ControlControl TreTreatedatedChallenge: Challenge: Cancer metastatic cascade

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Tony Ng

Randall Division of Cell & Molecular Biophysics

Cancer Cell Biology and Imaging, Division of Cancer Studies

‘‘Principles of cellular organization: Principles of cellular organization: genes, proteins, and global functiongenes, proteins, and global function’’

Chemistry/Chemical Biology

Mathematical/networkModelling

Bio / Nano-Photonics

Instrumentation & Data Processing

HTP PLATFORM

Bioinformatics

Interaction prediction

Molecular & Cell Biology

Understanding cell biology quantitatively requires multidiscipliUnderstanding cell biology quantitatively requires multidisciplinary collaboration nary collaboration

AmeerAmeer--Beg, Barber, Coolen, Festy, Beg, Barber, Coolen, Festy, Fraternali, Fraternali,

HailesHailes, , HeintzmannHeintzmann, Irving, Ng, , Irving, Ng, Richards, Richards, SuhlingSuhling, Tabor, Thomas, , Tabor, Thomas,

Vojnovic, et al.Vojnovic, et al.

http:www.opticalproteomics.orghttp:www.opticalproteomics.orgBiostatistics/stratification

Biomedical application

CancerAlterations in gene expression induces formation of a primarytumour.

1. Outside factors- Smoking- UV light

2. Genetic disease

3. Viral infection

Cancer• Understand the cellular processes causing

cancer– Identify changes in genes and protein

expression– Identify mutations in candidate genes and

proteins altering their function

Methods to study cancer

• Molecular Biology

• Protein Chemistry

• Cell Biology

• Molecular Biology

– Monitoring transcription and translation of DNA to RNA to protein

– Regulation of Gene expression

Methods to study cancer

• Protein Chemistry

– Physical behaviour of proteins

– Determination of protein expression

Methods to study cancer

• Cell Biology

– Study of cell behaviour

– Characterise protein function in cells

Methods to study cancer

Molecular Biology

Background:• Several whole genomes have been fully

sequenced, allow comparison of entire genomes, Genomics, and thus predict all proteins, as well as evolution of species Bioinformatics.

• Human Genome contains 23.000 genes compared to 28.500 in mouse.

Translate DNA to protein

Monitor gene expression

• Real-time Polymerase Chain Reaction

• cDNA array

PCRPolymerase Chain Reaction:

PCRPolymerase Chain Reaction:

PCR

- Monitor the incorporation of fluorescent dye during the reaction as an indication of production/amplification of gene of interest at each PCR cycle

cDNA array• Attach probes corresponding to larger amount of mRNA of interest• Commercially available

cDNA array

cDNA array

Gene array

Gene array1. Isolate four different cell lines with various

degree of metastatic probabilities for lung cancer

Breast CarcinomaCell line

Gene array2. Compare genotype of the four cell lines

Upregulated

Downregulated

Four cell lines

Gene array3. List of 54 genes with altered expression specific for

primary breast cancer to metastasise to lung.

• Compared expression of these 54 genes in primary tumours obtained from human breast cancer patients and developed a prognostic “gene signature” for breast-to-lung metastasis

Gene expression in cancer

• Very powerful technique, highly sensitive• Screen numerous genes in high-

throughput fashion. Large amount of data.

• Downside:– Gene expression levels is not always comparable to

protein expression levels. mRNA vs protein stability. – Protein function is regulated “post-translationally”

Protein Chemistry

Background:• Large macromolecules • The main machinery for all functions within the cell and

perform highly specialised duties determined by their amino acid composition

• Regulated function by modifications, phosphorylation, GDP/GTP loading, lipid anchoring, cellular localisation (membrane, cytoplasm, nucleus).

