Protocol for the creation and utilization of 3D pancreatic cancer models from circulating tumor cells

Jiajia Tang; Quan Zheng; Qi Wang; Yaru Zhao; Preeta Ananthanarayanan; Chiara Reina; Berina Šabanović; Ke Jiang; Ming-Hsin Yang; Clara Csilla Meny; Huimin Wang; Mette Ø Agerbaek; Thomas Mandel Clausen; Tobias Gustavsson; Chenlei Wen; Felice Borghi; Alfredo Mellano; Elisabetta Fenocchio; Vanesa Gregorc; Anna Sapino; Thor G Theander; Da Fu; Alexandra Aicher; Ali Salanti; Baiyong Shen; Christopher Heeschen

doi:10.1016/j.xpro.2025.103635

Protocol for the creation and utilization of 3D pancreatic cancer models from circulating tumor cells

STAR Protoc. 2025 Mar 21;6(1):103635. doi: 10.1016/j.xpro.2025.103635. Epub 2025 Feb 11.

Authors

Jiajia Tang¹, Quan Zheng², Qi Wang², Yaru Zhao², Preeta Ananthanarayanan³, Chiara Reina³, Berina Šabanović³, Ke Jiang², Ming-Hsin Yang⁴, Clara Csilla Meny⁵, Huimin Wang², Mette Ø Agerbaek⁶, Thomas Mandel Clausen⁷, Tobias Gustavsson⁸, Chenlei Wen⁹, Felice Borghi¹⁰, Alfredo Mellano¹⁰, Elisabetta Fenocchio¹¹, Vanesa Gregorc¹¹, Anna Sapino¹², Thor G Theander⁷, Da Fu⁹, Alexandra Aicher¹³, Ali Salanti⁷, Baiyong Shen⁹, Christopher Heeschen¹⁴

Affiliations

¹ Center for Single-Cell Omics, School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; State Key Laboratory of Systems Medicine for Cancer, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. Electronic address: jiajia.tang@shsmu.edu.cn.
² Center for Single-Cell Omics, School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; State Key Laboratory of Systems Medicine for Cancer, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
³ Pancreatic Cancer Heterogeneity, Candiolo Cancer Institute - FPO - IRCCS, 10060 Candiolo (TO), Italy.
⁴ Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan.
⁵ 2(nd) Institute for Pathology and Experimental Oncology Research, Semmelweis University, 1085 Budapest, Hungary.
⁶ Centre for Translational Medicine and Parasitology (CMP) at Department of Immunology and Microbiology, University of Copenhagen, 2200 Copenhagen N, Denmark; VarCT Diagnostics, Ole Maaloes Vej 3, 2200 Copenhagen, Denmark.
⁷ Centre for Translational Medicine and Parasitology (CMP) at Department of Immunology and Microbiology, University of Copenhagen, 2200 Copenhagen N, Denmark.
⁸ Centre for Translational Medicine and Parasitology (CMP) at Department of Immunology and Microbiology, University of Copenhagen, 2200 Copenhagen N, Denmark; VAR2Pharmaceuticals, Ole Maaloes Vej 3, 2200 Copenhagen, Denmark.
⁹ Research Institute of Pancreatic Disease, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; Pancreatic Disease Centre, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
¹⁰ Department of Surgical Oncology, Candiolo Cancer Institute - FPO - IRCCS, 10060 Candiolo (TO), Italy.
¹¹ Department of Medical Oncology, Candiolo Cancer Institute - FPO - IRCCS, 10060 Candiolo (TO), Italy.
¹² Department of Pathology, Candiolo Cancer Institute - FPO - IRCCS, 10060 Candiolo (TO), Italy.
¹³ Precision Immunotherapy, Graduate Institute of Biomedical Sciences, China Medical University, Taichung 404328, Taiwan.
¹⁴ Center for Single-Cell Omics, School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; State Key Laboratory of Systems Medicine for Cancer, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Pancreatic Cancer Heterogeneity, Candiolo Cancer Institute - FPO - IRCCS, 10060 Candiolo (TO), Italy. Electronic address: christopher.heeschen@icloud.com.

Abstract

We introduce a protocol for generating 3D organoids from circulating tumor cells (CTCs), enabling longitudinal functional and molecular analyses in pancreatic cancer patients, including those with unresectable disease, which constitutes the majority of cases. We outline the process for isolating and characterizing CTCs from the blood of pancreatic cancer patients and provide detailed instructions for initiating, passaging, and phenotyping CTC-derived organoids. Additionally, we describe techniques for utilizing these organoids in drug screening with a focus on stemness-related pathways. For complete details on the use and execution of this protocol, please refer to Tang et al.¹.

Keywords: Cancer; Cell-based Assays; Single Cell.

MeSH terms

Cell Culture Techniques, Three Dimensional* / methods
Cell Line, Tumor
Humans
Neoplastic Cells, Circulating* / metabolism
Neoplastic Cells, Circulating* / pathology
Organoids* / pathology
Pancreatic Neoplasms* / metabolism
Pancreatic Neoplasms* / pathology