Studies On The Immune Microenvironment Of Biliary Tract Cancer Based On Single-cell RNA Sequencing

Background&Aims:Immunotherapy has been widely used in the treatment of solid tumors,although only a small number of patients can get a durable response from immunotherapy and the efficacy of immunotherapy varies among different tumor types.Several studies have shown that the biological characteristics of immune cells in tumor microenvironment,such as cell types,distribution,crosstalk network,dynamic differentiation,etc.,will have a major influence on the immunotherapy,which can partly explain the vary efficacy of the immunotherapy.Therefore,exploring the tumor immune microenvironment in depth may help to dissect the potential mechanism of immunotherapy,discover new biomarkers for predicting the efficacy and even develop innovative immunotherapteutics.Single-cell RNA sequencing(sc RNA-seq)is an emerging method with high throughput and resolution,which can accurately depict the expressed genes in every single cell and play an essential role in understanding the heterogeneity and functions of various cell populations in tumor microenvironment,discovering less defined cell subsets,and portraying the dynamic differentiation and communication between cell types.At present,a large number of high-quality studies on immune cells within tumor microenvironment based on sc RNA-seq have been published,and these studies have deciphered significant enrichment of immunosuppressive cells in tumor microenvironment,such as exhausted CD8~+T cells and Regulatory T cells(Tregs),tumor-associated macrophages(TAMs),etc.Combined with T cell receptor(TCR)sequencing,it was extensively found that the effective CD8~+T cells within the tumor microenvironment were gradually activated and differentiated into exhausted CD8~+T cells under the stimulation of tumor specific antigens.In addition,a high degree of clonal expansion and proliferation of the exhausted CD8~+T cells can be observed in the tumor microenvironment,indicating a highly immunosuppressive microenvironment.Crosstalk analysis is conductive to analyzing the biological communication between various cell subsets through ligand-receptor axis,which plays an essential role in the formation of an immunosuppressive microenvironment.What’s more,sc RNA-seq studies on different time points before and after immunotherapy,in order to explore the influence of immunotherapy on immune cells in tumor microenvironment,which is instrumental to deeply depict the potential mechanisms of immunotherapy.Because of the difficulty in obtaining tissue samples,these studies predominantly focus on tumor types with easy sample accessibility and optimal immunotherapy efficacy,such as melanoma and breast cancer.Therefore,the number of these studies is significantly less than that of investigating untreated patients at the initial time point.Notably,the research of sc RNA-seq in biliary tract cancer(BTC)is still insufficient,which may be closely related to many factors,such as the low incidence of BTC,a high proportion of advanced diseases and the low chance of radical surgery.In addition,the efficacy of immunotherapy in BTC is dismal and the immunotherapeutics are only approved for patients with mismatch repair defects(d MMR)or high microsatellite instability(MVI).At present,radical resection is still the only curative treatment for patients with biliary tract cancer.It is noteworthy that only a fraction of patients in the middle or early stage can receive surgery,with a high recurrence rate.Instead,most patients with local invasive or advanced states lose the chance of operation due to the dismal efficacy of surgery.Palliative local and systemic therapies are recommended for patients with advanced diseases.Among them,the efficacy of gemcitabine combined with cisplatin recommended by National Comprehensive Cancer Network(NCCN)guidelines is still unstatisied.The newly developed targeted therapy and immunotherapy has brought hope to some patients with advanced diseases,and a fraction of patients have obtained durable therapeutic responses.In turn,how to identify patients who can benefit from treatment,improve the efficacy of therapies,and even develop innovative immunotherapeutics is still a clinical issue in urgent need.In this regard,we conducted a study on the immune cells within tumor microenvironment of patients with