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Quantitative spatiotemporal analysis of antibody fragment diffusion and endocytic consumption in tumor spheroids.

ABSTRACT: Antibody-based cancer treatment depends upon distribution of the targeting macromolecule throughout tumor tissue, and spatial heterogeneity could significantly limit efficacy in many cases. Antibody distribution in tumor tissue is a function of drug dosage, antigen concentration, binding affinity, antigen internalization, drug extravasation from blood vessels, diffusion in the tumor extracellular matrix, and systemic clearance rates. We have isolated the effects of a subset of these variables by live-cell microscopic imaging of single-chain antibody fragments against carcinoembryonic antigen in LS174T tumor spheroids. The measured rates of scFv penetration and retention were compared with theoretical predictions based on simple scaling criteria. The theory predicts that antibody dose must be large enough to drive a sufficient diffusive flux of antibody to overcome cellular internalization, and exposure time must be long enough to allow penetration to the spheroid center. The experimental results in spheroids are quantitatively consistent with these predictions. Therefore, simple scaling criteria can be applied to accurately predict antibody and antibody fragment penetration distance in tumor tissue.

SUBMITTER: Thurber GM

PROVIDER: S-EPMC2831542 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Quantitative spatiotemporal analysis of antibody fragment diffusion and endocytic consumption in tumor spheroids.

Thurber Greg M GM Wittrup K Dane KD

Cancer research 20080501 9

Antibody-based cancer treatment depends upon distribution of the targeting macromolecule throughout tumor tissue, and spatial heterogeneity could significantly limit efficacy in many cases. Antibody distribution in tumor tissue is a function of drug dosage, antigen concentration, binding affinity, antigen internalization, drug extravasation from blood vessels, diffusion in the tumor extracellular matrix, and systemic clearance rates. We have isolated the effects of a subset of these variables by ...[more]

PMID: 18451160

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Project description:Drug resistance remains a major clinical problem despite advances in targeted therapies. In recent years, methods to culture cancer cells in three-dimensional (3D) environments to better mimic native tumors have gained increasing popularity. Nevertheless, unlike traditional two-dimensional (2D) cell cultures, analysis of 3D cultures is not straightforward. Most biochemical assays developed for 2D cultures have to be optimized for use with 3D cultures. We addressed this important problem by presenting a simple method of quantitative size-based analysis of growth and drug responses of 3D cultures of cancer cells as tumor spheroids. We used an aqueous two-phase system to form consistently sized tumor spheroids of colorectal cancer cells. Using spheroid images, we computed the size of spheroids over time and demonstrated that growth of spheroids from this analysis strongly correlates with that using a PrestoBlue biochemical assay optimized for 3D cultures. Next, we cyclically treated the tumor spheroids with a MEK inhibitor, trametinib, for 6-day periods with a recovery phase in between. This inhibitor was selected because of mutation of colon cancer cells in the MEK/ERK pathway. We used size measurements to evaluate the efficacy of trametinib and predict development of resistance of colon cancer cells during the cyclical treatment and recovery regimen. This size-based analysis closely matched the biochemical analysis of drug responses of spheroids. We performed molecular analysis and showed that resistance to trametinib emerged due to feedback activation of the PI3K/AKT signaling pathway. Therefore, we combined trametinib with a PI3K/AKT inhibitor, dactolisib, and demonstrated that size-based analysis of spheroids reliably allowed quantifying the effect of the combination treatment to prevent drug resistance. This study established that size measurements of spheroids can be used as a straightforward method for quantitative studies of drug responses of tumor spheroids and identifying drug combinations that block resistance.

Project description:BackgroundAntibodies revolutionized cancer treatment over the past decades. Despite their successfully application, there are still challenges to overcome to improve efficacy, such as the heterogeneous distribution of antibodies within tumors. Tumor microenvironment features, such as the distribution of tumor and other cell types and the composition of the extracellular matrix may work together to hinder antibodies from reaching the target tumor cells. To understand these interactions, we propose a framework combining in vitro and in silico models. We took advantage of in vitro cancer models previously developed by our group, consisting of tumor cells and fibroblasts co-cultured in 3D within alginate capsules, for reconstruction of tumor microenvironment features.ResultsIn this work, an experimental-computational framework of antibody transport within alginate capsules was established, assuming a purely diffusive transport, combined with an exponential saturation effect that mimics the saturation of binding sites on the cell surface. Our tumor microenvironment in vitro models were challenged with a fluorescent antibody and its transport recorded using light sheet fluorescence microscopy. Diffusion and saturation parameters of the computational model were adjusted to reproduce the experimental antibody distribution, with root mean square error under 5%. This computational framework is flexible and can simulate different random distributions of tumor microenvironment elements (fibroblasts, cancer cells and collagen fibers) within the capsule. The random distribution algorithm can be tuned to follow the general patterns observed in the experimental models.ConclusionsWe present a computational and microscopy framework to track and simulate antibody transport within the tumor microenvironment that complements the previously established in vitro models platform. This framework paves the way to the development of a valuable tool to study the influence of different components of the tumor microenvironment on antibody transport.

Dataset Information

Quantitative spatiotemporal analysis of antibody fragment diffusion and endocytic consumption in tumor spheroids.

Publications

Quantitative spatiotemporal analysis of antibody fragment diffusion and endocytic consumption in tumor spheroids.

Similar Datasets

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