背景和目的

胰腺癌是恶性程度极高的消化系统肿瘤，具有早期临床症状不典型、容易发生转移、化疗易耐药和靶向与免疫治疗不敏感等特点，其患者的五年总体生存率仅为12%。因此，亟需进一步探索胰腺癌容易发生侵袭转移的分子机制，进而寻找新型的治疗靶点。Entosis，即细胞套亡，是由cell-in-cell（CIC）结构形成介导的一种非凋亡性的程序性细胞死亡方式，已被证实与包括胰腺癌在内的多种恶性肿瘤的进展有关。但是，Entosis在胰腺癌进展中发挥的具体作用与机制目前尚不清楚。本研究旨在：（1）探索胰腺癌组织和细胞系中是否存在Entosis现象，并研究其与胰腺癌进展的相关性；（2）筛选并验证胰腺癌发生Entosis的关键调控分子，并探究其在胰腺癌进展的作用；（3）探讨Entosis现象及其关键调控分子是否为推动胰腺癌进展的危险因素。

方法

    通过分析公共数据库中胰腺癌的测序数据和临床信息，探讨程序性细胞死亡的关键调控分子在胰腺癌和正常胰腺组织中的差异表达水平，及其与患者预后的相关性。通过悬浮培养的方式诱导胰腺癌细胞间形成CIC结构，利用细胞免疫荧光和透射电镜扫描观察CIC结构的形态特征，并利用活细胞延时显微成像观察内化细胞的命运结局。接下来，利用慢病毒载体构建敲低或过表达的稳转细胞株，并通过流式细胞荧光分选技术富集正在经历CIC形成过程的细胞群；利用单细胞转录组测序探索形成CIC结构后细胞亚群的基因表达特征。此外，在体外通过CCK-8细胞增殖实验、Transwell实验、失巢凋亡诱导实验和细胞竞争实验检测细胞增殖、迁移、侵袭、抵抗失巢凋亡和细胞间直接竞争的能力，并用实时荧光定量PCR和Western blot实验检测目的基因的RNA和蛋白表达水平。通过免疫组织化学染色和生存分析评估目的基因的表达水平与胰腺癌患者预后的相关性，并在裸鼠皮下移植瘤模型和胰腺原位移植瘤模型中比较肿瘤细胞在体内的成瘤和转移能力。

结果

对公共数据库中不同类型程序性细胞死亡相关通路代表性基因的转录组测序结果分析显示：在众多程序性细胞死亡通路中，Entosis通路的基因在癌与正常组织中的表达差异最为显著—相关基因在胰腺癌组织中普遍表达上调，并且与患者的预后不良有关；并且，与其他恶性肿瘤相比而言，这一通路的基因表达差异在胰腺癌中最为显著。对胰腺癌组织样本中CIC结构的统计结果提示，CIC现象与患者的预后不良有关，并且在胰腺癌肝转移灶中较原发灶更为多见，由此推测这可能代表了一种侵袭性的病理特征。

通过单细胞转录组测序分析发现了数个具有促癌作用的基因在经历了CIC过程后的细胞亚群中表达上调，如NET1, ALDH3A1, PLAT, GALNT5,和FAM3C。随后的细胞功能实验结果表明，形成CIC结构的这群细胞具有更强的细胞增殖、迁移侵袭和失巢凋亡抵抗的能力；动物实验的结果同时显示，这群细胞的体内成瘤和转移能力更强。

功能实验的结果提示，NET1是Entosis的重要调控基因，该基因的过表达能够促进胰腺癌细胞间形成CIC结构，且NET1高表达的胰腺癌细胞表现出更强的恶性生物学特性。最后，临床队列的结果表明，NET1在蛋白水平的高表达与胰腺癌患者更差的预后显著相关，可能是促进胰腺癌进展的危险因素。

