Podoplanin (PDPN) is expressed on many tumors and is involved in tumor metastasis. The objective of the present study was to develop an ELISA for determining soluble PDPN (sPDPN) levels as a potential novel tumor marker in plasma of patients with cancers for detection of tumor occurrence and metastasis. Mouse monoclonal antibodies (mAb) against human PDPN were developed and characterized. Two anti‐PDPN mAb, SZ‐163 and SZ‐168, were used in a sandwich ELISA to detect plasma sPDPN in patients with cancers and in normal individuals. The levels of sPDPN were detected in patients with adenocarcinoma (87 cases, 31.09 ± 5.48 ng/ml), squamous cell carcinoma (86 cases, 6.91 ± 0.59 ng/ml), lung cancer (45 cases, 26.10 ± 7.62 ng/ml), gastric cancer (38 cases, 23.71 ± 6.90 ng/ml) and rectal cancer (27 cases, 32.98 ± 9.88 ng/ml), which were significantly higher than those in normal individuals (99 cases, 1.31 ± 0.13 ng/ml) (P < .0001). Moreover, the sPDPN levels in patients with metastatic cancers were higher (192 cases, 30.35 ± 3.63 ng/ml) than those in non‐metastatic cancer patients (92 cases, 6.28 ± 0.77 ng/ml) (P < .0001). The post‐treatment sPDPN levels of cancer patients (n = 156) (4.47 ± 0.35 ng/ml) were significantly lower compared with those seen pre‐treatment (n = 128) (43.74 ± 4.97 ng/ml) (P < .0001). These results showed that an ELISA method was successfully established for quantitation of plasma sPDPN and plasma sPDPN levels correlate significantly with tumor occurrence and metastasis.

Raised levels of von Willebrand factor (VWF) and reduced levels of a disintegrin and a metalloproteinase with a thrombospondin type I motif, member 13 (ADAMTS13) activity are associated with thrombosis. We aimed to investigate the relationships between plasma levels of VWF and ADAMTS13, their ratios, and the occurrence of cerebral infarction and to understand the roles of VWF and ADAMTS13 in cerebral infarction. Ninety-four patients with cerebral infarction and 103 controls were analyzed. Plasma levels of VWF antigen (VWF: Ag), VWF ristocetin cofactor activity (VWF: Rcof), and VWF collagen binding activity (VWF: CB) were measured by enzyme-linked immunosorbent assay (ELISA). The ADAMTS13 activity (ADAMTS13) was measured with FRETS-VWF73. The relationship between plasma levels and ratios of VWF and ADAMTS13 and the occurrence of cerebral infarction were analyzed. Patients with cerebral infarction displayed higher VWF: Ag and VWF: Rcof levels and lower ADAMTS13, VWF: CB/VWF: Ag, ADAMTS13/VWF: Ag, and ADAMTS13/VWF: Rcof levels compared to controls (P < .01). The highest quartiles of VWF: Ag (odds ratio [OR] = 5.11, 95% confidence interval [CI], 1.49-17.50) and VWF: Rcof (OR = 5.04, 95% CI, 1.62-15.66) and the lowest quartiles of VWF: CB/VWF: Ag (OR = 5.91, 95% CI, 1.95-17.93), ADAMTS13/VWF: Ag (OR = 9.11, 95% CI, 2.49-33.33), and ADAMTS13/VWF: Rcof (OR = 3.73, 95% CI, 1.39-10.03) are associated with cerebral infarction. An association was found between reduced levels of VWF: CB/VWF: Ag, ADAMTS13/VWF: Ag, and ADAMTS13/VWF: Rcof ratios and cerebral infarction. Our data suggest that increased levels of VWF and reduced levels of ADAMTS13 activity may contribute to the pathogenesis of cerebral infarction.

Podoplanin (PDPN), a transmembrane O-glycoprotein, is up-regulated in many tumors and is involved in tumor metastasis and malignant progression. In previous studies, we generated a functional blocking monoclonal antibody (mAb, SZ168) against the extracellular domain of human PDPN. This study is aimed to investigate whether blocking PDPN by SZ168 inhibits tumor growth and metastasis. Malignant melanoma xenograft model by inoculating subcutaneously human malignant melanoma cell line C8161 into the back of BALB/c nude mice was used. Endogenous PDPN expression in C8161 cells and nasopharyngeal cancer cell line CNE-2 was detected using western blot and flow cytometry. SZ168 significantly inhibited C8161 or CNE-2 cell-induced platelet aggregation in a dose-dependent manner with a maximal inhibition of 73.9 ± 3.0% in C8161 cells or 77.1 ± 2.7% in CNE-2 cells. Moreover, SZ168 inhibited the growth and pulmonary metastasis of C8161cells in vivo. The number of lung metastatic foci in the SZ168-treated group was significantly decreased compared with that in the control mouse IgG group (1.61 ± 0.44 vs.3.83 ± 0.60, P < 0.01). Subcutaneous tumor volume, weight, and incidence were also significantly reduced in the SZ168-treated group compared to the control group (P < 0.05). Additionally, SZ168 recognized PDPN in immunohistochemical analyses of tumor tissue sections. SZ168 blocks growth and pulmonary metastasis of human malignant melanoma by inhibiting the interaction between tumor PDPN and platelet CLEC-2 and therefore is a promising antibody for therapeutic development against malignant melanoma.

Background: Podoplanin (PDPN) is a type-1 membrane sialoglycoprotein that is expressed in many cancer tumors including breast cancer; nonetheless, its roles in tumor occurrence, development, and metastasis are unclear. In this study, we aimed to investigate the clinical significance of plasma soluble PDPN (sPDPN) levels in patients with breast cancer and its significance in the diagnosis and metastasis. Materials and Methods: Blood samples from healthy controls (CTL), patients with fibroadenomas of breast (FOB), and breast cancer (pathological type: invasive ductal carcinoma, IDC) were collected. sPDPN levels in the plasma of CTL and patients with FOB and IDC were measured by the ELISA. Results: The plasma sPDPN levels in IDC patients (159 cases, 22.59± 3.70 ng/mL) were higher than those in FOB patients (50 cases, 8.29± 1.09 ng/mL; P< 0.05) and CTL (100 cases, 1.21± 0.12 ng/mL; P< 0.0001). The sPDPN levels in patients at stage III and stage IV (30.08± 4.66 ng/mL) were higher than in patients at stage I and stage II (11.84± 1.12 ng/mL; P=0.005). The sPDPN levels in patients with high-moderate and moderate differentiation (17.50± 3.02 ng/mL) were lower than those in patients with moderately low and low differentiation (35.73± 4.26 ng/mL; P=0.026). The sPDPN levels in patients with metastasis (30.60± 4.27 ng/mL) were much higher than those in patients without metastasis (13.02± 1.30 ng/mL; P=0.017). Conclusion: Plasma sPDPN may be used as a new marker for the determination of the clinical stage, differentiation degree, and metastasis status of breast cancer.

• Immunohistochemistry results directly show VWF is increased during tumor progression. • VWF is expressed as low molecular weight multimer in OS cell line SAOS2. • VWF promotes platelet-induced metastasis of OS through VWF-GPIb pathway.