Understanding cell state with quantitative live cell imagingUnderstanding cell state with quantitative live cell imaging

Copyright © 2000 Cell Press. The Hallmarks of Cancer

Douglas Hanahan and Robert A. Weinberg  

Gain pathway knowledge: mechanistic understanding of diseaseidentification/validation of cellular biomarkerstherapeutic intervention, drug discovery, toxicity

Copyright © 2000 Cell Press. The Hallmarks of Cancer

Douglas Hanahan and Robert A. Weinberg  

Single cell measurementsSingle cell measurements

Average expression

Biological variabilitySubpopulation information

GFP distribution from Tenascin-C GFP reporter cellsGFP distribution from Tenascin-C GFP reporter cells

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800

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10-2-07 p.16

10-9-07 p.18

Flow Cytometry

GFP fluorescence intensity

Cel

l #

• GFP intensity distributions are stable in culture

• Genetically identical cells exhibit a wide-range of intensities

gene promoter sequence GFP

Tenascin-C promoter

CONCEPT: GFP is produced when gene is active

Phase Contrast

GFP

Quantify GFP intensities from live cellsQuantify GFP intensities from live cells

GFP FluorescencePhase

12 fields * 3 replicates

•Collaboration with Institute for Systems Biology, Seattle, WA for automating live cell image analysis•Approximately 200 cells were hand segmented and tracked•Manual analysis has been the result of a collaboration with Dr. Ben Stottrup, (Augsburg College, MN)

Single cell GFP intensities over time indicate tenascin-C regulation is coupled to the cell cycle

Single cell GFP intensities over time indicate tenascin-C regulation is coupled to the cell cycle

Normalizing the intensity data and averaging over >30 cells suggests that tenascin-C production is upregulated before division and is directly coupled to cell cycle progression

Inte

nsity

Fraction of cell cycle

Averaging Cell Intensity Profiles

Averaging over all cells

Time (fraction of cell cycle)

Re

lativ

e G

FP

flu

ore

scen

ce

inte

nsity

relative GFP between daughter cells after division; length of cell cycle vs. TNC expression; TNC expression vs. parent cell expression

Automated Segmentation and tracking: collaboration with ITL

ITL: Joe Chalfoun, Antonio Cardone, Alden Dima, Marcin Kociolek

(Chapados et al., Circulation Research. 2006;99:837.)

Tenascin-C: an ECM proteinTenascin-C: an ECM protein

•Tenascin-C (TNC) is a high molecular weight (250kDa - 300kDa ) extracellular matrix (ECM) glycoprotein that has a complex spatial and temporal pattern of expression during embryogenesis, wound healing and neoplastic processes.

•TNC levels are high during embryogenesis, but almost absent during normal postnatal life with some basal expression detectable in tendons and ligaments only.

•Tenascin-C expression is upregulated during inflammation, wound healing, and in many cancers

•Tenascin-C is thought to have anti-adhesive properties and play a role in signaling

•The expression of TNC is often correlated with cell spreading

LED illumination stabilityLED illumination stability

LED intensity (@470 nm) vs. time

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Time (hrs)

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tal

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wer

(n

W)

LED intensity measured over 7 days

non-transfected NIH3T3’s

transfected cellsavg=610 +/- 240

avg=5 +/- 7.5

flow cytometry

Cell intensitiesCell intensities

•Single cell clones from NIH-3T3 cell population transfected with a destabilized EGFP reporter driven by the tenascin-C gene•Spread area, shape, GFP expression…these are dynamic parameters