背景和目的

胰腺由内分泌部和外分泌部组成，其交错排列的组织结构表明胰腺内分泌细胞和外分泌细胞之间可能存在潜在的相互作用。尽管胰腺癌的肿瘤微环境已被逐步揭示，但既往研究多数聚焦在成纤维细胞和各类免疫细胞在胰腺癌发生发展中的作用，而作为胰腺独特成分的胰腺内分泌细胞在一定程度上被忽视。因此，本研究旨在探索在胰腺癌发生发展过程中胰腺内分泌细胞是否发生改变，并深入探讨其发生改变的上游机制以及这种改变对胰腺癌发生发展的影响。

方法

我们通过单细胞转录组测序、空间转录组测序和多重荧光免疫组化染色，在临床标本水平描绘了胰腺癌中内分泌细胞的变化及潜在的分化轨迹。随后，我们对胰腺癌发生不同阶段的Kras^LSL-G12Dp53^LoxPPdx1-CreER小鼠进行胰岛提取纯化、单细胞转录组测序和多重荧光免疫组化染色，从而进一步揭示胰腺癌发生过程中胰腺内分泌细胞的主要变化。同时，我们对Kras^LSL-G12DPdx1-Cre小鼠、胰腺原位移植瘤小鼠不同阶段的胰腺组织进行多重荧光免疫组化染色，并在Gcg-cre;Rosa26-loxP-tdTomato-loxP-EGFP小鼠上进行谱系示踪实验，从而对上述主要发现进行了进一步的验证。此外，我们通过对胰腺癌细胞或胰腺内分泌细胞进行关键基因的敲降并进行体外共培养、体内共注射等实验，观察胰腺癌细胞的恶性生物学行为变化以及胰腺内分泌细胞的变化，从而揭示胰腺内分泌部和外分泌部之间的相互作用机制。

结果

我们发现，胰腺癌中的胰腺内分泌细胞呈现出显著不同的转录组学特征，并且胰腺内分泌细胞的分化轨迹从邻近相对正常胰腺组织指向肿瘤内部，并与外分泌细胞的相互作用显著增强，提示胰腺内分泌细胞在胰腺癌发生过程中发生了系统性的改变。其中，胰多肽（Pancreatic polypeptide，PPY）阳性内分泌细胞随着胰腺癌病变的进展显著增加，并且细胞拟时序分析及谱系示踪实验结果表明PPY⁺细胞的增加可能源于胰腺α细胞和β细胞的转分化。从机制层面，胰腺癌细胞能够通过旁分泌生长分化因子15（Growth Differentiation Factor 15，GDF15）的方式诱导胰腺α细胞和β细胞转分化为PPY⁺内分泌细胞。这些细胞进一步以旁分泌PPY的方式作用于胰腺癌细胞表面的神经肽Y受体Y4（Neuropeptide Y Receptor Y4，NPY4R），激活胰腺癌细胞中的蛋白激酶C（Protein Kinase C，PKC）-丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase，MAPK）信号通路，从而促进胰腺癌的发生和发展。

结论

本研究系统地描绘了胰腺癌中胰腺内分泌细胞的改变，并阐明了胰腺癌发生过程中潜在的内分泌-外分泌相互作用机制。胰腺癌细胞通过旁分泌GDF15的方式诱导胰腺α细胞和β细胞转分化为PPY⁺内分泌细胞，这些PPY⁺内分泌细胞进一步通过激活胰腺癌细胞的NPY4R-PKC-MAPK信号通路促进胰腺癌的发生发展。同时，我们定义了癌症相关内分泌细胞（Cancer-associated Endocrine cells，CAEs）的概念并描绘其主要特征，从而进一步扩展了胰腺癌肿瘤微环境的主要成分，为胰腺癌的早期诊断和精准治疗提供了新思路。

Background and Aims

The pancreas is composed of endocrine and exocrine parts, and its interlacing structure suggests potential interactions between endocrine and exocrine cells. Although the tumor microenvironment of pancreatic cancer has been gradually revealed, most previous studies have focused on the roles of fibroblasts and immune cells in the development of pancreatic cancer. The unique component of the pancreas, pancreatic endocrine cells, has been neglected to some extent. Therefore, this study aims to explore whether pancreatic endocrine cells undergo changes during the carcinogenesis and development of pancreatic cancer. Meanwhile, we also investigate the upstream mechanisms of these changes and their impact on the carcinogenesis and development of pancreatic cancer.

Methods

We used single-cell RNA sequencing, spatial transcriptomic sequencing, and multiplex immunohistochemistry to depict changes of endocrine cells and their potential differentiation trajectories in pancreatic cancer at the clinical specimen level. Subsequently, we performed islet extraction and purification, single-cell RNA sequencing, and multiplex immunohistochemistry on Kras^LSL-G12Dp53^LoxPPdx1-CreER mice at different stages of pancreatic cancer to further reveal the main changes in pancreatic endocrine cells during pancreatic carcinogenesis. Meanwhile, we conducted multiplex immunohistochemistry on pancreatic tissues from Kras^LSL-G12DPdx1-Cre mice and orthotopic transplantation mice at different stages and performed lineage tracing experiments in Gcg-cre;Rosa26-loxP-tdTomato-loxP-EGFP mice to further validate the above findings. In addition, we knocked down key genes in pancreatic cancer cells and pancreatic endocrine cells. Then we conducted co-culture in vitro and co-injection in vivo experiments to observe changes of pancreatic cancer cells and pancreatic endocrine cells, thereby revealing the potential interaction mechanisms between the endocrine and exocrine compartments of the pancreas.

Results

We found that pancreatic endocrine cells in pancreatic cancer exhibited significantly different transcriptomic characteristics. The differentiation trajectory of pancreatic endocrine cells was from the adjacent normal tissue to the interior of the cancer. And there was a significantly elevated frequency of interactions between the endocrine and exocrine parts of the pancreas in pancreatic cancer compared with the adjacent normal tissue, indicating that pancreatic endocrine cells underwent systematic changes during pancreatic carcinogenesis. Among all changes, pancreatic polypeptide (PPY)-positive endocrine cells significantly increased with the progression of pancreatic cancer lesions. Pseudotime trajectory analysis and lineage tracing experiments suggested that the increase of PPY⁺ cells might originate from the transdifferentiation of pancreatic α and β cells. Mechanistically, pancreatic cancer cells could induce the transdifferentiation of pancreatic α and β cells into PPY⁺ endocrine cells through the paracrine action of growth differentiation factor 15 (GDF15). These cells further acted on the neuropeptide Y receptor Y4 (NPY4R) on the surface of pancreatic cancer cells through paracrine PPY, activating the protein kinase C (PKC)-mitogen-activated protein kinase (MAPK) signaling pathway in pancreatic cancer cells, thereby promoting the carcinogenesis and development of pancreatic cancer.

Conclusions

This study systematically depicted changes of pancreatic endocrine cells in pancreatic cancer and elucidated the potential mechanisms of endocrine-exocrine interactions in pancreatic cancer. Pancreatic cancer cells induce the transdifferentiation of pancreatic α and β cells into PPY⁺ endocrine cells through paracrine GDF15. These PPY⁺ endocrine cells further promote the carcinogenesis and development of pancreatic cancer by activating the NPY4R-PKC-MAPK signaling pathway in pancreatic cancer cells. Meanwhile, we defined and characterized cancer-associated endocrine cells (CAEs), thereby further broadening the composition of pancreatic cancer microenvironment, which might provide novel insights for the early diagnosis and precise treatment of pancreatic cancer.