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Pharma&Biotech BioResearch Primary Cells: Optimizing Cancer Research and Drug Discovery 18 March 2014 / Speaker: Sean Fuerst 19 March 2014 / Speaker: Dr. Heiko Bueth Lonza Walkersville, Inc., Walkersville, MD 21793 / 2014© Lonza Clonetics™ Primary Cells & Media Topics What Is a Primary Cell Culture? Primary Cells in Cancer Research & Drug Discovery Introduction to Clonetics™ Primary Cells Poietics™ Stem Cells Nucleofector™ Transfection Cancer Research Product Portfolio 2 Cancer Research Market Approximately 12.5 mn new cases of cancer are diagnosed worldwide each year The global market for Biomarkers is forecast to reach $34 bn by the year 20171 Considerable research is in progress for drug discovery for cancer1 Innovative cancer therapies based on cancer molecular biology Antisense oligonucleotides in clinical trials for cancer Small interfering RNAs (siRNAs) can be targeted to tumors NCI’s Developmental Therapeutics Program (DTP) has over 400,000 drugs that have gone through screening process Half a million lives saved in 30 years thanks to cancer research2 1. 2. PRWEB Cancer Research UK 3 Clonetics™ Primary Cells & Media Topics What Is a Primary Cell Culture? Primary Cells in Cancer Research & Drug Discovery Introduction to Clonetics™ Primary Cells Poietics™ Stem Cells Nucleofector™ Transfection Cancer Research Product Portfolio 4 What Are Primary Cells? Isolated from an animal or human tissue Limited Lifespan (with the exception of some derived from tumors) Senescence Do not contain any man-made changes to their structure or genetic make-up Most physiologically relevant in vitro tool without in vivo studies Primary cells are ideal models for cancer biomarker discovery, drug screening, cancer development, cancer stem cells 5 Primary Cells vs. Cell Lines Cell Lines Primary Cells Permanently established culture Limited lifespan High mutation/modification rate Low mutation/modification rate Different genetic makeup from original tissue Isolated directly from human or animal tissue Extensive pre-characterization required Minimal pre-characterization needed Cheaper to work upfront but > 3 cell lines required at minimum for comprehensive study Priced slightly higher but limited samples needed Not as physiologically relevant Stepping stone to in vivo model 6 Concerns About Cell Lines NIH highlights authentication of cultured cell lines as critical for grant applications to be considered of the highest quality1 Some cell lines may not be the cell type they have historically been reported In a study of over 500 leukemia-lymphoma cell lines, 15% of the cell lines were misidentified2 18-36% of cell lines are misidentified or cross-contaminated3 Cell lines in cancer research do not provide complete or accurate data Nature News: Some argue tumor cells obtained directly from patients are the best way to study cancer 4 Review Breast Cancer Cell Lines: An alternative method is to use cultures from primary breast tumors5 1. 2. 3. 4. 5. Notice Number: NOT-OD-08-017 <grants.nih.gov/grants/guide/notice-files/NOT-OD-08-017.html> Drexler et al., (2003) Hughes P et al. (2007) Brendan Borrell. Nature News: How accurate are cancer cell lines?. Vol 463.18 February 2010. Burdall et al., Breast Cancer Cell Lines: friend or foe?. (2003). Review 7 Primary Cells Provide More Relevant Data D M Peehl. Review: Primary cell cultures as models of prostate cancer development. Endocr Relat Cancer. 2005 Mar;12(1):19-47 “Recent studies revealed that many of these [prostate cancer cell lines] are in fact derivatives of the three aforementioned cell lines [PC-3, DU145 and LNCaP] or other non-prostatic lines (van Bokhoven et al. 2003). The few bona fide cell lines, almost all derived from metastases, do not span the range of prostate cancer phenotypes, and in particular are not representative of primary adenocarcinomas of the prostate. Furthermore, the question of how extensively long-term culture alters the biological properties of cell lines is always lurking. For these and other reasons, primary cultures of malignant prostatic cells and their normal epithelial counterparts are sought. During the past 20 years, many of the technical hurdles