Large anti-HER2/neu liposomes for potential targeted intraperitoneal therapy of micrometastatic cancer.

UNLABELLED: Effective targeting and killing of intraperitoneally disseminated micrometastases remains a challenge. OBJECTIVE/METHODS: In this work, we evaluated the potential of antibody-labeled PEGylated large liposomes as vehicles for direct intraperitoneal (i.p.) drug delivery with the aim to enhance the tumor-to-normal organ ratio and to improve the bioexposure of cancer cells to the delivered therapeutics while shifting the toxicities toward the spleen. These targeted liposomes are designed to combine: (1) specific targeting to and internalization by cancer cells mediated by liposome-conjugated tumor-specific antibodies, (2) slow clearance from the peritoneal cavity, and (3) shift of normal organ toxicities from the liver to the spleen due to their relatively large size. RESULTS: Conjugation of anti-HER2/neu antibodies to the surface of large (approximately 600 nm in diameter) PEGylated liposomes results in fast, specific binding of targeted liposomes to cancer cells in vitro, followed by considerable cellular internalization. In vivo, after i.p. administration, these liposomes exhibit fast, specific binding to i.p. cancerous tumors. Large liposomes are slowly cleared from the peritoneal cavity, and they exhibit increased uptake by the spleen relative to the liver, while targeted large liposomes demonstrate specific tumor uptake at early times. Although tissue and tumor uptake are greater for cationic liposomes, the tumor-to-liver and spleen-to-liver ratios are similar for both membrane compositions, suggesting a primary role for the liposome's size, compared to the liposome's surface charge. CONCLUSIONS: The findings of this study suggest that large targeted liposomes administered i.p. could be a potent drug-delivery strategy for locoregional therapy of i.p. micrometastatic tumors.

Alpha-particle emitting atomic generator (Actinium-225)-labeled trastuzumab (herceptin) targeting of breast cancer spheroids: efficacy versus HER2/neu expression.

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 alpha-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 alpha-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 alpha-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.

Restructuring dynamics of DU 145 and LNCaP prostate cancer spheroids.

Neoplastic cells acquire multidrug resistance as they assemble into multicellular spheroids. Image analysis and Monte Carlo simulation provided an insight into the adhesion and motility events during spheroid restructuring in liquid-overlay culture of DU 145 and LNCaP human prostate cancer cells. Irregularly shaped, two-dimensional aggregates restructured through incremental cell movements into three-dimensional spheroids. Of the two cultures examined, restructuring was more pronounced for DU 145 aggregates. Motile DU 145 cells formed spheroids with a minimum cell overlay of 30% for 25-mers as estimated by simulation versus 5% for adhesive LNCaP cells in aggregates of the same size. Over 72 h, the texture ratio increased from 0.55 +/- 0.05 for DU 145 aggregates with projected areas exceeding 2000 microm2 to a value approaching 0.75 +/- 0.02 (P < 0.05). For LNCaP aggregates of comparable size, the increase in texture ratio was more modest, less than 15% during the same time period (P < 0.05). Combined, these data suggest that motility events govern the overall rate of spheroid restructuring. This information has application to the chemosensitization of solid tumors and kinetic modeling of spheroid production.

Monte Carlo simulation of LNCaP human prostate cancer cell aggregation in liquid-overlay culture.

Neoplastic cells self-assemble in liquid-overlay cultures into multicellular spheroids that resemble micrometastases and avascular regions of larger tumors. A Monte Carlo simulation based on Meakin's cluster-cluster aggregation model resolved the physical mechanisms by which LNCaP human prostate cancer cells aggregate in this environment. The best-fit solution suggests that LNCaP cells aggregate with an adhesion probability of 0.5% when they migrate within a radius of influence between cell centers of 180 microm, 10 times the cell diameter. The sweeping radius of influence is indicative of cell tethering and/or chemotaxis and results in an intrinsic rate of self-aggregation that increases from k(11) = 1.5 h(-1) for single cells to k(1010) = 17.5 h(-1) for 10-mers. Similar rates are predicted by Smoluchowski's collision theory (1), suggesting that they are inherent properties of LNCaP liquid-overlay culture. Aggregates form more compact structures in culture than during simulation as measured by the fractal dimension: D(F) = 1.74 +/- 0.04 for 10-mers in culture vs D(F) = 1.25 +/- 0.10 for simulated 10-mers. Additional restructuring would further extend the radius of influence and diminish adhesion. Applications of this work include the production of highly viable spheroids for drug testing and basic oncological research.

