Study On The Effect And Mechanism Of Herceptin-armed Nk Cells To Breast Cancer Cells In Vitro And In Vivo

Background:Nowadays,breast cancer becomes the most common type of cancer among women worldwide.The higher expression of human epidermal growth factor receptor-2?HER2?on cancer cells,the poorer prognoses of the patients have.Experienced Herceptin is a humanized anti-human epidermal growth factor receptor-2?HER2?monoclonal antibody and has been commonly used as first-line drug to treat positive HER2 positive breast carcinoma in clinic.It co?ld increase clinical response rates and prolong the time of tumor progression and the survival time of patients when combining with chemotherapy.However,Herceptin is limited for sever intolerance cardiac cytotoxicity and drug resistance.Natural killer?NK?cell therapy has gradually become the most promising clinical treatment option and it is an emerging approach for patients with a wide range of tumors.Our previous studies have shown that interleukin?IL?-2 and IL-15 co?ld successf?lly amplify NK cells but less increase in cytotoxicity.Recently,some studies implied Herceptin co?ld stim?late the cytotoxicity of lymphocytes in vitro.Object:Our study focused on Hercpetin's effects on the amplification,cytotoxicity,migration and adhesion of NK cells in vitro and further investigate the feasibility of HA-NKs?Herceptin-armed NK cells?in clinical therapy.Methods:We collected 20 enriched blood samples of breast cancer patients and isolated peripheral blood mononuclear cells?PBMCs?.Then we amplified the NK cells for 15 days in the flasks coated with or without Herceptin.Human immunoglob?lin G1 protein?IgG1?was used as isotype control group at present of IL-2 and IL-15.At day 15,we enriched NK cells used MACS MicroBeads.At Day 0and Day 15,we counted the total cell numbers of the final products.At Day 0,Day 5,Day 10 and Day 15,we used flow cytometry to examine the percentage of NK cells,especially the percentage of CD56^dimNK cells.We co-c?ltured NK cells with HER2positive or negative breast cancer cells to evaluate the NK cell-mediated cytotoxicity,including the levels of lactate dehydrogenase?LDH?release level in supernatants,the surface expression CD107a and secretion of interferon?IFN?-?.Chemokine chip were used to detect the secretion of m?ltiple chemokines in the supernatants.Living cell image station was applied to observe the migration and adhesion ability of different groups of NK cells co-c?lturing with HER2 positive or negative breast cancer cells.At Day 0,Day 5,Day 10 and Day 15,we examined the expression level of NKp30,NKp44,NKp46,NKG2D,DNAM-1,CD16 and Herceptin on the surface of NK cells via flow cytometry.Western blotting?WB?assay was used to investigate the expression and phosphorylation of proteins on the CD16 downstream signal pathway,such as PI3K,VAV,RAC,and ERK.At last,we conducted a pilot study on six patients using Herceptin-armed NKs?HA-NKs?to observe the feasiblility of HA-NKs in clinic.Results:After 15 days,total cell number in HA-NK group amplified?29.26±19.38?times as much as Day 0,which displayed no significant difference compared to either blank control or isotype control group.But the percentage and number of NK cells in Herceptin group?65.71±16.72%,29.26±19.38×10⁷?were higher than the blank control group?43±13.66%.18.57±14.99×107,p<0.0001?and the isotype control group?55.47±12.75%,22.22+19.47×107,p<0.0001?.We also found in final products of HA-NKs,the CD3^-CD56^dimim cells were expanded?5±1.12?times as much as Day 0,significantly in higher folds than the blank control group?1.61±0.43,p=0.01?and isotype control group?2.47±1.15,p=0.02?.In LDH assays,the cytotoxicity of Herceptin-armed NKs co-c?ltured with HER2 positive breast cancer cells was?60.00±20.10%?,which was significantly higher than the isotype control group?41.15±14.40%,p<0.0001?and blank control group?38.95±14.10%,p<0.0001?.However,when the target cells were HER2 negative,similar cytotoxicity was achieved among 3 groups,which was?43.10±24.14%?,?28.65±9.56%,p=0.18?and?35.11±21.90%,p=0.41?.Same res?lts obtained researching to expression level of CD107a and the secretion of IFN-?which implied higher cytotoxicity of HA-NK in vitro compared to the other 2 groups.When co-c?ltured with breast cancer cells,Herceptin-armed NKs transformed and moved more frequently and quickly than NK cells of isotype and blank control group.The number of Herceptin-armed NKs adhered to tumor cells increased dramatically.When the breast cancer cells were HER2 negative,those phenomena were not found.Protein chip analysis showed that the levels of various chemokines in the supernatants secretion,such as monocyte chemotactic protein?MCP?.The expression level of cell surface activated receptors on NK cells,such as NKp30,NKp44,NKp46,DNAM-1 and NKG2D displayed no significant difference compared with blank control group.But the expression level of CD16 in HA-NKs at Day 15 was?33.01±15.93%?,significantly lower than the blank control group?66.56±11.79%,p=0.0046?,but more Herceptin left on NK cell surface was indentified that implied a long-term occupation of CD16 by Herceptin.WB res?lts showed that Herceptin co?ld increase the phosphorylation levels of m?ltiple the CD16 downstream proteins,such as PI-3K,VAV-RAC and ERK.After repeat stim?lation of Herceptin,the phosphorylation levels of above proteins increased dramatically which implied a continuous activation of CD16 down-stream signal pathway upon Herceptin stim?lation.Finally,HA-NKs were amplified and infused into 6 patients for pilot study,in which one patient achieved PR.Conclusion:HA-NKs showed comparably higher proliferation,cytotoxicity migration and adhesion either by cytokine or IgG1 isotype induced NK cells.Herceptin amplified specially CD3^-CD56^dimim subset in NKs that showed high cytotoxicity.These significant increases of cytotoxicity and migration exclusively correlated with longer term of occupation of CD16 rather than other activated surface receptors.The CD16-downstream proteins,PI-3K,VAV-RAC and ERK were intensively activated.More importantly,CD16 occupied by Herceptin enhanced migration and adhesive functions of NK cells which dramatically promoted anti-tumor cytotoxicity of Herceptin-armed NKs both in vitro and in vivo.HA-NKs were toleranted well in clinic that might be a feasible immunotherapy strategy for HER2 positive breast cancer patients in future.