understanding cell state with quantitative live cell imaging copyright © 2000 cell press. the...
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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
0
400
800
0 2,000 4,000 6,000 8,000 10,000 12,000
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
0
100
200
300
0 50 100 150 200
Time (hrs)
To
tal
Po
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