Combination treatment with 17-N-allylamino-17-demethoxy geldanamycin and acute irradiation produces supra-additive growth suppression in human prostate carcinoma spheroids.

Failure to control localized prostate cancer can result not only in localized disease progression but also distant metastatic spread. Whereas significant advances in both surgical technique and radiation therapy have improved local control rates with decreased morbidity, consistent long-term control remains elusive. This study investigates the potential of 17-N-allylamino-17-demethoxy geldanamycin (17AAG), a geldanamycin derivative, to sensitize tumor cells to ionizing radiation, permitting a significant improvement to targeted radiotherapies of prostate carcinoma. As a monotherapeutic, 17AAG functions to modulate the action of heat shock protein 90, ultimately affecting a multitude of cellular signaling pathways. It is in Phase I trial and has shown promise in controlling prostate cancer progression. Human prostate tumor cells (LNCaP and CWR22Rv1) were grown as spheroids and incubated for 96 h with increasing doses of 17AAG immediately before and after 2 or 6 Gy low linear energy transfer (LET), high dose-rate irradiation (Cs-137 irradiator). Twelve or 24 spheroids (initial diameter, 150-200 microm) were used per experiment. Response was determined by spheroid volume measurements taken over at least 40 days, after treatment. Incubation of either cell line with 17AAG (

Pharmacokinetics and Biodistribution of (86)Y-Trastuzumab for (90)Y dosimetry in an ovarian carcinoma model: correlative MicroPET and MRI.

UNLABELLED: Preclinical biodistribution and pharmacokinetics of investigational radiopharmaceuticals are typically obtained by longitudinal animal studies. These have required the sacrifice of multiple animals at each time point. Advances in small-animal imaging have made it possible to evaluate the biodistribution of radiopharmaceuticals across time in individual animals, in vivo. MicroPET and MRI-based preclinical biodistribution and localization data were obtained and used to assess the therapeutic potential of (90)Y-trastuzumab monoclonal antibody (mAb) (anti-HER2/neu) against ovarian carcinoma. METHODS: Female nude mice were inoculated intraperitoneally with 5.10(6) ovarian carcinoma cells (SKOV3). Fourteen days after inoculation, 12-18 MBq (86)Y-labeled trastuzumab mAb was injected intraperitoneally. Tumor-free mice, injected with (86)Y-trastuzumab, and tumor-bearing mice injected with labeled, irrelevant mAb or (86)Y-trastuzumab + 100-fold excess unlabeled trastuzumab were used as controls. Eight microPET studies per animal were collected over 72 h. Standard and background images were collected for calibration. MicroPET images were registered with MR images acquired on a 1.5-T whole-body MR scanner. For selected time points, 4.7-T small-animal MR images were also obtained. Images were analyzed and registered using software developed in-house. At completion of imaging, suspected tumor lesions were dissected for histopathologic confirmation. Blood, excised normal organs, and tumor nodules were measured by gamma-counting. Tissue uptake was expressed relative to the blood concentration (percentage of injected activity per gram of tissue [%IA/g]/%IA/g blood). (86)Y-Trastuzumab pharmacokinetics were used to perform (90)Y-trastuzumab dosimetry. RESULTS: Intraperitoneal injection of mAb led to rapid blood-pool uptake (5-9 h) followed by tumor localization (26-32 h), as confirmed by registered MR images. Tumor uptake was greatest for (86)Y-trastuzumab (7 +/- 1); excess unlabeled trastuzumab yielded a 70% reduction. Tumor uptake for the irrelevant mAb was 0.4 +/- 0.1. The concentration in normal organs relative to blood ranged from 0 to 1.4 across all studies, with maximum uptake in spleen. The absorbed dose to the kidneys was 0.31 Gy/MBq (90)Y-trastuzumab. The liver received 0.48 Gy/MBq, and the spleen received 0.56 Gy/MBq. Absorbed dose to tumors varied from 0.10 Gy/MBq for radius = 0.1 mm to 3.7 Gy/MBq for radius = 5 mm. CONCLUSION: For all injected compounds, the relative microPET image intensity of the tumor matched the subsequently determined (86)Y uptake. Coregistration with MR images confirmed the position of (86)Y uptake relative to various organs. Radiolabeled trastuzumab mAb was shown to localize to sites of disease with minimal normal organ uptake. Dosimetry calculations showed a strong dependence on tumor size. These results demonstrate the usefulness of combined microPET and MRI for the evaluation of novel therapeutics.

Aggregation kinetics of well and poorly differentiated human prostate cancer cells.

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.