Clinical Data Analyses,Interaction Mechanisms Of Tumor Microenvironment And Novel Immunotherapy Strategies On Heterogeneity Of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is considered as a malignancy with characteristics of high incidence and low survival rates globally.HCC patients have an insidious onset and long disease course.Early selection of effective treatment options is a key step towards controlling HCC progression.Individualized precision treatment may help HCC patients achieve long-term survival with high quality of life.Liver function assessment is a prerequisite for patients in treatment selection.Currently,Child-Pugh score is the most commonly used indictor for liver function evaluation in clinical practice.Albumin-bilirubin(ALBI)classification has been proposed as a new objective method for assessing liver function,elucidating whether could assess liver function of HCC patients more precisely when compared with Child-Pugh score,is of great significance for treatment decision making and prognosis evaluation.Barcelona Clinic Liver Cancer(BCLC)staging is a widely used HCC diagnosis and treatment guideline.However,in clinical practice,we found that most HCC patients accompanied with abnormal liver function when firstly diagnosed.The recommendation for these patients in BCLC treatment guideline is still not clear.Therefore,it is important to identify HCC patients with abnormal liver function based on objective and accurate liver function indictors and find effective treatment strategies for improving the patients’survival benefit.In addition to liver function,the prognosis of HCC patients is closely related to other factors such as tumor burden and performance status.A large number of clinical real-world studies have shown that treatment beyond guidelines recommend could also bring greater survival benefits to some patients,suggesting the importance of establishing an individualized prognostic model to assist treatment decisions.Currently,there are various treatment methods and emerging treatment concepts related to HCC.However,the idealized prognosis of HCC patients under various treatment methods is far from being reached.The reason for this phenomenon is due to the high heterogeneity among HCC individuals.The tumor microenvironment(TME)is an important factor leading to significant heterogeneity in HCC.The immune suppressive TME formed by the crosstalk between tumor cells and immune cells in the microenvironment is the main reason for poor prognosis in patients.In consequence,exploring the mechanism of tumor cells in shaping the immune suppressive TME of HCC is of great significance for finding diagnostic and therapeutic targets for HCC.The complex and dynamic interaction between tumor cells and TME regulates the occurrence and development of tumors.Glucose and fatty acid,as important components of energy substances,signaling molecules,and cell membrane structure,are crucial for tumor progression.The energy metabolism reprogramming between tumor cells and immune cells plays a crucial role in the formation of immune suppressive TME and malignant biological behavior of tumors.Therefore,targeted glucose and fatty acid metabolism reprogramming is a promising novel strategy for tumor treatment.Objective1.To explore novel methods for liver function evaluation and personalized treatment strategies for HCC patients through clinical retrospective studies.More precisely,to assess the application value of ALBI grading in the evaluation of liver function and prognosis of HCC patients.To compare the safety and efficacy of liver resection(LR)and transarterial chemoembolization(TACE)in HCC patients with ALBI grade 2.To establish a new prognosis model by integrating multiple machine learning methods and recommend the treatment plan with higher survival benefit by the"self-control"method for assisting clinical decision-making.2.To explore new mechanisms of crosstalk between HCC cells and immune microenvironment through basic experimental studies.More precisely,to further explore the causes of HCC immune suppressive TME at the level of molecular,cellular,animal and human clinical samples for clarifying the key molecular interaction networks between HCC cells and immune microenvironment.3.To develop a new strategy of immune remodeling therapy targeting TME through the integration of medicine and industry studies.More precisely,to propose a novel biocatalytic therapy scheme based on nanodrug delivery system focused on HCC energy metabolism reprogramming mechanism.Method1.Independent sample T test,Mann-Whitney U test,χ~2 test or Fisher exact test were used to compare the baseline characteristics of patients.Survival curves were plotted by Kaplan-Meier method,and the difference in survival was compared by Log-rank test.Logistic regression was used to calculate the propensity score(PS)and patients were subjected to inverse probability of treatment weighting(IPTW).Univariate Cox and three multivariate(traditional Cox,PS,and IPTW adjusted)regression models were used to evaluate baseline characteristics and adjusted survival outcomes.Restricted cubic spline(RCS)is used to describe the nonlinear relationship between continuous variables and outcome risk.Individualized prognostic model for HCC patients was constructed by integrating the traditional Cox regression model and five machine learning methods(random survival forest,conditional survival forest,deep neural network,extremely random tree and Gompertz model).The 10-fold cross-validation method is used to optimize the hyperparameters through random grid search and repeated cross-validation.Multiple indicators including time-dependent ROC curve,C-index and Brier score were used to measure the predictive performance of survival prognosis models.2.The sequencing results of human HCC specimens,TCGA database and GEO database were integrated and analyzed to find miRNA related to prognosis of HCC patients.miR-144/451a overexpressed plasmid was constructed and transfected into cells to establish orthotopic HCC model in mouse.The proliferation and apoptosis levels were detected by CCK-8,cell cycle and AV-PI staining.The proportion of immune cells was analyzed by flow cytometry.The