC) are a small subpopulation of neoplastic cells within a tumor that theoretically possess the capacity to self-renew and develop into the heterogeneous lineages of cancer cells that comprise the tumor (1) and may contribute to tumor angiogenesis, tumor heterogeneity, resistance to multiple therapies (2, 3), recurrence and metastasis (2, 4, 5). Thus, it would be ideal to identify common yet specific surface target molecules and to development of corresponding targeted therapies so that the same targeted therapeutic agent can simultaneously target all three important tumor compartments.

 

Tissue factor (TF) is previously known for overexpression on many types of slid cancer cells and leukemic cancer cells (6) and for selective expression on tumor neovascular endothelial cells (TVEC) in patients’ tumors (7) and in tumor xenografts in mice (8-12), but not on normal resting vascular endothelial cells (VECs). But it was unknown whether cancer stem cells express TF. In the January 2017 issue of Oncotarget, Hu and colleagues (13) reported for the first time 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 (Figure 1). TF-targeted agents, i.e., a factor VII (fVII)-conjugated photosensitizer (fVII-PS for targeted photodynamic therapy, fVII-tPDT) and fVII-IgG1Fc (Immunoconjugate or called an ICON for immunotherapy), can eradicate CSCs without drug resistance (Figure 2) via the induction of apoptosis and necrosis and via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, respectively.

 

 

Figure 1. TF expression in CD133+ cancer stem cells (CSCs) isolated from in vitro cultured human lung cancer H460 cell line (a), human triple-negative breast cancer MDA-MB-231 line (b) and from patients’ breast tumor tissues (c). a: CD133+ CSCs from H460 lung cancer cell line 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. 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). Picture from “Hu et al. Oncotarget. 2017”

 

 

Figure 2. Tissue factor-targeted ICON and fVII-tPDT are effective in eradicating CSCs and non-CSC cancer cells without drug resistance via ADCC and CDC (a-b) or via by inducing apoptosis and necrosis (c-d), respectively. a-b: ICON can kill CD24-CD44+ CSCs (a) and CD133+ CSCs (b), as well as non-CSCs (CD24-CD44- in a and CD133- in b), isolated from MDA-MB-231 line separately using CD24- and CD44-based breast cancer stem cell kit and CD133-PE positive selection protocol. Data presented as Mean ± SEM. Each experiment was repeated 2-3 times. 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 X. e-f: Representative imaging of CD133+ CSCs from OVCAR5 line were stained with crystal violet dye after fVII-tPDT (showing that cancer cells were completed eradicated, only residual membrane remained) (e), and the control CSCs (showing that untreated cancer cells were intact) were not treated (f). Original magnification: 400 X. Picture from “Hu et al. Oncotarget. 2017”

 

 

Figure 3. TF is an angiogenic specific receptor on angiogenic endothelial cells. Representative confocal imaging of TF (green) and endothelial marker CD31 (red) expression on HMVEC before (0 hr) and 4 hrs after VEGF stimulation (4-6 hours reaching peak expression). Cell nuclei were counterstained by DAPI (blue). Scale bars: 20 μm. Picture from “Hu et al. Angiogenesis. 2017”

 

In addition, identification of target molecules specific for angiogenic vascular endothelial cells (VEC), the inner layer of pathological neovasculature, is critical for discovery and development of neovascular-targeting therapy for angiogenesis-dependent human diseases, notably cancer, macular degeneration and endometriosis, in which vascular endothelial growth factor (VEGF) plays a central pathophysiological role. Using VEGF-stimulated VECs isolated from micro-, venous and arterial blood vessels as in vitro angiogenic models and unstimulated VECs as a quiescent VEC model, Hu and colleagues (14) examined the expression of tissue factor (TF), a membrane-bound receptor on the angiogenic VEC models compared with quiescent VEC controls. Hu and colleagues reported in the February 2017 issue of Angiogenesis (14) that TF is specifically expressed on angiogenic VECs in a time-dependent manner in micro-, vein and artery vessels. TF-targeted therapeutic agents, including ICON and fVII-conjugated photosensitizer can selectively bind angiogenic VECs, but not the quiescent VECs. Moreover, fVII-targeted photodynamic therapy can selectively and completely eradicate angiogenic VECs. We conclude that TF is an angiogenic-specific receptor (Figure 3) and the target molecule for fVII-targeted therapeutics (Figure 4). This study supports clinical trials of TF-targeted therapeutics for the treatment of angiogenesis-dependent diseases such as cancer, macular degeneration and endometriosis.

