Background
The association between elevated platelet number and malignant tumors was initially reported in 1872 [
1,
2] and has been demonstrated in several common cancers [
3‐
6]. Tumor cells are capable of activating and aggregating platelets to form tumor thrombus—a process referred to as tumor cell-induced platelet aggregation (TCIPA) [
7]. Extensive evidence indicates that the formation of tumor thrombus contributes to critical steps in cancer metastasis, including shielding cancer cells from physiological clearance and immune surveillance and facilitating the migration, invasion, and arrestment of tumor cells within the vasculature [
2,
8]. It is increasingly recognized that the formation of tumor thrombus involving platelets is the first and one of the most important steps in cancer metastasis.
Two important platelet membrane receptors, glycoprotein Ib-IX-V (GPIb-IX-V) and glycoprotein IIb-IIIa (GPIIb-IIIa, also known as integrin α
IIbβ
3), are essential for tumor cell-platelet adhesion and aggregation when tumor cells invade into vasculature [
2]. An increasing number of studies have focused on the role of platelet membrane receptors in tumor metastasis [
7,
9‐
11]. Although it is generally believed that the deficiency of GPIIb-IIIa or blockade of GPIIb-IIIa by monoclonal antibodies may lead to severe bleeding complications [
11], which is the main reason to limit the clinical use of these anti-GPIIb-IIIa agents in cancer therapy, the anti-metastatic agent anti-GPIIIa49-66 scFv Ab A11 that has a slight effect on platelet count and vein bleeding time was found to have therapeutic potential in metastasis [
7,
12]. While the mechanism of GPIIb-IIIa involvement in tumor metastasis is largely clarified, the role of another important adhesion receptor GPIb-IX-V in metastasis remains debatable [
13]. Here, we evaluated the role of GPIb-IX-V and its therapeutic potential in metastasis.
The GPIb-IX-V complex consists of four subunits: GPIbα, GPIbβ, GPIX, and GPV. It interacts with many important extracellular ligands. GPIbα is the largest and most important component of the complex. The N-terminal domain of GPIbα contains the binding sites for several molecules, including vWF [
14], P-selectin (CD62P) [
15], and thrombin [
16], which are essential for primary hemostasis and blood coagulation. The interaction between vWF and GPIbα was found to be particularly critical in the formation of thrombus [
17]. Although there are studies that showed that knocking out the mouse GPIbα or replacing mouse GPIbα extracellular domain could significantly inhibit tumor cell metastasis [
18], the deletion of GPIbα extracellular domain unfortunately induced platelet depletion, leading to severe bleeding complications [
18]. In addition, blockage of GPIbα by monoclonal antibody p0p/B did not have the same influence on tumor metastasis as GPIbα knock out models [
9], raising the concern if GPIbα truly participates in the metastatic process. To address this question, we screened out three anti-GPIbα mAbs with minimal effect on platelet activation as the tools to dissect the therapeutic value of GPIbα in cancer metastasis.
Methods
Materials and animals
Platelet agonist ADP and collagen (equine tendon) were from HELENA laboratories (USA). Ristocetin was from Sigma (R7752, USA). Anti-human GPIbα monoclonal antibody SZ2, VM16d, and AK2 were from GenTex (GTX28822, USA), YO Proteins (656, USA), and Bio-Rad (MCA740T, USA), respectively. Secondary antibody anti-human/mouse CD62P (P-selectin) APC was from Thermo Fisher scientific (17-0626, USA), and FITC-conjugated anti-human PAC-1 was from Biolegend (362803, USA). Peptides of GPIbα fragments were synthesized by GL Biochen (China) Ltd. Recombinant mouse vWF protein was from Creative BioMart (VWF-1432 M, USA), and human vWF protein was from Sino Biological (10973-H08C, USA). C57BL/6J mice, BCLB/C mice, and Wistar rat were from JSJ laboratories (China) and were bred and housed at Putuo animal care facility. Transgenic mice expressing no mouse but only human GPIbα (hTg) were described previously [
19]. Animal experiments were conducted in mice or rats using protocols approved by the IACUC of Putuo Hospital. Six- to eight-week-old mice or rats were used in the study, and investigators were blinded to group allocation during data collection.
