| Background: Cancer is the second leading cause of death in the world.Cancer is the most deadly disease in both developed and developing countries.In recent years,the overall incidence of malignant tumors in China is still rising,especially in patients with metastasis,and the mortality rate will be significantly increased.In spite of the continuous improvement of modern medical care,there is still no good therapeutic schedule for metastatic patients clinically.Therefore,to clarify the mechanism of tumor metastasis and to intervene becomes the hotspot of global research.Tumor growth and metastasis is a highly complex process involving many molecules and organs and is affected by many factors.At present,many basic and clinical researchers carry out a large number of experimental studies and summarize the relevant experimental data,found that platelets involved in tumor growth and metastasis,and artificially induced thrombocytopenia can inhibit the occurrence of metastasis.Among them,tumor cell-induced platelet aggregation(TCIPA)plays an important role in tumor metastasis.One of the mechanisms is the expression of podoplanin(PDPN)on many tumor cells,through the interaction with platelet C-type lectin-like receptor-2(CLEC-2)to activated platelets.Activated platelets are coated on the surface of tumor cells to form a physical barrier that protects tumor cells from immune system capture and shear stress.It also releases a series of growth factors to promote tumor cells to form new metastases.PDPN,a type-I transmembrane glycoprotein with a molecular weight of 38 k Da,is expressed on a variety of tumor cells,such as lung squamous cell carcinoma,breast cancer,oral cancer,and central nervous system tumors,and is also found on human and mouse malignant melanoma cells.At present,many studies have shown that the expression of PDPN is closely related to the malignancy,invasiveness and metastasis of the tumor.CLEC-2 is a 32 KDa molecular transmembrane glycoprotein with high expression in platelets and megakaryocytes.To date,PDPN is the only endogenous ligand for CLEC-2.The interaction with PDPN activates signaling pathways to promote platelet activation and aggregation.There are many known monoclonal antibodies against PDPN,but most of them do not block the interaction between PDPN and CLEC-2.NZ-1 can inhibit the interaction of PDPN and CLEC-2,suggesting that PDPN can be a new target for the treatment of tumor metastasis.Our laboratory has prepared anti-PDPN monoclonal antibodies SZ163 and SZ168,have been identified its specificity and affinity.Previous experimental studies have shown that SZ163 and SZ168 can specifically interact with CHO/h PDPN,human astrocytic U87 and human lung squamous cell carcinoma NCI-H226 in flow cytometry and Western blot analysis(reported human tumor cells with high PDPN expression).In addition,we used SZ163 as the coated antibody,SZ168 as the enzyme secondary antibody,established a enzyme-linked immunosorbent assay(ELISA)to detect the plasma PDPN levels in tumor patients.Purpose: Our laboratory has successfully established anti-PDPN monoclonal antibodies SZ163 and SZ168,which can specifically recognize human PDPN(h PDPN),but whether the interaction between tumor cell PDPN and platelet CLEC-2 can be blocked remains unclear.The aim of this study is to investigate whether anti-PDPN antibodies can inhibit platelet aggregation induced by human tumor cells in vitro and further investigate whether anti-PDPN antibodies can inhibit tumor growth and metastasis in vivo.The application of two anti-PDPN antibodies has been established ELISA method to detect plasma levels of PDPN in patients with cancer,to assess the effect of PDPN in the development of tumors.Method:(1)The human tumor cells with high PDPN expression were detected with SZ163 and SZ168 by using normal mouse Ig G as a negative control by flow Cytometry and Western Blot.(2)In vitro biological functional studies of anti-PDPN m Abs.Tumor cells highly expressing PDPN were harvested and incubated with platelet-rich plasma(PRP)and the aggregation rate was measured in a lumi-aggregometer.In addition,different concentrations of anti-PDPN antibodies were incubated with tumor cells in advance,and then the treated tumor cells were incubated with PRP to detect the aggregation rate on a platelet lumi-aggregometer.The effect of SZ163 and SZ168 on platelet aggregation induced by tumor cells was observed.(3)In vivo biological functional studies of anti-PDPN m Abs.CHO/h PDPN cells were harvested and incubated with Mouse Ig G or SZ168 Ig G respectively,then injected into tail vein of nude mice.Thirty days later the lung tissues and subcutaneous tumors of the mice were completely excised and retained,weighed and counted.Another CHO/h PDPN and human melanoma cells were harvested respectively,subcutaneously by nude mice,one week after the injection of antibodies(Mouse Ig G or SZ168),once a week for three weeks in a row.C8161 is same as the CHO/h PDPN.After 30 or 27 days,the lung tissue and the subcutaneous tumor of the mouse are completely excised and retained for weighing and counting.(4)Histological analysis of nude mice.The lung and subcutaneous tumors of mice were fixed with 4% paraformaldehyde and then embedded in paraffin for hematoxylin-Eosin staining(HE)and immunohistochemical analysis,IHC).18H5 as a positive antibody,the same sections were incubated with 18H5 and SZ168,respectively,to compare the diagnostic sensitivity of IHC staining.(5)A total of 159 cases of firstly diagnosed breast cancer patients were collected from the First Affiliated Hospital of Soochow University.Fifty patients with breast benign tumor(fibroadenoma of the breast)and 100 healthy controls were collected in the first hospital affiliated to Soochow University.The established ELISA was used to detect the levels of soluble PDPN(s PDPN)in cancer patients.