Cancer Pharmacology Lecture Notes PDF

LSM4214: Cancer Pharmacology Models for drug target discovery and evaluation Lingzhi Wang, PhD Associate Professor Department of Pharmacology Cancer Science Institute of Singapore Yong Loo Lin School of Medicine, National Un...

LSM4214: Cancer Pharmacology Models for drug target discovery and evaluation Lingzhi Wang, PhD Associate Professor Department of Pharmacology Cancer Science Institute of Singapore Yong Loo Lin School of Medicine, National University of Singapore E-mail: [email protected]  Learning outcomes o Define and explain the process of target selection in cancer research o Describe various in vitro assays used to validate cancer targets o Understand the importance of in vivo assays in cancer target validation, be able to discuss animal models used in these studies, and assess the efficacy o Explain the principles of in vitro drug evaluation assays o Gain an understanding of in vivo drug evaluation methods o Understand advantages and disadvantages of the above-mentioned in vitro and in vivo models for drug target discovery and evaluation  Overview ❑ Target selection ❑ In vitro cancer target validation assays ❑ In vivo cancer target validation assays ❑ In vitro drug evaluation assays ❑ In vivo drug evaluation assays  Part 1 ❑ Target selection ❑ In vitro cancer target validation assays ❑ In vivo cancer target validation assays ❑ In vitro drug evaluation assays ❑ In vivo drug evaluation assays  Target selection ▪ Rational approach –Identifying likely targets from known cancer promoting pathways –“reverse genetics”(from gene to phenotype): specific genes are targeted for mutation to observe what phenotypes are produced. ▪ Unbiased approach –Uses a screen or a expression profile to identify probable targets without considering their known functions –“forward genetics”(from phenotype to gene): mutations are generated randomly to identify unknown genes responsible for the phenotypes Rational approach  Unbiased approach-RNAi RNA interference (RNAi) is a natural cellular process where small RNA molecules, such as small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), induce the silencing of specific genes. These molecules bind to complementary mRNA sequences, leading to their degradation or the inhibition of translation, effectively "knocking down" the expression of the target gene. Specific, individual genes are “knocked down” to find genes that regulate key cancer processes but limited to studies in cell lines. Types of RNAi Screens: ▪ siRNA Screens: 1. Use synthetic siRNAs to transiently knock down target genes. 2. siRNAs are delivered into cells, where they degrade target mRNA, leading to reduced protein expression. ▪ shRNA Screens: 1. Use plasmid or viral vectors to introduce shRNA sequences into cells. 2. shRNAs are processed into siRNAs within the cell, providing more stable and long-term gene knockdown compared to siRNA.  Rational approach: Loss-of-function screens An oncogene may be identified by decreased cell growth (blue) A tumor suppressor gene may be identified by increased cell growth (green) A drug resistance gene my be identified by decreased growth in the presence of a drug (yellow) NATURE REVIEWS GENETICS 7 (5) , pp.373-384  Part 2 ❑ Target selection ❑ In vitro cancer target validation assays ❑ In vivo cancer target validation assays ❑ In vitro drug evaluation assays ❑ In vivo drug evaluation assays Target validation –In vitro Target validation –In vitro Gene/protein expression analysis Apoptosis Annexin V assay  Cell Viability–In vitro Increasing number of cells per well The MTT assay is a colorimetric assay for assessing cell metabolic activity  Cell Proliferation–In vitro Measuring DNA replication by incorporation of base analogues such as 5-bromo-2′-deoxyuridine (BrdU), an analog of the nucleoside thymidine Annexin V translocation–In vitro Annexin-PI Assay For Apoptosis Cell Motility–In vitro The process of making wound Scratch assay (wound healing)-Motility  Cell Invasion–In vitro WL Thuya, et al. Theranostics 13 (2), 621 Matrigel invasion assay  3D in vitro models : spheroids ▪ Organoids are self-organized three-dimensional (3D) multicellular tissue cultures which derive from cancerous and healthy stem cells, sharing a highly similarity to the corresponding in vivo organs. ▪ Organoids can now be established for various tissues, including brain, retina, thyroid, gastrointestinal, lung, liver, pancreas, and kidney. ▪ Organoids have emerged as a valuable model for studying early embryogenesis, organ and tissue development, as well as tools in drug screening, disease modeling and personalized therapy. 3D Organoid Proliferation Assays– High Content Analysis Her2+ Her2+ SLAS Discov.2017 Mar;22(3):309-315. 3D Organoid Apoptosis Assays– Co-Culture Assays 3D Organoid Assays–potential of invasion Methods Mol Biol. 2017;1612:433-445.  Limitations of in vitro studies ▪ Behavior of cultured cells is often different from tumors in vivo ▪ Monocultures do not have cellular diversity of heterogeneous tumors ▪ Monocultures cannot evaluate contribution of immune system or stromal cells ▪ Absence of controls for toxicity  Part 3 ❑ Target selection ❑ In vitro cancer target validation assays ❑ In vivo cancer target validation assays ❑ In vitro drug evaluation assays ❑ In vivo drug evaluation assays Target validation –In vivo  Heterotopic Human Xenografts lack of T cells, B cells, the complement component C5, lack T cells and defective natural killer (NK) cell and antigen- presenting cell functions Overexpression of target gene Orthotopic Placement (lungs)  Orthotopic Placement (bladder) SKMES-1 H2170 a b Comparison between subcutaneous vs orthotopic implantation of cancer cells Pros and cons of Human Tumor Xenografts Murine Tumor Allograft/Isograft Pros and cons of Murine Tumor Allograft/Isograft Transgenic Models Pros and cons of Transgenic models Genetic models Pros and cons of Genetic models Evaluation of Tumors (Weight) Tumor Weight Evaluation of Tumors (Volume) Tumor Volume Evaluation of Tumors (Survival) Survival Time Labeled Tumor Models Pros and cons of Labeled Tumor Models Selection of a proper tumor model Micalizzi et al. J. Clin. Invest. 2009;119:9 Bioluminescence Bioluminescence Imaging BLI in orthotopic bladder cancer B J U I N T E R N A T I O N A L, 2007. 1 0 0 , 1 3 7 7 – 1 3 8 4 Pros and cons of mouse models  Part 4 ❑ Target selection ❑ In vitro cancer target validation assays ❑ In vivo cancer target validation assays ❑ In vitro drug evaluation assays ❑ In vivo drug evaluation assays  In vitro drug evaluation assays In vitro drug evaluation assays are similar to those used in Part 2  Part 5 ❑ Target selection ❑ In vitro cancer target validation assays ❑ In vivo cancer target validation assays ❑ In vitro drug evaluation assays ❑ In vivo drug evaluation assays Pre-clinical studies Drug Evaluation in PDXs: Addressing Patient Heterogeneity Breast Cancer Res. 2014 Apr 7;16(2):R36. Evaluation of Immune-based Therapy PDX in humanised mice Gut. 2018 Oct;67(10):1845-1854. Summary of in vivo drug-evaluation using mouse models Thank you!