1. Aggregation Kinetics of Well- and Poorly Differentiated Prostate Cancer Cells R. Enmon, K. O’Connor, H. Song, D. Lacks and D. Schwartz Tulane University and Medical School University of Colorado, Boulder

2. Objectives Evaluate responsiveness of kinetic model to: –Phenotype –Aggregate size –Cell Concentration Gain insight into aggregation mechanisms Tulane

3. Previous Research Attachment Independent Attachment Dependent Bovine Corneal Endothelial Cells (Muhitch et al., 2000.Cytotechnol. 32: 253-263) Chondrocytes (Vunjak-Novakovic et al., 1998. Biotechnol. Prog. 14: 193-202) DU 145 Human Prostate Cells ( Enmon et al., 2001. Biotechnol. Bioeng. 72: 579-91 ) Jurkat Human T-Cells (Neelamegham and Zygourakis, 1997. Ann. Biomed. Eng. 25: 180-9) Blood Platelets (Huang and Hellums, 1993. Biophys. J. 65: 344-355) Tulane

4. Phases of Spheroid Assembly 1 hr 0 hr 24 hr end of culture Inoculation: random cell distribution Redistribution: loss of cell-cell interaction, random cell movement Aggregation: no dissociation, no growth, inter-spheroid interactions, constant rate parameter Ripening: cell growth, differentiation, intra-spheroid interactions Tulane

5. Human Prostate Cancer Cell Lines LNCaP: retains an epithelial phenotype and is weakly invasive DU 145: poorly differentiated and moderately invasive PC 3: poorly differentiated and highly invasive Tulane

6. Transition in Spheroid Assembly Legend: Aggregation Ripening DU 145 LNCaP PC 3 Tulane

7. Aggregation of DU 145 Cells 1 hr 1 hr 40 min 5 hr 16 hr Tulane

8. Area/Cell Number Correlations DU 145 LNCaP PC 3 Tulane

9. Kinetic Model Accumulation = Input - Output dC 1 /dt = - C 1  C j k ij (1+  1j ) single cells dC i /dt = 0.5  C j C i-j k j,i-j (1+  j,i-j ) - C i  C j k ij (1+  ij ) spheroids dC i /dt = 0.5  C j C i-j k j,i-j (1+  j,i-j ) - C i  C j k ij (1+  ij ) spheroids Tulane

10. Kinetic Rate Constants k ii = a + bi+ ci 2 + di 3 symmetric across diagonal: k i,j = k j,i k i,j = (k i,i + k j,j )/2 k i,j = (k i,i k j,j ) 1/2 Rate Constant Matrix k 1,1     k 10,1     k 20,1    k 1,10     k 10,10     k 20,10    k 1,20     k 10,20     k 20,20 Tulane

11. Model Predictions [Spheroids] X 10 5 Time (hours) DU 145 LNCaP PC 3 Tulane

12. Cell Concentration Effects Rate Constants for 2 x 10 4 cells/cm 2 : k ii (h -1 ) = -0.4 + 1.1i - 0.11i 2 + 0.01i 3 k ij = (k ii k jj ) 1/2 Cell Concentration: 1 x 10 4 cells/cm 2 Cell/Volume Ratio: 1.6 x 10 5 cells/ml DU 145 Tulane

13. Self-Aggregation Rates Tulane

14. Physical Interpretation of Rate Parameter Smoluchowski Expression: k IJ =  (R I + R J ) (D I +D J ) Ideal Case spheroid is perfect sphere spheroid is perfect sphere max. radius = r I max. radius = r I  = 1  = 1 R I = r I D I  1/r I Spheroid Self-Assembly spheroid is not perfect sphere spheroid is not perfect sphere max. radius > r I  < 1  < 1 R I  f(cell number) D I  f(cell number) Tulane

15. Cell Motility Tulane

16. Adhesion Probability Tulane

17. Intra-Spheroidal Adhesion Tulane

18. E-Cadherin Expression Tulane T-Flask Liquid-Overlay Culture DU 145 LNCaP PC 3 FluorescenceMicroscopy Phase-ContrastMicroscopy

19. E-Cadherin Expression Tulane

20. Collagen IV Expression Tulane Liquid-Overlay Culture DU 145 LNCaP PC 3 Phase-ContrastMicroscopy FluorescenceMicroscopy

21. Collagen IV Expression Tulane

22. Conclusions Aggregation rates –Responsive to cell concentration –Consistent with adhesive properties than with motilities –Sensitive to phenotypic differences in cell lines –Described size-dependent changes in aggregation at a fine resolution Tulane

23. Applications Spheroid production for tissue engineering and in vitro drug testing Assay to evaluate adhesive capacity Cell flocculation in suspension cultures and for bioseparation Tulane

24. Acknowledgements This research was funded with grants from NASA and the Tulane Cancer Center. Time lapse images and additional information is available at our web site: http://www.tulane.edu/~kim/oconnor.html Tulane