Studies On Spatial Heterogeneity Of Immune Microenvironment In Hepatocellular Carcinoma

BACKGROUND&AIMSThe spatial heterogeneity of the immune microenvironment in tumors is one of the main problems faced in cancer immunotherapy,and it is also a research hotspot in recent years.A large number of reports have proved that there is spatial immune heterogeneity in lung cancer,breast cancer,colorectal cancer,liver cancer and many other tumors.In liver cancer,epigenetic changes,viral infection,alcohol consumption and metabolic abnormalities are involved in the pathogenesis process.Hepatocellular carcinoma（HCC）is also considered to be one of the most heterogeneous cancers.Recent studies have found that the transitional area（L）between cancerous tissues and para-cancerous tissues is also a transitional area of immune microenvironmental cells.PD-1+B cells enriched in the L area can promote HCC progression through the IL-10signaling pathway.The neutrophils residing in the L area can promote tumor progression by inducing angiogenesis.Therefore,an in-depth study of the immune microenvironmental changes from non-tumor adjacent tissues to liver cancer tissues,searching for key immune cell subgroups,exploring their origin and potential biological functions,and linking them with the clinical phenotype and prognosis of patients,will help us to establish more personalized treatment strategies and new immunotherapy evaluation models for different liver cancer patients.This project aims to explore the spatial heterogeneity of the immune microenvironment of liver cancer,analyze the composition of special immune subgroups in the liver cancer transitional area（L）and provide new targets and ideas for liver cancer immunotherapy.METHODS1.Obtain freshly resected liver cancer samples and strictly follow the principle of collecting half of the cancer tissue and half of the adjacent cancer.The sample size is6cm（length）*4cm（width）*3cm（thickness）.A total of 13 samples were collected.Each sample is cut into three parts,cancer（T）,transitional area（L）,and adjacent（N）.L area is defined as plus or minus about 0.5cm distant to the boundary between the cancer and the adjacent cancer tissue.Then extract the infiltrating lymphocytes in the T/L/N area using ficoll reagent extraction method;2.According to the characteristics of T cell surface grouping markers,design a metal-labeled antibody panel containing 35 T cell surface markers,and perform antibody staining on the infiltrating lymphocytes extracted from the T/L/N region,and then subject the stained cells to mass cytometry platform（Helios,Fluidigm）;3.Through mass cytometry,we obtained single-cell level proteomic expression data and collected data of 500,000 cells from different areas of each sample;4.After standardization of the obtained data,we use the cytobank platform to pre-gate the data,and then use the"R"（Cytofkit package）to perform in-depth analysis of the data after the gating;5.Flow cytometric separation of T cell subpopulations specifically enriched in the junction area and incubate the cells with Phorbol 12-myristate 13-acetate（PMA）and Ionomycin for 4.5 hours in vitro,while adding protein transport inhibitor（Brefeldin A）,and finally use flow cytometry to detect the expression level of a series of cytokines;6.Use multiple staining immunofluorescence technology to verify the presence of characteristic T cell subsets in the transitional area（L）in situ.And by staining the tissue chip,explore the relationship between this subset and the prognosis of liver cancer;7.Construct animal models of liver cancer by liver in situ implantation and subcutaneous implantation then isolate the the infiltrated lymphocytes of animal model liver cancer,and verify the existence of characteristic T cell subsets in the transitional area;8.Reveal the internal heterogeneity and lineage origin of the characteristic T cell subpopulations enriched in the transitional area in HCC by using single-cell transcriptome and TCR sequencing.RESULTS1.The immune microenvironment of liver cancer exhibits obvious heterogeneity in terms of lymphocytes.The transitional area（L）has its unique composition of immune cell subgroups,and the difference in the composition of T cell subgroups is particularly obvious;2.By analyzing the composition and abundance of T cell subsets in the three regions of T/L/N,we found that Double positive T cells（DPT）are specifically enriched in the transitional area（L）and based on the expression abundance of the fatigue molecule PD-1,we can divide these DPT cells into PD-1^highDPT cells and PD-1^lowDPT cells;3.Through further research,we found that PD-1^highDPT cells enriched in the transitional area（L）not only highly express fatigue molecules,such as PD-1,TIM-3,LAG-3,CTLA-4,but also highly express T cell activation molecules such as HLA-DR,CD28,and its expression level is much higher than PD-1^lowDPT cells and single positive T cells;4.By isolating DPT cells from the transitional area and performing stimulation and flow cytokine detection in vitro,we found that PD-1^highDPT cells secrete high levels of IFNγand TNFα,suggesting they are functional T cells with killing activity;5.By performing multiple immunofluorescence staining on a tissue chip containing 46sets of T/L/N,we not only verified the presence of PD-1^highDPT cells in situ in the tissue,but also found PD-1^highDPT cells in the transitional area（L）is positively correlated with the prognosis of liver cancer patients;6.Through mass cytometry data of liver cancer animal models and other cancer types（cholangiocarcinoma,kidney cancer）,we verified that PD-1^highDPT cells not only exist in human liver cancer tissues,but also exist in liver cancer animal models and other cancer types;7.By performing single-cell transcriptome sequencing on 5018 DPT cells derived from liver cancer,we divided the DPT cells into 11 subsets,among which subset No.10 was identified resembling the PD-1^highDPT cells with similar phenotypes,simultaneously expressing fatigue molecules such as LAG-3,CTLA-4 and granzyme such as GZMH,GZMB.Using the difference in transcription profile expression,we calculated the activation score and exhaustion score on 11 DPT cell subsets.The results showed that subset No.10 had the highest activation score and fatigue score and also highly express PD-1;8.In order to explore the lineage origin of DPT cells derived from liver cancer,we also performed TCR sequencing on 5018 DPT cells.At the same time,we also sorted 3676intratumoral CD4 single-positive T cells,4,470 intratumoral CD8 single-positive T cells and 4652 CD3 positive T cells derived from blood.Through TCR comparison,we found that the DPT cells derived from liver cancer originated from the local tumor environment rather than the peripheral blood.The PD-1^highDPT cells and the PD-1^highCD8-positive T cells in the tumor originated from the same precursor cells.CONCLUSIONSOur research found that the transitional area（L）of liver cancer has its unique immune cell composition.Double-positive T cells are specifically enriched in the transitional area of liver cancer and are divided into two groups based on the level of PD-1 expression.Among them,PD-1^highDPT cells highly express fatigue molecules and activation molecules,suggesting they are in an activated state and further investigation revealed PD-1^highDPT cells was a favorable prognosis factor for HCC patients.Through single-cell transcription profiling and TCR sequencing technology,we learned that DPT cells have internal heterogeneity and can be further divided into 11 subsets.Among them,PD-1^highDPT cells and PD-1^highCD8-positive T cells originated from the same precursor cells,and L area-enriched DPT cells are derived from local tumor environment rather than peripheral blood.Our research deeply explored the complex functions of DPT cells derived from liver cancer and provided new insights into the origin and differentiation of DPT cells.