L12: Models of drug target discovery and evaluation Flashcards
What are the characteristics of a good target?
- Highly expressed in cancer cells: Ideally, broadly expressed in cancers: To help most patients with that cancer, cost effective to research
- Selectively expressed in cancer cells
- Contribute metastatically to cancer pathology: either GOF/LOF
- Expression correlates with cancer stage/clinical outcome: Eg people with this BM have poorer prognosis etc, monitoring BM
What is the process of drug discovery?
Target selection -> In vitro cancer target validation assays -> In vivo cancer target validation assays -> In vitro drug evaluation assays -> In vivo drug evaluation assays
How do you select a target?
Rational approach
- Identifying likely targets from known cancer promoting pathways (Would require a lot of information)
- “Reverse genetics” (from gene to phenotype)
- time consuming
Unbiased approach eg. SKY painting
- Uses a screen or an expression profile to identify probable targets without considering their known functions
- “forward genetics” (from phenotype (what you observe) to gene)
How is unbiased approach done?
RNAi screens/CRISPR screen
- Specific, individual genes are “knocked down” to find genes that regulate key cancer processes
- Unlike expression studies, can identify potential targets by function (whether the protein is functional)
- Limited to studies in cell lines.
- Tell you which gene/protein plays the most important role in cancer growth
How to do it?
- If oncogene is KO, decreased cell growth
- If TSG is KO, increased cell growth eg. DEATH receptor
- If drug resistance gene KO, decreased growth in the presence of drug
How is in vitro cancer target validation performed?
- Performed in cells isolated from human tumours and grown in monoculture
- To perform 5 assays: Gene expression analysis, cell rounding assay, proliferation assay, motility (migration) assay, in vitro metastasis assay
1) Engineer cells lines (GOF/LOF): - LOF: using siRNA, shRNAi to silence the gene
- GOF: overexpress the gene
- Allows analysis of specific role of target
- If OG; LOF would return to normal phenotype and GOF will cause cancer
2) Cell-based assays - Viability - MTT (reagent): measures cell metabolism
- If cell is alive after KO target, metabolism occurs -> MTT turns blue (mitochondrial reductase) -> means it may not be our target since we want cancer cells to die after KO the target gene
3) Cell-based assays - Proliferation - If we KO the target/add a drug -> how do label the newly proliferating cells
- BrdU incorporation -> label actively dividing cell (incorporated into DNA when it is actively dividing) -> labels S-phase cells
4) Cell-based assays - Death - To test if a drug kills cells/target kills cells -> measure apoptosis using Annexin V translocation (lipids flip during apoptosis, recognising the flip annexin V)
5) Cell-based assays - Motility - to show that my target affects cells and whether it plays a role in metastasis (one of the goals for cancer research is to prevent metastasis)
- Using scratch assay -> how fast the cancer cells fills the gap after scratching
6) Cell-based assays - Metastatic potential - Matrigel invasion assay
- [Refer to slides] Cell suspension placed in upper chamber (no GF in the gel chamber but GF in solution) -> Invasive cells pass through basement membrane layer and cling to the bottom of the Boyden chamber membrane. Non-invasive cells stay in upper chamber -> After removal of non-invasive cells, invaded cells are stained and quantified
- To see how many cells/how long the cells take to eat through the gel and move to GF solution
What are some 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
What assays can you do with 3D in vitro models and some advantages?
- Using spheroids, possible to recreate the TME (Include cancer cell lines, primary tumour cells and cancer stem cells) to validate targets: Efficacy Testing, Cell Migration, Toxicity Testing, Cell Differentiation, Embryoid Body Formation, Angiogenesis, Colony Formation, Cell Expansion, Spheroid Formation, Cell-to-Cell Interactions, Genomic Expressions, Cell-to- ECM Interactions, Proteomic Expressions
- 3D Organoids proliferation assays - High content analysis: label organoids with Edu (similar to BrdU) -> Test GOF/LOF or drug -> Image Edu+ Green and Edu- Red to look at the ratio of red vs green -> Quantify green signal compared to control
- 3D Organoids Apoptosis assays - Co culture assays: Can create the tumour immune environment -> To test how the immune cells react to drug/target
- 3D Organoid Assays - Metastatic potential: Spherical/Spiky (looking for blood vessel for invasion, highly metastatic) + Using biotech chips to test invasion (invading gel interface)
What assays are done for in vivo cancer target validation?