expression levels of related molecules were detected by q PCR,Western Blot and immunofluorescence staining.ELISA was used to detect the expression level of cytokines.LDH and NO assay kits were used to evaluate the cytolytic activity of T cells and the polarization phenotype of macrophages.3.Lactate oxidase(LOx)and mononuclear carboxylate transporter 4 inhibitor Syrosingopine(Syro),Etomoxir and Serine palmitoyl transferase(Sptlc2)overexpression plasmids were synthesized by hydrothermal method and biological mineralization method,respectively.The morphological characteristics of USL and ESZ were analyzed by high-resolution transmission electron microscopy.USL and ESZ were characterized by elements mapping,X-ray diffraction,Fourier infrared spectroscopy,UV-VIS spectroscopy,N2adsorption isothermal curve,specific surface area analysis and thermogravimetric curve.The biosafety of USL and ESZ was confirmed by H&E staining,serum biochemical and inflammatory factor detection.The proliferation and apoptosis levels were detected by CCK-8 and AV-PI staining and clonal formation assay.The expression levels of macrophage markers were evaluated by q PCR.The polarization subtypes and ROS levels of macrophages were detected by immunofluorescence staining.Use relevant reagent kits to detect the concentrations of lactic acid and pyruvate,ceramides and fatty acids,JC-1 levels,ATP/ADP and NADPH/NADP ratios.ELISA detects the concentration of cytokines in the cell supernatant or mouse serum.Flow cytometry was used to detect the proportion of immune cells in liver,spleen and blood of mice.The regulatory effects of USL and ESZ on cell metabolism and immune response were analyzed by RNA sequencing.Result1.Child-Pugh score and ALBI grading are independent prognostic factors for HCC patients treated with LR and TACE.Kaplan-Meier curve showed that patients with higher Child-Pugh score and ALBI grade had worse prognosis.ALBI grading could further divide Child-Pugh A5 patients into ALBI grade 1 and ALBI grade 2,while two prognostic cohorts were with significant differences.However,the Child-Pugh score could not further distinguish the patients with ALBI grade 2.ALBI score predicted survival and prognosis of the overall population and subsets after LR and TACE treatment with higher C-index compared with Child-Pugh score.2.In addition to treatment methods,tumor burden,ascites,alpha-fetoprotein,albumin,total bilirubin and physical status score are independent prognostic factors for HCC patients with ALBI grade 2 liver function.Patients treated with LR had lower mortality at 90 days and 6 months than those treated with TACE.Heavy tumor burden,high m ALBI grade and poor performance score significantly increase short-term postoperative mortality.Kaplan-Meier curve shows that compared with TACE treatment,HCC patients in LR group have a better prognosis,with 1-year,3-year and 5-year survival rates of 81.4%,66.9%and 44.3%,respectively.After adjusting for multivariate Cox,PS,and IPTW in the overall population and subsets,LR treatment reduced the risk of death by 2 to 4 times.3.Individualized prognostic model for HCC patients were constructed through integrating the traditional Cox regression model and five machine learning algorithms,which has a C-index greater than 0.8 in both the training set and the validation set,and an area under the ROC curve greater than 0.9 in years 1,3 and 5 years.Compared with BCLC stage,the difference in survival curves between the recommended and unrecommended populations of this model was more significant in the LR or TACE treated cohorts.4.The expression of miR-144/451a in HCC cells/tissues is lower than that in normal cells/tissues.Low expression of miR-144/451a is associated with advanced tumor stage,poor survival prognosis and strong invasion ability.miR-144/451a can significantly promote the polarization of M1-type macrophages and the expression of related markers.miR-144/451a increase CD8~+T cells and decrease the proportion of Treg cells.HGF/MIF are the target of miR-144/451a in HCC,respectively.Moreover,the expression of HGF/MIF level in HCC cells/tissues is increased,which is correlated with the pro-tumor phenotype of immune cells and poor prognosis of HCC patients.5.USL and ESZ,as the nano-catalytic systems,have good stability,biosafety,and tumor targeting properties.USL and ESZ can catalyze the oxidation of lactic acid to pyruvate and the synthesis of ceramides from fatty acids,respectively.After the intervention of USL and ESZ,the tumor proliferation ability was weakened and apoptosis ability was enhanced.USL and ESZ remodeled local and systemic immunity,and significantly increased the proportion of anti-tumor M1 macrophages and cytotoxic CD8~+T cells.In addition,ESZ and USL can cause significant changes in tumor gene expression profiles,with significant enrichment in pathways related to energy metabolism,immune response,and cell death.Conclusion1.Compared to the Child-Pugh score,ALBI grade is more objective,accurate and reproducible for preoperative liver function assessment of HCC patients.ALBI grade is better than Child-Pugh score in predicting the prognosis of HCC patients treated with LR or TACE.2.Compared with TACE,LR has higher safety and greater survival benefit for HCC patients with ALBI grade 2 liver function.3.Individualized prognostic model for HCC patients can predict the prognosis of HCC patients treated by LR or TACE with high efficiency and good fitting.The web-based prediction tool helps the clinical practice of individualized precision therapy for HCC patients.4.miR-144/451a inhibits HCC progression through paracrine HGF/MIF mediated immune microenvironment remodeling,which could be expected to be a novel biomarker for early diagnosis and prognosis of patients.The remodeling of TME provides a new idea for clinical treatment of HCC patients.5.Nano-catalyst USL and ESZ have high biosafety and good tumor targeting.USL and ESZ regulating local and systemic immune responses and inhibiting HCC progression through metabolic reprogramming of lactic acid and fatty acid.Catalytic therapy targeting biological metabolism opens up new strategies for the treatment of tumors and other metabolic diseases.