Purpose: The humanized monoclonal antibody, trastuzumab (Herceptin), directed against HER2/neu, has been effective in the treatment of breast cancer malignancies. However, clinical activity has depended on HER2/neu expression. Radiolabeled trastuzumab has been considered previously as a potential agent for radioimmunotherapy. The objective of this study was to investigate the efficacy of trastuzumab labeled with the ␣-particle emitting atomic generator, actinium-225 ( 225 Ac), against breast cancer spheroids with different HER2/neu expression levels.225 Ac has a 10-day half-life and a decay scheme yielding four ␣-particles.Experimental Design: The breast carcinoma cell lines MCF7, MDA-MB-361 (MDA), and BT-474 (BT) with relative HER2/neu expression (by flow cytometry) of 1:4:18 were used. Spheroids of these cell lines were incubated with different concentrations of 225 Ac-trastuzumab, and spheroid growth was measured by light microscopy over a 50-day period.Results: The activity concentration required to yield a 50% reduction in spheroid volume at day 35 was 18.1, 1.9, and 0.6 kBq/ml (490, 52, 14 nCi/ml) for MCF7, MDA, and BT spheroids, respectively. MCF7 spheroids continued growing but with a 20 -30 day growth delay at 18.5 kBq/ml. MDA spheroid growth was delayed by 30 -40 days at 3.7 kBq/ml; at 18.5 kBq/ml, 12 of 12 spheroids disaggregated after 70, days and cells remaining from each spheroid failed to form colonies within 2 weeks of being transferred to adherent dishes. Eight of 10 BT spheroids failed to regrow at 1.85 kBq/ml. All of the BT spheroids at activity concentrations 3.7 kBq/ml failed to regrow and to form colonies. The radiosensitivity of these three lines as spheroids was evaluated as the activity concentration required to reduce the treated to untreated spheroid volume ratio to 0.37, denoted DVR 37 . An external beam radiosensitivity of 2 Gy was found for spheroids of all three of the cell lines. After ␣-particle irradiation a DVR 37 of 1.5, 3.0, and 2.0 kBq/ml was determined for MCF7, MDA, and BT, respectively.Conclusion: These studies suggest that 225 Ac-labeled trastuzumab may be a potent therapeutic agent against metastatic breast cancer cells exhibiting intermediate to high HER2/neu expression.
Aggregation of attachment-dependent animal cells represents a series of motility, collision, and adhesion events applicable to such diverse fields as tissue engineering, bioseparations, and drug testing. Aggregation of human prostate cancer cells in liquid-overlay culture was modeled using Smoluchowski's collision theory. Using well (LNCaP) and poorly differentiated (DU 145 and PC 3) cell lines, the biological relevance of the model was assessed by comparing aggregation rates with diffusive and adhesive properties. Diffusion coefficients ranged from 5 to 90 microm(2)/min for single LNCaP and PC 3 cells, respectively. Similar diffusivities were predicted by the persistent random walk model and Einstein relation, indicating random motion. LNCaP cells were the most adhesive in our study with reduced cell shedding, 100% adhesion probability, and enhanced expression of E-cadherin. There was an increase in DU 145 cells staining positive for E-cadherin from nearly 20% of single cells to uniform staining across the surface of all aggregates; under 30% of PC 3 aggregates stained positive. Aggregation rates were more consistent with adhesive properties than with motilities, suggesting that aggregation in our study was reaction-controlled. Relative to other assays employed here, aggregation rates were more sensitive to phenotypic differences in cell lines and described size-dependent changes in aggregation at a finer resolution. In particular, model results suggest similar aggregation rates for two-dimensional DU 145 and PC 3 aggregates and upwards of 4-fold higher rates for larger three-dimensional DU 145 spheroids, consistent with expression of E-cadherin. The kinetic model has application to spheroid production, to cell flocculation and as an adhesion assay.
The in vitro self‐assembly of multicellular spheroids generates highly organized structures in which the three‐dimensional structure and differentiated function frequently mimic that of in vivo tissues. This has led to their use in such diverse applications as tissue regeneration and drug therapy. Using Smoluchowski‐like rate equations, herein we present a model of the self‐aggregation of DU 145 human prostate carcinoma cells in liquid‐overlay culture to elucidate some of the physical parameters affecting homotypic aggregation in attachment‐dependent cells. Experimental results indicate that self‐aggregation in our system is divided into three distinct phases: a transient reorganization of initial cell clusters, an active aggregation characterized by constant rate coefficients, and a ripening phase of established spheroid growth. In contrast to the diffusion‐controlled aggregation previously observed for attachment‐independent cells, the model suggests that active aggregation in our system is reaction‐controlled. The rate equations accurately predict the aggregation kinetics of spheroids containing up to 30 cells and are dominated by spheroid adhesive potential with lesser con‐ tributions from the radius of influence. The adhesion probability increases with spheroid size so that spheroid–spheroid adhesions are a minimum of 2.5 times more likely than those of cell–cell, possibly due to the upregulation of extracellular matrix proteins and cell‐adhesion molecules. The radius of influence is at least 1.5 to 3 times greater than expected for spherical geometry as a result of ellipsoidal shape and possible chemotactic or Fröhlich interactions. Brownian‐type behavior was noted for spheroids larger than 30 μm in diameter, but smaller aggregates were more motile by as much as a factor of 10 for single cells. The model may improve spheroid fidelity for existing applications of spheroids and form the basis of a simple assay for quantitatively evaluating cellular metastatic potential as well as therapies that seek to alter this potential. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 579–591, 2001.
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