Integrative single-cell and spatial transcriptomics approaches to identify key regulators in lung diseases

Abstract

The past 10 years have seen significant advancements in sequencing technology, enabling both spatial transcriptomic analysis and transcript-level analysis via long-read sequencing. However, numerous sequencing technologies have emerged in the meantime, making it difficult to select the appropriate technology for the research objective. Moreover, indiscriminate quality control during analysis, leading to a loss of biological relevance and inaccuracies in data interpretation, undermines the reliability and reproducibility of research using transcriptomic data. Therefore, we investigated two lung diseases using suitable sequencing technology with knowledge of sequencing technology and present them in two parts. The first part is Post COVID-19 Pulmonary Fibrosis (PCPF), which refers to pulmonary fibrosis (PF) developed following a COVID-19 infection, and PF is classified under interstitial lung diseases (ILDs), with Idiopathic Pulmonary Fibrosis (IPF) being the most prevalent condition within ILD. To identify potential therapeutic targets for PCPF derived from IPF, shared characteristics were extracted from both PCPF and IPF, analyzing scRNA-seq data from public repositories related to PCPF, IPF, and control donor lungs. In the second part, we examined cellular communication within the tumor microenvironment (TME) using spatial transcriptomic data from small cell lung cancer (SCLC). In SCLC, it has been demonstrated that CD8+ T cell infiltration exhibits an improved survival rate, but the cell-cell communication within the infiltrated environment remains poorly understood. To address this, we applied Xenium, a spatial transcriptomic platform with high resolution, to analyze the interactions within the TME. Spatial analysis revealed increased interaction in CD4+ T cells and Antigen Presenting Cells (APCs) in tumors with high CD8+ T cell infiltration, as well as CD4+ T cells were spatially located close to various immune cells. In addition, it was confirmed that CD8+ T cells were more clustered with immune cells such as CD4+ T and conventional Dendritic Cell (cDC). Furthermore, CD4+ T cells showed enhanced expression of genes related to antigen presentation, T cell activation. These findings suggest that the improved survival rate mediated by CD8+ T infiltration in SCLC is related to antigen presentation and T cell activation at the spatial and transcriptomic levels. Collectively, we demonstrated at both the spatial and transcriptomic levels that improved survival rate in SCLC, mediated by CD8+ T infiltration, is closely associated with APCs and CD4+ T cells. In summary, this research identified key regulatory factors in lung diseases by employing sequencing techniques and suitable analytical approaches for the research objectives and biological context.