背景及目的：胰腺癌，尤其是胰腺导管腺癌（Pancreatic ductal adenocarcinoma，PDAC），是消化系统中最具致命性的恶性肿瘤之一，其发展迅速且预后极为不良。由于其独特而复杂的肿瘤微环境（Tumor microenvironment，TME），临床上PDAC患者的治疗效果十分有限。在这一环境中，活化的胰腺星形细胞（Pancreatic stellate cells，PSCs）是最主要的间质细胞类型之一，随着肿瘤的恶性演化，PSCs发生动态变化。不同亚群的PSCs与胰腺癌细胞（Pancreatic cancer cells，PCCs）之间建立复杂的信号转导，促进胰腺癌生长和转移。课题组前期构建了胰腺癌演进的单细胞转录组图谱，挖掘出半胱氨酸结分泌型蛋白（Gremlin 1，GREM1）阳性的关键PSCs亚群，并且体外实验发现PSCs旁分泌GREM1促进胰腺癌细胞的侵袭转移。本实验旨在研究GREM1⁺PSCs介导的间质与肿瘤细胞的相互作用及其具体机制，从代谢重编程及表观遗传修饰两个维度深入解析胰腺癌侵袭转移的新机制。

方法：

本研究收集胰腺癌演进的新鲜样本，绘制单细胞转录组（scRNA-seq）图谱，深入挖掘GREM1与组织激肽释放酶10（Kallikrein-related peptidase 10，KLK10）分子在各个细胞类型的表达特征、与胰腺癌演进的关系，对GREM1、KLK10表达进行相关性分析并对强相关基因进行富集分析，对scRNA-seq进行非负矩阵分解（Non-negative matrix factorization，NMF）分析并计算Jaccard相似性系数以识别关键基因表达模块，进行SCENIC分析以识别与胰腺癌演进相关的转录因子。

收集外部PDAC患者数据集，包括TCGA转录组数据和CPTAC蛋白组数据，分析GREM1、KLK10与临床病理特征、预后的相关性。

收集临床PDAC患者的福尔马林固定和石蜡包埋（Formalin-fixed paraffin-embedded，FFPE）样本，进行KLK10蛋白的免疫组织化学（Immunohistochemistry，IHC）检测和多重荧光免疫组化（Multiplex immunohistochemical，mIHC）检测。

采用人PDAC组织分离的活化PSCs、ATRA处理的静止PSCs与PCCs，比较GREM1、KLK10分别在PSCs与PCCs的表达水平、GREM1在活化与静止PSCs之间的表达差异。

使用GREM1重组蛋白、PSCs条件培养基、PSCs-PCCs直接共培养处理PCCs建立体外共培养模型，检测下游分子的表达，针对PCCs增殖、迁移以及侵袭的恶性表型进行探究，并进一步检测了其对上皮间质转化（Epithelial-mesenchymal transition，EMT）表型的影响。

使用GREM1重组蛋白、梯度葡萄糖、乳酸钠及糖酵解抑制剂处理PCCs，检测泛乳酸化（Pan-Kla）、组蛋白H3K18乳酸化（H3K18la）、KLK10的表达变化。综合利用H3K18A突变质粒、CUT&Tag测序、CUT-qPCR等技术，明确H3K18la对KLK10的转录调控作用及直接结合位点。

通过Jaspar网站预测转录因子STAT1与GLUT1启动子区域的结合位点，并使用双荧光素酶报告基因实验验证STAT1与GLUT1启动子的直接结合作用。

结果：

scRNA-seq分析显示，GREM1在PSCs中特异性高表达，且GREM1⁺PSCs表现出肌成纤维细胞样活化特征，是胰腺癌演进的关键细胞亚群。

将随胰腺癌演进而在恶性导管上皮中显著上调的基因与GREM1⁺PSCs表达强相关的基因进行交集分析，筛选出GREM1的下游潜在效应分子KLK10。scRNA-seq分析显示，KLK10仅在恶性导管上皮细胞中特异性高表达，且其表达水平随胰腺癌演进显著上调。

PSCs通过旁分泌GREM1显著上调肿瘤细胞中KLK10的表达，进而驱动胰腺癌侵袭和转移。

临床队列分析显示，GREM1、KLK10高表达均与不良病理特征、较差的OS和PFS显著相关，且GREM1与KLK10的联合高表达是PDAC患者预后的独立危险因素。

肿瘤细胞糖酵解活性与GREM1⁺PSCs、KLK10表达均显著相关，GREM1处理上调葡萄糖转运蛋白1（Glucose transporter type 1，GLUT1）表达、促进乳酸产生，回复实验证实GLUT1是GREM1调控KLK10表达的必要条件。

肿瘤细胞糖酵解通过AARS1介导组蛋白乳酸化修饰调控KLK10高表达，机制上，H3K18A突变质粒、CUT&Tag测序和CUT-qPCR实验明确H3K18la与KLK10启动子区域结合。

GREM1激活肿瘤细胞的FGFR1/JAK1-2/STAT1信号通路调控GLUT1的表达，双荧光素酶报告基因实验明确转录因子STAT1与GLUT1启动子区域直接结合、并促进其转录活性。

结论：深入探索TME中PSCs与肿瘤细胞的相互作用机制，有助于挖掘联合靶向PSCs-PCCs轴的新策略，为胰腺癌精准治疗提供科学依据。本研究发现，在胰腺癌演进过程中，GREM1⁺PSCs旁分泌GREM1增多，通过肿瘤细胞FGFR1/JAK1-2/STAT1信号通路促进GLUT1表达，激活有氧糖酵解表型，促进AARS1介导的组蛋白乳酸化修饰，上调KLK10的表达，最终推动胰腺癌的侵袭与转移。