(6)Using SPSS19.0 software for data analysis and statistics and using mean ± standard deviation(x-±s)for date measurement,P <0.05 for the difference was statistically significant.Result:(1)By FCM,human melanoma cell line C8161 and human nasopharyngeal carcinoma cell line CNE-2,were successfully detected.Western blot analysis of cell lysates of C8161 and CNE-2 showed that both SZ163 and SZ168 specifically recognize C8161 and CNE-2(36 or 25 k Da),confirming the high expression of PDPN in C8161 and CNE-2.(2)C8161,CNE-2,CHO/h PDPN and NCI-H226 could induce platelet aggregation in vitro,the maximum aggregation rate of 50-60%.The tumor cells were incubated with antibodies(SZ163,SZ168,Mouse Ig G)and then detect the aggregation rate.The results show that only SZ168 can inhibit tumor cell induced platelet aggregation in a concentration dependent manner,whereas SZ163 could not.The maximal inhibition ratio of SZ168 was 73.9±3.0% in C8161 cells at 20?g/m L of SZ168 and 77.1±2.7% in CNE-2 at 25?g/m L of SZ168.Furthermore,SZ168(15 ?g/m L)also inhibited platelet aggregation induced by CHO/h PDPN and NCI-H226 cells with maximal inhibition ratios of 72.6±3.4% and 74.2±3.1%,respectively.Therefore,SZ168 is an anti-h PDPN m Ab that inhibits platelet aggregation induced by tumor cells that express PDPN.(3)Suppression of experimental pulmonary metastasis by SZ168 in vivo.CHO/h PDPN cells incubated with Mouse Ig G and SZ168 Ig G respectively and injectied to two groups.After injection,30 days after the mice were euthanasia.The CHO/h PDPN cell-induced lung metastasis was nearly inhibited by administration of SZ168,and the number of metastatic foci(32.50±5.70)was significantly lower than that of the control mouse Ig G group(231.10±27.25;P<0.001).Moreover,the number of tumor events in the mouse Ig G-treated group(6.17±0.95)was higher than that of the SZ168-treated group(1.44±0.38;P<0.001).Subcutaneous tumor formation was confirmed by H&E staining,and lung micro-metastasis foci and subcutaneous tumors were confirmed by immunostaining of PDPN using SZ168 and 18H5 antibodies.H&E staining revealed more metastatic foci in the lungs of the mouse Ig G control group than in those of the SZ168-treated group.IHC staining showed that SZ168 and 18H5 can show the positive results,suggesting that SZ168 may also useful in IHC staining.(4)Anti-tumor effect of SZ168 in CHO/h PDPN xenografts.CHO/h PDPN cells were injected subcutaneously into nude mice.Antibody(Mouse Ig G or SZ168 Ig G)was injected through tail vein one week later.The results showed that the tumor of nude mice in Mouse Ig G group was higher than that in SZ168 Ig G group.The tumor weight of SZ168 Ig G group(0.09 ± 0.04g)was significantly lower than that of Mouse Ig G group(0.63±0.16 g),(P <0.01).In addition,the tumor volume of SZ168 Ig G group was lower than that of Mouse Ig G group(P <0.05).(5)Anti-tumor effect of SZ168 on primary tumor growth and spontaneous pulmonary metastasis of C8161 xenografts.C8161 cells were used in the established tumor model,as described above.The results showed that SZ168 Ig G mice weight,tumor volume,tumor formation rate were significantly reduced(P < 0.05),the difference was statistically significant.In the anatomy of the lung surface nodules under the microscope counting.The results showed that the number of nodules of Mouse Ig G group in the lung surface was more than SZ168 Ig G group [(3.83 ±0.60)vs(1.61 ± 0.44),P < 0.01].Furthermore,the lung tissue paraffin embedded tissue sections after HE and IHC staining,the results obtained are similar with CHO/h PDPN orthotopic xenografts model.(8)The ELISA method established by SZ163 and SZ168 was used to detect the level of s PDPN in the plasma of breast cancer patients.The results showed that the s PDPN of breast cancer was higher than that of the breast fibroadenoma and healthy controls.159 patients with breast cancer were classified according to age,clinical stage,degree of differentiation,tumor size,etc.The results showed that plasma levels of s PDPN in patients with stage III and IV were higher than those in patients with stage I and II[(30.08 ± 4.66)vs(11.84±1.12),P=0.005];patients with metastasis were significantly higher than those without metastasis[(30.60±4.27)vs(13.02±1.30),P=0.017];patients with(high-moderately differentiated + moderately differentiated)patients had lower s PDPN than those with(moderately-poorly differentiated + poorly differentiated)[(17.50±3.02)vs(35.73±4.26),P =0.026]?(6)s PDPN as a new tumor marker,with other commonly tumor markers CA125,CA153 and CEA compared the AUC in the diagnosis of breast cancer.Results showed that the area under the curve was 0.961,0.741,0.860,0.716,and showed that the diagnostic efficiency of s PDPN is the highest,its sensitivity and specificity is the highest.The Logistic regression analysis results showed that after adjusting for confounding factors,the s PDPN value of OR was higher than CA153,suggesting that exposure to the risk factors in the proportion was 4.024 times of the control.Conclusion:(1)Human cancer cell lines C8161 and CNE-2 are confirmed with high PDPN expressing by FCM and Western blot.(2)SZ168 can inhibit human platelet aggregation induced by tumor cells in vitro in a dose-dependent manner.(3)SZ168 not only inhibited tumor growth and metastasis of CHO/h PDPN,but also inhibited tumor growth and metastasis of human tumor cell line C8161,which endogenous expresses PDPN in vivo experiments.These results suggested that SZ168 is expected to become a novel antibody-dependent targeted therapy for tumors that expressing PDPN,such as human malignant melanoma.(4)The s PDPN has a certain reference value of patients with breast cancer staging,differentiation,metastasis and other clinical state.(5)The s PDPN has a certain clinical value in the diagnosis of breast cancer. |
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