- Xenograft (either through subcutaenous or orthotopic)
- Allograft/isograft
- Genetic models (for OG/TSG): KO or overexpression of candidate target
- For genes that modify tumour progression eg. VEGF: Crossing KO mice and transgenic mice -> transgenic mice that spontaneously form tumour
How do you validate using xenograft mice?
- Xenograft models: the inoculation of cells (dispersed, organs, tissues) from one individual (source) to another from a different species.
- Usually involves the use of models with an impaired immune system for avoiding rejection
- Eg. Nude mice (Immunosuppressed, athymic), NOD-SCID mice (immunodeficient, no acquired immunity) -> No immune response due to human cells
Can be done through:
Subcutaneous: Injecting empty vector on one side (control, no tumour) and overexpress target gene on another side (forms tumour)
Orthotopic: Injecting at the place where cancer takes plc eg. lung cancer cells at lung, bladder cancer cells at bladder -> forming orthotopic tumours
What are the adv and disadv of subcutaneous and orthotopic?
Subcutaneous:
Adv:
- Easy
- Monitor growth with calipers
Disadv
- Usually do not metastasize (Cannot study where it goes to)
- Non-physiologic extracellular environment
Orthotopic:
Adv:
- Often metastasize
- Similar extracellular environment -> more close to what happens
Disadv:
- Require more technical skill -> a lot of training
- Usually cannot use calipers -> cannot use ruler to measure the tumour
What are the advantages and disadvantages of using xenograft?
Adv:
- More clinically relevant tumor model. (Compared to cell culture)
- Malignant cells are human.
- Reproducible models.
- Variety of tumor lines available.
- Validated model for the predictive assessment of cytotoxics.
- In vivo studies of acquired drug resistance and its circumvention.
Disadv:
- Stromal component of tumors is rodent (no communication between cancer cells and TME/immune cells)
- Hosts are immunodeficient.
- Tumors growth in non-natural sites (heterotopic).
- The subcutaneous model generally does not metastasize: not a good model for studying anti-metastatic strategies.
What is allograft and how is it done?
- the inoculation of cells (dispersed, organs, tissues) from one to another individual (source) from the same species. For cancer research the mostly used models are Mice and rats.
- Taking mouse cells in vitro/vivo -> transfer to another mouse of same/diff species
- syngeneic: genetically identical, or sufficiently identical and immunologically compatible
- allogenic: different individual from same species (genomic identity slightly different -> to identify which cells are host/new
What is the adv and disadv of allograft?
Adv:
- Can be perfomed in immunocompetent mice (allows analysis of immune contribution)
- More accurately represents tumor stromal interactions
Disadv:
- Tumor cells are murine and less representative of human disease -> more like solving mouse cancer
What are transgenic models?
[Refer to slides]
- Mouse in which its genetic material has been altered using genetic engineering techniques
- Genetically altered mouse models (GAMM) or genetically engineered models (GEM) for human cancers have been critical to the investigation and characterization of OG/TSG expression and function and the associated cancer phenotype.
- Forming genetic models with tissue-specific overexpression of target gene
What are some adv and disadv of transgenic model?
Adv:
- Tumor arises “naturally”.
- Can be followed for a long time course -> can study as normal cancer progression
Disadv:
- Breeding and maintenance: Expensive
- Tumors arise at variable stages from animal-animal and nodule- nodule. (may not develop cancer/only know 1 year later)
- The tumor cells are rodent.
- The stroma is rodent.
- Tumors difficult to follow.
- End-points is frequently survival.
- Often a good model for biology study. Not always useful for drug discovery -> Humans usually experience more genetic changes (Eg. not just HER2 but other genetic mutations too, hence not the same reaction)
