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Preclinical Models of Neuroendocrine Neoplasia. Cancers (Basel) 2022; 14:cancers14225646. [PMID: 36428741 PMCID: PMC9688518 DOI: 10.3390/cancers14225646] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2022] [Revised: 11/15/2022] [Accepted: 11/15/2022] [Indexed: 11/18/2022] Open
Abstract
Neuroendocrine neoplasia (NENs) are a complex and heterogeneous group of cancers that can arise from neuroendocrine tissues throughout the body and differentiate them from other tumors. Their low incidence and high diversity make many of them orphan conditions characterized by a low incidence and few dedicated clinical trials. Study of the molecular and genetic nature of these diseases is limited in comparison to more common cancers and more dependent on preclinical models, including both in vitro models (such as cell lines and 3D models) and in vivo models (such as patient derived xenografts (PDXs) and genetically-engineered mouse models (GEMMs)). While preclinical models do not fully recapitulate the nature of these cancers in patients, they are useful tools in investigation of the basic biology and early-stage investigation for evaluation of treatments for these cancers. We review available preclinical models for each type of NEN and discuss their history as well as their current use and translation.
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Establishment of Novel Neuroendocrine Carcinoma Patient-Derived Xenograft Models for Receptor Peptide-Targeted Therapy. Cancers (Basel) 2022; 14:cancers14081910. [PMID: 35454817 PMCID: PMC9033026 DOI: 10.3390/cancers14081910] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Accepted: 03/30/2022] [Indexed: 02/01/2023] Open
Abstract
Simple Summary Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are a family of rare cancers with rising incidence in recent years. GEP NEN tumor cells are difficult to propagate, and few cellular and patient-derived xenograft (PDX) models are available for testing new therapies and studying the heterogeneous nature of these cancers. Here, we described the establishment and characterization of two novel NEC cellular and PDX models (NEC913 and NEC1452). NEC913 PDX tumors express somatostatin receptor 2 (SSTR2), whereas NEC1452 PDX tumors are SSTR2 negative. As a proof-of-concept study, we demonstrated how these PDX models can be used for peptide imaging experiments targeting SSTR2 using fluorescently labelled octreotide. The NEC913 and NEC1452 PDX lines represent valuable new tools for accelerating the process of drug discovery for GEP NENs. Abstract Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.
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Sasaki N, Shinji S, Shichi Y, Ishiwata T, Arai T, Yamada T, Takahashi G, Ohta R, Sonoda H, Matsuda A, Iwai T, Takeda K, Yonaga K, Ueda K, Kuriyama S, Miyasaka T, Yoshida H. TGF-β1 increases cellular invasion of colorectal neuroendocrine carcinoma cell line through partial epithelial-mesenchymal transition. Biochem Biophys Rep 2022; 30:101239. [PMID: 35252596 PMCID: PMC8891970 DOI: 10.1016/j.bbrep.2022.101239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2021] [Revised: 12/20/2021] [Accepted: 02/24/2022] [Indexed: 02/07/2023] Open
Abstract
Epithelial–mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis in many types of malignancies, including colorectal cancer. Although the importance of EMT is also considered in colorectal neuroendocrine carcinoma (NEC), its regulatory mechanisms have not been elucidated. We recently established a human colorectal NEC cell line, SS-2. In this study, we aimed to clarify whether these cells were sensitive to transforming growth factor beta 1 (TGF-β1) and whether EMT could be induced through TGF-β1/Smad signaling, with the corresponding NEC cell-specific changes in invasiveness. In SS-2 cells, activation of TGF-β1 signaling, as indicated by phosphorylation of Smad2/3, was dose-dependent, demonstrating that SS-2 cells were responsive to TGF-β1. Analysis of EMT markers showed that mRNA levels changed with TGF-β1 treatment and that E-cadherin, an EMT marker, was expressed in cell-cell junctions even after TGF-β1 treatment. Invasion assays showed that TGF-β1-treated SS-2 cells invaded more rapidly than non-treated cells, and these cells demonstrated increased metalloproteinase activity and cell adhesion. Among integrins involved in cell-to-matrix adhesion, α2-integrin was exclusively upregulated in TGF-β1-treated SS-2 cells, but not in other colon cancer cell lines, and adhesion and invasion were inhibited by an anti-α2-integrin blocking antibody. Our findings suggest that α2-integrin may represent a novel therapeutic target for the metastasis of colorectal NEC cells.
