Establishment of pancreatic cancer cell lines with endoscopic ultrasound‐guided biopsy via conditionally reprogrammed cell culture

Abstract

Recent studies have identified the mutational landscape of pancreatic cancer and suggested tumor‐specific subtypes. However, the major hurdle against personalized treatment is the difficulty to obtain sufficient cancer tissues from most inoperable cases. We investigated whether patient‐derived conditionally reprogrammed cells (CRCs) can be constructed using a small piece of tumor tissue using endoscopic ultrasound (EUS)‐guided fine needle biopsy (FNB). Thirty patients with pancreatic solid mass (mean size, 34.6 mm) were enrolled prospectively. Among 22 patients who were diagnosed with pancreatic ductal adenocarcinoma, we established patient‐derived pancreatic cancer cell lines from eight patients (36.4%). Immunofluorescence colony staining for CRCs showed that the cytoplasm of cancer cells was clearly stained with anti‐cytokeratin 19 monoclonal antibody. In the soft agar colony formation assay, CRCs formed colonies compared with the negative control by day 15. In vivo, implanted CRCs showed tumor engraftment and hematoxylin and eosin staining showed pancreatic cancer ductal structure. All established CRCs showed a KRAS mutation. In conclusion, we established patient‐derived pancreatic cancer cell lines with a small tumor tissue obtained by EUS‐FNB. With in vitro drug sensitivity and genomic studies, established patient‐derived cell lines can be used in identification of new targets for diagnosis and treatment of pancreatic cancer.

Preclinical models derived from endoscopic ultrasound-guided tissue acquisition for individualized treatment of pancreatic ductal adenocarcinoma

“…Since there is no study report on the number of cells, grams, milliliters, or size required to successfully culture the preclinical model, we used the number of passes as a criterion to assess the amount of samples required to culture the preclinical model. The study of Lee et al showed that there was no statistically significant difference (87.5 vs. 100%, P = 0.58) between two passes and three passes in acquiring the histological core when used a 20-gauge (G) FNB needle (EchoTip ProCore™ Endoscopic Ultrasound Needle, Cook Medical Inc., Bloomington, IN, USA), both of which could successfully establish the patient-derived cell (PDC) model ( 43 ). In another study, the endoscopists determined the number of passes according to the results of macroscopic on-site evaluation (MOSE), and compared the different success rates of PDAC organoid isolation (P0) and establishment (P5) in cell culture from a single FNB (22-gauge, SharkCore FNB Exchange System, Medtronic Inc., Minneapolis, MN, USA; or Acquire FNB Device, Boston Scientific Inc., Marlborough, MA, USA) pass and a double FNB pass.…”

“…In different models, the success rate in establishing patient-derived preclinical models ranges from 36.4 to 87% ( Table 2 ). Previously, Lee et al showed that the establishment rate of PDC was 36.4% (8/22) using the FNB samples ( 43 ). Hermans et al showed that the engraftment rate of FNB samples (6/10, 60%) was lower than that of surgical samples (4/4, 100%).…”

Conditional Cell Reprogramming in Modeling Digestive System Diseases

“…Research by Lee et al (2019Lee et al ( , 2021 showed that CR technology can be used to establish pancreatic cancer cell lines through biopsies guided by endoscopic ultrasound. CRCs from pancreatic cancer tissues have the same genetic characteristics as those from the primary tumor and can be used for genomic research and drug sensitivity studies and can identify new diagnostic and therapeutic targets for pancreatic cancer (Lee et al, 2019(Lee et al, , 2021. The PDX model has become an important tool for translational research, but there are some limitations, including unsustainable in vitro growth.…”

“…However, tissue samples can only be obtained from many advanced cancer patients through fine-needle or core needle biopsy. Research by Lee et al (2019 , 2021) showed that CR technology can be used to establish pancreatic cancer cell lines through biopsies guided by endoscopic ultrasound. CRCs from pancreatic cancer tissues have the same genetic characteristics as those from the primary tumor and can be used for genomic research and drug sensitivity studies and can identify new diagnostic and therapeutic targets for pancreatic cancer ( Lee et al, 2019 , 2021 ).…”

Culture and application of conditionally reprogrammed primary tumor cells

“…Conditional reprogramming (CR) is a novel method of co-culturing epithelial cells with irradiated feeder cells in the presence of a Rho-associated coiled-coil kinase (ROCK) inhibitor, which achieves rapid and sustained expansion of primary cancerous and normal epithelial cells [ 8 , 9 ]. These reprogrammed immortalized cells of malignant tumors [ 10 ], such as bladder cancer [ 11 ], prostate cancer [ 12 ], pancreatic cancer [ 13 ], breast carcinoma [ 14 ], and hepatocellular carcinoma [ 15 ], without genetic manipulation or chromosomal abnormalities, represent an adult stem-cell-like state but express fairly low levels of Sox2 , Oct4 , and Klf4 [ 16 ], which are the pluripotent progenitor markers [ 17 ]. What is more, these non-tumorigenic cells can maintain intra-tumor heterogeneity [ 18 ] in addition to keeping their molecular features [ 19 , 20 ], and are only capable of differentiating into the native tissues in which they originated [ 16 , 21 ].…”