Attempts to establish a subcutaneous ABCG2 overexpressing tumor model

To proof the therapeutic benefit of a combination of an ABCG2 modulator with a cytostatic, which is a substrate of this transporter, in vivo, the reversal of ABCG2 mediated chemoresistance was investigated in vitro. Furthermore, it is essential that the ABCG2 transporter status is retained in vivo over a long period of time.

4.4.7.1 In vitro assays to determine stable ABCG2 transporter expression

For investigations on a stable transporter expression, MCF-7/Topo cells were subdivided into 2 groups. Whereas the first one was supplemented with topotecan as described (see materials and methods), the second group was cultured in the absence of the cytostatic. The cells were investigated for expression of functional ABCG2 in the flow cytometric mitoxantrone efflux assay over several passages after co-incubation with either DMSO (vehicle control) or 10 µM of fumitremorgin C, respectively. As a criterion for stable transporter expression and protein functionality, the obtained fluorescence intensities were analyzed according to the following equation:

, with GeoMeancorr. (MCF-7/Topo)non-suppl. representing the vehicle-corrected geometric means of fluorescence intensities for the non-topotecan supplemented MCF-7/Topo cells. The corrected GeoMean values obtained for the cells cultured in the presence of topotecan are abbreviated as GeoMeancorr. (MCF-7/Topo)suppl.. The results of the flow cytometric assays are shown in Figure 4.20.

Flow cytometry confirmed a stable expression of the functionally active efflux pump ABCG2 even in the absence of topotecan. The investigations were arranged over 9 passages corresponding to a period of several weeks in which the overexpression of ABCG2 efflux pumps was preserved. This is an important result with respect to in vivo studies, as the lack of selection pressure due to leaving out topotecan treatment during tumor growth, should not affect the transporter levels.

Passage

126 127 128 129 130 135

ABCG2 inhibition [%]

0 20 40 60 80 100 120 140

Figure 4.20: ABCG2 inhibition [%] by 10 µM of fumitremorgin C of MCF-7/Topo cells cultured in the presence of topotecan. Transporter modulation is expressed as the ratio of ABCG2 inhibition in non-topotecan supplemented and topotecan-treated MCF-7/Topo cells. The cells were analyzed over a period of 9 in vitro passages. 

Modulation of the efflux pump ABCG2 115

4.4.7.2 Overcoming ABCG2-mediated drug-resistance in vitro by co-administration of efflux pump inhibitors

The crystal violet chemosensitivity assays were performed as previously described [Bernhardt et al., 1992a]. To examine the potential of ABCG2 modulators to reverse the efflux pump-mediated drug resistance, the assays were arranged as follows:

2-3 days after seeding, MCF-7/Topo cells were either treated with 100 nM of topotecan alone or combined with various concentrations of the known ABCG2 modulator Ko143 (Figure 4.21). In an additional experiment the cells were incubated with the ABCG2 substrate mitoxantrone alone or combined with the new tariquidar analog 2 (Figure 4.22). Moreover, as a control, the effect of the transporter modulators alone against the cells was determined in a concentration-dependent manner.

Figure 4.21: Effect of Ko143 alone (a) and in combination with 100 nM topotecan (b) on proliferating MCF-7/Topo cells (passage 138); vehicle (open stars), 100 nM topotecan (open circles) and Ko143 at different concentrations: 10 nM (filled squares), 50 nM (filled triangles), 100 nM (filled inverted triangles) and 500 nM (filled diamonds). 

Incubation of MCF-7/Topo breast cancer cells with the ABCG2 inhibitor Ko143 alone caused no toxic effects up to concentrations of 500 nM (Figure 4.21a). The combination of 100 nM Ko143 with topotecan at a concentration of 100 nM, which was non-toxic in the absence of a ABCG2 modulator, resulted in total reversal of the ABCG2-mediated drug-resistance, i.e. a remarkable cytostatic effect (Figure 4.21b).

There was no cytotoxic effect detectable when MCF-7/Topo cells were incubated with the new tariquidar analog 2 alone up to a concentration of 500 nM (Figure 4.22a). However, compound 2 at a concentration of 10 nM combined with 100 nM mitoxantrone, a per se non-toxic concentration of the cytostatic in the ABCG2-overexpressing cells, yielded a strong antiproliferative effect (Figure 4.22b).

These in vitro investigations demonstrate that inhibition of the ABCG2 efflux pump is effective to reverse the transporter-mediated drug resistance against the cytostatics topotecan and mitoxantrone.

Figure 4.22: Effect of compound 2 alone (a) and in combination with 100 nM mitoxantrone (b) on proliferating MCF-7/Topo cells (passage 193); vehicle (open stars), 100 nM mitoxantrone (open circles) and compound 2 at different concentrations: 10 nM (filled squares), 50 nM (filled triangles), 100 nM (filled inverted triangles) and 500 nM (filled diamonds). 

Modulation of the efflux pump ABCG2 117

4.4.7.3 Subcutaneous injection of MCF-7/Topo cells into nude mice

Stimulated by the promising in vitro results, MCF-7/Topo cells were subcutaneously injected into nude mice to establish a subcutaneous tumor model. For this purpose, the human breast cancer cells were injected into both, male and female nude mice in order to check sex-dependent variations in tumor growth. Furthermore, female nude mice were subdivided into 2 groups:

One group of animals was left without additional hormone supplementation; in the other group estradiol depots were implanted to exclude growth retardations of MCF-7/Topo tumors due to estradiol deficiency.

Figure 4.23 shows the implantation procedure of estradiol depots according to Bernhardt et al. [Bernhardt et al., 1992b].

A total of 22 mice were included in this investigation on the in vivo growth of MCF-7/Topo cells. Over a period of more than 3 months, in none group, the subcutaneously injected breast cancer cells resulted in tumor growth. Therefore, these attempts to establish a subcutaneous model of ABCG2-overexpressing MCF-7/Topo tumors, failed.

Figure 4.23: Implantation procedure of estradiol depots. a) Opening of skin on anesthetized female nude mice, b) subcutaneous placement of estradiol depots and c) mice with subcutaneous implanted estradiol depots after skin closure by brackets. Tumor cell suspensions were subcutaneously injected 7 days after estradiol depot implantation.