involved in growing primary cultures of human prostatic epithelial cells have been overcome, and a variety of more or less similar methods have been reported” 8 Clonetics™ Primary Cells & Media Topics What Is a Primary Cell Culture? Primary Cells in Cancer Research & Drug Discovery Introduction to Clonetics™ Primary Cells Poietics™ Stem Cells Nucleofector™ Transfection Cancer Research Product Portfolio 9 Cell Lines Do Not Always Behave the Same As Primary Cells Effect of Camptothecin on primary and continuous renal cells measured using ViaLight™ Plus % maximal response 110 100 90 80 70 60 50 40 30 20 10 0 HEK 293 primary HRE primary HRCE Control 10 1 10 2 10 3 10 4 [Camptothecin] nM EC 50 HEK 293 HRE HRCE 68.10 537.5 557.4 10 Some Pathways Only Work in Primary Cells Localization of hMUNC13-4/RFP in cytolytic T lymphocytes (CTLs) CTLs recognize foreign cells and lyse them via the “immunological synapse” At the immunological synapse CTLs secrete cytolytic granules containing Perforin hMUNC13-4 is involved in an activation step preceeding vesicle membrane fusion in cytolytic granule secretion Nucleofection™ of a plasmid expressing hMUNC13-4/RFP fusion Upon target cell contact, (L, M) hMUNC13-4 polarizes and extensively co-localizes with Perforin-containing cytolytic granules at the immunological synapse Isolated CTL CTL docked to target cell Reprinted from: Feldmann J et al. (2003) Cell 115(4):461-73; with permission of Elsevier. 11 Primary Endothelial Cells Diversity Cannot Be Captured with Cell Lines Endothelial cell diversity revealed by global expression profiling Gene expression analysis Endothelial cells 14 distinct locations 53 different lots Conclusions Different expression based on vessel size Different expression between veins and arteries Differences are conserved over some passages Chi JT. et al. (2003) PNAS 100:10623-8. By permission of the author. Copyright 2003 National Academy of Sciences. 12 Primary Cells and Cancer Biomarker Discovery Cancer Biomarkers: Biological molecules predictive of tumor development Challenges Incomplete or misleading profile: Cell lines have altered profile than actual tumor tissue. Mutations, copy number variations, chromosome aberrations, lack of characterization High costs: 3-10 cell lines required at minimum from same origin for comprehensive study Resolution Primary cells as controls for complete and validated profile Cost-effective: No pre-characterization, minimal samples required 13 Clonetics™ Human Umbilical Vein Endothelial Cells (HUVECs) – Cancer Biomarker Discovery NGAL has been proposed as an early biomarker in pancreatic cancer (PaCa) Clonetics™ HUVEC cells were assayed for tube formation in presence of high and low levels of NGAL. The tube formation was reduced by 69% with high levels of NGAL Additional data suggested NGAL potently blocks pancreatic cancer angiogenesis in vitro in part by reducing VEGF secretion Zhimin Tong et al. Neutrophil Gelatinase–Associated Lipocalin: A Novel Suppressor of Invasion and Angiogenesis in Pancreatic Cancer. Cancer Res 2008;68:6100-6108 HUVEC diluted in conditioned medium (1:1 dilution) from NGAL overexpression or shNGAL clones were seeded onto 96-well plates that were precoated with Matrigel. After 16 h, the total number of vascular tubes were counted. The levels of VEGF in the culture medium were assayed by using a commercially available VEGF ELISA kit 14 Primary Cells and Drug Screening Applications Challenges New drug targets, Drug resistance assays, chemotherapy drugs Assays developed with cell lines cannot easily be replicated in vivo In vivo models are expensive for prelim drug screens Resolution Biological relevance: Assays with primary cells are more representative of in vivo models Cost-effective: Use primary cells for refinement studies before stepping into animal work 15 Clonetics™ Human Prostate Epithelial Cells – Drug Screening HTS of 4910 drugs to determine efficacy for prostate cancer Clonetics PrECs (Cat. No. CC-2555) utilized as control with malignant and non-malignant cell lines 3 drug compounds (see below) were identified to inhibit prostate cancer cell proliferation, but had little effect on control cells Kristiina Iljin et al. High-Throughput