Fluorescence Imaging Study Of Activation Of Hepatic Stellate Cells Promoting Immune Evasion Of Hepatocellular Carcinoma Cells

Hepatic stellate cells（HSCs）are improtant members of the liver.After being stimulated and activated,HSCs secrete a large number of active molecules.These molecules regulate the physiological and pathological processes in the liver.HSCs play a vital role in maintaining liver homeostasis.Current studies have shown that during the development and progression of liver cancer,abnormally activated hepatic stellate cells（a HSCs）enhance the immune suppression of liver cancer,leading to immune evasion of hepatocellular carcinoma（HCC）cells.a HSCs promote the proliferation and metastasis of HCC cells and further weaken the efficacy of immunotherapy for liver cancer.However,the detailed molecular mechanism of how a HSCs regulate immune evasion of HCC is unclear.Considering that the activation process of HSCs is accompanied by lipid metabolism and massive production of ROS,which can cause oxidative stress in the hepatocellular carcinoma microenvironment.This process would profoundly affect the immune evasion of HCC.Superoxide anion(O₂^·-)is the first ROS produced in large quantities during lipid metabolism of a HSCs.O₂^·-plays key roles in oxidative stress.Therefore,we speculate that O₂^·-generated by a HSCs may play an important role in the regulation of immune evasion of HCC.However,due to the lack of suitable research methods,no research work has revealed the changes of O₂^·-levels in a HSCs and the detailed molecular mechanism of how to regulate the immune escape of HCC.Two-photon fluorescence imaging has made great progresses in recent years due to its advantages of high sensitivity and selectivity,strong tissue penetration and weak biological fluorescence background.The liver is located in the deep tissue,and the development of liver cancer and the activation of HSCs are dynamic processes.So,two-photon fluorescence imaging technology is a powerful means to image the real-time and dynamic changes of O₂^·-in a HSCs in deep tissues.It can provide effective information for revealing the detailed molecular mechanism of a HSCs regulating the immune evasion of HCC.However,suitable imaging materials are currently lacking.To solve the above problems,we designed and synthesized a two-photon fluorescence probe TPH for imaging O₂^·-in HSCs.Then,with TPH,we established a new imaging analysis method for detecting O₂^·-level changes during HSCs activation.We further explored the effect of O₂^·-on immune evasion of HCC and related molecular mechanisms using the established strategy.The work carried out in this paper is as follows:1.O₂^·-imaging during hepatic stellate cells activationWe designed and synthesized a two-photon fluorescent probe TPH for O₂^·-detection in HSCs.TPH uses caffeic acid as the O₂^·-recognition group and fluorophore.The polypeptide（CGPTAKYIC-）is utilized as the HSCs targeting group.Thus,TPH realizes sensitive and specific imaging detection of O₂^·-in HSCs.Using TPH,we found that O₂^·-levels were significantly increased in activated HSCs after TGF-βstimulation compared to quiescent HSCs.Imaging of mouse models with different degrees of HSCs activation using TPH shown that there was a positive correlation between the changes of HSCs activation and O₂^·-level in the liver.We constructed mouse orthotopic model of HCC with different degrees of HSCs activation.The results of TPH imaging showed that:at the same progression of HCC,the level of O₂^·-in HCC with a higher degree of activation was significantly increased,and the number of CD8⁺T cells,a marker related to the immunosuppressive microenvironment decreased,leading to aggravation of immunosuppression.2.Molecular mechanism of HSCs activation promoting immune evasion of HCCThe above work suggested that the activation of a HSCs produce a large amount of O₂^·-,resulting in oxidative stress in a HSCs,thus enhancing the immunosuppressive ability.These results indicate that O₂^·-is an important factor in regulating a HSCs to promote the immune evasion of HCC.Through fluorescence imaging and western blotting experiments,we found that HSCs with higher degree of activation can produce more O₂^·-and PD-L1.Further experimental results show that:O₂^·-destroys the phosphorylation function of CDK4 through oxidative modification of the active site of CDK4.This changes the phosphorylation level of downstream signaling molecules,and increases the expression of PD-L1,promoted the immune evasion of HCC.This work reveals the molecular mechanism by which O₂^·-produced by HSCs activation regulates PD-L1 level and promotes immune evasion of HCC,providing a new knowledge for revealing the detailed mechanism of occurrence and development of HCC and developing new immunotherapy methods for HCC.