NEC cell line SS-2 is responsive to TGF-β1. TGF-β1 stimulation induces partial EMT, maintaining E-cadherin in SS-2 cells. TGF-β1-treated SS-2 cells exhibit increase in metalloproteinase activity and cell adhesion. α2-integrin is exclusively upregulated in TGF-β1-treated SS-2 cells. Use of anti-α2-integrin blocking antibody inhibits enhanced adhesion and invasion.
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Key Words
- Adhesion
- BSA, bovine serum albumin
- CSC, cancer stem cell
- EMT
- EMT, epithelial-to-mesenchymal transition
- FACS, fluorescence activated cell sorter
- Invasion
- MFI, mean fluorescence intensity
- MMP, matrix metalloproteinase
- NEC, neuroendocrine carcinoma
- NENs, neuroendocrine neoplasms
- Neuroendocrine carcinoma
- SD, standard deviation
- SEM, scanning electron microscopic
- TGF, transforming growth factor-beta
- TGF-β1
- qRT-PCR, quantitative reverse transcription-polymerase chain reaction
- α2-integrin
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Affiliation(s)
- Norihiko Sasaki
- Research Team for Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Tokyo, 173-0015, Japan
| | - Seiichi Shinji
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan.,Division of Aging and Carcinogenesis, Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, 173-0015, Japan
| | - Yuuki Shichi
- Division of Aging and Carcinogenesis, Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, 173-0015, Japan
| | - Toshiyuki Ishiwata
- Division of Aging and Carcinogenesis, Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, 173-0015, Japan
| | - Tomio Arai
- Department of Pathology, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tokyo, 173-0015, Japan
| | - Takeshi Yamada
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Goro Takahashi
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Ryo Ohta
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Hiromichi Sonoda
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Akihisa Matsuda
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Takuma Iwai
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Kohki Takeda
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Kazuhide Yonaga
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Koji Ueda
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Sho Kuriyama
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Toshimitsu Miyasaka
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
| | - Hiroshi Yoshida
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan
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Lu L, Przybylla R, Shang Y, Dai M, Krohn M, Krämer OH, Mullins CS, Linnebacher M. Microsatellite Status and IκBα Expression Levels Predict Sensitivity to Pharmaceutical Curcumin in Colorectal Cancer Cells. Cancers (Basel) 2022; 14:1032. [PMID: 35205780 PMCID: PMC8870219 DOI: 10.3390/cancers14041032] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2021] [Revised: 02/11/2022] [Accepted: 02/14/2022] [Indexed: 02/06/2023] Open
Abstract
Clinical utilization of curcumin in colorectal cancer (CRC) was revived as a result of the development of novel curcumin formulations with improved bioavailability. Additionally, identification of biomarkers for curcumin sensitivity would also promote successful clinical applications. Here, we wanted to identify such biomarkers in order to establish a predictive model for curcumin sensitivity. Thirty-two low-passage CRC cell lines with specified tumor characteristics were included. Curcumin suppressed cell proliferation, yet sensitivity levels were distinct. Most curcumin-sensitive CRC cell lines were microsatellite stable and expressed high levels of IκBα. The predictive capacity of this biomarker combination possessed a statistical significance of 72% probability to distinguish correctly between curcumin-sensitive and -resistant CRC cell lines. Detailed functional analyses were performed with three sensitive and three resistant CRC cell lines. As curcumin's mode of action, inhibition of NF-κB p65 activation via IκBα was identified. In consequence, we hypothesize that novel curcumin formulations-either alone or, more likely, in combination with standard therapeutics-can be expected to prove clinically beneficial for CRC patients with high IκBα expression levels.
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Affiliation(s)
- Lili Lu
- Department of General Surgery, Molecular Oncology and Immunotherapy, Rostock University Medical Center, 18057 Rostock, Germany; (L.L.); (R.P.); (Y.S.); (M.D.); (M.K.); (C.S.M.)
| | - Randy Przybylla
- Department of General Surgery, Molecular Oncology and Immunotherapy, Rostock University Medical Center, 18057 Rostock, Germany; (L.L.); (R.P.); (Y.S.); (M.D.); (M.K.); (C.S.M.)
| | - Yuru Shang
- Department of General Surgery, Molecular Oncology and Immunotherapy, Rostock University Medical Center, 18057 Rostock, Germany; (L.L.); (R.P.); (Y.S.); (M.D.); (M.K.); (C.S.M.)