Protein Chemistry

Background:• Chain of amino acids (polypeptide)

– Number of amino acids varies from 10s to 20,000aa, average ~ 500 aa

• 20 different amino acids• Each protein has it’s own specifications

– Molecular weight (Daltons)– Isoelectric point (pH at which the molecule is neutral)

• Dimensions: ~ 10 nm3

Protein Chemistry

Protein ChemistrySDS Poly Acryl Gel Electrophoresis (SDS-Page):

- Analyse Protein Expression

Protein Chemistry

Mw

SDS Poly Acryl Gel Electrophoresis

Visualised by Dye (Coomasie)

Western Blot

Visualised by Chemiluminescence

Transfer to membrane

Incubate with antibodies of interest

- Analyse Protein Expression

Antibodies• Source for immune response in

order to neutralise foreign objects • Each antibody recognises

one specific antigen• Important tool in biochemistry

due to specificity• Produced in mouse or rabbit

by repeatedly injecting antigen (i.e. protein) of interest for a immune response, and isolate the serum.

Protein Chemistry- Analyse Protein Expression

Isoelectric focusing

Mw2 Dimensional SDS Page:

Protein Chemistry- Analyse Protein Expression

Control

Treated

Protein identification by Mass spectrometry

• Monitor protein expression, which is the cell machinery and ultimately the end-product of gene expression.

• Downside:– Low sensitivity, i.e. needs any cells for

detection– Limited by antibody specificity

Protein Chemistry

>Cancer Cell Biology

• Characterise the cellular function of expressed proteins:

– Alter cell proliferation

– Induce metastasis,i.e. increased cell migration

Structures within the cell

Cells in tissue culture• Grows at 37 degrees at controlled pH, supplied with calf serum,which contains vital supplement of various proteins and growth factors

• Divides every 16-24 hours

Imaging proteins in cells

• Immunofluorescent staining– detect localisation of endogenous proteins

• Fusion proteins with fluorescent proteins– detect spatial and temporal localisation of

exogenous proteins (live imaging)

• Immunofluorescent staining– detect localisation of endogenous proteins

• Specific fluorescent dyes or fluorescently conjugated antibodies against protein of interests on fixed samples

– DAPI, FITC/Cy2, TRITC/Cy3, Cy5

Imaging proteinsin cells

Imaging proteinsin cells

• Fusion proteins with fluorescent proteins– detect spatial and temporal localisation of

exogenous proteins (live imaging)

• Expression from plasmid after lipid transfection or microinjection

– Cyan, Yellow, Green, Red fluorescent proteins

Imaging proteinsin cells

18min18min

60min60min

88min88min

120min120min

131min131min

180min180min

260min260min

260+min260+min

EGFREGFR--GFPGFP Activated Activated ββ1 1 IntegrinIntegrin((mAbmAb 12G10)12G10)--Cy3Cy3

EGFEGF

Plasmid•Circular DNA approximately 5000 nucleotides•Easy to produce in large quantities in bacteria•Easy to “manipulate” (=cloning)

GFP-fusion protein

Excitation maximum at 488nm

Emission maximum at 507nm

GFP-fusion protein

CXCR4 is upregulated in many tumours. We wanted to characterise the recycling, and possibly manipulate and induce downregulationof the receptor

Receptor recycling using CXCR4-GFP

Cell migration• in vitro models for cancer cell metastasis

– characterise proteins promoting migration– identify signals expressed in tissues, which

attract cancer cells

Cell migration• Identify and characterise proteins involved

in cell migration– Detachment– Translocation– Protrusion

Cell migration“Wound assay”- easy to perform- measuring “wound closure”

Cell migrationRandom migration- observe behaviour- measuring cell speed

Cell migrationChemotaxsis- what attracts cells/ promotes migration - measuring cell direction

Gradient

Altering protein expression levels

• Knock out mice. – Delete single genes within the mouse

genome.

• RNA interference– Decrease the mRNA level in tissue culture

cells.

