| ObjectHepatocellular carcinoma is the fifth most common malignant tumor and the second leading cause of cancer-related death in the world.At present,the treatment of patients with liver cancer mainly relies on surgical resection,chemotherapeutic drug treatment,and radiotherapy.Sorafenib,an oral multikinase inhibitor targeting vascular endothelial growth factor receptor VEGFR,is currently the first-line anti-liver cancer drug in clinical.Sorafenib can normalize tumor neovascularization by inhibiting VEGFR,thereby increasing the infiltration of immune cells into the tumor.However,sorafenib is only beneficial to about 30%of liver cancer patients,and acquired drug resistance usually occurs within half a year of treatment,which severely hinders its clinical efficacy.Natural killer（NK）cells are an important part of the liver’s natural immune system and participate in liver immune defense and pathological processes.Reduced NK cell infiltration and impaired functional activity can often be observed in patients with advanced liver cancer.After ablation or resection of hepatocellular carcinoma,infusion of NK cells which activated by cytokines can improve the survival of patients.The cytokine IL-15 and humanized antibodies targeting NKG2D ligand can restore the function of NK cells in patients with liver cancer.In addition,sorafenib can enhance the killing function of NK cells on tumor cells by activating tumor-associated macrophages（TAM）.There are many mature cell lines of NK cells,including NK-92,NKG,NKL,NK-YS,HANK-1,YTS and YT.The NK-92 cell line is currently the most widely studied cell line among NK cells.It is isolated from male patients with non-Hodgkin’s lymphoma,and its growth depends on IL-2.Compared with primary NK cells,the biggest advantage of the NK-92 cell line is that the expression of inhibitory receptors（such as KIR）on its surface is very low.Which makes its killing ability against many tumors better than primary NK cells or other killer cells activated by cytokines.The NKL cell line is derived from the peripheral blood of patients with CD3-CD16+CD56+large granular lymphocytic leukemia,and its growth is also dependent on IL-2.NKL has natural killing activity and ADCC effect.The killing spectrum of NKL cell line and NK-92 cell line is not completely consistent.Some tumor cells that are not sensitive to NK-92 can be effectively killed by NKL cells,such as human gastric cancer cell line.It can be used as a cell supplement in the cell treatment of tumors,which also makes the NKL cell line an important choice for adoptive immunotherapy.Based on the above studies,we used lentiviral vectors to construct NKL cells overexpressing hIL-15 to improve the killing ability of NKL cells on liver cancer cell lines.The present study aims to observe the effect of the combination therapy on liver cancer cells.Meanwhile,we study the therapeutic mechanism of the combination therapy.To explore the feasibility of combination therapy.Aims1.To evaluate the therapeutic effect of NKL-IL15 cells combined with sorafenib on liver cancer cells;2.To explore the therapeutic mechanism of NKL-IL15 cells combined with sorafenib on liver cancer cells;3.To provide new strategies for clinical liver cancer treatment.Methods1.Amplify hIL-15 fragments from activated human peripheral blood mononuclear cells by RT-PCR technology to construct a lentiviral vector containing hIL-15;2.Infect the NKL cell line with the lentiviral supernatant,and construct the NKL cell line overexpressing hIL-15;3.Detect the expression level of hIL-15 in NKL-IL15 cells by RT-PCR and enzyme-linked immunosorbent assay;4.CCK8 method to detect the effect of hIL-15 gene modification on the killing effect of NKL cells;5.CCK8 method to detect the dose-response curve of sorafenib on liver cancer cells Huh7 and HepG2,calculate the IC50 value,the effect of sorafenib on the proliferation of NKL cells and NKL-IL15 cells,and the killing effect of NKL-IL15 combined with sorafenib on liver cancer cells;6.Liver cancer cell lines were treated with NKL/NKL-IL15 cells or sorafenib alone or their combination.At 24 h after treatment,the activation and killing-related genes of NKL/NKL-IL15 cells were detected by qPCR.Meanwhile,the expression of apoptotic molecules of liver cancer cells such as Mcl-1 and Bcl-2 were detected by