Classification of anticancer drugs based on tissue penetration using a novel in vitro screening assay

A213 Background: The failure of many anticancer drugs to control the growth of solid cancers may stem in part from inadequate delivery to tumor regions distant from vasculature. However, gaining an understanding of diffusion limitations of existing drugs and developing new drugs with improved tumor penetration is often limited by a lack of techniques with which to evaluate drugs. In this study we employ multilayered cell culture (MCC) in combination with a biological endpoint to assess the tissue penetration of a panel of 18 commonly used anti-cancer drugs. MCC is a planar analogue of spheroidal cell culture, in which tumor cells are instead grown into discs of tissue. Due to their 3-D conformation they model many characteristics of the tumor extravascular compartment. A unique property of MCC is that it possesses two populations of rapidly proliferating cells, one on each side of the culture, that are separated by a known thickness of tissue. In tumors and spheroids proliferation status falls off with distance from the vasculature (tumors) or tissue edge (spheroids), which in turn modifies cellular response to drugs. Hence, visualizing the distribution of a drug’s effect within these tissues cannot be used to directly determine actual drug distribution.
 Method: In this study, we exploit the symmetrical growth that occurs within MCCs by exposing them to drugs from one side and then comparing drug effect on the exposed side versus the far side of the cultures. This approach circumvents issues that normally arise from the biochemical gradients that occur with distance into tissue (e.g. changing intrinsic sensitivity of cells to drugs with depth into tissue) and in effect uses the cells themselves as the drug detection endpoint. Using this technique we examined the tissue penetration of representative anti-cancer drugs from a selection of classes, including anthracyclines, microtubule agents, anti-metabolites, platinum based agents and others. The distribution of drug activity within HCT-116 MCCs was assessed 1 to 3 days after a 1-h drug exposure via immunodetection of S-phase cells using bromodeoxyuridine. Using an automated computer analysis routine, the effect of the drugs in the first 30 µm of tissue located on either edge of the cultures relative to controls was assessed.
 Results: Penetration of the agents through HCT-116 MCCs was grouped into four classes: near uniform tissue distribution (cisplatin, 5-FU and vinorelbine), 1-5 fold decrease in drug exposure to cells on the far side versus the exposed side of the cultures (vincristine, vinblastine, paclitaxel, mitomycin C and etoposide), ~10-fold difference (doxorubicin, epirubicin, docetaxel and gemcitabine) and greater than 10-fold (daunorubicin and mitoxantrone). In the case of the anthracyclines and taxanes, the MCC-based assay was validated by comparing the predicted drug distributions with direct visualization of the drugs themselves.
 Conclusion: This model could be applied as a screening system for the discovery of biologically active drugs which exhibit desirable penetration properties.
 This research was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society, the Michael Smith Foundation For Health Research and the Canadian Institutes for Health Research.

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