Issue: Q1, 2011

In Vitro Assays Available

• Proliferation– Metabolic read-out– Biomass– BrdU– Dual live/dead

• Colony Formation (2D)• Colony Formation (3D soft agar)• Combination Assays• Matrigel Phenotypic Assays• 3D Spheroid Assays• FACS• Invasion Assays• Migration Assays• Apoptosis Assays

• Western Blotting• Cellular ELISA

– In-cell– Lysates

• Senescence Assays• Autophagy Assay• siRNA• Comet Assays• Hypoxic Assays• RNA and cell pellet/lysate preparation• Cell Panels

– Growth properties, condition scouting– Compound profiling– Protein expression profiling

Proliferation Assay – Standard Cell Lines

The effect of compounds on cell viability are investigated over a time course of 24-96h

Proliferation assays can be run using the method of your choice. Current assays running at HDS include alamar blue, WST-1, Sulforhodamine B (SRB), BrdU and Cell Titer-Glo

Proliferation assays can also be multiplexed with live/dead viability readouts and a variety of apoptosis assays

We offer proliferation assays in cancer cell lines, primary cell lines, or growth factor-induced assays for specific targets

The effect of the VEGFR inhibitor SU5416 on VEGF-stimulated cell growth

HUVEC cells are seeded in full growth media overnight followed by 48h compound incubation in basal media containing 10ng/ml VEGF. Viability is measured using alamar blue.

X-MAN Proliferation Assay - Profiling Sensitive Patient Populations

Assays are available using the full panel of genetically defined and patient relevant X-MAN human cancer models

Cell backgrounds include non-transformed MCF10A and tumourigenic lines e.g. HCT116, DLD-1 and SW48

Each cell type comprises a panel of mutant or normal target gene knock-ins or knock-outs, covering important therapeutic targets e.g. PI3K, PTEN, B-Raf, K-Ras, EGFR, p53 and many others

Assays are performed under standard or defined conditions to mimic the tumour microenvironment:• Reduced or defined serum• Low energy• Hypoxia

Using defined assay conditions, selectivity profiles of targeted agents are often maximally revealed

Gefitinib profiling on the HME X-MAN panel

A panel of HME lines containing knock-in mutations of EGFR, K-Ras, B-Raf or PI3K were seeded in 96-well plates overnight followed by 96h compound incubation. Viability was measured using SRB.

EGFR knock-in lines are ~1000 fold more sensitive to gefitinib.

X-MAN Proliferation Assay - Profiling Resistant Patient Populations

The effects of secondary patient mutations on targeted drugs can be investigated using an expanding array of double knock-in X-MAN cell models such as:

• EGFR & B-Raf• EGFR & PI3K• PI3K & K-Ras• ATR/p53

Results have been shown to recapitulate known patient data

Cetuximab profiling on HME X-MAN single and double knock-in cells

An HME panel of wild type, EGFR, EGFR & B-Raf and EGFR & PI3K knock-in lines were seeded in 96-well plates overnight followed by 7 days compound treatment. Viability was measured using SRB.

Cetuximab is sensitive in EGFR single knock-in cells, with resistance seen in the double mutant lines.

Colony Formation Assay

Using standard or X-MAN cell lines, the effect of compounds over extended assay times can be investigated in colony formation assays

Colony forming assays are particularly useful when investigating compounds that act via a cytostatic mechanism or require many cell doublings

Assays are fully quantitative allowing IC50 values to be generated

In addition to 2D colony formation assays, quantitative soft agar assays for colony formation in 3D can also be investigated

DLD-1 BRCA2 null cells show selective sensitivity to the PARP inhibitor olaparib

DLD-1 parental and BRCA2 null lines were seeded in 24-well plates overnight, followed by compound incubation with cells for 10 days. Colonies were stained with crystal violet and absorbance of solubilised stain measured.

Target Validation: siRNA Studies

Target Expression

β-actin Expression

1 2 3

1 = Cells Only2 = Non-Target siRNA3 = On-Target siRNA

Validation of a target using siRNA allows targeted, gene-specific loss-of-function phenotypes to be explored in human cells.

