LSM4214 Lecture 9

Cancer Pharmacology: Models for Drug Target Discovery and Evaluation

• Learning Outcomes: Define and explain target selection in cancer research; describe in vitro assays for cancer target validation, including their importance and animal models used; explain in vitro drug evaluation assays and in vivo drug evaluation methods; understand the advantages and disadvantages of in vitro and in vivo models for drug target discovery and evaluation.

Target Selection

• Rational Approach: Identify likely targets from known cancer-promoting pathways using "reverse genetics" (gene to phenotype). Specific genes are targeted for mutation to observe resulting phenotypes.

• Unbiased Approach: Use expression profiles to identify probable targets without prior knowledge of their function using "forward genetics" (phenotype to gene). Randomly generated mutations identify unknown genes responsible for observed phenotypes.

In Vitro Target Validation Assays

• Engineered Cell Lines: Use siRNA, shRNAi for loss-of-function studies & overexpression for gain-of-function studies.

• Assays: Analyze gene/protein expression, use Apoptosis (Annexin V) assays, Proliferation assays, Motility (migration) assays, and in vitro metastasis assays.

In Vitro Assays: Cell Viability

• MTT Assay: Cell-based assay to measure cell metabolism. MTT is a tetrazolium salt that converts to formazan inside active cells.

In Vitro Assays: Cell Proliferation

• BrdU Incorporation (BrdU) Assay: Cell-based assay for measuring DNA replication by incorporating BrdU (an analogue of thymidine) into S-phase cells.

In Vitro Assays: Cell Apoptosis

• Annexin-V/PI Assay: Detects apoptotic cells using flow cytometry.

• Mechanism: Annexin V binds to phosphatidylserine, which is externalized on the surface of apoptotic cells; PI stains the nuclei of dead cells.

In Vitro Assays: Cell Motility

• Scratch Assay: Measures cell migration by creating a wound in a cell culture and measuring its closure over time.

In Vitro Assays: Cell Invasion

• Matrigel Invasion Assay: Measures the ability of cells to invade through a Matrigel-coated membrane.

In Vitro Models: Organoids

• Description: Self-organized three-dimensional (3D) multicellular tissue cultures derived from healthy/cancerous stem cells, resembling in vivo organs.
• Applications: Study early embryogenesis, organ and tissue development, drug screening, disease modeling, and personalized therapy.

In Vivo Cancer Target Validation Assays

• Xenograft: Human cancer cell lines implanted into immunocompromised mice.
• Allograft/Isograft: Cancer cells from one mouse or rat transplanted into another (same species): used for immune system contribution and interactions with tumor stroma.
• Genetic Models: Genetically modified mice for oncogenes, tumor suppressors, or modifications in tumor progression (e.g., VEGF).

In Vivo Cancer Models: Heterotopic Human Xenografts, Murine Tumor Allograft/Isograft, Transgenic Models

• Heterotopic/Xenograft: Transplanting cells or tissues from one species to another species or individual. Immunosuppressed hosts are typically used to prevent rejection; often used with human cells.

• Murine Tumor Allograft/Isograft: Cancer cells from one mouse or rat transplanted into another (same species) to evaluate immune contribution and tumor stroma interactions.

• Transgenic Models: Generating genetically altered mice to study oncogene and tumor suppressor gene expression, function, and associated cancer phenotypes.

Evaluation of Tumors (Weight, Volume, Survival)

• Tumor Weight: Measured as a means of evaluating tumor growth.

• Tumor Volume: Calculated as length x width2 to measure tumour growth rate.

• Tumor Survival: Measured using Kaplan-Meier survival plots to analyze the duration/prolongation of life of a group with a specific treatment or condition.

In Vivo Drug Evaluation Assays

• In vivo: Assays use whole living organisms, including mice or other animals.
• Specificity: Evaluation of drug activity in animals/immunocompromised mice is generally done via drug vs. no-drug studies, rather than engineered models vs. wild-type models.

Labeled Tumor Models

• Technique: Engineer tumor cells to express fluorescent/luminescent proteins for tracking, metastasis, and imaging purposes to visualize tumour spread or response to a specific treatment.
• Advantages: Study tumor response, visualize metastasis, and assess internal tumor nodules.
• Disadvantages: Clonal cell lines might not completely represent the original tumor, and costly equipment is needed for visualization.

Bioluminescence Imaging

• Application in research: Use of Bioluminescence to visualize the presence and localization of tumors and their response to treatment.
• Process: Use luciferase genes and a luciferin substrate to produce readily detectable bioluminescent signals to study tumour extent.
• Advantages: Non-invasive methods for studying tumor growth, response to treatment, and metastasis in live animals.

Patient-Derived Xenografts (PDXs)

• Concept: Fresh tumor tissues from patients are implanted into immunocompromised mice to create personalized cancer models that better mimic the human condition.

Summary of Mouse Models

• General Advantages: Fast, Reproducible, Relatively inexpensive, relevant to clinical studies
• General Limitations: Short lifespan, different contexts (cellular, physiology, immune), different tumor growth/environment.

Limitations of In Vitro Studies

• Behavior: Cultured cells differ significantly from in vivo tumor behavior.
• Diversity: Monocultures lack the cellular diversity found in heterogeneous tumors.
• Immune System/Stroma: Cannot evaluate contributions from immune system and stromal cells.
• Toxicity: Lack of controls for toxicity evaluation.