Generation and genome analysis of patient-derived pancreatic cancer cell line using conditionally reprogrammed cell method
Other Titles
조건부 재편집 세포 배양법을 이용한 췌장암 환자 유래 종양 세포주의 형성 및 유전체 분석
Authors
이희승
College
Graduate School, Yonsei University
Department
Dept. of Medicine
Degree
박사
Issue Date
2017
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.