| | - Meng Dai
- Department of General Surgery, Molecular Oncology and Immunotherapy, Rostock University Medical Center, 18057 Rostock, Germany; (L.L.); (R.P.); (Y.S.); (M.D.); (M.K.); (C.S.M.)
| | - Mathias Krohn
- Department of General Surgery, Molecular Oncology and Immunotherapy, Rostock University Medical Center, 18057 Rostock, Germany; (L.L.); (R.P.); (Y.S.); (M.D.); (M.K.); (C.S.M.)
| | | | - Christina Susanne Mullins
- Department of General Surgery, Molecular Oncology and Immunotherapy, Rostock University Medical Center, 18057 Rostock, Germany; (L.L.); (R.P.); (Y.S.); (M.D.); (M.K.); (C.S.M.)
| | - Michael Linnebacher
- Department of General Surgery, Molecular Oncology and Immunotherapy, Rostock University Medical Center, 18057 Rostock, Germany; (L.L.); (R.P.); (Y.S.); (M.D.); (M.K.); (C.S.M.)
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Detjen K, Hammerich L, Özdirik B, Demir M, Wiedenmann B, Tacke F, Jann H, Roderburg C. Models of Gastroenteropancreatic Neuroendocrine Neoplasms: Current Status and Future Directions. Neuroendocrinology 2021; 111:217-236. [PMID: 32615560 DOI: 10.1159/000509864] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/22/2020] [Accepted: 06/23/2020] [Indexed: 11/19/2022]
Abstract
Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are a rare, heterogeneous group of tumors that originate from the endocrine system of the gastrointestinal tract and pancreas. GEP-NENs are subdivided according to their differentiation into well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Since GEP-NENs represent rare diseases, only limited data from large prospective, randomized clinical trials are available, and recommendations for treatment of GEP-NEN are in part based on data from retrospective analyses or case series. In this context, tractable disease models that reflect the situation in humans and that allow to recapitulate the different clinical aspects and disease stages of GEP-NET or GEP-NEC are urgently needed. In this review, we highlight available data on mouse models for GEP-NEN. We discuss how these models reflect tumor biology of human disease and whether these models could serve as a tool for understanding the pathogenesis of GEP-NEN and for disease modeling and pharmacosensitivity assays, facilitating prediction of treatment response in patients. In addition, open issues applicable for future developments will be discussed.
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Affiliation(s)
- Katharina Detjen
- Department of Hepatology and Gastroenterology, Charité - University Medicine Berlin, Campus Virchow Klinikum and Charité Campus Mitte, Berlin, Germany
| | - Linda Hammerich
- Department of Hepatology and Gastroenterology, Charité - University Medicine Berlin, Campus Virchow Klinikum and Charité Campus Mitte, Berlin, Germany
| | - Burcin Özdirik
- Department of Hepatology and Gastroenterology, Charité - University Medicine Berlin, Campus Virchow Klinikum and Charité Campus Mitte, Berlin, Germany
| | - Münevver Demir
- Department of Hepatology and Gastroenterology, Charité - University Medicine Berlin, Campus Virchow Klinikum and Charité Campus Mitte, Berlin, Germany
| | - Bertram Wiedenmann
- Department of Hepatology and Gastroenterology, Charité - University Medicine Berlin, Campus Virchow Klinikum and Charité Campus Mitte, Berlin, Germany
| | - Frank Tacke
- Department of Hepatology and Gastroenterology, Charité - University Medicine Berlin, Campus Virchow Klinikum and Charité Campus Mitte, Berlin, Germany
| | - Henning Jann
- Department of Hepatology and Gastroenterology, Charité - University Medicine Berlin, Campus Virchow Klinikum and Charité Campus Mitte, Berlin, Germany
| | - Christoph Roderburg
- Department of Hepatology and Gastroenterology, Charité - University Medicine Berlin, Campus Virchow Klinikum and Charité Campus Mitte, Berlin, Germany,
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Schulte am Esch J, Windmöller BA, Hanewinkel J, Storm J, Förster C, Wilkens L, Krüger M, Kaltschmidt B, Kaltschmidt C. Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition. Cancers (Basel) 2020; 12:cancers12092582. [PMID: 32927768 PMCID: PMC7564713 DOI: 10.3390/cancers12092582] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Revised: 09/04/2020] [Accepted: 09/08/2020] [Indexed: 12/24/2022] Open
Abstract
Simple Summary The aim of this study was to gain a better understanding of cancer stem cells, which are a small subpopulation of tumor cells with high plasticity driving tumor growth and metastasis. Here we isolated two novel colorectal cancer cell lines originating from a rectal neuroendocrine carcinoma and a colorectal adenocarcinoma, depicting stem-like properties. These in vitro models offer the possibility to evaluate pathophysiological mechanisms in order to develop tailored therapeutic strategies for distinct colorectal malignancies. Investigations revealed gene copy number gain of the N-myc proto-oncogene for both. Accordingly, inhibition of the protein–protein interaction of myc and N-myc proto-oncogenes with the myc-associated factor X utilizing small molecule KJ-Pyr-9, exhibited a significant reduction in survival of both cell lines by the induction