 

 

Figure 4. TF is the target for fVII-targeted ICON. Representative Western blots using mouse ICON (mfVII/hIgG1Fc) and human ICON (hfVII/hIgG1Fc) to immune-precipitate their cognate receptor TF that was detected by monoclonal antibody against human TF (HTF) (clone HTF1). The negative control was the untransfected CHO-K1 cells. Human IgG was an isotype control. Cell lysates were derived from CHO-K1 cells expressing tissue factor (TF), endothelial protein C receptor (EPCR) or both (TF+EPCR). Picture from “Hu et al. Angiogenesis. 2017”

 

 

Figure 5. fVII-tPDT is selective and effective in eradicating angiogenic VEC. a. The fVII-tPDT is effective and selective in killing angiogenic VEC (angiogenic HMVEC) with an EC50 of 0.031 μM SnCe6 in fVII/NLS-SnCe6, whereas it has no side effects to quiescent VEC (HMVEC). The fVII-tPDT conditions were as follows: 635 nm laser light at 36 J/cm2 and the SnCe6 concentrations in the fVII/NLS-SnCe6 conjugate (x axis) were 0.0 (buffer only), 0.5, 1 and 2 μM, respectively. Note that the VEC cells without fVII/NLS-SnCe6 (0.0 μM) also served as the light only control as they were also irradiated with 635 nm laser light (36 J/cm2). b-c. Representative imaging of crystal violet-stained VEGF-stimulated and unstimulated HMVECs right after being treated with fVII-tPDT or ntPDT (2 μM and 635 nm laser light at 36 J/cm2) (b). Control HMVECs include an untreated control and a maximal killing control (completely lysed by 1% Triton X-100) (c). Original magnification: 400 x phase contrast. Picture from “Hu et al. Angiogenesis. 2017”

 

In conclusion, these two recent studies (13, 14) demonstrate that TF is a novel surface therapeutic oncotarget for CSC (Figure 1), an angiogenic-specific receptor (Figure 3) and the target for fVII-targeted immunotherapy and photodynamic therapy (Figures 2, 3 and 5). Moreover, these research highlights that TF-targeting therapeutics can effectively and selectively eradicate angiogenic VECs without harming normal quiescent VECs (Figure 5) and CSCs without drug resistance (Figure 2) (hopefully to overcome CSC’s drug resistance and prevent recurrence and metastasis), in addition to targeting TF-expressing cancer cells. Since Vascular endothelial growth factor (VEGF) can induce TF expression on angiogenic VECs (Figure 3) and plays a central role in angiogenesis-dependent cancer and non-malignant human diseases (15), such as age-related macular degeneration (AMD) (16), rheumatoid arthritis (RA) (17) and endometriosis (18), the findings reported in the February 2017 issue of Angiogenesis may be applied not only to cancer, but also to other angiogenesis-dependent human diseases. Thus, we anticipate that TF-targeted immunotherapy and fVII-tPDT could have broad potential to achieve optimal therapeutic efficacy not only for the treatment of cancer, including solid cancers, acute myeloid leukemia and acute lymphocytic leukemia, in which their cancer cells, tumor neovascular endothelial cells and/or cancer stem cells express TF (6, 7, 13, 14), but also for the treatment of angiogenesis-dependent non-cancerous diseases, notably AMD, RA and endometriosis, etc, in which TF is selectively expressed on in vitro VEGF-stimulated angiogenic VEC models (14) and on their angiogenic VECs in vivo in animal models of AMD (19, 20) and endometriosis (21).

 

Acknowledgments: This work was supported by the Dr. Ralph and Marian Falk Medical Research Trust Awards Programs. The project described was also partly supported by Award Number UL1TR001070 from the National Center for Advancing Translational Sciences through a Phase 1 L-Pilot Award and a voucher award from the Ohio State University Center for Clinical and Translational Science.

 

Conflicts of Interest: Z.H. is co-inventor of the first generation tissue factor-targeting “neovascular-targeted immunoconjugates” (ICON) and is the inventor of a second and a third generation tissue factor-targeting ICONs, named L-ICON1 and L-ICON3 (Patents Pending).

 

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