结论

    本研究首次报道，胰腺癌细胞利用Entosis产生了一群具有更强“转移性”的细胞亚群，这可能是导致胰腺癌预后较差的原因之一。本研究通过一系列细胞功能实验和动物实验首次确认NET1是Entosis的重要调控分子，可能在胰腺癌进展中起促进作用。靶向Entosis通路的关键分子有望成为胰腺癌的新型治疗选择。

Background & Objective

Pancreatic cancer is a deadly malignancy of the digestive system, with atypical early clinical symptoms and metastatic potentials, as well as drug resistance to chemotherapy and low response rate to targeted therapy and immunotherapy, resulting in the worse prognosis and low five-year overall survival rate which is only 12%. Entosis, a non-apoptotic programmed cell death mediated by cell-in-cell (CIC) structures, has been shown to be associated with the progression of multiple malignancies, including pancreatic cancer. However, the function and mechanism of Entosis in the progression of pancreatic cancer is currently unknown. The purposes of this study were: (1) to investigate whether Entosis exists in pancreatic cancer tissues and cell lines, and the correlation between Entosis and prognosis; (2) to screen key regulators of Entosis in pancreatic cancer progression, as well as the effect on phenotype after cells experienced Entosis; and (3) to validate whether Entosis and its key regulators are risk factors of promoting pancreatic cancer progression.

Methods

We investigated the expression levels of key regulators of programmed cell death in pancreatic cancer samples and the correlation with patient’s prognosis by analyzing RNA-sequencing data and clinicopathological features of those samples from public datasets. We induced the formation of CIC structures among pancreatic cancer cells via suspended cell culture, and observed the fate outcome of internalized cells in CIC structures through time-lapse microscopy. We used lentiviral vectors to construct stable knockdown or overexpression cells. Fluorescence-activated cell sorting was used to sort cancer cells undergoing entotic CIC. Single-cell RNA sequencing analysis was used to delineate gene expression changes of CIC-forming cancer cells. Next, we examined the ability of cell proliferation, migration, invasion, anoikis-resistance, and direct competition among cancer cells by using CCK-8 assay, Transwell assay, anoikis assay and cell competition assay. Real-time fluorescence quantitative PCR and Western blot assays were used to validate mRNA and protein levels of targeted genes. Immunohistochemical staining and overall survival analysis were used to assess the correlation between the expression levels of target genes and the prognosis of pancreatic cancer patients. We further performed subcutaneous xenograft and orthotopic xenograft to assess the tumorigenesis and metastatic potential of cancer cells in vivo respectively.

Results

In this study, we found that Entosis-related regulators were commonly overexpressed in pancreatic cancer tissues compared with other cancers and associated with worse prognosis of pancreatic cancer patients. CIC structures detected in pancreatic cancer specimens have a significant correlation with liver metastases, suggesting that CIC phenomenon in pancreatic cancer may represent an aggressive pathological feature.

The results of our single-cell transcriptome analysis revealed that several oncogenes, such as NET1, ALDH3A1, PLAT, GALNT5, and FAM3C, were upregulated in CIC-forming cancer cells compared with non-CIC-forming cancer cells. Moreover, the results of functional assays demonstrated that these CIC-forming cancer cells had enhanced oncogenic characteristics, for instance, cell proliferation, migration, invasion and anoikis resistance in vitro.

The results of nude mice xenograft also showed that these cells had enhanced tumorigenic and metastatic abilities in vivo. Furthermore, NET1 (neuroepithelial cell transforming gene 1) overexpression promoted the formation of CIC structures among pancreatic cancer cells, and cancer cells with higher NET1 expression levels exhibited enhanced malignant behaviors. Finally, we found that NET1 protein level was associated with unfavorable prognosis for patients, which may play a pro-tumorigenic role in pancreatic cancer progression.

Conclusion

These findings support the hypothesis that entosis may play a vital oncogenic role in pancreatic cancer progression. Pancreatic cancer cells generate a highly “progressive” subpopulation marked by increased expression of NET1 via entotic CIC. NET1 is a newly validated gene involved in entosis. Targeting entotic processes may be a potential therapeutic option for pancreatic cancer.