Generation and genome analysis of patient-derived pancreatic cancer cell line using conditionally reprogrammed cell method

Abstract

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy. One of the greatest challenges in PDAC research has been the generation of stable cancer cell lines from primary tumors. Conditionally reprogrammed cell (CRC) technology is a novel cell-culture system enabling the generation of stable cell cultures. CRCs can be grown indefinitely under defined conditions without the use of genetic immortalization techniques. The objective of this study was to utilize the CRC technology in human PDAC specimens to develop cancer cell lines that phenotypically represent native tumors. METHODS: We obtained the tumor specimens from resection specimens for patients with operable PDAC and from endoscopic ultrasound-guided biopsy or percutaneous liver biopsy for patients with inoperable PDAC. Cancer cells were co-cultured with irradiated feeder cells and the Rho-associated kinase inhibitor Y-27632 to develop CRCs. To enable the rational design and testing of patient-origin cancer cell lines for patients with PDAC, we analyzed the CRCs at the genetic level by whole exome sequencing. In vivo, NOD/SCID mice (5-week-old male) were injected with CRCs (2 × 106) in the right and left flank. After sacrifice, the tumor size was measured and implanted tumor tissue was fixed in paraformaldehyde for histologic analysis. RESULTS: Sixteen (34.8%) CRCs were established from 46 PDAC patients. We confirmed that the genetic characteristics of cancer tissues were preserved in CRCs by comparing the AFs of somatic mutations found in cancer tissues and CRCs. Mutation profiles were 100% concordant between original PDAC tissue and CRCs for 3 patients with PDAC. In vivo, 3 CRCs were implanted in NOD/SCID mice, and routine hematoxylin and eosin histology revealed that the implanted tumor tissue showed identical morphology to the parent tumor tissue. The allele frequency of key oncogenic mutations common to both tumor and CRCs, including TP53, SMAD4, and KRAS, was either higher in CRCs or similar in both groups. CONCLUSIONS: We established the first PDAC cell line system to represent original PDAC tissue. The ability to rapidly generate patient-origin cancer cells from small tumor specimens would facilitate the development of individualized treatment and could be used to identify effective drug combinations for PDAC.