of apoptosis. Consequently, the blockage of these interactions may serve as a possible treatment strategy for colorectal cancer cell lines with gene copy number gain of the N-myc proto-oncogene. Abstract Cancer stem cells (CSC) are crucial mediators of cancer relapse. Here, we isolated two primary human colorectal cancer cell lines derived from a rectal neuroendocrine carcinoma (BKZ-2) and a colorectal adenocarcinoma (BKZ-3), both containing subpopulations with potential stem-like properties. Protein expression of CSC-markers prominin-1 and CD44 antigen was significantly higher for BKZ-2 and BKZ-3 in comparison to well-established colon carcinoma cell lines. High sphere-formation capacity further confirmed the existence of a subpopulation with potential stem-like phenotype. Epithelial–mesenchymal transition markers as well as immune checkpoint ligands were expressed more pronounced in BKZ-2. Both cell populations demonstrated N-myc proto-oncogene (NMYC) copy number gain. Myc proto-oncogene (MYC)/NMYC activity inhibitor all-trans retinoic acid (ATRA) significantly reduced the number of tumor spheres for both and the volume of BKZ-2 spheres. In contrast, the sphere volume of ATRA-treated BKZ-3 was increased, and only BKZ-2 cell proliferation was reduced in monolayer culture. Treatment with KJ-Pyr-9, a specific inhibitor of MYC/NMYC-myc-associated factor X interaction, decreased survival by the induction of apoptosis of both. In summary, here, we present the novel colorectal cancer cell lines BKZ-2 and BKZ-3 as promising cellular in vitro models for colorectal carcinomas and identify the MYC/NMYC molecular pathway involved in CSC-induced carcinogenesis with relevant therapeutic potential.
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Affiliation(s)
- Jan Schulte am Esch
- Department of General and Visceral Surgery, Protestant Hospital of Bethel Foundation, 33611 Bielefeld, Germany;
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
| | - Beatrice Ariane Windmöller
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
- Correspondence: ; Tel.: +49-0521-106-5629
| | - Johannes Hanewinkel
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
| | - Jonathan Storm
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
| | - Christine Förster
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Institute of Pathology, KRH Hospital Nordstadt, affiliated with the Protestant Hospital of Bethel Foundation, 30167 Hannover, Germany
| | - Ludwig Wilkens
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Institute of Pathology, KRH Hospital Nordstadt, affiliated with the Protestant Hospital of Bethel Foundation, 30167 Hannover, Germany
| | - Martin Krüger
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Internal Medicine and Gastroenterology, Protestant Hospital of Bethel Foundation, 33611 Bielefeld, Germany
| | - Barbara Kaltschmidt
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
- Molecular Neurobiology, University of Bielefeld, 33615 Bielefeld, Germany
| | - Christian Kaltschmidt
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
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Song L, Cui Z, Guo X. Comprehensive analysis of circular RNA expression profiles in cisplatin-resistant non-small cell lung cancer cell lines. Acta Biochim Biophys Sin (Shanghai) 2020; 52:944-953. [PMID: 32716023 DOI: 10.1093/abbs/gmaa085] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Revised: 03/01/2020] [Accepted: 06/05/2020] [Indexed: 12/18/2022] Open
Abstract
Platinum-based drugs such as cisplatin are widely used in combination chemotherapy for non-small cell lung cancer (NSCLC) owing to their high clinical response rate; however, acquired resistance to cisplatin is eventually inevitable. Circular RNAs (circRNAs) are involved in the development of diverse types of cancers, but their connection to cisplatin-resistance in NSCLC has not been studied. In the present study, two cisplatin-resistant NSCLC cell lines (A549/DDP and PC9/DDP) were established by gradually increasing concentrations of cisplatin in the media. The resulting cell lines possessed high resistance to cisplatin and strong proliferation, migration, and colony formation abilities compared to the parental cells. Microarray analysis identified 19,161 circRNAs that were dysregulated in cisplatin-resistant cell lines (fold change abs>2), including 11,915 up-regulated and 7246 down-regulated circRNAs. The expression of the top five up-regulated and down-regulated circRNAs was validated using real-time quantitative polymerase chain reaction. A circRNA-micro RNA (miRNA) network of the top 20 dysregulated circRNAs and their predicted miRNAs was constructed using Cytoscape. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the host genes of the identified circRNAs were involved in the regulation of MAP kinase kinase kinase kinase activity, 6-phosphofructo-2-kinase activity, focal adhesion, ErbB signaling, and ECM-receptor interactions, which may contribute to cisplatin resistance in NSCLC. In summary, this is the first report on circRNA profiling in cisplatin-resistant NSCLC cells and it provides new potential targets for the reversal of cisplatin resistance in NSCLC.
