An integrated bioinformatic analysis of bulk and single-cell sequencing clarifies immune microenvironment and metabolic profiles of lung adenocarcinoma to predict immunotherapy efficacy

Abstract

Targeting the tumor microenvironment is increasingly recognized as an effective treatment of advanced lung adenocarcinoma (LUAD). However, few studies have addressed the efficacy of immunotherapy for LUAD. Here, a novel method for predicting immunotherapy efficacy has been proposed, which combines single-cell and bulk sequencing to characterize the immune microenvironment and metabolic profile of LUAD. TCGA bulk dataset was used to cluster two immune subtypes: C1 with "cold" tumor characteristics and C2 with "hot" tumor characteristics, with different prognosis. The Scissor algorithm, which is based on these two immune subtypes, identified GSE131907 single cell dataset into two groups of epithelial cells, labeled as Scissor_C1 and Scissor_C2. The enrichment revealed that Scissor_C1 was characterized by hypoxia, and a hypoxic microenvironment is a potential inducing factor for tumor invasion, metastasis, and immune therapy non-response. Furthermore, single cell analysis was performed to investigate the molecular mechanism of hypoxic microenvironment-induced invasion, metastasis, and immune therapy non-response in LUAD. Notably, Scissor_C1 cells significantly interacted with T cells and cancer-associated fibroblasts (CAF), and exhibited epithelial-mesenchymal transition and immunosuppressive features. CellChat analysis revealed that a hypoxic microenvironment in Scissor_C1elevated TGFβ signaling and induced ANGPTL4 and SEMA3C secretion. Interaction with endothelial cells with ANGPTL4, which increases vascular permeability and achieves distant metastasis across the vascular endothelium. Additionally, interaction of tumor-associated macrophages (TAM) and Scissor_C1 via the EREG/EFGR pathway induces tyrosine kinase inhibitor drug-resistance in patients with LAUD. Thereafter, a subgroup of CAF cells that exhibited same features as those of Scissor_C1 that exert immunosuppressive functions in the tumor microenvironment were identified. Moreover, the key genes (EPHB2 and COL1A1) in the Scissor_C1 gene network were explored and their expressions were verified using immunohistochemistry. Finally, the metabolism dysfunction in cells crosstalk was determined, which is characterized by glutamine secretion by TAM and uptake by Scissor_C1 via SLC38A2 transporter, which may induce glutamine addiction in LUAD cells. Overall, single-cell sequencing clarifies how the tumor microenvironment affects immunotherapy efficacy via molecular mechanisms and biological processes, whereas bulk sequencing explains immunotherapy efficacy based on clinical information.

Keywords: cancer-associated fibroblasts; epithelia-mesenchymal transition; immunotherapy; lung adenocarcinoma; metabolism; single-cell multi-omics.