| Melanoma is a highly aggressive malignant tumor that occurs on the surface of the skin,such as the back,arms and legs.Skin cancers of the patients diagnosed each year,only a small percentage(3%)patients have melanoma,but it results in the majority of deaths(65%).A century ago,melanoma was a rare cancer,but now,the average lifetime risk of melanoma has reached 1 in 50 in many Western countries.Unlike the other solid tumors,melanoma mainly affects young and middle-aged people.It has been observed that the incidence of melanoma increases linearly between the ages of 25 and 50 and then slows down.In recent years,the morbidity and mortality of melanoma are on the rise,and the age of onset is getting earlier and earlier.It has received widespread attention due to the recurrence and metastasis of melanoma after surgical resection.Currently,the treatment of melanoma includes surgery,radiotherapy,chemotherapy and targeted therapy,etc.Although surgical treatment is still the current gold standard,recent advances in immunotherapy and targeted molecular therapy for metastatic melanoma have shown great hope for the development of medical treatment of melanoma.Immunotherapy of tumors,such as immune checkpoint blockade,adoptive immunity,CAR-T,cancer vaccine,etc.,has shown good therapeutic effects in experimental and clinical studies.Among them,melanoma vaccine research has been under discussion and development for many years.The vaccines can activate specific CTL(Cytotoxic T Lymphocyte,Tc cells)and specific antibody in the immunized body,and play a role in inhibiting and killing cancer cells.However,owing to the weak immunogenicity of tumor cells and the complex immune escape mechanism of tumors,the actual effect of vaccines is far from the expected results.In order to improve the immune effect of cancer vaccines and screen effective target antigens,researchers are also making efforts to solve this critical scientific problem.Recent studies have shown that the receptor tyrosine kinase like orphan receptor 1(ROR1)is desirable target point for cancer therapy.Previous work of our group in the ovarian cancer research has found that the ROR1 is an important target antigen,and that the immunogenicity of tumor cell vaccine is associated with the expression of ROR1.However,there is a little of report that the effect of ROR1 molecule on the immunogenicity of melanoma vaccine now.For this reasons,we investigated the melanoma vaccine’s immunogenicity based on the ROR1 molecule,and protective efficacy against melanoma in a murine model.Objective:To investigate the effect of down-regulation of the expression of ROR1 on the anti-tumor effect of mouse melanoma vaccine,and to preliminary study its molecular mechanism,so as to provide experimental basis for validating ROR1 as an effective candidate target antigen.Methods:1.We used the constructed recombinant plasmids of pHBLV-U6-shRORl-ZGreen and pHBLV-U6-shNC-ZGreen,the lentivirus ROR1(pLV-shRORl)in our laboratory to transiently transfect the HEK293T cells together with psPAX2,and pMD2.G plasmid DNA plus Lipofectamine 2000.Forty-eight hours after the co-transfection,the lentivirus-bearing supernatants were collected and the viral titers were determined by multiple dilution method,then B16F10 cells were infected with lentiviruses containing pLV-shRORl and pLV-shNC.The stable down-regulation of ROR1 colonies was screened by limiting dilution method and expanded culture.Then real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blotting were used to detect the expression of ROR1 at the levels of RNA and protein.2.To initially investigate the effects and mechanisms of down-regulated ROR1 expression on the biological characteristics of B16F10 cells.The CCK-8 assay was used to detect the effect on Lv-shROR1-B16F10 cell proliferation.Transwell and Scratch assays were used to detect the ability of cell migration.qRT-PCR was used to detect the mRNA level of indicator molecules for EMT(E-cadherin,N-cadherin,Vimentin).All the assays were carried out in B16F10,Scrambled and Lv-shRORl-B16F10 cells.The 1×106 B16F10 cells,Lv-shROR1-B16F10 cells and Scrambled cells were respectively subcutaneously injected into C57BL/6 mice at left-inguen Groina site.The survival status and the tumor-free survival period,tumor size and body weight change of the mice were monitored.The expression of ROR1 in the melanoma tissue was detected by qRT-PCR and Western Blot,and the expression levels of E-cadherin,N-cadherin and vimentin were detected by qRT-PCR.3.The uninfected B16F10 cells,Lv-shROR1-B16710 cells and