Cell-Based Screening of 4910 Known Drugs and Drug-like Small Molecules Identifies Disulfiram as an Inhibitor of Prostate Cancer Cell Growth. Clin Cancer Res October 1, 2009 15; 6070 16 Primary Cells and Cancer Formation Understand how cancer forms Challenges Primary cells can be transformed into cancer cells through genetic mutations and introduction of oncogenes Cell lines cannot offer same application as they are not representative of actual human tissue Well-characterized primary cells needed Resolution Primary cells from Lonza are extensively characterized and publication validated 17 Clonetics™ Human Mammary Epithelial Cells (HMECs) – Cancer Formation Clonetics™ HMECs were infected with retrovirus and express LT, hTERT and H-rasV12 Introduced in mice by injection HMECs have successfully been transformed to tumorigenicity through the introduction of a limited set of oncogenes Brian Elenbaas, Lisa Spirio et.al. Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial. Genes Dev. 2001 Jan 1;15(1):50-65 18 Primary Cells and Stem Cell Biology Cancer Stem Cells (CSCs) Hypothesis Challenges Cancer stem cells originate from normal tissue stem cells CSCs share several properties with normal stem cells, such as their capacity for self-renewal and their ability to differentiate Cell lines are difficult to re-program as they may not contain stemcell like components so CSC study becomes difficult Resolution Primary cells contain subpopulations of stem cell markers Primary cells can be re-programmed into iPSCs 19 Clonetics™ Human Prostate Epithelial Cells (PrECs) and Media – Cancer Stem Cells Prostate cancer arises within the epithelial compartment and generally believed to begin within the stem-cells In the adult human prostate, CD133/ABCG2 expression is indicative of small population of putative stem cells that suppress differentiation Clonetics™ PrECs (Cat. No. CC-2555) utilized alongside in-house isolated PrEC’s to assess stemcell like properties. Both cell types were cultured in Clonetics™ PrEGM Growth Media BulletKit (Cat. No. CC-3166). Both exhibit high expression of stem-cell markers CD133/ABCG2 Expression of stem cell markers in PrEC cultures. A: ABCG2-FITC and CD133-PE (AC141) expression in PrECs. B: dual-label flow cytometry showing that CD133+ cells are also ABCG2+ Donald J. Vander Griend et.al. The Role of CD133 in Normal Human Prostate Stem Cells and Malignant Cancer-Initiating Cells. Cancer Res 2008;68:9703-9711 20 Clonetics™ Primary Cells & Media Topics What Is a Primary Cell Culture? Primary Cells in Cancer Research & Drug Discovery Introduction to Clonetics™ Primary Cells Poietics™ Stem Cells Nucleofector™ Transfection Cancer Research Product Portfolio 21 Why Use Clonetics™ Primary Cells? Customer Needs Lonza Solutions Product quality Product availability Reliability Cost-effective Easy to culture Convenience Superior to cell lines, Minimum specs guarantee, ISO-certified, extensive QC & consistency 100+ cell types (human and animal), donor variety, steady supply schedule 40+ years experience, market leader, 10X more publications than competitors No pre-characterization needed; No repeat analyses; Refine experiments and reduce animal work Specialized support team to assist, optimized protocols guaranteed to work Cryopreserved amp or proliferating in flasks/plates 22 Clonetics Growth Media Systems Developed for each cell type and tested against our primary cells BulletKit™ Media: Clonetics™ SingleQuots™ Kits and basal media sold together as a “package deal” but can be bought separately as well Optimized protocols supplied with all media systems Basal Media (Water, Amino Acids, Vitamins, Sugar) + SingleQuots™ Kit (Growth Factors: Serum, Insulin, Antibacterial Agents, etc) = BulletKit™ Media 23 Clonetics™ Primary Normal Human Cells Breast Cancer: Mammary Epithelial Cells (130 unique publications for breast cancer research) Prostate Cancer: Epithelial, Smooth Muscle and Stromal cells Pancreatic Cancer: Pancreatic Islets and Acinar Tissue Lung Cancer: airway fibroblasts, epithelial and smooth muscle cells from various tissues Skin Cancer: Endothelial cells, fibroblasts, keratinocytes, melanocytes All related growth media kits available for each cell type with optimized protocols Additional cell types available. Visit www.lonza.com/primary for complete listing. 