Investigation into the Spatial Heterogeneity of Lung Composite Large-Cell Neuroendocrine Carcinoma Spatial Transcriptomic Analysis of Combined Large-Cell Neuroendocrine Carcinoma.

Abstract

Background: Lung combined large-cell neuroendocrine carcinoma (CoLCNEC) refers to lung regions exhibiting both the features of large-cell neuroendocrine carcinoma (LCNEC) and the defined components of nonsmall cell lung cancer (NSCLC), with a relatively high mitotic rate. Diagnosing and predicting the prognosis of CoLCNEC are challenging. This study aimed to explore spatial transcriptomic expression patterns and identify crucial genes. Methods: We utilized a sample from a CoLCNEC patient containing three distinct components, namely, LCNEC, adenocarcinoma, and squamous cell carcinoma, with the former being predominant. Spatial transcriptomics (ST) technology, which employs the 10× Genomics Visium formalin-fixed paraffin-embedded ST kit, was applied along with high-throughput sequencing to obtain gene expression information and spatial locations for each spot. Subsequent analysis included differentially gene expression and functional enrichment. Finally, immunohistochemistry was employed to validate the marker protein structural maintenance of chromosomes 1A (SMC1A). Then, SMC1A was overexpressed and silenced in NCI-H661 and LTEP-a-2 cells, and the migration and invasion ability of the cells were detected by scratch assay and Transwell, respectively. The role of SMC1A in cancer cell cycle was detected by Real-time Reverse Transcription-PCR(RT-qPCR), Western blot, and flow cytometry, the apoptosis was detected by flow cytometry. Results: The results revealed that tumor tissue regions had higher unique molecular identifiers and gene counts than nontumor regions did. Unsupervised clustering identified four clusters, revealing the uniform distribution of unique transcripts, which were mapped onto slices to display apparent spatial separation. Differentially gene expression analysis revealed genes highly expressed in cancer cells. Further analysis of different regions revealed distinct cellular subgroups enriched through differentially gene expression analysis in various pathways, such as the cell cycle and DNA replication. Finally, SMC1A was chosen as a candidate gene, and immunohistochemistry confirmed its elevated expression in tumor regions. In addition, compared with oe-NC, oe-SMC1A can significantly promote the migration, invasion and G1/S phase transition of lung cancer cells, and promote the inhibition of apoptosis of cancer cells, while sh-SMC1A is completely opposite. Conclusions: In the tumor region of CoLCNEC, SMC1A is significantly upregulated. Moreover, silencing SMC1A effectively inhibits lung cancer cell invasion, migration, and G1/S phase transition, while promoting apoptosis. These findings indicate that SMC1A has the potential to be a new therapeutic target for CoLCNEC treatment.