Identification of macrophage-related genes in bladder cancer patients using single-cell sequencing and construction of a prognostic model.

Am J Clin Exp Immunol 2024;13(3):88-104 www.ajcei.us /ISSN:2164-7712/AJCEI0156764 Original Article Identification of macrophage-related genes in bladder cancer patients using single-cell sequencing and construction of a prognostic model Weizhuo Wang1*, Junheng Shen2*, Dalong Song3*, Kai Fu4, Xu Fu5 Department of Urology, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou 215002, Jiangsu, PR China; 2Department of Reproduction, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou 215009, Jiangsu, PR China; 3 Department of Urology, Guizhou Provincial People’s Hospital, Guiyang 550002, Guizhou, PR China; 4Center of Reproduction, Second Affiliated Hospital of Soochow University, Suzhou 215002, Jiangsu, PR China; 5Center of Reproduction, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou 215002, Jiangsu, PR China. *Equal contributors. 1 Received March 24, 2024; Accepted June 19, 2024; Epub June 25, 2024; Published June 30, 2024 Abstract: Single-cell sequencing is an emerging technology that can effectively identify cell types in tumors. In the tumor microenvironment of bladder cancer, macrophages play a crucial role in invasion and immune escape. This study aimed to assess the expression of macrophage-related genes (MRGs) in the tumor microenvironment of bladder cancer patients and construct a prognostic model based on MRGs using bioinformatics methods. Methods: Single-cell sequencing data from bladder cancer patients was downloaded from the GEO. After quality control and cell type identification, macrophages in the samples were extracted for re-clustering. Feature genes were then identified, and MRGs were assessed. Genetic data from TCGA database bladder cancer patients was also downloaded and organized. The intersection of MRGs and the TCGA gene set was determined. Clinical information was connected with this intersection, and the data was divided into training and validation sets. The training set was used for model construction and the validation set for model verification. A prognostic model based on MRGs was built using the LASSO algorithm and Cox regression. Patients were divided into high-risk and low-risk groups based on their prognostic features, and survival information in the training and validation sets was observed. The predictive ability of the model was assessed using a ROC curve, followed by a calibration plot to predict 1-, 3-, and 5-year survival rates. Results: Four cell types were identified, and after extracting macrophages, three cell subgroups were clustered, resulting in 1,078 feature genes. The top 100 feature genes from each macrophage subgroup were extracted and intersected with TCGA expressed genes to construct the model. A risk prediction model composed of CD74, METRN, PTPRR, and CDC42EP5 was obtained. The survival and ROC curves showed that this model had good predictive ability. A calibration curve also demonstrated good prognostic ability for patients. Conclusion: This study, based on single-cell data, TCGA data, and clinical information, constructed an MRG-based prognostic model for bladder cancer using multi-omics methods. This model has good accuracy and reliability in predicting the survival and prognosis of patients with bladder cancer, providing a reference for understanding the interaction between MRGs and bladder cancer. Keywords: Single-cell sequencing, macrophages, bladder cancer, prognostic model Introduction Single-cell sequencing is a powerful technique to measure gene expression, DNA methylation, chromatin accessibility, and various other molecular features at the level of individual cells. Through single-cell RNA sequencing (scRNA-seq), various cell subtypes present in bladder cancer can be identified, which includes both cancer cells and non-tumor cells within the tumor microenvironment. This technique could potentially aid in understanding the heterogeneity of bladder cancer, as well as the roles of different cell subtypes in disease progression. Concurrently, with single-cell DNA sequencing (scDNA-seq), the evolutionary history of bladder https://doi.org/10.62347/VLDZ7581 Prognostic model of BCa cancer can be depicted, including the accumulation of mutations, variations in gene copy numbers, and the expansion of clones. Through scRNA-seq, researchers can gain deep insights into the cellular components and interactions within the tumor microenvironment, such as immune cells, fibroblasts, and vascular cells, etc. [1]. Furthermore, single-cell sequencing can help researchers understand the mechanisms of resistance to chemotherapy or immunotherapy in bladder cancer, and how such resistance evolves within cell populations [2, 3]. Bladder cancer is one of the top ten most common malignant tumors worldwide. The incidence is significantly higher in men, and disease risk increases with age, with the majority of newly diagnosed cases being >65 years old. Smoking, occupational exposure (especially to aromatic amines and other chemicals), chronic cystitis, certain hereditary diseases, and the use of certain anticancer drugs or radiation therapy are all considered major risk factors for bladder cancer [4]. Regarding the treatment options for bladder cancer, these mainly depend on the stage and grade of the tumor, as well as the overall health status of the patient. Common treatments include surgery, chemotherapy, radiotherapy, and immunotherapy. Surgery is the primary treatment, including transurethral resection of the bladder tumor (TURBT) and radical cystectomy. Chemotherapy can be used before or after surgery, or as the main treatment for metastatic bladder cancer. Radiotherapy is usually used in patients who cannot undergo surgery, or as a supplementary treatment to surgery [5]. More recently, immunotherapy, particularly immune checkpoint inhibitors, has become an important part of the treatment for bladder cancer; it has shown significant value, especially in the treatment of metastatic bladder cancer [6]. Immunotherapy works by activating the patient’s immune system, enabling it to more effectively recognize and attack cancer cells. Notably, although existing treatments can improve survival and quality of life in some patients, several challenges remain in the treatment of bladder cancer, including disease recurrence, treatment resistance, and side effects. Therefore, basic research and clinical trials are currently being conducted to identify more effective and safer treatments. This includes the development of new drugs and treatment strategies, as 89 well as the use of molecular diagnostics and precision medicine technologies to treat bladder cancer in a personalized manner [6]. Macrophages are an important part of the immune system that are capable of engulfing and eliminating invading pathogens and damaged cells. Macroph (...truncated)