The standard therapy for advanced hepatocellular carcinoma (HCC) is sorafenib, with most patients experiencing disease progression within 6 months. Label-retaining cancer cells (LRCC) represent a novel subpopulation of cancer stem cells (CSC). The objective was to test whether LRCC are resistant to sorafenib.
Methods
We tested human HCC derived LRCC and non-LRCC before and after treatment with sorafenib.
Results
LRCC derived from human HCC are relatively resistant to sorafenib. The proportion of LRCC in HCC cell lines is increased after sorafenib while the general population of cancer cells undergoes growth suppression. We show that LRCC demonstrate improved viability and toxicity profiles, and reduced apoptosis, over non-LRCC. We show that after treatment with sorafenib, LRCC upregulate the CSC marker aldehyde dehydrogenase 1 family, wingless-type MMTV-integration-site family, cell survival and proliferation genes, and downregulate apoptosis, cell cycle arrest, cell adhesion and stem cells differentiation genes. This phenomenon was accompanied by non-uniform activation of specific isoforms of the sorafenib target proteins extracellular-signal-regulated kinases and v-akt-murine-thymoma-viral-oncogene homologue (AKT) in LRCC but not in non-LRCC. A molecular pathway map for sorafenib treated LRCC is proposed.
Conclusions
Our results suggest that HCC derived LRCC are relatively resistant to sorafenib. Since LRCC can generate tumours with as few as 10 cells, our data suggest a potential role for these cells in disease recurrence. Further investigation of this phenomenon might provide novel insights into cancer biology, cancer recurrence and drug resistance with important implications for the development of novel cancer therapies based on targeting LRCC.
Abstract Cancer stem cells (CSC) are thought to be responsible for cancer initiation, maintenance, and therapeutic failure. A hallmark of stem cells is self-renewal via asymmetric cell division (ACD) into daughter stem cells and cells predestined for differentiation. In addition, recent reports questioned the ability of the gold standard for testing CSC (xenotransplantation into immunodeficient mice) to truly detect and test CSC. The aim of this study was to demonstrate fundamental stem cell's traits such as ACD with non-random-chromosomal cosegregation (ACD-NRCC) in cancers. Here, we show that a unique self-renewal mechanism i.e. ACD-NRCC occurs in various human cancers using DNA double labeling and confocal microscopy. ACD-NRCC was found exclusively in a subpopulation of CSC (side population and CD133+ cells) and not in the non-cancer-stem-cells fractions. Cells that demonstrated ACD-NRCC also exhibited superior tumor initiation capacity in nude mice (p=0.028). Furthermore, we found that the niche provided by the non-stem cancer cells directly regulates self-renewal via ACD-NRCC. This regulation is dependent on a potentially novel heat sensitive soluble factor (Self Renewal Factor, SRF). Gene expression microarray data showed down-regulation of genes associated with ACD in tested cancers. Detection of ACD-NRCC in various human cancers provides direct evidence for the existence of cancer cells with unique stem cells traits. This is the first report to demonstrate the fundamental stem cells trait of ACD-NRCC in human cancer. Detection of cells capable of undergoing ACD-NRCC in various cancers potentially defines a novel class of CSC with superior tumor initiating capacity. Using ACD-NRCC, we propose a universal method for testing, isolating and targeting CSC. The gene expression microarray data suggested that faulty mechanisms of ACD-NRCC might result in stem cells derived cancers. Further purification of the niche's self-renewal-signals and utilizing our methodology for isolation of CSC have far reaching implications in regenerative medicine, cancer genetics and potential novel cancer therapeutics targeting CSC via ACD-NRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 11.