24 Clonetics™ Primary Cells & Media Topics What Is a Primary Cell Culture? Primary Cells in Cancer Research & Drug Discovery Introduction to Clonetics™ Primary Cells Poietics™ Stem Cells Nucleofector™ Transfection Cancer Research Product Portfolio 25 Poietics™ Stem Cells and Media Human Mesenchymal Stem Cells with Growth and Differentiation Media Kits Human Hematopoietic Stem Cells with Growth Media and EasyDiff™ Kits Study cancer progression in bone marrow, blood and lymph tissues Isolated from cord blood and bone marrow for blood cancer and transplantation studies Osteoclast Precursors, Preadipocytes, Adipose-derived Stem Cells and Media Kits Study various bone and lipoma tumors 26 Clonetics™ Primary Cells & Media Topics What Is a Primary Cell Culture? Primary Cells in Cancer Research & Drug Discovery Introduction to Clonetics™ Primary Cells Poietics™ Stem Cells Nucleofector™ Transfection Cancer Research Product Portfolio 27 Nucleofector™ Technology Benefits for Cancer Research Non-viral transfection of primary cells and difficult-to-transfect cancer cell lines with up to 90% efficiency High cell viability and preservation of cell functionality High flexibility in cell types, substrates or sample throughput Option of adherent Nucleofection™ adding more convenience More than 100 ready-to-use protocols and more than 800 publications with cancer cells 28 Nucleofector™ Technology Proven DNA Transport Into the Nucleus Transport DNA Expression GFP Nucleus DAPI Merge Primary NHDF-neo (Neonatal normal human dermal fibroblasts) were transfected with labeled plasmid DNA encoding GFP, fixed after 2h in 3.5% PFA and analyzed by confocal microscopy 29 Transfection of Primary Cells or Cancer Cells Lines by Nucleofection™ Blood Breast Colon Liver Lung Neurological Prostate Other 30 Clonetics™ Primary Cells & Media Topics What Is a Primary Cell Culture? Primary Cells in Cancer Research & Drug Discovery Introduction to Clonetics™ Primary Cells Poietics™ Stem Cells Nucleofector™ Transfection Cancer Research Product Portfolio 31 Lonza Cancer Product Portfolio Blood & Lymph Blood & lymphatic micro-vascular endothelial cells Hematopoietic stem cells from cord blood Mesenchymal stem cells Fresh bone marrow Pulmonary Bronchial epithelial & smooth muscle cells Fibroblasts Micro-vascular endothelial cells Pulmonary artery endothelial and smooth muscle cells Small airway epithelial cells Related Clonetics™ and Poietics™ Media tested and guaranteed to perform www.lonza.com/primary Nucleofector™ Transfection for primary cells and cancer cell lines Clonetics™ Conditionally Immortalized Cell Lines (CCICs) Bone Osteoblasts Osteoclast precursors Hematopoietic stem cells from bone marrow Fresh bone marrow Cell Function assays for bone mineralization and resorption Mammary Mammary epithelial Pancreatic Fresh Human Pancreatic Islets Acinar tissue (custom isolation) Reproductive Prostate epithelial, smooth muscle and stromal cells Uterine endothelial and smooth muscle cells Bladder endothelial and smooth muscle cells Biowhittaker™ liquid and powder media for cancer research and therapeutic applications Nucleic Acid and Protein Electrophoresis products Skin Blood & lymphatic microvascular endothelial cells Adult and neonatal MV endothelial cells Fibroblasts Keratinocytes Melanocytes Pre-adipocytes and Adiposederived stem cells 32 How to Order or Pose Questions Email order to: [email protected] or [email protected] For order-related questions, contact Customer Service For scientific support please contact U.S : 800 638 8174 (toll free) or [email protected] Europe: +32 87 321 611 or [email protected] U.S: +1 800 521 0390 or [email protected] Europe: +32 87 321 611 or [email protected] Visit our website at www.lonza.com/cancer for more information 33 Interested to Learn More? Join our upcoming webinar: Clonetics™ Cells in Pancreatic Cancer Research Slot 1: Tuesday, 1 April 2014 2PM EDT (New York) / 11 AM PDT (Los Angeles) Slot 2: Wednesday, 2 April 2014 10 AM CEST (Berlin) / 9 AM BST (London) / 5 PM JST (Tokyo) Register at: www.lonza.com/webinar14 34 Thank You for Your Kind Attention