患者来源的CSF和腹水类器官揭示晚期胃癌中不同的转移机制

Gastric cancer most frequently metastasizes to the peritoneum (PM), whereas leptomeningeal metastasis (LM) is extremely rare but highly lethal. Although LM shows markedly worse outcomes than PM, the mechanisms driving LM remain poorly understood due to the absence of appropriate experimental models. To address this limitation, we established organoid models derived from PM and LM from the same patient and compared their morphological and genomic characteristics. Organoids were generated from ascites and Cerebrospinal fluid obtained from a 50-years old female patient with stage IV diffuse-type gastric cancer, with passage 5 defined as the establishment point. Organoid cultures were initiated under optimized 3D conditions and maintained through multiple passages to ensure stability and expansion. Morphological and phenotypic features were characterized using 3D bright-field imaging, scanning electron microscopy (SEM), and hematoxylin and eosin (H&E) staining. For molecular characterization, targeted panel sequencing and whole-genome sequencing were performed. Copy number variations of selected genes were validated by qPCR. The ascites-derived organoids (AS2 and AS4) required approximately 34-39 days to reach establishment, while the Cerebrospinal fluid-derived organoid (CSFO) required about 39 days. At morphological characterization, AS2 formed a lumen structure and displayed pronounced pseudopodia with elongated cellular shape, indicative of stronger cell-cell interactions. In contrast, CSFO and AS4 exhibited rounder cellular shapes with grape-like clustered architectures and weaker intercellular adhesion. Despite the different metastatic microenvironments, 136 mutations common among all organoids, 20 mutations were CSFO-specific, 19 mutations were AS2-specific, and 28 mutations were AS4-specific. Subsequently, we conducted Gene Ontology analysis. CSFOs were enriched for mutations associated with vascular morphogenesis. In contrast, ASOs harbored mutations related to epithelial proliferation and organ development. Patient-derived peritoneal tissues exhibited mutations predominantly in immune-regulatory pathways, including MHC class I/II-mediated antigen presentation. Notably, MET amplification and CDKN2A deletion were consistently detected in all organoid models but not in the original patient tissues, highlighting culture-associated genomic selection. MAP2K4 deletion was observed in CSFOs and GO15-5, while MYCN and ADGRA2 amplification were detected only in GO15-5, suggesting additional clonal evolution during extended in vitro passage, and genomic variants were validated using qPCR. Overall, this study demonstrates the feasibility of using patient-derived organoids to model atypical metastatic routes of gastric cancer and provides novel insights into the biology of LM through integrative morphological and genomic analyses.