Human blood collection
Written, informed consent was obtained from all participants prior to their inclusion in studies. Venous blood was collected from healthy adult volunteers at East China University of Science and Technology, as well as lung cancer patients at Shanghai Pulmonary Hospital. In addition, the use of donor-derived human platelets was approved by IRB in Shanghai Pulmonary Hospital.
Production and characterization of GPIbα mAbs
Rat anti-mouse GPIbα monoclonal antibodies and mouse anti-human GPIbα monoclonal antibodies were prepared according to the methods described by Koehler and Milstein [
20]. Briefly, to develop mouse anti-human antibodies, BALB/C female mouse (6 to 8 weeks) were immunized by four injections of human platelet lysate at a 28-day interval. To develop rat anti-mouse antibodies, Wistar female rats were given an intraperitoneal injection of washed mouse platelets four times at a 20-day interval. Three days after the fourth immunization, mouse or rat splenocytes were fused with Sp2/0-Ag14 myeloma cells and cultured in HAT selection medium. IgGs were purified from hybridoma supernatants using a protein G-Sepharose 4B column (6518-1, Biovision, CA, USA).
The ELISA assay was then used to characterize these antibodies. Ninety-six well microtiter plates were coated overnight at 4 °C with 50 μl of 4 μg/ml anti-human/mouse CD42a Ab (MBS9206081, MyBioSource). After washing three times, the wells were blocked with 2% (w/v) BSA-PBS for 2 h. The wells were then incubated with lysate of human or mouse platelets for 2 h. After that, hybridoma supernatants or purified mAbs were added to the wells and incubated for 60 min. The bound antibodies were detected by HRP-conjugated goat anti-mouse IgG or goat anti-rat IgG. After the background signal had been subtracted, the binding curve was fitted to the eq. Y = Bmax × [ligand]/(Kd + [ligand])
where Y is the specific binding, [ligand] the ligand concentration, Bmax the binding maximum, and Kd the equilibrium dissociation constant.
The specificities of the antibodies were also determined by Western blot as previously described [
21].
Preparation of Fab fragment
The generation of Fab fragment was following previously described protocol with the modification in incubation time with immobilized papain (20341, Thermo Scientific) [
21]. After papain was removed via centrifugation, the generated Fab fragment was purified using Protein A beads (6501-5, Biovision, CA, USA).
Platelet activation
Washed platelets (1.2 × 107 cells/ml) were treated with hybridoma supernatants or purified mAbs at room temperature (RT) for 20 min and detected with FITC or APC-conjugated antibody. Platelet activation induced by tumor cells was by adding 1 × 105 cells/100 μl tumor cells to 1.2 × 106 cells/100 μl washed platelets. When needed, the antibody was added to the platelets and incubated for at least 20 min before their stimulation by tumor cells. The signal of platelet activation was quantitated by the mean fluorescence intensity for the entire cell population (10,000 cells) on a Becton-Dickinson FACS Canto II instrument (BD Biosciences, San Jose, CA, USA).
Platelet aggregometry
Platelet-rich plasma (PRP) was generated as previously described [
21]. The final platelet count in PRP was adjusted to 2.5 × 10
8 cells/ml. Aggregation was initiated in 300 μl of stirred PRP by the addition of noted agonists or 1 × 10
5 cells/50 μl MCF-7 cells to form the mixture of platelets and tumor cells. When required, the antibody was added to PRP and incubated for at least 5 min before stimulation with either agonists or tumor cells. Agonist-induced platelet aggregation was monitored in dual-channel Chrono-Log aggregometer (Havertown, PA, USA)
Assay of tumor cells adhesion to platelets, platelet adhesion to endothelial cells, or tumor cell adhesion to endothelial cells
The adhesion between tumor cells, platelets, and endothelial cells was measured as previously described [
7].