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Affiliation(s)
- Lin Song
- Department of Respiratory Medicine, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200090, China
| | - Zhilei Cui
- Department of Respiratory Medicine, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200090, China
| | - Xuejun Guo
- Department of Respiratory Medicine, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200090, China
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Shinji S, Sasaki N, Yamada T, Koizumi M, Ohta R, Matsuda A, Yokoyama Y, Takahashi G, Hotta M, Hara K, Takeda K, Ueda K, Kuriyama S, Ishiwata T, Ueda Y, Murakami T, Kanazawa Y, Yoshida H. Establishment and characterization of a novel neuroendocrine carcinoma cell line derived from a human ascending colon tumor. Cancer Sci 2019; 110:3708-3717. [PMID: 31648389 PMCID: PMC6890439 DOI: 10.1111/cas.14221] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2019] [Revised: 10/19/2019] [Accepted: 10/21/2019] [Indexed: 01/15/2023] Open
Abstract
The incidence of rare neuroendocrine tumors (NET) is rapidly increasing. Neuroendocrine carcinoma (NEC) is a NET with poorly differentiated histological features, high proliferative properties and associated poor prognoses. As these carcinomas are so rare and, thus, affect only a small number of patients allowing for few cell lines to be derived from patient biopsies, the histological, immunohistochemical, and clinical characteristics associated with colorectal NEC and NEC in other organs have yet to be clearly defined. Herein, we describe the establishment of a novel NEC cell line (SS‐2) derived from a tumor resection of the ascending colon from a 59‐year‐old Japanese woman. The histological, electron microscopic and immunohistochemical features of chromogranin A (CgA) as well as confirmation of synaptophysin positivity in this tumor were typical of those commonly observed in surgically resected colorectal NEC. Further, the Ki‐67 labeling index of the resected tumor was >20% and, thus, the tumor was diagnosed as an NEC of the ascending colon. The SS‐2 cell line maintained characteristic features to those of the resected tumor, which were further retained following implantation into subcutaneous tissues of nude mice. Additionally, when SS‐2 cells were seeded into ultra‐low attachment plates, they formed spheres that expressed higher levels of the cancer stem cell (CSC) marker CD133 compared to SS‐2 cells cultured under adherent conditions. SS‐2 cells may, therefore, contribute to the current knowledge on midgut NEC biological function while providing a novel platform for examining the effects of colorectal NEC drugs, including CSC.