Confirmation of target knock-down and functional consequence can be measured, proving invaluable information regarding gene functions and maximum effects that can be achieved.

Confirmation of knock-down of the target protein

Knockdown of the target of interest was confirmed by Western blot, and the anti-proliferative effect of the targeted and non-targeted siRNA studied using SRB

Functional consequence of target knock-down: Cell viability assessed by SRB

Drug Combination Assays

Using standard or X-MAN cell lines, the effect of drugs in combination can be investigated in a quantitative manner

Drug combinations can be run using any assay format, most commonly proliferation assays, colony forming assays and apoptosis assays

Such combinations can be used to screen for agents that are only effective as combination therapies, such as Chk1 inhibitors

Assays can also be used to identify novel combinations of existing drugs

The Chk1 inhibitor UCN-01 sensitises HT29 cells to the topoisomerase inhibitor SN38

HT29 cells were seeded in 96-well plates overnight followed by 72h exposure to a matrix of concentrations of UCN01 and SN38. Viability is measured using alamar blue.

Signal Transduction Assays

Signal transduction events can be measured in numerous ways within the cell

Panels of Western blots can qualitatively investigate multiple signalling pathways and phosphorylation events from one cell sample

Quantitative cell ELISAs can be used to investigate specific mechanism of action of compounds in a 96-well format, permitting rapid evaluation of compound effects and ranking of compounds

Multiple cell ELISAs have been established at HDS including phospho-S6 ribosomal protein, phospho-histone H3 and phospho-aurora A

The ability of the PI3K/mTOR inhibitor BEZ-235 to inhibit phosphorylation of the downstream protein S6

MCF7 cells were treated with BEZ-235 for 2h and phosphorylation of S6 ribosomal protein measured by ELISA.

Apoptosis Assays

A variety of different apoptosis detection methods are available including:

• Caspase activity• PARP cleavage• TUNEL• Flow cytometry

Assays can be multiplexed with cytotoxicity assays providing multiple read-outs from one well

Staurosporine induces apoptosis in NRK-52E cells

Above: NRK-52E cells were exposed to staurosporine for 24h and apoptosis and cell viability measured using the Caspase-Glo luminescence assay (apoptosis) multiplexed with the CellTiter Blue assay (viability)

Left: PARP Cleavage by Western blotting

Full length PARP Cleaved PARP

Cleaved PARP

C 10 30 100 300

β-Actin

Paclitaxel, nM

DNA Damage Assays

The single cell gel electrophoresis assay, or comet assay, is the gold standard technique used to directly measure DNA damage at the level of the individual cell

Evaluation of the DNA comet tail shape and migration pattern allows assessment of DNA damage

Additional assays available include evaluation of histone H2AX protein phosphorylation (γ-H2AX) following DNA damage stimulus

Hydrogen peroxide causes DNA damage as detected by increased tail length in the alkaline comet assay

H460 cells were treated with hydrogen peroxide for 10min before lysis and running in the alkaline comet assay.

Control Compound Treated

Western Blotting

Western blotting allows detection of proteins from cell and tissue samples

Panels of Western blots can qualitatively investigate multiple signalling pathways from one cell sample

Events such as phosphorylation of target and non-target proteins, apoptosis and cell cycle progression can be monitored simultaneously

Nuclear and cytoplasmic extracts can be independently investigated

PI3K and ERK pathway activation in X-MAN HCT116 cells containing PI3K and K-Ras mutations

Cells were grown overnight in normal or low serum conditions and whole cell lysates separated by SDS-PAGE and Western blotted for components of the PI3K and K-Ras pathways.

Flow Cytometry Assays

A wide range of applications for flow cytometry are available, where physical and chemical characteristics of cells can be measured

The most common use of flow cytometry at HDS includes the analysis of cell cycle and apoptosis

Other assays include the detection of multiple cell markers, and measurement of telomere length by fluorescent in-situ hybridization

Combination effects of SN38 and a Chk1 inhibitor on cell cycle

HT29 cells were seeded into 6-well plates overnight, followed by 24h drug treatment as single agents or in combination. Cells were stained with propidium iodide and measured using the FACS Calibur.

