The Hollow Fibre Assay as a model for in vivo pharmacodynamics of fluoropyrimidines in colon cancer cells

Abstract

The Hollow Fibre Assay (HFA) is usually applied as an early in vivo model for anti-cancer drug screening, but is potentially an excellent model for short-term in vivo pharmacodynamic studies. We used the model to study the in vivo role of thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) in the cytotoxicity and pharmacodynamics of TAS-102 in colon cancer cells. TAS-102 is a new oral drug formulation, which is composed of trifluorothymidine (TFT) and thymidine phosphorylase inhibitor (TPI), which prevents TFT degradation. We compared the activity with Xeloda (capecitabine), which is activated by TP into 5FU. Hollow fibres filled with human Colo320 or Colo320TP1 colorectal cancer cells with deficient or high TP expression, respectively, were implanted subcutaneously (s.c.) at both flanks of BALB/c mice. The mice were treated orally over 5 days with TAS-102, TFT alone, 5'DFUR+/-TPI or capecitabine at their maximum tolerated dose (MTD). The cells were retrieved from the fibres and assayed for growth (MTT assay), cell cycle distribution (flow cytometry) and apoptosis induction (FragEL method). TAS-102 induced considerable growth inhibition (50%, P<0.01) to both cell lines, which was completely abolished in the absence of TPI. Capecitabine and its metabolite 5'DFUR reduced proliferation of Colo320TP1 cells in the fibres significantly (down to 25-40%), but much less in Colo320 cells, whereas addition of TPI reduced the effect of 5'DFUR, although not completely. These differences in cytotoxic effects were reflected in the pharmacodynamic evaluation. TAS-102 induced a G2M-phase arrest (from 25 to 40%) and apoptosis (>8-fold), which was more pronounced in Colo320 than in Colo320TP1. Again, omission of TPI neutralised the effect of TAS-102. Similarly, 5'DFUR and capecitabine induced a significant G2M-phase arrest (up to 45%) in the Colo320TP1 cell line, but less pronounced in the parental Colo320. Addition of TPI to 5'DFUR reduced this effect to control levels. Also induction of apoptosis was reduced in the presence of TPI. The data demonstrated that the HFA is excellently suited for studying short-term pharmacodynamic effects of fluoropyrimidines in vivo. TAS-102 is only effective in inducing cytotoxicity when systemic TPI is present, but acts against both low and high TP expressing colon cancer cells, while 5'DFUR needs cellular TP to exert significant activity.

Figure 1

Growth inhibition of Colo320 and Colo320TP1 cells in the hollow fibres implanted s.c. in mice. The mice were treated with the fluoropyrimidines as described in Materials and methods. Four to five fibres per cell line implanted in different mice were assayed. Values are means±s.e. In vitro control: fibres were incubated in DMEM culture medium. Compared to in vivo fibre control: ^*P<0.05; to Colo320: ^#P<0.05.

Figure 2

Cell cycle distribution for Colo320 and Colo320TP1 after retrieval from the hollow fibres. Per cell line three to five fibres from different mice were assayed using flow cytometry analysis. Values are means (%)±s.e.m. Total viable cell population was set at 100%. In vitro control: fibres were incubated in DMEM culture medium. (%) G2M compared to in vivo fibre control: ^*P<0.05; to Colo320: ^#P<0.05.

Figure 3

Representative DNA histograms to determine cell cycle distribution. The mice were treated with the fluoropyrimidines as described in Materials and Methods. After retrieval, the fibres were assayed to measure the percentage of nonapoptotic Colo320TP1 cells in the G1-, S- and G2M-phases. A clear G2M-arrest was observed for mice treated with TAS-102, 5′DFUR or Capecitabine.

Figure 4

Induction of apoptosis in Colo320 and Colo320TP1 cells grown in hollow fibres. Per cell line three to five fibres from different mice were assayed for apoptosis. Values are means±s.e.m. and were expressed as (%) apoptosis treated/(%) apoptosis control (was set at 1). Compared to fibre control: ^*P<0.05; to Colo320: ^#P<0.05.