Objective: To explore the heterogeneity of pancreatic cancer and new methods for tumor cell molecular subtyping and identify the signature genes in pancreatic cancer progression.
Methods: Based on the single-cell sequencing data of 16 pancreatic cancer tissues from the GSE155698 dataset, the single pancreatic cancer cells were classified according to EPCAM gene expression after preliminary clustering, re-clustering, and subgrouping to identify the signature genes, followed by pathway enrichment analysis and pseudo-time analysis. The key genes identified were validated using the clinical and tissue gene and protein expression data from 179 pancreatic cancer patients and 171 healthy controls. The impact of CEACAM5, LGALS1, and CENPF on proliferation, migration and invasion of pancreatic cancer cells were analyzed.
Results: Analysis of 48 570 cells from 16 pancreatic cancer samples revealed a total of 22 clusters, including 5 clusters of pancreatic cancer cells, which were classified into Subtype 1, Subtype 2, and Subtype 3, each exhibiting distinct gene expression patterns and functions. The signature genes were enriched in negatively regulated protein metabolic processes, ferroptosis, and antigen processing and presentation-related pathways in Subtype 1 pancreatic cancer cells; in peptide synthesis processes, translation, and ribosome-related pathways in Subtype 2; and in ATP metabolic processes, glycolysis/gluconeogenesis, and cell cyclerelated pathways in Subtype 3. Subtypes 2 and 3 were potentially derived from Subtype 1, and Subtype 3 possibly represented the final developmental stage of pancreatic cancer cells. The key signature genes (CEACAM5, LGALS1, and CENPF) also exhibited different expression patterns in the developmental trajectory and showed high expressions in pancreatic cancer in association with poor prognoses. In pancreatic cancer cells, downregulation of CEACAM5, LGALS1, and CENPF significantly inhibited the proliferation, migration, and invasion abilities of the cells (P<0.05).
Conclusion: Pancreatic cancer cells exhibit significant heterogeneity, and CEACAM5, LGALS1, and CENPF gene expressions, which affect pancreatic cancer cell proliferation, invasion, and metastasis, can be used to identify distinct molecular subtypes during tumor cell development.
目的: 探讨胰腺癌的异质性和肿瘤细胞分子亚型鉴定新方法，鉴定胰腺癌发展过程中的特征基因。
方法: 分析GSE155698数据集中16例胰腺癌组织单细胞测序数据，初步聚类后，根据各细胞EPCAM基因的表达情况分离胰腺癌细胞，再聚类、分群后鉴定特征基因，进行通路GO和KEGG通路富集分析，并进行拟时序分析。提取癌症基因组图谱179例胰腺癌患者和正常胰腺组织基因表达库中171例正常人基因表达数据、临床信息及预后情况，结合人类蛋白表达图谱蛋白表达情况对各亚型关键基因进行验证。最后通过细胞功能实验验证CEACAM5、LGALS1、CENPF对胰腺癌细胞的增殖、侵袭与迁移等功能的影响。
结果: 对16例胰腺癌样本48 570个细胞进行分析后发现，细胞总共被聚类为22群，其中包含5群胰腺癌细胞。这些胰腺癌细胞被分为3群：亚型1、亚型2和亚型3，分别具有完全不同的基因表达模式和功能。亚型1胰腺癌细胞的特征基因主要富集于蛋白代谢过程的负调节、铁死亡和抗原提呈相关通路；亚型2胰腺癌细胞的特征基因主要于肽合成过程、翻译和核糖体相关通路；亚型3胰腺癌细胞的特征基因主要富集于ATP代谢过程、糖酵解/葡萄糖生成和细胞周期相关通路。亚型2和亚型3可能是由亚型1发展而来，亚型3可能是胰腺癌细胞发育的最终状态。各亚型的关键特征基因CEACAM5、LGALS1、CENPF的表达情况也在发育轨迹中展现了不同状态。基因和蛋白表达层面均显示各亚型的关键特征基因CEACAM5、LGALS1、CENPF在胰腺癌中高表达，且与患者的不良预后相关，当下调CEACAM5、LGALS1、CENPF的表达后，胰腺癌细胞的增殖（P<0.05）、迁移（P<0.05）和侵袭（P<0.05）能力均被抑制。
结论: 胰腺癌细胞有较强的异质性，基于CEACAM5、LGALS1、CENPF基因表达可鉴定肿瘤细胞发展过程中的不同分子亚型，且影响胰腺癌细胞的增殖、侵袭和转移。
Keywords: heterogeneity; molecular subtypes; pancreatic cancer; single cell sequencing.