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Affiliation(s)
- Seiichi Shinji
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Norihiko Sasaki
- Research Team for Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
| | - Takeshi Yamada
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Michihiro Koizumi
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Ryo Ohta
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Akihisa Matsuda
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Yasuyuki Yokoyama
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Goro Takahashi
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Masahiro Hotta
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Keisuke Hara
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Kohki Takeda
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Koji Ueda
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Sho Kuriyama
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Toshiyuki Ishiwata
- Division of Aging and Carcinogenesis, Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
| | - Yoshibumi Ueda
- Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan
| | - Takashi Murakami
- Department of Microbiology, Saitama Medical University, Saitama, Japan
| | - Yoshikazu Kanazawa
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
| | - Hiroshi Yoshida
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
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Yip H, Haupt C, Maresh G, Zhang X, Li L. Humanized mice for immune checkpoint blockade in human solid tumors. AMERICAN JOURNAL OF CLINICAL AND EXPERIMENTAL UROLOGY 2019; 7:313-320. [PMID: 31763362 PMCID: PMC6872471] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 09/25/2019] [Accepted: 10/13/2019] [Indexed: 06/10/2023]
Abstract
Immunotherapy, specifically research involving immune checkpoint blockers (ICBs), has become a popular trend in anticancer research over the last three years. Due to the difficulties and often poor translation of results from in-vitro models, in-vivo models have become more relevant than ever. With the discovery of NOD, Prkdcscid , and Il2rγ-/- mutations, patient-derived xenograft (PDX) mouse models were developed, providing an ideal environment for ICBs testing. By implanting a PDX with either CD34+ or peripheral blood mononuclear cells, we can create a human immune system capable of mounting a response against tumor burden. These animal models are currently being used to study molecular mechanisms, test drug efficacy, and trial drug combinations. Others have found use for these humanized mouse models as surrogates to represent otherwise uncommon diseases. Limitations remain with regards to what the models are capable of, but in the short amount of time between the development of these models and heightened interest in ICBs, these mice have already shown utility for future developments in the field of immunotherapy.
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Affiliation(s)
- Henry Yip
- UQ-Ochsner Clinical School, Institute for Translational Research, Ochsner Clinic Foundation New Orleans, LA, USA
| | - Carl Haupt
- UQ-Ochsner Clinical School, Institute for Translational Research, Ochsner Clinic Foundation New Orleans, LA, USA
| | - Grace Maresh
- UQ-Ochsner Clinical School, Institute for Translational Research, Ochsner Clinic Foundation New Orleans, LA, USA
| | - Xin Zhang
- UQ-Ochsner Clinical School, Institute for Translational Research, Ochsner Clinic Foundation New Orleans, LA, USA
| | - Li Li
- UQ-Ochsner Clinical School, Institute for Translational Research, Ochsner Clinic Foundation New Orleans, LA, USA
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Mullins CS, Micheel B, Matschos S, Leuchter M, Bürtin F, Krohn M, Hühns M, Klar E, Prall F, Linnebacher M. Integrated Biobanking and Tumor Model Establishment of Human Colorectal Carcinoma Provides Excellent Tools for Preclinical Research. Cancers (Basel) 2019; 11:1520. [PMID: 31601052 PMCID: PMC6826890 DOI: 10.3390/cancers11101520] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Revised: 09/26/2019] [Accepted: 09/30/2019] [Indexed: 12/22/2022] Open
Abstract
Over the time period from 2006 to 2017, consecutive patients operated on at the University Medical Center Rostock participated in the comprehensive biobanking and tumor-modelling approach known as the HROC collection. Samples were collected using strict standard operating procedures including blood (serum and lymphocytes), tumor tissue (vital and snap frozen), and adjacent normal epithelium. Patient and tumor data including classification, molecular type, clinical outcome, and results of the model establishment are the essential pillars. Overall, 149 patient-derived xenografts with 34 primary and 35 secondary cell lines were successfully established and encompass all colorectal carcinoma anatomic sites, grading and staging types, and molecular classes. The HROC collection represents one of the largest model assortments from consecutive clinical colorectal carcinoma (CRC) cases worldwide. Statistical analysis identified a variety of clinicopathological and molecular factors associated with model success in univariate analysis. Several of them not identified before include localization, mutational status of K-Ras and B-Raf, MSI-status, and grading and staging parameters. In a multivariate analysis model, success solely correlated positively with the nodal status N1 and mutations in the genes K-Ras and B-Raf. These results imply that generating CRC tumor models on the individual patient level is worth considering especially for advanced tumor cases with a dismal prognosis.
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Affiliation(s)
- Christina S Mullins
- Molecular Oncology and Immunotherapy, Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, Schillingallee 69, 18057 Rostock, Germany.
| | - Bianca Micheel
- Molecular Oncology and Immunotherapy, Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, Schillingallee 69, 18057 Rostock, Germany.
| | - Stephanie Matschos
- Molecular Oncology and Immunotherapy, Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, Schillingallee 69, 18057 Rostock, Germany.
| | - Matthias Leuchter
- Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, University of Rostock, Schillingallee 35, 18057 Rostock, Germany.
| | - Florian Bürtin
- Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, University of Rostock, Schillingallee 35, 18057 Rostock, Germany.
| | - Mathias Krohn
- Molecular Oncology and Immunotherapy, Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, Schillingallee 69, 18057 Rostock, Germany.
| | - Maja Hühns
- Institute of Pathology, University Medical Center Rostock, Strempelstraße 10, 18057 Rostock, Germany.
| | - Ernst Klar
- Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, University of Rostock, Schillingallee 35, 18057 Rostock, Germany.
| | - Friedrich Prall
- Institute of Pathology, University Medical Center Rostock, Strempelstraße 10, 18057 Rostock, Germany.
| | - Michael Linnebacher
- Molecular Oncology and Immunotherapy, Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, Schillingallee 69, 18057 Rostock, Germany.
