Background
Tumor growth and metastasis are highly complex processes that are affected by a wide variety of factors. Extensive evidence suggests that platelets play a key role in tumor cell proliferation and metastasis [
1,
2]. One of the mechanisms is tumor cell-induced platelet aggregation (TCIPA) [
3,
4], which may enhance embolism in the microvasculature and prevents elimination by host immune system.
Podoplanin (PDPN) is a transmembrane sialo-glycoprotein and its overexpression has been detected in many types of tumors, including squamous cell carcinoma [
5‐
7], malignant mesothelioma [
8,
9], Kaposi’s sarcoma [
10], testicular seminoma [
11], and brain tumors [
12]. Recent studies suggested that the role of PDPN is associated with tumor metastasis, malignancy, and poor prognosis [
13‐
18]. The extracellular domain of PDPN contains a heavily glycosylated amino terminal of approximately 130 amino acids, and conserved amino acid sequence EDXXVTPG is designated as the platelet aggregation stimulating (PLAG) domain [
19]. PDPN is the only known endogenous ligand of the C-type lectin-like receptor 2 (CLEC-2) expressed on platelets [
20]. The binding of tumor cell PDPN to platelet CLEC-2 triggers platelet activation and aggregation [
21,
22]. To date, a number of anti-human PDPN monoclonal antibodies (mAbs) have been established; however, other than the rat anti-hPDPN mAb NZ-1 and a few mAbs that inhibit PDPN-induced platelet aggregation [
23], most fail to block the interaction between PDPN and CLEC-2.
We have produced mAbs (SZ163 and SZ168) against the extracellular domain of human PDPN, and both exhibited high specificity and sensitivity [
24]. An SZ163/SZ168-double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantitate plasma-soluble PDPN in cancer patients and evaluate the correlation between PDPN and tumor occurrence and metastasis [
24], although it is unknown whether SZ163 and SZ168 inhibit the growth and metastases in PDPN-expressing human tumors.
In this study, we showed that SZ168 inhibited platelet aggregation induced by PDPN-expressing human cancer cells in a dose-dependent manner. Furthermore, we found that SZ168 inhibited tumor growth and suppresses pulmonary metastasis in PDPN-expressing tumors in vivo.
Methods
Mice
Female BALB/c nude mice (4–5 weeks old) were purchased from Shanghai SLRC Experimental Animal Co. Ltd. (Shanghai, China) and maintained under specific pathogen-free conditions. Compressed CO2 asphyxiation was used to sacrifice mice in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. All animal procedures were approved by the Animal Use and Ethics Committee of Soochow University (Suzhou, China).
Cell lines
The Chinese hamster ovary (CHO) cell lines, nasopharyngeal carcinoma cells line CNE-2, and C8161 melanoma cell line were purchased from American Type Culture Collection (Gaithersburg, MD, USA). NCI-H226 human non-small cell lung tumor cell line was purchased from Jiangsu KeyGEN BioTECH Co. Ltd. (Nanjing, China). Mycoplasma Stain Assay Kit (Beyotime Institute of Biotechnology, Beijing, China) was used for testing mycoplasma contamination. None of the cell cultures were contaminated with mycoplasma. CHO cells expressing human podoplanin (CHO/hPDPN) were established as described previously [
25]. CHO/hPDPN and C8161 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, Logan, UT, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA). CNE-2 and NCI-H226 cells were cultured in RPMI 1640 medium (HyClone), supplemented with 10% FBS. These cell lines were cultured at 37 °C in a humidified atmosphere of 5% CO
2. All human materials related studies were approved the Ethics Committee of the First Affiliated Hospital of Soochow University.
Antibodies
SZ163 and SZ168, two mouse anti-hPDPN mAbs, were developed as described previously [
24]. A mouse anti-hPDPN mAb (18H5), a normal mouse IgG (ab188776), and a rabbit anti-hPDPN mAb (EPR7072) were purchased from Abcam (Cambridge, UK). A mouse beta-actin antibody was purchased from ProteinTech (Wuhan, China). Fluorescein isothiocyanate-conjugated goat anti-mouse IgG polyclonal antibodies (FITC-GAM IgG) were purchased from Beckman-Coulter (Suzhou, China).
Flow cytometry
Flow cytometry was performed as previously described [
24]. Cultured cells were harvested by brief exposure to trypsin-ethylenediaminetetraacetic acid (EDTA) treatment and then incubated with 18H5 as a positive control, mouse IgG as a negative control, or anti-PDPN antibodies (SZ163 and SZ168, 2 μg) for 30 min at room temperature, followed by FITC-GAM IgG as secondary antibody for 30 min at room temperature. Flow cytometry was performed using a Cytomics FC500 machine (Beckman Coulter, CA, USA).
