Abstract
Abstract Introduction: Elevated interstitial fluid pressure (IFP) is a characteristic feature of many solid tumours. One consequence of elevated IFP is that the normal process of convective fluid flow through tissues is impeded, and this is now recognised as a major barrier to drug uptake. Therapeutic approaches designed to reduce tumour IFP with the aim of restoring convective fluid flow thereby enhancing the delivery of partner therapeutics, are currently of considerable interest. To date, studies of intratumoural IFP have almost exclusively used in vivo models; the aim of this study was to develop an in vitro model that could be used to measure convective fluid flow. Methods: The standard approach to analysing drug penetration through multicell layers involves culturing cells on transwell membranes which are then inserted into one well of a 24 well plate. The level of medium is the same in both top and bottom chambers, so there is no pressure gradient across the membrane. In a modification of this model, a graduated tube is attached to the top chamber of the transwell; the level of medium in the top chamber can be systematically raised above that in the bottom chamber thereby creating a pressure gradient across the membrane. Convective flow is determined by measuring the weight of medium in the bottom chamber as a function of time, expressed as ml/min. DLD-1 human colorectal cells were grown on the transwell membrane to various thicknesses and convective flow was determined using this model. Results: Collagen coated membranes with a pore size of 3 μm provided a consistent flow rate of 5.5 ml/min when the hydrostatic pressure gradient applied across the membrane was 28.8 mmHg (13mls of medium in the graduated tube). DLD-1 cells seeded 1 × 105 cells per well were cultured at 37oC for up to 6 days with daily changes in medium. Histological analysis allowed the thickness of the multicell layer to be determined using a graduated eyepiece calibrated with a stage micrometer. The thickness of the multicell layer increased from 6.17 ± 2.12 μm to 19.62 ± 4.21 μm on days 1 and 5 of culture respectively. With a pressure gradient of 28.8 mmHg, convective flow rates ranged from 0.192 ml/min to 0.09 ml/min on days 1 to 4 of culture respectively; beyond day 4, the convective flow rate was less than 0.001 ml/min. Conclusions: A novel model for measuring the convective flow of fluid across multicell layers in vitro has been developed. Initial validation of this model has demonstrated that as the thickness of the multicell layer increases, convective flow decreases. In the case of DLD-1 cells, multicell layers of approximately 20 μm completely prevented convective flow. Further characterisation of this model is required but these initial results suggest it could be used to assess therapeutic strategies designed to modulate the tumour microenvironment with the aim of restoring or modifying convective fluid flow. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5507.
Published Version
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