| Background Hepatocellular carcinoma(HCC)is one of the most common primary liver tumor.Its morbidity and mortality are one of the highest in the world.Because of the high postsurgical recurrence and/or metastasis,the prognosis of HCC is often poor.Liver NK cells play an important role in immune surveillance,but the depletion and dysfunction of NK cells are closely related to the occurrence and development of HCC.And,with the development of liver cancer,it gradually produce immune resistance to NK cells,resulting in immune escape,more prone to recurrence and metastasis.Lots of studies have shown that epithelial-mesenchymal transition(EMT)plays a major role in HCC progression.MAML3(mastermind-like 3)is a transcriptional regulator in Notch signal transduction and is involved in cell development,proliferation and differentiation.Some studies have shown that MAML3 plays an important role in the metastasis and invasion of cancer cells.However,at present,the role of MAML3 in liver cancer is not known.As one of the most critical molecules in EMT,many studies have found that ZEB1 can promote the development of EMT by inhibiting E-cadheirn protein in a variety of tumors.Purpose We established immune-resistant hepatocellular carcinoma cells(HCC-R)by a unique co-culture and stimulating protocol in vitro,which is NK cells-resistant.We clarify the expression of MAML3 in HCC-R and its mechanism in the metastasis and invasion of HCC through the basic research of molecular biology,so as to provide new ideas for the treatment of HCC after immune resistanceMethods We co-cultured hepatoma cells with NK cells for a long time to induce immune-resistant hepatoma cells(HCC-R),and the tolerance of HCC-R to NK cells was verified by lactate dehydrogenase cytotoxicity test(LDH cytotoxicity assay).At the same time,we studied the changes of proliferation,migration and invasion of HCC-R by CCK-8 and migration and invasion assays.With qPCR and Western Blot,it was found that the expression of MAML3 in HCC-R was significantly increased,and it was related to EMT markers.Finally,we detected the EMT markers of cells after knockout of MAML3 to explore the effect of MAML3 on the invasion and metastasis of HCC-R.Results We successfully constructed two immune-resistant hepatocellular carcinoma cell lines and found that they had stronger metastatic and invasive ability.After knockout of MAML3 protein,the metastatic and invasive ability of immune-resistant hepatocellular carcinoma cell lines was significantly weakenedConclusion For the first time,we constructed immune-resistant hepatocellular carcinoma cell line resistant to NK cells in vitro.We found that MAML3 can enhance its ability of metastasis and invasion in immune-resistant hepatocellular carcinoma cells MAML3 inhibits E-cadherin by promoting the expression of ZEB1,thus promoting the invasion and metastasis of HCC.MAML3 also has the potential to become a new target for the treatment of liver cancerBackground Hepatocellular carcinoma is one of the most malignant tumors and occupies the second place in the death rate in the world.Presently,surgical resection is the main treatment for liver cancer.But some patients are in the middle or late stage when diagnosed,most patients lose the opportunity of operation due to diffusion,metastasis and immune resistance.Even in those who have undergone surgical treatment,recurrence and metastasis of cancer are still the main causes of deathIn recent years,because of its remarkable curative effect,immunotherapy has rapidly developed into one of the new treatments after surgery,radiotherapy and chemotherapy,especially for metastatic tumors.Checkpoint blockade immunotherapy shows great potential in cancer treatment.Photothermal therapy(PTT)can kill tumor by producing high temperature locally by external laser,which avoids systemic toxicity,has little side effects,and achieves local accurate treatment.At the same time,it can activate the immune response of the body and improve the curative effect combining with immune checkpoint blockade therapyWith the integration of nanotechnology and new materials,various types of nano-drug carriers have been constructed into a multi-functional cooperative phototherapy platform,which has brought a new change to the diagnosis and treatment of cancer,including liver cancerPurpose In this study,we propose to construct a multi-functional nano-drug loading system,which can regulate the tumor immune environment while killing tumor cells with PTT,and combine with immune checkpoint blockade therapy to achieve superposition and coordination among different treatment methods,so as to effectively exert the anti-tumor effect of immunityMethods Firstly,MoO2-R837 nanomaterials were prepared,and the microstructure characteristics of the materials were studied by field emission scanning electron microscope(FESEM,Hitachi SU-70)and transmission electron microscope(TEM,Tecnai F20).The structure of the nanomaterials was characterized by X-ray diffraction(XRD,X’pert pro MPD).Immune resistant mouse liver tumor cells were constructed(Hep1-6 R),we verified the effect of MoO2-R837 on 293T,HEP1-6 wt,Hepl-6 R by CCK8 cell proliferation test.All of them are non-toxic and meet the conditions for treatment experiments.Through in vitro experiments,it was proved that PTT triggered by near-infrared(NIR)can kill tumor cells.We also find the combination of PTT and immune checkpoint blockade therapy contributed to the treatment of metastatic tumor,through the construction of in situ tumor model in mice and the establishment of bilateral subcutaneous tumor modelResults On the basis of good experimental results in vitro,we used mouse hepatoma cells Hepl-6 wt,Hepl-6 R in situ tumor model and bilateral subcutaneous tumor model on C57BL/6 to carry out a series of experiments.The study found that the body weight of all mice was maintained at the same level,indicating that MoO2-R837 nanomaterials did not produce significant toxic reactions in vivo.The volume of tumors treated with MoO2-R837 nanomaterials decreased rapidly after PTT treatment.The volume of metastatic tumors also reduced with PD-L1 checkpoint blocking immunotherapy after PTT treatment,and remained at a low level for 14 days,which proved that there was no recurrenceConclusion We found that MoO2 nanoparticles have a high efficiency of photothermal effect,which can directly kill mouse hepatoma cells(Hep1-6 wt)and Hep1-6 R cells by photothermal therapy of(PTT)under 808 nm of near-infrared light Tumor-associated antigens are produced in the process.With the help of immune adjuvant R837,the released tumor-associated antigens can stimulate a strong systemic anti-tumor immune response and promote PD-L1 checkpoints blockade immunotherapy And the synergistic therapy has obvious killing and inhibitory effect on the residual unirradiated distant tumors in mice. |
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