advanced biliary tract tumors using sc RNA-seq,in order to dissect the causes of high invasion and dissemination of BTC and explore potential new immunotherapy strategies.Methods:1.In this study,we enrolled 5 advanced BTC patients.Among them,there were 2patients with intrahepatic cholangiocarcinoma,2 patients with gallbladder carcinoma and 1patient with distal cholangiocarcinoma.In order to comprehensively describe the immune cells within tumor microenvironment of patients with advanced biliary tract cancer,we obtained matched tissue samples,including the primary tumor focus,metastatic tissues(liver or/and lymph nodes)and peripheral blood.In addition,we obtained paraffin slices from 18patients with intrahepatic cholangiocarcinoma who received immunotherapy treatment and had sufficient follow-up and efficacy evaluation data.Through the multiplex Immunohistochemistry(m IHC)staining in paraffin,we can verify the results of our sc RNA-seq.2.We performed Chromium Next GEM Single Cell 5’Library Construction and V(D)J Enrichment provided by 10X Genomics.3.The algorithm of Seurat,Monocle,Cellphone DB was used to define the cell clusters and explore the biological characteristics in tumor microenvironment,such as dynamic differentiation,cell-cell crosstalk network,et al.4.We verified two main subsets of exhausted CD8~+T cells:XBP1~+CD8~+T cells and PD1~+CD8~+T cells by m IHC in another 18 BTC patients’paraffin slices.In addition,through in vitro experiments,we verified the potential role of tumor microenvironment in T cell dysfunction.Results:1.A total of 45,851 cells were obtained from all tissues,and 19 cell subsets of 4 types of immune cells(T,B,NK and myeloid cells)were identified.Among which,exhausted CD8~+T cells,macrophages and dendritic cells were significantly enriched in the tumor microenvironment.2.Cell-cell crosstalk analysis indicated that exhausted CD8~+T cells showed frequent interaction with myeloid cells,including dendritic cells and macrophages,which indicated their potential role in inducing an immunosuppressive microenvironment in advanced biliary tract cancer.3.Combined with TCR sequencing,the exhausted CD8~+T cells enriched in tumor microenvironment were clonally expanded,and had a high proliferation ability,which indicated that the cells played a major role in modeling immunosuppressive microenvironment and might be regarded as a potential target population of immunotherapy.4.Further analysis of exhausted CD8~+T cells showed that there are two main kinds of cell types in the tumor microenvironment,one of which is PD-1~+CD8~+T cells highly expressing various immunosuppressive receptors,which may represent a class of severe exhausted CD8~+T cells,and the other one especially express XBP1,suggesting the potential role of endoplasmic reticulum(ER)stress in T cell dysfunction.These two cell types were verified by m IHC in another 18 BTC patients’paraffin slices.5.In vitro,the potential role of endoplasmic reticulum stress in promoting T cell dysfunction has been confirmed.When CD8~+T cells isolated from healthy individuals were co-cultured with the supernatants of biliary tumor cell lines(GBC-SD,HCCC-9810),the expression of immune checkpoint receptors(PD1,TIGIT)and XBP1 in CD8+T cells was parallelly upregulated,which was significantly higher than that of the control group.As expected,when treating the coculture groups with the specific inhibitor of IRE1α-XBP1(4μ8C),we observed the downregulation of TIGIT in the treatment group,suggesting that XBP1 might serve as an underlying target for novel antitumor therapy.Conclusion:Our transcriptional map of immune cells from advanced BTC provided a framework for understanding the various immune status and portrayed the dynamic characteristics of immune cells in the BTC setting.Coupled with TCR sequencing,we identified highly proliferative and clonally expanded dysfunctional T cells in the tumor microenvironment,indicating an immune-suppressive state and potential targets for developing immunotherapies in BTC.Finally,we observed a less described dysfunctional marker,XBP1(an ER stress sensor),enriched in exhausted CD8+T cells,which may help us to stratify patients and evaluate therapeutic efficacy.Collectively,our results revealed an atlas of suppressive immune cells in the TME and set the horizon for the application and development of immunotherapies for biliary tract cancer.