The ability to retain DNA labels over time is a property proposed to be associated with adult stem cells. Recently, label retaining cells (LRC) were indentified in cancer. LRC were suggested to be the result of either slow-cycling or asymmetric-cell-division with nonrandom-chromosomal-cosegregation (ACD-NRCC). ACD-NRCC is proposed to segregate the older template DNA strands into daughter stem cells and newly synthesized DNA into daughter cells destined for differentiation. The existence of cells undergoing ACD-NRCC and the stem-like nature of LRC remain controversial. Currently, to detect LRC and ACD-NRCC, cells need to undergo fixation. Therefore, testing the stem-cell nature and other functional traits of LRC and cells undergoing ACD-NRCC has been limited. Here, we show a method for labeling DNA with single and dual-color nucleotides in live human liver cancer cells avoiding the need for fixation. We describe a novel methodology for both the isolation of live LRC and cells undergoing ACD-NRCC via fluorescence-activated cell sorting with confocal microscopy validation. This has the potential to be a powerful adjunct to stem-cell and cancer research.
Choledochal cysts are rare congenital dilations of the biliary system. They are more common in East Asia than in the West. Choledochal cysts have been traditionally classified into 5 types.1Todani T. Watanabe Y. Narusue M. et al.Congenital bile duct cysts: classification, operative procedures, and review of thirty-seven cases including cancer arising from choledochal cyst.Am J Surg. 1977; 134: 263-269Abstract Full Text PDF PubMed Scopus (1147) Google Scholar A 6th type of choledochal cyst has been described as either a cyst arising off the cystic duct or a dilation of the cystic duct (Fig. 1). Data on type VI choledochal cysts are limited, with fewer than 25 cases reported. Malignant transformation of these cysts has been described; from a review of the literature, malignancy was found in 2 of 15 adult patients and 0 of 9 pediatric patients.2Bode W.E. Aust J.B. Isolated cystic dilatation of the cystic duct.Am J Surg. 1983; 145: 828-829Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 3Champetier P. Partensky C. Ponchon T. Cystic malformations of the cystic duct.Surg Radiol Anat. 1987; 9: 287-291Crossref PubMed Scopus (10) Google Scholar, 4Kise Y. Uetsuji S. Takada H. et al.Dilatation of the cystic duct with its congenital low entry into the common hepatic duct.Am J Gastroenterol. 1990; 85: 769-770PubMed Google Scholar, 5Serena Serradel A.F. Santamaría Linares E. Herrera Goepfert R. Cystic dilatation of the cystic duct: a new type of biliary cyst.Surgery. 1991; 109: 320-322PubMed Google Scholar, 6Bresciani C. Gama-Rodrigues J. Santos V.R. Video-laparoscopic treatment of a sizeable cyst of the cystic duct: a case report.Surg Laparosc Endosc. 1998; 8: 376-379Crossref PubMed Scopus (15) Google Scholar, 7Loke T.K. Lam S.H. Chan C.S. Choledochal cyst: an unusual type of cystic dilatation of the cystic duct.AJR Am J Roentgenol. 1999; 173: 619-620Crossref PubMed Scopus (36) Google Scholar, 8Conway W.C. Telian S.H. Wasif N. et al.Type VI biliary cyst: report of a case.Surg Today. 2009; 39: 77-79Crossref PubMed Scopus (20) Google Scholar, 9Chan E.S. Auyang E.D. Hungness E.S. Laparoscopic management of a cystic duct cyst.JSLS. 2009; 13: 436-440PubMed Google Scholar, 10Yoon J.H. Magnetic resonance cholangiopancreatography diagnosis of choledochal cyst involving the cystic duct: report of three cases.Br J Radiol. 2011; 84: e18-e22Crossref PubMed Scopus (22) Google Scholar, 11De U. Das S. Sarkar S. Type VI choledochal cyst revisited.Singapore Med J. 2011; 52: e91-e93PubMed Google Scholar, 12Maheshwari P. Cystic malformation of cystic duct: 10 cases and review of literature.World J Radiol. 