Western blot analysis
Cells were solubilized using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The lysates were separated with 10% reduced SDS-PAGE and transferred onto a nitrocellulose membrane (Pall Corporation, New York, USA). After blocking with 5% skim milk in 0.1% PBST, the membrane was incubated with SZ163 or SZ168 (3 μg/mL), a rabbit anti-hPDPN mAb (EPR7072; 1:2000), or beta-actin antibody (1:2000) for 2 h at room temperature. Specifically-bound primary antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies (1:10000, Immunotech, Marseille, France; 1:2000, Abcam, Cambridge, UK, respectively) and enhanced chemiluminescence (ECL) substrate (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.
Platelet aggregation assay
Human venous blood was collected from healthy donors in compliance with the Declaration of Helsinki, which was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University. All participants gave written informed consent for this study. Platelet-rich plasma (PRP) was obtained from the whole blood supernatants by centrifugation at 100 x g for 10 min. The cells were harvested, washed, and resuspended in phosphate-buffered saline (PBS; 1 × 107 cells/mL). For the antibody inhibition assay, cells were incubated with different concentrations of SZ163, SZ168, or control mouse IgG for 15 min on ice, followed by addition of 250 μL PRP. Platelet aggregation was measured in a Lumi-Aggregometer Model 700 (Chrono-log, Havertown, PA, USA). Data are provided as means ± SD of three independent experiments.
CHO/hPDPN cells were harvested from culture using trypsin, washed, and resuspended in PBS (1 × 107 cells/mL). The cells were incubated with SZ168 or mouse IgG and inoculated intravenously (1 × 106 cells/mouse) into the lateral tail vein of (4–5 weeks old) female BALB/c nude mice. A total of 36 mice were used for three independent experiments, and each experiment involved 6 mice in the mouse IgG negative control group and 6 mice in the SZ168 treatment group. After 30 d, the mice were euthanized, and the number of lung surface metastatic foci was counted. The lungs and primary tumor tissues were also harvested for hematoxylin and eosin (H&E) staining.
Immunohistochemical (IHC)
All tissue samples were fixed in formalin and embedded in paraffin, and 5-μm sections were cut out. After dewaxing, hydration, and antigen retrieval, sections were incubated with 2 μg/mL SZ168 or 18H5 overnight at 4 °C, followed by treatment with the Envision+ kit (MBX, Fuzhou, China) for 30 min and 3,3-diaminobenzidine tetrahydrochloride (DAB; MBX) for 1 min. Sections were subsequently counterstained with hematoxylin (MBX).
Xenografts
CHO/hPDPN or C8161 cells were trypsinized, washed, and suspended with PBS (5 × 106 cells/mL). The cells were inoculated subcutaneously into the backs of BALB/c nude mice (4–5 weeks old) at a dose of 200 μL per mouse. After 1 d, 30 μL of 1 mg/mL SZ168 or control mouse IgG were intravenously injected once a week for 3 weeks. Thirty-six mice were carried out for three independent experiments at different times, and each experiment was divided into mouse IgG negative control group mice and SZ168 treatment group, 6 mice per group. Tumor volumes were calculated every 3 d from caliper measurements of tumor dimensions using the formula (L × W2)/2, where L is the longer measurement. The mice were euthanized 27 or 30 d after tumor cell implantation. The lung tissues were harvested for H&E staining.
Quantification of growth factors
C8161 cells (2.5 × 105 cells/mL) were incubated with washed platelets (5 × 105 platelets/mL) for 8 h at 37 °C in a 96-well culture plate (n = 6). After centrifuging at 3000 rpm for 5 min, the supernatants of the reaction mixtures were designated C8161-platelet reactants. The quantification of human growth factors, including PDGF and TGF-β-1, in the C8161-platelet reactants was conducted using enzyme-linked immunosorbent assays (ELISAs). All ELISA kits were purchased from R&D Systems (Wiesbaden, Germany) and were used according to the manufacturer’s instructions.
Statistical analysis
Data are presented as the mean ± SD. Mann-Whitney U-test and two-way analysis of variance (ANOVA) were used to determine the statistical significance of the results in the tumor growth and metastasis models in vivo. *P < 0.05 was considered to be statistically significant. All statistical tests were two-sided.
