Fluorescence Imaging Study Of Immune Evasion-related Biomarkers Of Hepatocellular Carcinoma In Tumor Microenvironment

The immune system is the key means for the body to remove tumors.Immunotherapy based on this development has shown great potential in the treatment of liver cancer.However,liver cancer can evade the surveillance and killing of the immune system through a variety of mechanisms to complete immune evasion,resulting in ineffective treatment and making liver cancer immunotherapy face great challenges.Therefore,it is urgent to explore the immune evasion process and related molecular mechanisms of liver cancer in detail.Oxidative stress is involved in the regulation of immune response and is closely related to immune evasion.Oxidative stress is closely related to the occurrence and development of immune evasion in liver cancer,but there is a lack of relevant research work.The tumor microenvironment（TME）is the primary site of immune evasion in liver cancer,and oxidative stress is an important characteristic.Liver cancer mostly develops from diseases such as chronic hepatitis,liver fibrosis,and cirrhosis.During this process,fibroblasts,macrophages,and various inflammatory factors cause oxidative stress,deepen immune suppression,and create a favorable microenvironment for immune evasion in liver cancer.As liver cancer progresses,it plays a key role in promoting immune evasion in the liver cancer TME.Therefore,the occurrence and development of immune evasion in liver cancer are largely influenced by fibroblasts,macrophages,and various inflammatory factors in the liver cancer TME,and their roles in immune evasion in liver cancer must be elucidated.Current research indicates that the activation process of fibroblasts in the TME of liver cancer is the key event leading to immune evasion in liver cancer,which is mediated by oxidative stress,arginase-mediated communication between macrophages and fibroblasts,and the Caspase-6-mediated inflammatory process.However,the mechanism of action has not been fully explored,and the relevant signaling pathways are unclear.The above processes occur in dynamic events within living cells and organisms and are deeply influenced by the concentration,distribution,and activity of relevant marker cells and active molecules.Therefore,to elucidate the detailed information of cell-to-cell communication mediated by disease markers,oxidative stress,and other factors leading to immune evasion in liver cancer,it is necessary to explore the in situ,real-time,and dynamic changes of cells and active molecules in cells.However,there is currently a lack of suitable research methods.Fluorescence imaging technology has advantages such as high sensitivity,high temporal and spatial resolution,non-invasiveness,and easy operation.In addition,by increasing the wavelength of the excitation or emission light,the imaging depth of tissues can be significantly enhanced,which has great advantages in exploring the dynamic changes of cells and active molecules in the TME.Currently,a large number of specific fluorescent probes for oxidative stress-related active molecules have been developed.Researchers use fluorescence imaging technology to visualize the real-time and dynamic changes of oxidative stress-related active molecules at the level of live cells and organisms,and deeply explore various disease-related molecular mechanisms.However,there is still a lack of fluorescent imaging probes for studying the activation markers of fibroblasts,oxidative stress markers,arginase,and Caspase-6 in the TME of liver cancer.Based on the above reasons,this thesis designs specific fluorescent probes for various liver cancer immune evasion markers,including fibroblast activation protein（FAP）,cancer-associated fibroblasts（CAFs）oxidative stress markers H₂O₂and GGT,arginase and Caspase-6,which are activated during CAFs proliferation.The design aims to construct various imaging and detection methods to reveal the dynamic changes of the above markers in liver cancer immune evasion.Moreover,this thesis explores the relationship between oxidative stress and the liver cancer immune evasion process,and investigates the related signaling pathways.Effective information is provided for revealing the detailed molecular mechanisms of liver cancer immune evasion.Specifically,this thesis conducted the following work:First,to explore the dynamic changes of CAFs promoting HCC immune evasion and reveal the relevant molecular mechanism,a new imaging method Cy-FAP was established based on the CAFs marker FAP.The Cy-FAP is composed of FAP-specific valine-proline dipeptide and half-monocyanine structure.When the dipeptide is cut by FAP-specific cleavage,the ICT effect is enhanced,resulting in enhanced fluorescence intensity at 705 nm.Using this imaging method,the dynamic changes of CAFs in mouse HCC were explored.The results showed that as the activation degree of CAFs increased,the level of ROS in the cells also increased accordingly.By analyzing immune evasion-related markers,a new mechanism was further revealed:CAFs produced excess ROS during the activation process,which then damaged JAK2 protein,downregulated the JAK2/STAT3 signaling pathway that inhibits PD-L1 production,resulting in increased PD-L1 expression and ultimately promoting HCC immune evasion.Second,to further explore the types and sources of ROS produced by CAFs promoting HCC immune evasion,a new dual-color imaging and detection method was constructed for detecting GGT and H₂O₂in HCC CAFs.This material is composed of functionalized liposomes containing vitamin A,which encapsulate small-molecule fluorescent probes specific to GGT and H₂O₂.After targeting the vitamin A receptor on the surface of HCC CAFs,the internal small-molecule probes of GGT（Ex=550 nm,Em=620 nm）and H₂O₂（Ex=680 nm,Em=705nm）were released for simultaneous imaging and detection.Then,this method was used to investigate the real-time and dynamic changes of GGT and H₂O₂in HCC CAFs during immune evasion.The results showed that the activation of HCC CAFs produced excess GGT,which promoted the increase of glutamic acid content in the cells,resulting in an increase of H₂O₂and up-regulation of the expression of immune evasion-related active molecules through the H₂O₂/HIC-5/SMAD2 signaling pathway,ultimately promoting HCC immune evasion.Third,to explore the molecular mechanism of cell-to-cell communication between CAFs and TAMs promoting HCC immune evasion,a new detection strategy for arginase in TAMs was established using the arginase-specific fluorescent probe TPEARG.The probe is composed of arginine and tetraphenylethene（TPE）fluorophore.When TPE is restricted by arginine-specific targeting of the active site of arginase,aggregation-induced fluorescence phenomenon induces enhanced fluorescence intensity at 450 nm.This strategy was successfully used for the imaging and detection of arginase in TAMs.A combined Cy-FAP and TPEARG imaging revealed the mechanism by which interactions between TAMs and CAFs contribute to HCC immune evasion:TAMs promote the production of proline through arginase,thereby increasing the activation degree of CAFs and promoting HCC immune evasion through the JAK2-STAT3 signaling pathway.Fourth,to explore the detailed role of Caspase-6 in HCC immune evasion,a new real-time imaging method for Caspase-6 was constructed based on the Caspase-6-specific fluorescent probe Rh TDQ.Rh TDQ is composed of a peptide segment that specifically recognizes Caspase-6,a fluorescence quenching group,and a rhodamine fluorophore.When Rh TDQ is specifically cleaved by Caspase-6,the rhodamine fluorophore is released.Based on the fluorescence resonance energy transfer（FRET）principle,bright fluorescence is displayed at 550 nm.This imaging strategy revealed that compared with normal liver cells,the content of Caspase-6 in HCC cells is increased,and the expression of immune evasion markers PD-L1 and CTLA4 is also increased.Subsequently,we investigated the process of communication between CAFs and HCC cells promoting HCC immune evasion.The results showed that when the activation degree of CAFs increased,the content of Caspase-6 in HCC cells significantly increased,and the expression of PD-L1 and CTLA4 also increased.