2012; 4: 413-417Crossref PubMed Google Scholar, 13Shah O.J. Shera A. Shah P. et al.Cystic dilatation of the cystic duct: a type 6 biliary cyst.Indian J Surg. 2013; 75: 500-502Crossref PubMed Scopus (15) Google Scholar The overall rate of malignancy ranges from 5% to 7.5%, with the highest rates reported in types I and IV, and the lowest rates in types II and III.14Ten Hove A. de Meijer V.E. Hulscher J.B.F. et al.Meta-analysis of risk of developing malignancy in congenital choledochal malformation.Br J Surg. 2018; 105: 482-490Crossref PubMed Scopus (42) Google Scholar Type VI choledochal cysts are typically managed with cholecystectomy. The definitive diagnosis of type VI choledochal cysts is usually made intraoperatively; the differentiation between type VI and type II cysts can be challenging on preoperative imaging. Here we present the case of a type VI choledochal cyst diagnosed by ERCP with direct cholangioscopy, allowing for preoperative planning for laparoscopic cholecystectomy, instead of cyst resection with bile duct reconstruction by means of Roux-en-Y hepaticojejunostomy appropriate for a type II choledochal cyst. A 62-year-old Korean woman was evaluated for abdominal pain. Her liver function test results were as follows: alkaline phosphatase 84 U/L, aspartate aminotransferase 20 U/L, alanine aminotransferase 12 U/L, and total bilirubin 0.6 mg/dL. MRCP imaging revealed a 2- × 2-cm cystic structure adjacent to the cystic duct, common bile duct, and duodenum, suggestive of a type II choledochal cyst; however, a diagnosis could not be definitively made by radiology (Fig. 2). Attendees of a multidisciplinary meeting, which included gastroenterology, surgical oncology, and radiology services, made the decision to proceed with EUS and ERCP to further characterize the cyst, inasmuch as differentiation of a type II from a type VI choledochal cyst would significantly change surgical management. The patient underwent EUS and ERCP with direct cholangioscopy (SpyGlass DS II; Boston Scientific, Inc, Marlborough, Mass, USA) (Video 1, available online at www.VideoGIE.org). EUS demonstrated a 2-cm cystic structure adjacent to the common bile duct; no obvious mass was seen. ERCP with sphincterotomy and cholangioscopy identified an opening 4 cm distal to the bifurcation, which led to the cystic duct, and a cyst arising from the cystic duct; the ductal epithelium appeared normal, decreasing concerns for malignancy. A cholangiogram confirmed the cystic duct as the origin of the cyst and demonstrated contrast material filling the cyst followed by filling of the gallbladder immediately afterward. The patient tolerated EUS/ERCP without adverse events. Subsequently, the patient underwent outpatient robotic multiport cholecystectomy with an intraoperative cholangiogram with the use of indocyanine green and the Da Vinci FireFly fluorescence system without adverse events (Fig. 3). The gallbladder and choledochal cyst were resected en bloc (Fig. 4). The patient was seen 2 weeks postoperatively and was asymptomatic. The final pathologic report confirmed the diagnosis of type VI choledochal cyst without dysplasia (Fig. 5).Figure 4Resected en bloc specimen; Debakey forceps on the cystic duct (arrow), tonsil forceps inside the choledochal cyst, and gallbladder stones (*).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5A, Type VI choledochal cyst without dysplasia (H&E, orig. mag. ×20). B, Type VI choledochal cyst without dysplasia (H&E, orig. mag. ×200).View Large Image Figure ViewerDownload Hi-res image Download (PPT) All authors disclosed no financial relationships relevant to this publication. The authors thank Dr Michael J. Crabtree and Dr Samuel W. French of the Department of Pathology and Dr Alisa S. Han of the Department of Radiology for assisting with the interpretation of images for this case. https://www.videogie.org/cms/asset/bf13aa4d-2106-4f31-b66e-9e36ddfdf65f/mmc1.mp4Loading ... Download .mp4 (47.66 MB) Help with .mp4 files Video 1EUS and ERCP with direct cholangioscopy diagnosing type VI choledochal cyst.