qPCR;7.NKL/NKL-IL15 cells were treated with HCC or sorafenib alone or their combination.After treatment,the activation and killing-related genes of NKL/NKL-IL15 cells were detected by FACS;8.Liver cancer cell lines were treated with sorafenib in different concentrations.At 24 h after treatment,the apoptosis protein of liver cancer cell lines were detected by Western Blot;9.We established a subcutaneous tumor-bearing model of BALB/C/Nude mice with human-derived HCC cell line HepG2,sorafenib was injected intraperitoneally,and NKL cells or NKL-IL15 cells were injected via the tail vein for combined therapy.The tumor volume and tumor tissue apoptosis were compared.Infiltration of NKL cells in tumors were detected by FACS.Results1.Construction of hIL-15 gene modified NKL cell lineqPCR and enzyme-linked immunosorbent assay confirmed that NKL-IL15 cells could stably express hIL-15,and the positive rate of NKL-IL15 cells detected by flow cytometry was about 80%.2.The killing function of NKL-IL15 cells on liver cancer cells is enhancedCompared with unmodified NKL cells,the killing effect of NKL-IL15 cells on liver cancer cells such as Huh7 and HepG2 was significantly improved.3.The IC50 value of sorafenib on Huh7 and HepG2 cellsAccording to the dose-response curve of Huh7 and HepG2 cells treated with sorafenib for 48 h,the IC50 values of Huh7 and HepG2 cells were calculated to be 13 μM and 5.9 μM,respectively.4.Sorafenib induces the expression of anti-apoptotic molecules in liver cancer cellsAfter treated with 5 μM and 10 μM sorafenib for 24 h,qPCR results showed that the expression of anti-apoptotic genes Mcl-1 and Bcl-2 in liver cancer cells were up-regulated.5.Low-dose sorafenib has no significant toxicity on NKL/NKL-IL15 cellsNKL and NKL-IL15 cells were treated with 5 μM and 10 μM sorafenib for 24 h,sorafenib did not show significant effect on the viability of NKL cells and NKL-IL15 cells at the dose less than 5 μM.6.NKL-IL15 cells combined with low-dose sorafenib can inhibit the proliferation of HCC cellsAfter treated with 5 μM sorafenib for 48 h,qPCR results showed that the stemness and drug resistance of liver cancer cells were significantly enhanced;CCK8 experiments showed that the combined treatment effect was more effective than NKL/NKL-IL15 cells alone.Compared with NKL cells combination therapy,the killing effect of NKL-IL5 cells combined with sorafenib was more obvious.7.Low-dose sorafenib-treated liver cancer cells enhances the cytotoxicity of effector cells by changing the supernatant compositionLiver cancer cells were treated with low-dose of sorafenib for 24 h,then cultured with fresh medium for 24 h.The liver cancer cells supernatant was collected to incubate NKL/NKL-IL15 cells for 6 h.qPCR and flow cytometry results showed that the activating receptor NKG2D,killing-related molecules perfrion and IFN-y were up-regulated by supernatant from low-dose sorafenib-treated liver cancer cells.8.Low-dose sorafenib enhances the cytotoxicity of effector cells by up-regulating MICA of liver cancer cellsMICA expression was up-regulated in liver cancer cells treated with sorafenib and co-incubated with effector cells.Further experiments confirmed that sorafenib can promote the expression of MICA liver cancer cells by down-regulating the expression of ADAM9,thereby enhancing the sensitivity of liver cancer cells to NKL-IL15 cells.9.NKL-IL15 cell therapy inhibits tumor growth in tumor-bearing miceNKL and NKL-IL15 cells combined with sorafenib significantly inhibited tumor growth in tumor-bearing mice and increased the proportion of NK cells in tumor infiltration sites.Conclusions1.In vitro experiments show that NKL-IL15 cells combined with low-dose of sorafenib can significantly inhibit the proliferation of Huh7 cells and HepG2 cells.This process is related to the effector cell activation and up-regulation of MICA expression in Huh7 cells and HepG2 cells;2.In vivo experiments show that NKL-IL15 cells combined with low-dose of sorafenib can significantly inhibit tumor growth,and compared with NKL cells,NKL-IL15 cells infiltrate the tumor more;3.This study initially explored the effect of NKL-IL15 cells combined with the chemotherapy drug sorafenib in the treatment of liver cancer cells,which provides a new strategy for clinical liver cancer treatment. |