Discussion
Platelets have been reported to be involved in tumor cell-growth, metastasis, and invasiveness [
26‐
28]. The following possible mechanisms have been proposed: (i) tumor cells are coated by platelets in the microvasculature and form large tumor cell-platelet aggregates, (ii) tumor cell-platelet aggregates protect tumor cells by forming a physical shield to protect tumor cells, and (iii) activated platelets release soluble factors that enhance tumor motility [
29]. In addition, the administration of subcutaneous low molecular weight heparin has been found to prolong the survival of patients with advanced cancer [
30]; however, administration of antiplatelet or anticoagulant drugs to patients receiving cancer therapy is risky due to bleeding concerns, particularly in patients’ chemotherapy-induced thrombocytopenia. Thus, it is desirable to have a novel targeted therapy to block platelet-cancer cell interactions.
The interaction between tumor cell PDPN and platelet CLEC-2 drives tyrosine phosphorylation of the Src family kinases (Syk) and phospholipase C gamma 2 (PLCγ2), resulting in platelet activation and aggregation [
22,
31]. Activated platelets release secretory factors, such as transforming growth factor-β, vascular endothelial growth factor A, and platelet-derived growth factor, promoting tumor growth and angiogenesis [
32,
33]. CLEC-2-deficient platelets had normal adhesion and spreading on platelet agonists except for a snake venom protein rhodocytin, indicating that the inhibition of PDPN-CLEC-2 interaction does not interfere with physiological hemostasis [
31]. Thus, platelet-targeted therapy may be useful and biologically safe. PDPN is expressed in many types of tumors, as well as in normal tissues, including lymphatic vessels, type I alveolar epithelium, and kidney podocytes. The interaction between PDPN and CLEC-2 not only promotes cancer cell-induced platelet aggregation but also plays an essential role in physiological processes. PDPN knock-out mice exhibit impaired congenital lymphedema and lymphatic injury patterns [
31]. Since cancer cells may interfere with physiological interaction between PDPN and CLEC-2, targeting the PDPN and CLEC-2 interaction seems to be a reasonable cancer treatment.
SZ163 and SZ168, two mAbs against human PDPN previously produced in our laboratory, showed high reactivity with PDPN-expressing cell lines CHO/hPDPN, U87, and NCI-H226. In the present study, we found that SZ163 and SZ168 specifically recognize endogenous hPDPN in cancer cell lines C8161 and CNE-2 by flow cytometry and western blot. Furthermore, comparison of the reactivity between SZ168 and 18H5 against hPDPN showed that SZ168 may be more sensitive and useful than 18H5 in flow cytometry. Moreover, SZ168 significantly inhibited platelet aggregation induced by C8161, CNE-2, NCI-H226, or CHO/hPDPN in a dose-dependent manner, whereas SZ163 IgG did not. Furthermore, SZ168 suppressed tumor growth and metastasis in CHO/hPDPN and C8161cells in vivo. Both tumor weight and the number of lung micro-metastasis foci were significantly lower in the mice treated with SZ168 than in those treated with control mouse IgG group. It is reported that NZ-1 and MS-1 inhibit PDPN-CLEC-2 interaction in vitro, but whether these antibodies suppress human cancer growth and/or metastasis remains unknown [
23,
34]. We found that SZ168 not only suppresses PDPN-induced platelet aggregation in vitro but also inhibits tumor growth and metastasis in both mouse (CHO/hPDPN) and human (C8161) cancer cells. The experimental results confirmed that the PDGF and TGFβ-1 secreted by platelets can significantly promote the metastasis of tumor cells. Moreover, the migration-promoting effects of PDGF and TGF-β were partially inhibited by the mAb SZ-168. Furthermore, SZ168 may also be useful for diagnostics in immuno-histochemical analysis due to its high sensitivity, similar to another anti-PDPN antibody, D2–40, which has been used as a marker for the diagnosis of many different human tumors by immunohistochemical analysis [
5,
35].
Our data show that the growth of C8161 malignant melanoma is regulated via PDPN-CLEC-2-mediated platelet aggregation. Thus, SZ168 may suppress the growth of malignant melanoma cells in vivo by inhibiting platelet activation and reducing secretion of tumor growth factors. PDPN is up-regulated in skin melanoma, which is an aggressive tumor with an increasing incidence, a high degree of malignancy, and high metastatic rate [
36,
37]. Notably, distant metastases in melanoma patients are associated with a lower five-year survival rate [
38]. Our results indicate that SZ168 may be a promising antibody to be developed as targeted therapy for PDPN-expressing malignant melanoma.
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