Animal experiments
In the Lewis lung carcinoma (LLC) model, 6-week-old C57BL/6J mice were randomly divided into six groups. There were eight mice per group, and half of the mice are male and half are female in each group. Female and male mice were separately cultured to avoid mating. For groups 1–6, mice were injected with LLC tumor cells (2.5 × 105 cells/mouse) with control IgG, control Fab, 2B4, 1D12, 2B4 Fab, or 1D12 Fab, respectively, at the dose of 50 μg/mouse through the lateral tail vain, along with the tumor cells. After 14 days, the lungs were removed, rinsed with PBS, and the number of metastatic foci on the lung surface was counted. The pulmonary lobes were subsequently kept in 4% paraformaldehyde for later paraffin embedding and hematoxylin and eosin staining. The same experiment was repeated using B16F10 melanoma mouse model (1 × 106 B16F10 cells/mouse).
For spontaneous metastasis, 6-week-old BCLB/C female mice were subcutaneously injected with 1 × 105 4T1 tumor cells. The mice were treated with 50 μg/mouse 2B4, 2B4 Fab, 1D12, or 1D12 Fab, respectively, when the tumor volume reached 80 mm3. After 3 weeks, the mice were killed, and the surface metastatic nodules on the lung were counted and the volume of primary tumor was recorded.
Determination of mouse platelet count
The platelet number was quantitated by a Becton-Dickinson FACS Canto II instrument equipped with BD Trucount Tubes (340334). The subsequent steps were then carried out by following the manufacturers’ instructions.
Bleeding time
The bleeding time was measured as previously described [
22]. Briefly, the mouse tail vein was severed 2 mm from its tip and blotted every 30 s on a circular sheet of filter paper to obtain an objective measurement. Bleeding time was calculated when there was absence of blood on the filter paper. Bleeding time differences were recorded by an unbiased observer and confirmed by two other observers blinded to the experimental status of the mice.
Statistical analysis
Statistical analysis was performed using Prism 6 software. All experiments were carried out at least three times, and the results are presented as the mean ± standard deviation. Statistical significance was assessed by using the one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. P values < 0.05 were considered statistically significant.
Discussion
Multiple studies have shown that the platelet GPIbα is an important receptor in the process of tumor metastasis [
9,
18,
31,
32]. However, the biggest obstacle to use GPIbα inhibition for cancer treatment is potential severe bleeding complications. This is even more concerning when conventional chemotherapy is used since it almost universally affects platelet count. However, several antibodies specifically targeting platelet GPIbα and inhibiting vWF binding to platelet were reported not to influence the platelet count dramatically [
33‐
35]. Nancy et al. investigated the anti-thrombotic effect of human GPIbα mAb 6B4 Fab fragment in vivo and found that through inhibition of the binding of vWF to GPIbα, fewer platelets were activated, resulting in decreased risk of bleeding [
33]. Similarly, the anti-human vWF monoclonal antibody SZ-123 was found to inhibit vWF-collagen and vWF-platelet interactions in vivo and did not significantly prolong bleeding time [
34,
35]. These findings are important considering inhibition of collagen-vWF-GPIbα axis is therefore considered as a new strategy in anti-thrombotic therapy [
29]. Nevertheless, contradictory findings were reported that vWF deficiency could promote tumor metastasis instead [
36]. It remains debatable whether this was due to enhanced platelet GPIbα availability that promoted metastasis in the absence of vWF. If this is the case, then inhibiting the interactions among collagen, vWF, and GPIbα might be valuable. Indeed, in this study, we have confirmed such strategy is useful with minimal effect on platelet number and function.
Compared with traditional anti-platelet drugs, which prevent thrombosis by inhibiting normal platelet function, the monoclonal antibodies developed in this study utilize a different mechanism of action. In our report, we developed two novel rat anti-mouse GPIbα monoclonal antibodies, 2B4 and 1D12, and a mouse anti-human GPIbα monoclonal antibody YQ3. These antibodies exhibited inhibitory potential on cancer metastasis by blocking the vWF-GPIbα axis without affecting platelet activation and hemostatic function. Therefore, we proved that it is possible to use antibodies to inhibit metastasis without inducing thrombocytopenia by blocking the vWF-GPIbα interaction.