Background and Objective: Pancreatic cancer, especially pancreatic ductal adenocarcinoma (PDAC), is one of the most fatal malignant tumors in the digestive system. It develops rapidly and has an extremely poor prognosis. Due to its unique and complex tumor microenvironment (TME), the therapeutic effect of PDAC patients in clinical practice is very poor. In the microenvironment, activated pancreatic stellate cells (PSCs) are one of the main cell types. They exhibit dynamic changes and engage in continual crosstalk with pancreatic cancer cells (PCCs), contributing to tumor progression in PDAC. A single-cell transcriptomic (scRNA-seq) atlas of pancreatic cancer progression was established, revealing a critical subpopulation of PSCs characterized by Gremlin 1 (GREM1) expression. The paracrine secretion of GREM1 by PSCs significantly promoted the migration and invasion of PCCs. This study aims to investigate the stromal-PCCs interactions mediated by GREM1⁺PSCs, exploring novel molecular mechanisms underlying pancreatic cancer invasion and metastasis, with a focus on metabolic reprogramming and epigenetic modifications.

Methods:

1. In this study, using scRNA-seq atlas, the expression patterns of GREM1 and Kallikrein-related peptidase 10 (KLK10) across different cell types and their association with pancreatic cancer progression were investigated. Correlation analyses were performed to assess the relationship between GREM1 and KLK10 expression, followed by enrichment analysis of highly correlated genes. Additionally, NMF and Jaccard similarity coefficient analyses were applied to identify key gene expression modules. SCENIC analysis was performed to identify transcription factors associated with progression.

2. Transcriptomic data from The Cancer Genome Atlas (TCGA) and proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) for PDAC were collected and analyzed to evaluate the relationship between the expressions of GREM1 and KLK10, along with their association with clinicopathological characteristics and prognostic significance in PDAC.

3. Formalin-fixed, paraffin-embedded (FFPE) clinical PDAC samples were collected for immunohistochemistry (IHC) and multiplex immunohistochemical (mIHC) detecting.

4. Activated PSCs isolated from human PDAC fresh tissues, ATRA-treated quiescent PSCs, and PCCs were used to compare the expression levels of GREM1 and KLK10 in PSCs and PCCs, as well as the differential expression of GREM1 between activated and quiescent PSCs.

5. In vitro co-culture models were established by treating PCCs with recombinant GREM1, PSC-conditioned medium, and direct PSCs-PCCs co-culture. The expression of epithelial-mesenchymal transition (EMT) markers, along with the assessment of PCCs proliferation, migration, and invasion capabilities were evaluated.

6. PCCs were treated with recombinant GREM1, glucose, sodium lactate, and glycolysis inhibitors to evaluate the relationship between pan-lactylation (Pan-Kla), histone H3K18 lactylation (H3K18la), and KLK10 expression. H3K18A-mutant plasmid, CUT&Tag sequencing, and CUT-qPCR were used to explore the transcriptional regulatory role of H3K18la on KLK10, and identify its direct binding sites within the KLK10 promoter region.

7. The binding sites of the transcription factor STAT1 within the promoter region of GLUT1 were predicted using the Jaspar database. Dual-luciferase reporter gene assays were subsequently performed to validate the direct interaction between STAT1 and the promoter of GLUT1.

Results:

1. The analysis of scRNA-seq data revealed that GREM1 is specifically highly expressed in PSCs, which is a key cellular subpopulation in pancreatic cancer progression. GREM1⁺PSCs exhibited myofibroblast-like characteristics.

2. By intersecting genes that are significantly upregulated in malignant ductal epithelial cells during progression with those strongly positively correlated with GREM1⁺PSCs, we identified KLK10 as a potential downstream effector of GREM1. ScRNA-seq data revealed that KLK10 is specifically and highly expressed in malignant ductal epithelial cells, with its expression significantly increasing as pancreatic cancer progresses.

3. KLK10 expression in PCCs was significantly upregulate through paracrine secretion of GREM1 from PSCs, thereby promoting PCCs migration and invasion.

4. PDAC patients with high GREM1 or KLK10 expression were associated with higher pathological grades, more advanced T stages, and poorer prognoses.

5. Glycolysis activity in PCCs was significantly correlated with the presence of GREM1⁺PSCs and KLK10 expression. GREM1 treatment upregulated GLUT1 expression, thereby enhancing lactate production. Rescue experiments confirmed that GLUT1 is an essential factor for GREM1-induced KLK10 expression.

6. Glycolysis in PCCs induced the high expression of KLK10 through AARS1-mediated histone lactylation modification. Mechanistically, H3K18A mutant plasmids, CUT&Tag sequencing, and CUT-qPCR experiments confirmed that H3K18la binds to the promoter region of KLK10, leading to the upregulation of KLK10 expression in PCCs.

7. The FGFR1/JAK1-2/STAT1 signaling pathway in PCCs was activated by GREM1 to regulate the expression of GLUT1. Dual-luciferase reporter assays confirmed that the transcription factor STAT1 directly binds to the GLUT1 promoter region and enhances its transcriptional activity.

Conclusions: Elucidating the interaction mechanisms between PSCs and tumor cells within the TME may contribute to the development of promising therapeutic strategies for combined targeting of the PSCs-PCCs axis. In this study, GREM1⁺PSCs increased as pancreatic cancer progression and enhanced GLUT1 expression through the FGFR1/JAK1-2/STAT1 signaling pathway in PCCs. The upregulation of GLUT1 promoted the aerobic glycolysis phenotype, facilitated AARS1-mediated histone lactylation, upregulated KLK10 expression, and ultimately contributed to pancreatic cancer invasion and metastasis.