Scrambled cells were prepared by freezing and thawing for three times,and then inoculated into C57BL/6 mice(5×105 cells/each mouse)for three times,an interval of 10 days between the immunizations,and then all immunized mice were challenged subcutaneously with 2×106 wild-type B16F10 cells.By observing the tumor-bearing time,tumor volume,survival and body weight changes in the different immunized mice,we preliminarily evaluated the anti-tumor effect of the B16F10 cells,Lv-shROR1-B16F10 cells and Scrambled cell vaccines.The expression of ROR1 at the levels of mRNA and protein in the melanoma tissue was detected by qRT-PCR and Western Blot.The expression of T cell subsets(CD4/CD8)in spleen cells of immunized mice was analyzed by flow cytometry(FCM).In addition,the levels of IFN-γ and TGF-γcytokines of peripheral serum in immunized mice were detected by enzyme-linked immunosorbent assay(ELISA).The killing activity of immunized mice serum on wild type B16F10 cells(complement dependent cytotoxic(CDC))was detected by trypan blue staining.The cytotoxic activity of spleen cells and the killing ability of natural killer(NK)cells were detected by flow cytometry(FCM).Results:1.The recombinant lentivirus plasmid included with down-regulated ROR1 gene was successfully constructed.The lentivirus was packaged and concentrated with infected B16F10 cells,and stable infection clones were screened by a limited the dilution assay.The results of qRT-PCR and Western Blot showed that the expression of ROR1 in the B16F10 cells infected with Lv-shROR1 was significantly decreased than that in the control cells(P<0.05).2.The results of in vitro experiments showed that the cellular proliferation and migration of Lv-shROR1-B16F10 cells were respectively decreased compared with the control cells,and the expression of E-cadherin was increased and the expression of N-cadherin and vimentin was decreased in Lv-shROR1-B16F10 cells analyzed by qRT-PCR(P<0.05).3.The results of in vivo tumorigenesis experiment showed that the tumor growth was slower,tumor size was smaller,and the tumor-free life was longer in Lv-shROR1-B16F10 group than those of B16F10 and Scrambled groups.The results of qRT-PCR and western blotting showed that lower expression levels of ROR1 in the melanoma tissue than that in the control melanoma tissue(P<0.05).The results of qRT-PCR showed that higher levels of E-cadherin,and slower levels of N-cadherin and vimentin were found in the mouse tumor tissue from Lv-shROR1-B16F10 cells vaccination group compared with B16F10 cells and Scrambled cell vaccination groups(P<0.05).4.The B16F10,Scrambled and Lv-shROR1-B16F10 vaccines were developed by application of repeated freezing-thawing method,and then were respectively immunized into C57BL/6 mice.The in vivo tumor vaccine experiment results in the vaccine immunized mice showed that the anti-tumor effects were lower,the tumor grew were more quickly,tumor sizes were bigger and tumor-free survival time were shorter in Lv-shROR1-B16F10 vaccine group than those of control vaccine groups,which was statistically significant(p<0.05).Lv-shRORl-B16F10 vaccine group could decrease the level of serum IFN-γ and attenuate the activities of CDC,NK and Spleen cytotoxic but increase the level of serum TGF-β.Flow cytometry(FCM)analysis showed that the numbers of T lymphocyte subsets(CD4+/CD8+T cells)in spleen cells in the Lv-shROR1-B16F10 immunized mice were lower than that of control mice(p<0.05).The expression levels of ROR,N-cadherin and Vimentin detected by both qRT-PCR and Western blot assays were higher in the melanoma tissue of mice vaccinated with Lv-shROR1-B16F10 vaccine group,while the expression of E-cadherin was lower in the melanoma tissue than in the control melanoma tissue(P<0.05).These results confirmed that ROR1 may be an ideal target antigen for melanoma vaccine and play a key immunogenic role in B16F10 vaccine.Conclusions:1.The ROR1 molecule is clousely associated with the cell proliferation and migration as well as the tumorigenecity of B16F10 melanoma cells.These biological characteristics were weakened by down-regulation of ROR1 molecule.2.B16F10 andScrambled vaccines could induce C57BL/6 mice to produce anti-tumor immunity against melanoma tumor growth.The mechanisms were associated with the strong immune responses induced by high expression of the dominant antigen ROR1.Downregulation of ROR1 molecule can affect the immune responses and anti-melanoma effects of the vaccines.These findings suggested that ROR1 could be used as a potential target antigen for melanoma immunotherapy. |
