In Vivo Bioluminescent / Fluorescent Imaging


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The presentation is an introduction to using bioluminescent "reagents" to evaluate drug efficacy in tumor models. This presentation briefly highlights one of many research platforms available at Caliper Life Sciences\' Discovery Alliances\' In Vivo Division that performs contract research for the life science community.

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In Vivo Bioluminescent / Fluorescent Imaging

  1. 1. Bioluminescent Imaging for Oncology Studies 1
  2. 2. Bioluminescent/Fluorescent Imaging Introduction 2
  3. 3. Advances in Biology: Probe and Model Development and Validation Luciferase Fluorescent Proteins Fluorescent Probes 3
  4. 4. Integration with Biophotonic Imaging Assays Image 4
  5. 5. Current vs. IVIS Methodology Conventional Methodology = 24 animals are sacrificed over four time points Week 1 Week 2 Week 3 Week4 IVIS Methodology = 6 animals over four or more timepoints Week 1 Week 2 Week 3 Week4 The same group of animals is reused at each time point; far fewer animals are used in this experiment than using conventional technologies 5
  6. 6. Oncology Techniques Caliper measurements Non-invasive bioluminescent imaging of implanted light Only for S.C. models producing tumor cell growth in Limited to solid tumors the following models: >100 mm3 tumor size – Subcutaneous Tumor cell line does not require genetic manipulations – Orthotopic – Metastatic Histopathology More sensitive: as few as 100/500 cells can be detected Time consuming (depending on reporter system) Terminal procedure Light correlates with the number Requires a lot of animals of live cells in the tumor Tumor cell line does not require genetic manipulations 6
  7. 7. Applications of IVIS Technology Oncology Infectious Diseases Immunology, Inflammation and Autoimmune Diseases Cardiovascular Diabetes and Obesity Neurology Drug Metabolism Gene Therapy Vectors Formulations & Biodistribution Stem Cell Research Gene Therapy 7
  8. 8. Therapeutic Area Specific - Models and Applications Oncology - Spontaneous tumor model Inflammation – Arthritis model CNS - Stroke Models EL1-Tag- Luc Fluorescent conjugate detects GFAP-Luc Thrombin activity Stem Cells – grafting of Primate Models – Viral gene transfer Metabolic Disease – Diabetes model Neuronal stem cells Macaque Model RIP-Luc 8
  9. 9. Xenograft Models Human Tumor Cell Lines in Immuno- deficient Mice 9
  10. 10. Light Producing Human Tumor Cell Lines Constitutively expressing luciferase or a fluorescent protein used in primary and metastatic tumor growth models. Lung Prostate - A549-luc-C8 - PC-3M-luc-C6 - LoVo-6-luc-1 - LNCaP-luc-M6 Breast Colorectal - MDA-MB-231-luc-D3-H1 - HT-29-luc-D6 - MCF-7-luc-F5 - LoVo-6-luc-1 - MDA-MB-435-luc-G2 Ovarian Cervical -SKOV3-luc-D3 - HeLa-luc Customized light producing tumor cell line production also available. 10
  11. 11. Example of an Oncology Study Light producing (luciferase or fluorescent protein expressing) tumor cells are implanted into immuno-compromised or immuno-competent mice/rats. The tumor growth/metastasis formation is monitored once/twice a week using none-invasive in vivo imaging (IVIS) technology. Compound administration is initiated when tumor(s) are established based on the results of preliminary IVIS imaging. The efficacy of the compound is monitored by imaging using IVIS instrumentation and/or caliper measurements. Additionally, we have capabilities to perform toxicity and pharmacokinetic studies prior to conducting the efficacy study. 11
  12. 12. Subcutaneous Tumor Models 12
  13. 13. Subcutaneous Tumor Models PC3M-luc-6 Human Prostate Therapeutic Treatment Paradigm Adenocarcinoma Day 14 Day 21 Day 28 Day 35 No Treatment Mouse #6 2.2x109 n=7 Mouse #40 n=8 5-FU (treatment started 2.8x108 on Day 11) Mouse #31 Mitomycin C n=8 (treatment started 3.6x106 on Day 11) 13
  14. 14. Subcutaneous Tumor Models Drug Analysis In Vivo – PC-3M-luc 1x106 Cells s.c. 14
