Aggregation Kinetics of Well- and Poorly Differentiated Prostate Cancer Cells R. Enmon, K. O

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Aggregation Kinetics of Well-and Poorly
Differentiated Prostate Cancer CellsR. Enmon,
K. OConnor, H. Song, D. Lacksand D.
SchwartzTulane University and Medical
SchoolUniversity of Colorado, Boulder
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Objectives

• Evaluate responsiveness of kinetic model to
• Phenotype
• Aggregate size
• Cell Concentration
• Gain insight into aggregation mechanisms
  
  

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Previous Research
Attachment Independent
Attachment Dependent
Bovine Corneal Endothelial Cells (Muhitch et al.,
2000.Cytotechnol. 32 253-263) Chondrocytes (Vunja
k-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)
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Phases of Spheroid Assembly
end of culture
1 hr
0 hr
24 hr
Inoculation random cell distribution Redistribu
tion 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
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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

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Transition in Spheroid Assembly

• Legend
• Aggregation
• Ripening
• DU 145
• LNCaP
• PC 3

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Aggregation of DU 145 Cells
1 hr
1 hr 40 min
16 hr
5 hr
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Area/Cell Number Correlations
DU 145
PC 3
LNCaP
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Kinetic Model

• Accumulation Input - Output
• dC1/dt - C1 ?Cj kij(1?1j) single cells
• dCi/dt 0.5?CjCi-jkj,i-j(1?j,i-j) - Ci
  ?Cjkij(1?ij) spheroids

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Kinetic Rate Constants

• kii a bi ci2 di3
• symmetric across diagonal ki,j kj,i
• ki,j (ki,i kj,j )/2 ki,j (ki,ikj,j)1/2

Rate Constant Matrix
k1,1 ? ? ? ? k10,1 ? ? ? ? k20,1 ? ?
? k1,10 ? ? ? ? k10,10 ? ? ? ?
k20,10 ? ? ? k1,20? ? ?
? k10,20 ? ? ? ? k20,20
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Model Predictions
DU 145
LNCaP
PC 3
Spheroids X 105
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Time (hours)
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Cell Concentration Effects
DU 145

• Rate Constants for 2 x 104 cells/cm2
• kii (h-1) -0.4 1.1i - 0.11i2 0.01i3
• kij (kiikjj)1/2
• Cell Concentration 1 x 104 cells/cm2
• Cell/Volume Ratio 1.6 x 105 cells/ml

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Self-Aggregation Rates
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Physical Interpretation of Rate Parameter
Smoluchowski Expression kIJ ? (RI RJ) (DI
DJ)
Ideal Case spheroid is perfect sphere max.
radius rI ? 1 RI rI DI ?1/rI
Spheroid Self-Assembly spheroid is not perfect
sphere max. radius gt rI ? lt 1 RI ? f(cell
number) DI ? f(cell number)
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Cell Motility
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Adhesion Probability
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Intra-Spheroidal Adhesion
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E-Cadherin Expression
T-Flask
Liquid-Overlay Culture
DU 145
DU 145
LNCaP
PC 3
Phase-Contrast Microscopy
Fluorescence Microscopy
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E-Cadherin Expression
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Collagen IV Expression
Liquid-Overlay Culture
DU 145
LNCaP
PC 3
Phase-Contrast Microscopy
Fluorescence Microscopy
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Collagen IV Expression
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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

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Applications

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

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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

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