Several anti-GPIbα monoclonal antibodies which inhibit vWF binding to platelet have already been developed, these include p0p/B [
9,
28], SZ2 [
23], AK2 [
24], and VM16d [
37]. Luise et al. [
9] investigated the effect of Fab fragment of p0p/B, a mAb directed against the vWF-binding site on mouse GPIbα, on pulmonary metastasis. An unexpected increase in experimental metastasis after blockade of GPIbα was observed. The mechanism of p0p/B promotion on metastasis was thought that the blockade of GPIbα by p0p/B led to the decrease in platelet interaction with P-selectin, which then resulted in increased availability of P-selectin for the direct interaction of cancer cells with endothelium cells. In comparison, in our study, 1D12 and 2B4 were found to inhibit metastasis likely due to their capacity of inhibiting the vWF-GPIbα interaction. Therefore, despite using GPIbα as the same target, using antibodies binding to different sites of GPIbα, hence affecting its binding partners through the resultant spatial conformations, distinct influence on cancer metastasis could be observed.
Interestingly, the binding site of our YQ3 overlaps the previously reported monoclonal antibody SZ2, which binds to vWF-binding site aa 268-282 on mouse GPIbα [
23]. Leslie et al. [
38] reported previously that MCF-7-induced platelet aggregation was inhibited by 46% when tumor cells were pretreated with SZ2. However, SZ2 failed to affect the extent of platelet-LS174T cell hetero-aggregation [
39]. Furthermore, the human GPIbα monoclonal antibody AK2 [
24], which has an overlap-binding site aa 41-55 with YQ3, was not reported on its effect on metastasis. Based on the above reports, we tested the effect of SZ2 and AK2 on platelet-tumor cell binding. SZ2 did not affect the adhesion between patients’ platelets and tumor cells (Additional file
4: Figure S2F). In addition, AK2 also had no effect on the adhesion of platelet to HCT116 cells (data not shown). As spatial conformation is critical for function, for example, normal thrombus formation was performed primarily through tethering of GPIbα to the A1 domain of immobilized vWF
14, different antibodies may exert different effects despite their binding to similar sites (secondary structure) on GPIbα. We therefore propose that YQ3, SZ2, and AK2 may have distinct spatial conformation upon binding to GPIbα. Meanwhile, our data did suggest such possibility: YQ3 not only inhibited vWF binding (platelet aggregation induced by ristocetin) but also platelet aggregation induced by collagen. Similarly, 1D12 and 2B4 also inhibited the aggregation induced by collagen. Therefore, likely a broader effect on collagen-vWF-GPIbα interaction played the role in promoting metastasis, which is different from the mechanism of the other platelet antibodies used in anti-thrombotic therapy. Certainly, more definitive evidence is warranted.
It is interesting that injection of YQ3 full-length antibody to hTg mice induced severe thrombocytopenia similar to the traditional anti-platelet drugs, but injection of Fab fragment alone did not. Such phenomenon might be explained by a recently proposed theory that anti-GPIbα antibodies harboring bivalent structure may exert a pulling force on platelet GPIbα by crosslinking platelets under shear flow [
30]. The bivalent structure of YQ3 full-length antibody could therefore exert pulling force to induce platelet clearing, while the univalent structure of YQ3 Fab will not. The YQ3 Fab could serve as a prototype for further exploration.
Currently, metastasis inhibition potential of anti-human GPIIb-IIIa agents including oral antagonist XV454 [
10], abciximab, tirofiban, and eptifibatide [
40] have been investigated in murine models. However, as the key receptor in the most important and common final pathway of platelet aggregation, blockade of GPIIb-IIIa will likely influence the hemostasis and coagulation. Even though the humanized anti-GPIIIa49-66 scFv Ab A11 demonstrated significant inhibition in metastasis with prolonged bleeding time that could recover in 24 h, the precipitous drop of platelet count by about 70% is concerning [
12]. In addition, cancer metastasis still cannot be maximally inhibited because metastasis can be carried out by the adherence between tumor cells and GPIbα. Novel compounds such as YQ3 need to be pursued.
Various ligands of GPIbα, such as vWF, thrombin [
16], and P-selectin [
15,
41], are all essential for metastasis-promoting activity of platelets, resulting in a complex role of GPIbα in the process of tumor cell metastasis. This study demonstrated that targeting the interaction among collagen, vWF, and GPIbα in cancer therapy could attenuate the metastatic potential of tumor cells. We therefore reinforced the importance of GPIbα in metastasis, as well as the great potential in suppressing metastasis via novel targeting strategies.