Study On Mechanism Of Primary Resistance To Sorafenib Mediated By FcRn In Hepatocellular Carcinoma

The tendency of personalized medicine is proposed a new research direction for the development of pharmaceutical analysis.It is no longer limited to chemical examination and quality control of manufacturing and storage.Increased attention has been paid to providing precise medicine according to the diverse and complex status of patients.Primary resistance is an important basis for clinical drug selection.The response of different patients to treatment drugs varies greatly,and some patients will not benefit from the drugs.This phenomenon is particularly prominent in the selection of anti-tumor agents.Hepatocellular carcinoma(HCC)is the sixth most common cancer and the second leading cause of cancer-related mortality worldwide.The incidence of this deadly disease is increasing year by year.Currently,such as resection,liver transplantation and ablation are only feasible in patients with early-stage HCC.For patients with advanced HCC,there is still a lack of optimal treatment options.Sorafenib is a first-line drug approved by the Food and Drug Administration(FDA)for advanced HCC.Sorafenib can prolong the median survival time of patients with advanced HCC effectively.However,many patients were found to develop primary resistance or acquired resistance to sorafenib during treatment,which reduced the therapeutic effect of sorafenib in hepatocellular carcinoma.Primary resistance refers to patients who have no beneficial response to the drug at the beginning of treatment.Sorafenib is unable to inhibit tumor growth,leading to distant metastasis during treatment.Acquired resistance refers to the patient's sensitivity to sorafenib gradually decreased with increasing duration of treatment.At present,the mechanism of acquired resistance to sorafenib is relatively clear,such as crosstalk between PI3 K Akt and JAK-STAT pathways,the hypoxic tumor microenvironment.As the mechanism of primary resistance to sorafenib has not been elucidated,patients with advanced HCC cannot obtain accurate clinical medication guidance,leading to missed optimal treatment timing.This is one of the major challenges of precision medicine in HCC-related clinical pharmacy.There are two main reasons for the difficulty in researching the mechanism of primary resistance to sorafenib.1.Tumor tissue samples from patients with advanced HCC are difficult to obtain.So far,there is no tumor marker that can be used as a reference for primary resistance to sorafenib in HCC.The patients who needed to take sorafenib for treatment had already developed distal metastasis in the advanced stages of HCC.Most of patients are not consented for liver puncture.And it is not ethical to perform surgical resection for patients with advanced HCC.Therefore,it is difficult to obtain patient tumor tissue samples for the study of primary resistance.2.Lack of suitable methods for the spatial heterogeneity of HCC complex tissue samples.The study of primary resistance to sorafenib requires extensive bioinformatic data of HCC cells as a basis.HCC tissues include not only malignant and transformed cells,but also many different types of cells recruited from around tissues and the immune system.Only an average qualitative characterization of this highly spatial complex tissue can be achieved using traditional bulk sequencing techniques.It may ignore the unique phenotypic and functional characteristics of individual cells in tumor samples.The result of sequencing analysis is quite different from that of real tumor cells.To solve the above two difficulties in the study of the primary resistance mechanism of sorafenib,in chapter 2 of this thesis,Multiple groups of patient-derived tumor xenograft(PDX)models were constructed using tumor tissues from HCC patients who had not been treated with sorafenib as the source of tumor tissue samples.Compared with the traditional cell line transplantation model,the tumor tissue of the PDX model has not been cultured in vitro.It maintains the genetic characteristics,heterogeneity of the primary tumor and better clinical predictability.It could solve the problem that the tumor tissue samples of patients with advanced HCC were difficult to obtain.The PDX model was screened for efficacy assay,and the PDX models of the sorafenib primary resistance group and the sorafenib sensitive group were identified.In Chapter 3,single cell RNA sequencing(scRNA-seq)technology was used to sequence and characterize complex PDX tumor tissue samples.ScRNA-seq enabled high-throughput sequencing analysis of genomes,transcriptomes,and epigenomes at the single-cell level.The scRNA-seq data of the sorafenib primary resistance group and the sorafenib sensitive group were analyzed and compared.Single cell regulatory network inference and clustering(SCENIC)was applied to identify the specific cell clusters from scRNA-seq data.These cell clusters belong to the PDX model of primary resistance to sorafenib.SCENIC analysis showed that JUN,JUND and FOS(main regulatory genes of the HIF pathway)were activated abnormally in these cells.Next,the data were analyzed by quantitative set analysis for gene expression(QuSAGE).The results showed that the signal pathways related to hypoxic metabolism in these special cell clusters were up-regulated.In Chapter 4,through the analysis of intercellular communication data,it was found that the clusters have a high-intensity interaction with several fixed cell clusters and itself through the neonatal Fc receptor(FcRn)and albumin(ALB).This phenomenon was not observed in other cell clusters.Therefore,it was inferred that these cell clusters are the key clusters for primary resistance to sorafenib.Under the pressure of sorafenib,the HIF related transcription factors in these cells were highly activated through the ALB-FcRn interaction,and the cellular hypoxic metabolic pathway was up-regulated,resulting in sorafenib resistance.To investigate the role of ALB-FcRn interaction in primary resistance to sorafenib.Huh7-sorafenib resistant(SR)cell lines and Huh7 wild-type(WT)cell lines were serum-starved,then stimulated with bovine serum albumin(BSA),followed by transcriptome sequencing.Analysis of the sequencing results showed that after BSA stimulation in Huh7 SR cell lines,several signaling pathways related to the central dogma were up-regulated.However,ion channel related signaling pathways were up-regulated in Huh7 WT cell lines.In the efficacy assay,the FcRn of Huh7 cell line was knocked out.Compared with Huh7 WT cell line,the ability of Huh7 cell line to antagonize sorafenib after knockout of FcRn was weaker significantly.It was indicating the correlation between FcRn and sorafenib primary resistance.In summary,this study investigated the transcriptome of tumor tissues in the PDX model of the sorafenib primary resistance group and the sorafenib sensitive group at the single cell level.The results showed that in the primary resistance cell clusters,the transcription factor JUN could regulate the HIF signaling pathway to form hypoxic tumor microenvironment to antagonize sorafenib.ALB and its receptor FcRn were the core pathway for the interaction of these cell clusters with other cell clusters.The cell clusters could stimulate the differentiation of itself and other cell clusters in the direction of antagonizing sorafenib through the interaction of ALB and FcRn.The results of the study initially revealed the molecular mechanism of primary resistance to sorafenib at the cell cluster level.It provides a certain theoretical basis and research methods for HCC related indication drug development and clinical drug selection.