1. Understanding 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 disease identification/validation of cellular biomarkers therapeutic intervention, drug discovery, toxicity

2. Copyright © 2000 Cell Press. The Hallmarks of Cancer Douglas Hanahan and Robert A. Weinberg Single cell measurements Average expression Biological variability Subpopulation information

3. GFP distribution from Tenascin-C GFP reporter cells Flow Cytometry GFP fluorescence intensity Cell # GFP intensity distributions are stable in culture Genetically identical cells exhibit a wide-range of intensities gene promoter sequenceGFP Tenascin-C promoter CONCEPT: GFP is produced when gene is active Phase Contrast GFP

4. Quantify 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)

5. 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 Intensity Fraction of cell cycle Averaging Cell Intensity Profiles Averaging over all cells Time (fraction of cell cycle) Relative GFP fluorescence intensity relative GFP between daughter cells after division; length of cell cycle vs. TNC expression; TNC expression vs. parent cell expression

6. Automated Segmentation and tracking: collaboration with ITL ITL: Joe Chalfoun, Antonio Cardone, Alden Dima, Marcin Kociolek

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

8. LED illumination stability LED intensity measured over 7 days

9. non-transfected NIH3T3’s transfected cells avg=610 +/- 240 avg=5 +/- 7.5 flow cytometry Cell 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