{"doi":"10.1016/j.xpro.2025.104133","title":"Protocol for isolating patient-derived ascites cells and extracellular vesicles from gastric cancer peritoneal metastases","abstract":"Peritoneal carcinomatosis (PC) cells and extracellular vesicles (EVs) from gastric cancer ascites are valuable for studying tumor-stroma interactions. Here, we present a protocol for isolating PC cells and EVs from patient ascites. We describe steps for PC cell culture, iodixanol-based EV purification, electron microscopy, nanoparticle tracking analysis, flow cytometry, and proteomic profiling. This protocol also includes TD-139 loading into exosomes for functional assays to evaluate their role in modulating the tumor microenvironment. For complete details on the use and execution of this protocol, please refer to Fan et al. 1 • Isolation and culture of patient-derived PC cells from malignant ascites • Density gradient ultracentrifugation for extracellular vesicle purification • Extracellular vesicle characterization by TEM, NTA, and flow cytometry • Proteomic profiling of extracellular vesicles using timsTOF HTmass spectrometry (Bruker) Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Peritoneal carcinomatosis (PC) cells and extracellular vesicles (EVs) from gastric cancer ascites are valuable for studying tumor-stroma interactions. Here, we present a protocol for isolating PC cells and EVs from patient ascites. We describe steps for PC cell culture, iodixanol-based EV purification, electron microscopy, nanoparticle tracking analysis, flow cytometry, and proteomic profiling. This protocol also includes TD-139 loading into exosomes for functional assays to evaluate their role in modulating the tumor microenvironment.","journal":"STAR Protocols","year":2025,"id":547728,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9453,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":295085,"name":"Jody V. Vykoukal","orcid":"0000-0001-7797-627X","position":1,"is_corresponding":false},{"id":265913,"name":"Shumei Song","orcid":"0000-0002-8128-6287","position":2,"is_corresponding":false},{"id":342225,"name":"Melissa Pool Pizzi","orcid":"0000-0002-2373-0052","position":3,"is_corresponding":false},{"id":991030,"name":"Gengyi Zou","orcid":"0000-0002-5399-8674","position":4,"is_corresponding":false},{"id":1017343,"name":"Katsuhiro Yoshimura","orcid":null,"position":5,"is_corresponding":false},{"id":342227,"name":"Jiankang Jin","orcid":"0000-0001-9864-9817","position":6,"is_corresponding":false},{"id":265911,"name":"Hiroyuki Katayama","orcid":"0000-0003-3117-1390","position":7,"is_corresponding":false},{"id":44983,"name":"George A. Calin","orcid":"0000-0002-7427-0578","position":8,"is_corresponding":false},{"id":1205661,"name":"Rebecca Waters","orcid":"0000-0002-8163-7497","position":9,"is_corresponding":false},{"id":445196,"name":"Qiong Gan","orcid":"0000-0001-6563-5611","position":10,"is_corresponding":false},{"id":14192,"name":"Linghua Wang","orcid":"0000-0001-9380-0266","position":11,"is_corresponding":false},{"id":57918,"name":"Samir Hanash","orcid":"0000-0002-4210-1593","position":12,"is_corresponding":false},{"id":1034911,"name":"Shilpa S. Dhar","orcid":"0000-0002-5922-2164","position":13,"is_corresponding":false},{"id":1440947,"name":"J. A. Ajani","orcid":null,"position":14,"is_corresponding":false},{"id":719082,"name":"Yibo Fan","orcid":"0000-0001-7589-4158","position":0,"is_corresponding":true}],"reference_count":7,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:53:53.962932Z","pmid":"41105517","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}