  15. 15. Orthotopic Tumor Models 15
  16. 16. Orthotopic Models for Prostate and Breast Cancers Technique: Orthotopic injection of Tumor cell suspension MDA-MB-231-luc-D3H1 Human Mammary Gland Adenocarcinoma (pleural effusion) PC3M-luc-6 Human Prostate Adenocarcinoma 16
  17. 17. Orthotopic Colon Carcinoma Model LoVo6-luc-1 Human Colorectal Adenocarcinoma S.C. tumor excision Tumor fragment Orthotopic model transplant surgery Week 5 Day 0 Week 6 Week 7 Week 4 Compound Treatment Subcutaneous model Measurements: Bio-luminescence Endpoints: Day 0 Week 1 Week 2 Week 4 Tissue collection for histopathology and biochemical analysis Measurements: Bio-luminescence Tumor volume (caliper) S.C. Injection In left flank week 4 week 7 Luminescent LoVo-6-luc cells in vitro 17
  18. 18. Orthotopic Prostate Tumor Model Therapeutic Treatment Disease Relapse Week 1 Week 3 Week 5 Week 7 Week 8 n = 13 Saline Control Group Mouse #40 End Point iv, 3x/wk — 100 — 100 Week 5 Wk 2, 3 — 80 — 80 n = 6/7 5-FU x106 x106 — 60 — 60 Mouse #38 100mg/kg, iv, 1x/wk — 40 — 40 Wk 2, 3, 4 — 20 — 20 Mitomycin C n = 3/5 Mouse #4 2/mg/kg, iv, 3x/wk Wk 2, 3 PC-3M-luc-C6, 5x105 Cells; Male nu/nu CR 18
  19. 19. Metastatic Tumor Models Experimental and Spontaneous Metastases 19
  20. 20. PC3M-luc-6 Experimental Metastases Human Prostate Intravenous Models Adenocarcinoma nu/nu SCID-bg 20
  21. 21. PC3M-luc-6 Experimental Metastases Human Prostate Intracardiac Models Adenocarcinoma nu/nu 21
  22. 22. PC3M-luc-6 Spontaneous Metastases Human Prostate Subcutaneous Model Adenocarcinoma Primary Tumor Primary Tumor is Shielded 22
  23. 23. Spontaneous Metastases MDA-MB-231-luc-D3H1 Human Mammary Gland Orthotopic Model Adenocarcinoma (pleural effusion) 23
  24. 24. Metastatic Tumor Models Inhibition of Tumor Growth by SU11248 Experimental Breast Cancer Bone Metastasis Model Murray et al 2003 435/HAL-Luc Cells 24
  25. 25. Bioluminescent/Fluorescent Imaging Success Stories 25
  26. 26. Caliper Technology Played a Role in the Preclinical Development of the Following Therapies FDA Approved In Clinical Trials Pfizer, Sutent (sunitinib) Phase I: (kidney and stomach cancer) Cell Genesys, CG0070 Novartis, Tasigna (nilotinib) (Bladder and multiple indications) (CML, chronic myeloid leukemia, Gleveec resistance) Nereus Pharma, NPI-0052 Novartis, Zometa (zoledronic acid) (multiple myeloma) (metastasis of breast, lung, prostate and multiple myeloma) Novartis, AEE788 BMS, Sprycel (dasatinib) (Advanced Cancers) Millennium, PS-341 (combo) (CML, chronic myeloid leukemia, Gleveec resistance) (Non-Hodgkin’s Lymphoma, others) Insert Therapeutics, IT101 Cubist Pharma, Cubicin (daptomycin) (solid tumors) (S. aureus infections – MRSA treatment) Novartis, CHIR-258 (Metastatic melanoma) Phase II,III: Sanofi-Aventis, Aflibercept (Multiple indications) EntreMed, Panzem (Recurrent Glioblastoma) 26
  27. 27. Examples of Recent Oncology Drugs IVIS used in Preclinical Studies IT-101: Phase I clinical trials to Tasigna is an FDA approved AEE788 (Novartis) is in Phase I determine its safety and toxicity for drug for the treatment of clinical trial for patients with treatment of patients suffering from Philadelphia chromosome Advanced Cancer Advanced Solid Tumors positive chronic myeloid leukemia (CML) Schluep et al., Clin Cancer Res Weisberg et al., Cancer Cell : 2006;12(5)March1, 2006, p. 4908–4915 Lu et al., Clin Cancer Res 2007;13(14) Feb. 2005 — Vol. 7, p. 129-141 July 15, 2007, p. 4209–4217 27
  28. 28. Business Development Contacts Alex Chang, Ph.D. Associate Director, Business Development E-mail: Phone: 609-235-1413 Mobile: 609-969-8019 Stephen J. McAndrew, Ph.D. Vice President, Business Development E-mail: Phone: 609-235-1420 For more information about the Discovery Caliper Life Sciences Alliances & Services Division, please visit Discovery Alliances & Services . Division (Xenogen Biosciences) 5 Cedar Brook Drive Cranbury, New Jersey 08512 USA Phone: 609-860-0806 Fax: 609-860-8515 28
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