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Gock M, Mullins CS, Bergner C, Prall F, Ramer R, Göder A, Krämer OH, Lange F, Krause BJ, Klar E, Linnebacher M. Establishment, functional and genetic characterization of three novel patient-derived rectal cancer cell lines. World J Gastroenterol 2018; 24:4880-4892. [PMID: 30487698 PMCID: PMC6250916 DOI: 10.3748/wjg.v24.i43.4880] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/03/2018] [Revised: 10/22/2018] [Accepted: 11/02/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To establish patient-individual tumor models of rectal cancer for analyses of novel biomarkers, individual response prediction and individual therapy regimens. METHODS Establishment of cell lines was conducted by direct in vitro culturing and in vivo xenografting with subsequent in vitro culturing. Cell lines were in-depth characterized concerning morphological features, invasive and migratory behavior, phenotype, molecular profile including mutational analysis, protein expression, and confirmation of origin by DNA fingerprint. Assessment of chemosensitivity towards an extensive range of current chemotherapeutic drugs and of radiosensitivity was performed including analysis of a combined radio- and chemotherapeutic treatment. In addition, glucose metabolism was assessed with 18F-fluorodeoxyglucose (FDG) and proliferation with 18F-fluorothymidine. RESULTS We describe the establishment of ultra-low passage rectal cancer cell lines of three patients suffering from rectal cancer. Two cell lines (HROC126, HROC284Met) were established directly from tumor specimens while HROC239 T0 M1 was established subsequent to xenografting of the tumor. Molecular analysis classified all three cell lines as CIMP-0/ non-MSI-H (sporadic standard) type. Mutational analysis revealed following mutational profiles: HROC126: APCwt , TP53wt , KRASwt , BRAFwt , PTENwt ; HROC239 T0 M1: APCmut , P53wt , KRASmut , BRAFwt , PTENmut and HROC284Met: APCwt , P53mut , KRASmut , BRAFwt , PTENmut . All cell lines could be characterized as epithelial (EpCAM+) tumor cells with equivalent morphologic features and comparable growth kinetics. The cell lines displayed a heterogeneous response toward chemotherapy, radiotherapy and their combined application. HROC126 showed a highly radio-resistant phenotype and HROC284Met was more susceptible to a combined radiochemotherapy than HROC126 and HROC239 T0 M1. Analysis of 18F-FDG uptake displayed a markedly reduced FDG uptake of all three cell lines after combined radiochemotherapy. CONCLUSION These newly established and in-depth characterized ultra-low passage rectal cancer cell lines provide a useful instrument for analysis of biological characteristics of rectal cancer.
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Affiliation(s)
- Michael Gock
- Department of General Surgery, University Medical Center, Rostock 18055, Germany
| | - Christina S Mullins
- Section of Molecular Oncology and Immunotherapy, University Medical Center, Rostock 18055, Germany
| | - Carina Bergner
- Department of Nuclear Medicine, University Medical Center, Rostock 18055, Germany
| | - Friedrich Prall
- Institute of Pathology, University Medical Center, Rostock 18055, Germany
| | - Robert Ramer
- Institute of Pharmacology, University Medical Center, Rostock 18055, Germany
| | - Anja Göder
- Institute of Toxicology, University Medical Center Mainz, Mainz 55131, Germany
| | - Oliver H Krämer
- Institute of Toxicology, University Medical Center Mainz, Mainz 55131, Germany
| | - Falko Lange
- Oscar-Langendorff-Institute of Physiology, University Medical Center, Rostock 18055, Germany
| | - Bernd J Krause
- Department of Nuclear Medicine, University Medical Center, Rostock 18055, Germany
| | - Ernst Klar
- Department of General Surgery, University Medical Center, Rostock 18055, Germany
| | - Michael Linnebacher
- Section of Molecular Oncology and Immunotherapy, University Medical Center, Rostock 18055, Germany
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