Elevated tissue factor procoagulant activity in CD133-positive cancer cells

Authors


Janusz Rak, McGill University, Montreal Children’s Hospital Research Institute, Place Toulon, 4060 Ste Catherine West, PT-232, Montreal, Quebec, H3Z 2Z3, Canada.
Tel.: +1 514 412 4400 ext. 22342; fax: +1 514 412-4331; e-mail: janusz.rak@mcgill.ca

Clinical and experimental evidence suggests a parallel between increasing cancer aggressiveness and procoagulant tendencies, which are often attributable to high levels of tissue factor (TF). TF is upregulated on the surface of cancer cells and their derived microvesicles due to a combined impact of oncogenic events and tumor microenvironment [1–4] and thereby becomes involved in cancer progression, both as the principal initiator of the blood coagulation cascade and as a signaling receptor [1,2,5,6]. These TF activities are implicated in tumor growth, angiogenesis and metastasis [1,2,5,6], a finding congruent with the anticancer effects of anticoagulation revealed in recent clinical trials [1,7]. It remains unclear which cancer cells harbor biologically relevant TF.

It has recently come to light that the capacity of cancer cells to initiate tumor growth is not universal, but rather can only be executed by a small minority of specialized cancer cells (fewer than 1%), often referred to as cancer stem cells (CSCs) [8]. Identification of CSCs in several solid tumors has been made possible by the recent discovery of their molecular markers, of which expression of CD133 (prominin-1) represents one of the best-known paradigms [9–12]. CD133 belongs to a family of five-transmembrane cell-surface glycoproteins commonly localized to membrane protrusions of various progenitors [9,13]. While CD133-positive cells have been identified as CSCs in several solid tumors, e.g. of the brain [10], colon [12] and in melanoma [11], it is unclear whether CD133 expression plays a causative, contributive or correlative role in the formation of the CSC population. Still, CD133-expressing CSCs have been implicated in a number of key processes, including tumor repopulation, resistance to therapy [14], increased aggressiveness [10] and angiogenesis [15].

It is intriguing to note that various recent reports point either to cancer cells with increased expression of TF or to cancer cells harboring the CSC marker CD133 as particularly central to cancer progression [4,16]. We reasoned that this may signify a deeper interrelationship between these two properties. In order to examine this in more detail, we tested the expression of CD133 and TF in the highly tumorigenic squamous cell carcinoma cell line A431, which is known to express considerable procoagulant activity [17]. We used Caco-2 cells as a positive control due to their curiously high CD133 expression [coupled with a paradoxically poor tumor-forming capacity and undetectable TF expression (our unpubl. obs.)].

Interestingly, whole cell lysates of the pooled A431 cell population were found to contain appreciable amounts of TF [3], but surprisingly little detectable CD133 (Fig. 1A). However, flow cytometry analysis revealed that A431 are heterogeneous in that a small subset of these cells (0.5%) stained strongly with the CD133 antibody (Fig. 1B). In order to better understand the significance of this heterogeneity, A431 cells were separated immunomagnetically into CD133-positive and CD133-negative fractions (using the CD133 MACS system, Miltenyi Biotech, Auburn, CA, USA), each of which was then tested for TF content. Interestingly, CD133-positive A431 cells expressed a 5- to 6-fold greater amount of TF antigen on their surfaces than their CD133-negative counterparts did (Fig. 1C) [3]. This was paralleled by a corresponding increase in the TF-dependent procoagulant activity (TF-PCA) [3,18] of CD133-positive A431 cells relative to cells lacking this stem cell marker. This latter assay measures the ability of the TF/factor (F) VIIa complex to generate FXa-dependent, prothrombin-activating proteolytic activity [18] on the surface of A431 cancer cells.

Figure 1.

 Procoagulant phenotype of CD133-positive cancer cells. (A) Global Western analysis of CD133 (rabbit polyclonal to C-terminal domain; Abcam, Cambridge, MA, USA) and tissue factor (TF; rabbit antihuman TF, American Diagnostica, Greenwich, CT, USA) expression in A431 squamous cell carcinoma cell line (Caco-2 positive control). A431 cells are TF-high/CD133-low, while Caco-2 cells are TF-low/CD133-high. (B) Detection of high CD133 expression [monoclonal CD133/2 clone (293C3)-PE, Miltenyi Biotech, Auburn, CA, USA] in a small A431 cell subset (numbers indicate % positive cells). (C) High levels of cell surface TF (sheep antihuman TF, Affinity Biologicals, Ancaster, ON, Canada) in the CD133-positive subset of A431 cells [FACS; mean channel fluorescence (MCF)]. (D) Prominent TF-dependent procoagulant activity (TF-PCA) of CD133-positive A431 cells, but not CD133-negative A431 cells. (E) A431 cells (unfractionated) were s.c. injected into immunodeficient (SCID) mice in the presence of the anti-TF neutralizing antibody (CNTO 859) and this was followed by weekly i.p. injections of this agent (200 μg) until tumors reached a volume of 600 mm3. Treatment of tumors with CNTO 859 led to a reduction in tumor growth relative to vehicle-treated controls (day 20 post-tumor-cell injection).

Thus we found that in a subset of A431 cancer cells CD133 is coexpressed with high levels of TF. As the CD133-positive (CSC) cancer cell subset is thought to drive tumor initiation/formation events, we asked whether TF was required for the manifestation of these properties in vivo. Immunodeficient (SCID) mice were injected with A431 cells and treated with CNTO 859, a neutralizing TF-directed antibody that blocks FX activation [19]. Indeed, this therapy resulted in a marked inhibition of tumor growth (Fig. 1E).

In light of our observations, we propose that TF expression (and TF-PCA) probably contributes to the tumor growth-initiating/-regulating properties of CD133-positive CSCs. This could occur through recruitment of growth-promoting effectors of the coagulation system such as thrombin, fibrin and platelets, all of which could provide matrix and growth factor support for the emerging tumor, i.e. could act as a provisional stem cell niche.

Acknowledgements

This project was supported by grants from the National Cancer Institute of Canada and the Canadian Cancer Society to J. Rak, who is a recipient of the NCIC Scientist Award. J. Rak is also a Jack Cole Chair in Pediatric Oncology. We would like to thank our families and our colleagues at the Henderson Research Centre and McGill University for their continued support and encouragement.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest. J. Rak has a consulting relationship with NUVELO Inc. in the area of targeting TF in anticancer therapy.

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