Asynchronous S phase arrest Arrest over-ridden

Control SN38 SN38+Chk1 inhibitor

Angiogenesis Assays

A range of assays are available varying from proliferation of endothelial cells to invasion of cells through extracellular matrices

Migration and invasion assays are based on the ability of endothelial cells to move through extracellular matrices towards a chemoattractant

Tube formation assays and scratch assays mimic cell migration and the ability of endothelial cells to form 3D capillary-like tubular structures

Angiogenesis assays and conditions are tailored to meet the relevant requirements for your target and agent

The matrix metalloprotease inhibitor GM6001 inhibits invasion across basement membrane

extract

HT1080 cells were seeded into the top well of a Boyden chamber and allowed to invade across BME towards 10% serum. Invaded cells were stained with calcein AM and fluorescent product detected.

3D Assays – Matrigel Assay

The culture of cells in 3D on extracellular matrix allows crucial micro environmental cues that may be lost in 2D to be investigated

Cells are grown in a more physiological system and the effects of compounds on the morphology or migration of the cells can then be studied

Of particular relevance is the morphological phenotype of invasive cells which can be investigated in this assay

Semi-quantitative analysis of compound effects can be performed by measuring the branching phenotype and clumping of the cells

MCF10A parental or PI3K mutant cells grown in 3D matrigel phenotypic assay

MCF10A parental and PI3K mutant cells were treated with GDC-0941 and allowed to grow on matrigel for 24h. GDC-0941 prevents formation of the invasive phenotype seen in PI3K mutant cells.

Parental Cells PI3K Mutant Cells

Control

GDC-0941

Hypoxic Assays - I

The importance of hypoxia in cancer is firmly established, with hypoxic regions associated with altered cellular signalling, metabolism and increased resistance to radiation and chemotherapy

The ability to tightly control in vitro assay conditions permits investigation of cells under a range of oxygen concentrations

The Bactron anaerobic chamber allows investigation under complete anoxia, and assay manipulations to be performed under anoxic atmospheres

The BioSpherix OxyCycler allows oxygen profiling from 0.1% to 99.9% and can hold any set point for any length of time, allowing complete flexibility in oxygen profiling

Hypoxic Assays - II

The hypoxic tumour microenvironment can be exploited for therapy in a number of ways:

• Altered metabolism• Hypoxia inducible targets• Bio-reductive drugs

Over 30 cell lines have been evaluated for growth under different oxygen levels

Compounds can be profiled under tumour relevant oxygen levels

The bio-reductive drug tirapazamine shows hypoxic selectivity in an HCT116 cell proliferation assay

HCT116 cells were seeded in glass 96-well plates overnight before a 4h exposure to hypoxia/normoxia in the presence of tirapazamine. Cells were grown out in drug free media under normoxia for 5 days and viability determined using alamar blue.

3D Assays – Spheroids and Soft Agar

Cells can be grown in 3D independently of supportive matrix, to form a ball of cells termed a ‘spheroid’

Spheroids of uniform size are generated and grown in 96-well plates, allowing compounds to be studied in a quantitative manner

Multiple end points can be investigated, including viability, apoptosis, spheroid outgrowth, and sectioning of spheroids for immunocytochemistry

>20 cell lines evaluated for spheroid growth, including standard and X-MAN cell lines

Spheroids formed from different cancer cell backgrounds

HCT116 BT474 A549

HCT116 spheroids were treated with the hypoxic marker pimonidazole, embedded and sectioned. Cell nuclei were detected with Hoechst 33258, hypoxia using Hypoxyprobe, and the image merged to show a central hypoxic core.

Cell nuclei Hypoxia

Merged image

Immunocytochemical staining of HCT116 spheroids

Senescence Assays

Cellular senescence is an irreversible arrest of cell proliferation

The limiting dilution senescence assay is able to differentiate between compounds which induce cellular senescence and those which are cytotoxic is available

Mechanism of action studies including SA--gal staining, p16 and p21 expression can be run simultaneously

The ability of the test compound to induce a permanent growth arrest in IMR-90 cells

IMR-90 cells were seeded in 24-well plates overnight, compound treated for 3 days, then grown in drug free media for up to 6 days. Cells were stained with crystal violet then absorbance of the solubilized stain measured.

