LSM4214 Lecture 9

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the primary defining characteristic of the rational approach in target selection?

It focuses on non-specific gene manipulation methods.

It uses known cancer-promoting pathways to identify targets. (correct)

It relies on random genetic mutations to identify targets.

It screens environmental factors influencing gene expression.

How does RNA interference (RNAi) primarily function in the context of gene targeting?

It introduces mutations to genes randomly.

It degrades or inhibits translation of target mRNA. (correct)

It specifically amplifies target gene expression.

It activates silent genes to enhance their expression.

What do in vivo assays primarily evaluate in the context of cancer target validation?

The expression profiles associated with cancer genes.

The efficacy of treatment in whole organisms. (correct)

Cellular mechanisms in vitro.

The specific phenotypes produced through gene alteration.

What is a main disadvantage of using unbiased approaches in target selection?

They are not based on established cancer pathways. Signup and view all the answers

Which method is used in the rational approach to understand gene function?

Reverse genetics Signup and view all the answers

Which of the following is NOT a feature of in vitro drug evaluation assays?

They require animal models for validation. Signup and view all the answers

What role does 'reverse genetics' play in cancer pharmacology?

It links specific genes to known cancer traits through mutation study. Signup and view all the answers

What is a key advantage of using in vivo assays over in vitro assays?

They provide information about drug effects in an actual biological system. Signup and view all the answers

What is one significant limitation of monoculture models in cancer research?

They lack cellular diversity found in heterogeneous tumors. Signup and view all the answers

What is a key advantage of using organoids in drug screening?

They provide a model for personalized therapy and disease modeling. Signup and view all the answers

What is a primary feature of heterotopic human xenografts?

They lack certain immune components including T cells and B cells. Signup and view all the answers

When comparing subcutaneous and orthotopic placement of cancer cells, what is a potential drawback of subcutaneous implantation?

It may not replicate the tumor's natural microenvironment. Signup and view all the answers

Which of the following terms is used to describe the placement of human cancer cells in their original organ site?

Orthotopic placement Signup and view all the answers

What does an in vitro drug evaluation assay primarily assess?

The cellular response to drugs in a controlled environment. Signup and view all the answers

Which model is characterized by genetically modified organisms to study tumor behavior?

Transgenic models Signup and view all the answers

Which evaluation metric is NOT typically used in tumor assessments?

Drug toxicity levels Signup and view all the answers

What is the primary function of siRNAs in siRNA screens?

To degrade target mRNA and reduce protein expression. Signup and view all the answers

Which statement accurately describes shRNA screens?

They use plasmid or viral vectors for more stable gene knockdown. Signup and view all the answers

What might an increase in cell growth indicate when conducting loss-of-function screens?

The activity of a tumor suppressor gene. Signup and view all the answers

Which assay is used to assess cell metabolic activity in vitro?

MTT assay. Signup and view all the answers

What is the purpose of using base analogues like BrdU in cell proliferation assays?

To measure DNA replication. Signup and view all the answers

What type of assay is the Annexin-PI assay designed for?

Apoptosis detection. Signup and view all the answers

Which of the following accurately describes organoids?

They share a high similarity to corresponding in vivo organs. Signup and view all the answers

What does the scratch assay primarily measure?

Cell motility during wound healing. Signup and view all the answers

Study Notes

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.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.