Altering protein expression levels

• Knock out mice. 1. Gene isolated and

manipulated in vitro2. Isolate stem cells from

embryos3. Introduce “new” gene into

stem cells4. Insert stem cells into

blastocytes, and implant back into “pseudo”-pregnantfemale mouse

5. Chimera offspring, heterozygote6. Cross (breed) with other chimera

homozygote

Altering protein expression levels

• …. knock-out mice– Expression of …. is necessary for cell migration

Wildtype mouse

embryonic fibroblasts

Knock-out mouse

embryonic fibroblasts

Altering protein expression levels

• RNA interference

Scramble RNAi Cdc42 RNAi

Western blotfor expression levels

– RNA interference of Cdc42

Altering protein expression levels

Scramble RNAi Cdc42 RNAi

– RNA interference of Cdc42

Altering protein expression levels

Compare cell tracks of individual cells- expression of Cdc42 is important for cell migration

• Analyse cell behaviour upon altering expression of individual proteins

– Secondly, to identify signalling pathways involved, i.e. other proteins and their function affected due to altered expression

Altering protein expression levels

Analyse protein-protein interactions, proteomics

• Proteins function in larger complexes

– Identify known members within these complexes can indicate previous unknown functions of the protein of interest

Analyse protein-protein interactions

Complexity of the Proteome Complexity of the Proteome ––

The need for an imaging approachThe need for an imaging approach

Protein ‘modifications’ +

interactions in time and space

= Function

Dynamics!Dynamics!

Analyse protein-protein interactions

• Immunoprecipitation (-protein chemistry)– Analyse protein complexes

• FRET/FLIM (-cell imaging)– Analyse protein-protein interaction in cells

Analyse protein-protein interactions

• Immunoprecipitation– Analyse protein complexes

X

Y

What interacts withprotein X?

Analyse protein-protein interactions

• Immunoprecipitation– Analyse protein complexes

Mass spectrometry and/or

Western Blot analysisto identify protein Y

X

Y

Incubate with immobilised antibodies

towards protein Xand isolatecomplexes

Analyse protein-protein interactions

• FRET/FLIM Fluorescent/Förster resonance Energy Transfer, FRET by Fluorescent Life Time Measurements, FLIM– Analyse where protein-protein interactions

occur within cells

Analyse protein-protein interactions

• FRET, Fluorescent Resonance Energy Transfer– causes excitation of acceptor if within the proximity of 9 nm

Donor Acceptor

Förster Resonance Energy Transfer (FRET)

donor2-photon excitation

acceptor

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FRE

T Ef

ficie

ncy

RR

R/R0

Fraction of excited donors transferring to acceptor

FRET

Energy absorbed by donor can Energy absorbed by donor can resonantly transfer to acceptor.resonantly transfer to acceptor.

Energy appears as donor fluorescence, Energy appears as donor fluorescence, acceptor fluorescence, or are lost acceptor fluorescence, or are lost through nonthrough non--visible processes.visible processes.

Multi-photon fluorescence lifetime techniques

Laser

ScannerFilters &

DescannedDetectors

Non-descannedDetectors

Sample

Dichroic mirrors

Dichroic

TCSPC Histogram

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coun

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TCSPC Histogram

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time (ns)

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nsity

τ = 2 ns

Excitationpulse

Nextpulse

TRI2 software- analyse fluorescent lifetime

Photon count

Average fluorescentlifetime Time-Domain

Cell expressing ezrin-GFP

Developed by Dr Paul Barber at Gray Cancer Institute

Control

TRI2 software- analyse fluorescent lifetime

Photon count

Average fluorescentlifetime Time-Domain

Cell expressing ezrin-GFP

Treated

FRET

Active(low GFP lifetime,

low RFP anisotropy)

Inactive(high GFP lifetime,

high RFP anisotropy)

sensorligand

Conformation sensing - Two examples

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FRET population

time (s)

Rac/Cdc42

FRET

PAKInactive(high GFP lifetime

low FRET Pop)

Active(low GFP lifetime,high FRET Pop)

GDP-bound

Leo Carlin, Ton CoolenLeo Carlin, Ton Coolen

A delayed negative feedback loop in immunosurveillance