Mitochondrial Assays

Purified mitochondria can be isolated from cells allowing the effects of compounds on the mitochondria to be studied

Screening assays using isolated healthy versus tumour mitochondria can help to identify tumour-specific drugs and understand their mechanism of action

Functional or protein-localisation assays include:• Compound toxicity profiling• Compound efficacy profiling • Biomarker localisation

Localisation of specific biomarkers in mitochondria isolated from wild type vs. mutant PI3K HME cells

Mitochondria were isolated from HME wild type (WT) and PI3K mutant (H10475/+) cells and lysates separated by SDS-PAGE and Western blotted for components of the apoptotic pathway.

Synthetic Lethality Assays - I

Pooled shRNA-based screens are a powerful way to find novel targets that have selectively lethal effects on cells harbouring specific genetic backgrounds.

Using X-MAN cell lines in combination with Alan Ashworth’s latest generation shRNA libraries and ‘Next-Gen’ sequencing capabilities, we offer a premium service to screen for synthetically lethal target genes in specific patient relevant genotypes.

PARP inhibitor synthetic lethality screen performed by the Ashworth laboratory

Synthetic Lethality Assays - II

We also offer a service to screen compound libraries to directly identify, or ‘re-profile’ drug candidates on specific target patient genotypes.

This approach is becoming common to interrogate patient genotypes that are ‘undruggable’ by conventional means.

By screening the full complexity of the cancer cell’s biology, yet retaining a read-out to a specific patient population, one can rapidly de-orphanize previously undruggable cancer genes e.g. K-Ras, p53, and quickly ‘re-profile’ already approved treatments to new uses in cancer.

Effect of a hit on WT vs. mutant K-Ras X-MAN cells

30,000 compounds were screened against X-MAN cells harbouring WT vs mutant K-Ras and several mutant K-Ras selective compounds were identified (example shown above). Another hit was tested in vivo and showed good activity with minimal side-effects, and was licensed to a big pharma.

Pre-Clinical Discovery Services (via TD2)

ONCOLOGY DRUG DEVELOPMENT CONTINUUM

A druggable genome siRNA screen to identify genes that affect bortezomib sensitivity

(A) Heat map of raw data of all 384-well plates in primary screen. After 24h siRNA transfection, various doses of bortezomib were added and cell viability determined at 96h

(B) Dose response curves fitted to the data of siRNAs. Red= control siRNAs +bortezomibGrey= one siRNA +bortezomib

(C) Histogram of EC50 values derived from the dose response curves of all siRNAs

siRNA Screens - I

(A) A list of the strongest sensitizer hits that were defined as genes targeted by at least two distinct siRNA species that decrease EC50 by three standard deviations from cells treated with control siRNAs.

(B) Dose response curves fitted to the data of 6 top ranking hits in rescreen.

siRNA Screens - II

A druggable genome siRNA screen to identify genes that affect bortezomib sensitivity

Top ranking bortezomib sensitizers identified from drugable genome screens

Cells were infected with control virus and five different CDK5 shRNA. At day 2 and day 3 after infection, expression of CDK5 was assayed by western blot (A-B)

Three proteasome inhibitors were added at the indicated concentration. Cell viability was measured at day 3 post exposure and the dose responsive curve was made for each control and CDK5 shRNA infected cells (C).

Sensitization to proteasome inhibitors after introducing CDK5 shRNAs

shRNA Screens

In Vivo Assays Available - I

TD2 Tumour Cell Lines

TD2 Primary Tumourgrafts Isogenic Tumour Cell Lines

In Vivo Assays Available - II

Oral HDAC6 inhibitor shows synergistic effects at suboptimal doses when combined with standard of care treatments

Tumour Xenograft Efficacy Studies

• Validated assay systems

• Experienced handling of using isogenic and non-isogenic cell lines in both standard and non-standard conditions

• Extensive range of in vitro and in vivo assays

• Access to entire X-MAN panel on a per compound, per project or per FTE basis

• Opportunity to evaluate isogenic cell lines before bringing in-house

• License and transfer validated assays

Why Work With HDS?