The effects of taxanes, vorinostat and doxorubicin on growth and proliferation of Echinococcus multilocularis metacestodes assessed with magnetic resonance imaging and simultaneous positron emission tomography

Abstract

Cytostatic drugs used in cancer therapy were evaluated for their capacity to inhibit Echinococcus multilocularis metacestode growth and proliferation. Metacestode tissues were exposed in vitro to docetaxel, doxorubicin, navelbine, paclitaxel, and vorinostat for 1 week, then incubated in drug-free culture, and thereafter metacestodes were injected into the peritoneum of Meriones unguiculatus. Magnetic resonance imaging (MRI) and simultaneous positron emission tomography (PET) were applied to monitor in vivo growth of drug-exposed E. multilocularis in Meriones. At 3 month p.i., docetaxel (at 10 μM, 5 μM and 2 μM) inhibited in vivo growth and proliferation of E. multilocularis, and at 5 months p.i., only in the 2 μM docetaxel exposure group 0.3 cm ³ of parasite tissue was found. With paclitaxel and navelbine the in vivo growth of metacestodes was suppressed until 3 months p.i., thereafter, parasite tissues enlarged up to 3 cm ³ in both groups. E. multilocularis tissues of more than 10 g developed in Meriones injected with metacestodes which were previously exposed in vitro to doxorubicin, navelbine, paclitaxel or vorinostat. In Meriones infected with metacestodes previously exposed to docetaxel, the in vivo grown parasite tissues weighted 0.2 g. In vitro cultured E. multilocularis metacestodes exposed to docetaxel did not produce vesicles until 7 weeks post drug exposure, while metacestodes exposed to doxorubicin, navelbine and vorinostat proliferated continuously. In summary, docetaxel, and less efficaciously paclitaxel, inhibited in vivo and in vitro parasite growth and proliferation, and these observations suggest further experimental studies with selected drug combinations which may translate into new treatment options against alveolar echinococcosis.

Keywords: drug exposure; echinococcus multilocularis, metacestode; magnetic resonance imaging; positron emission tomography; taxanes, paclitaxel, docetaxel, histone deacetylase inhibitor, vorinostat, doxorubicin.

Figure 1. PET- and MR-imaging, and quantification of parasite growth, in docetaxel, paclitaxel and vorinostat (SAHA) exposed E.multilocularis metacestodes

In vivo quantification of parasite growth was performed at two time points in all tested animals. The in vivo grown volumes of docetaxel (A), paclitaxel (B) and navelbine (D) exposed E. multilocularis metacestodes is shown. At the second measurement time point (F) and simultaneously to the MR acquisition all animals were PET imaged with [¹⁸F]FDG. Quantification of the [¹⁸F]FDG uptake in parasite tissue is presented as percentage of the injected dose per cubic centimeter (%ID/cc) and error bars represent one SD. Results are shown for docetaxel (C), paclitaxel and navelbine (E) exposure. Coronal [¹⁸F]FDG maximum intensity projections (MIP) and fused PET/MR images from E. multilocularis metacestode infected gerbils are shown in the Figure 1F. Arrows indicate the positions of the [¹⁸F]FDG uptake in the metacestode tissue. (n.d., non detected).

Figure 2. PET- and MR-imaging, and quantification of parasite volumes, in doxorubicin, vorinostat (SAHA) and DMSO (control) exposed E. multilocularis metacestodes

In vivo quantification of parasite growth was performed at two time points in all tested animals. The parasite tissue volumes of doxorubicin (A), vorinostat (SAHA; C) and DMSO control (E) exposed E. multilocularis metacestodes is shown. At the second measurement time point and simultaneously to the MR acquisition all animals were PET imaged with [¹⁸F]FDG. Quantification of the [¹⁸F]FDG uptake in parasite tissue is presented as percentage of the injected dose per cubic centimeter (%ID/cc) and error bars represent one SD. Results are shown for doxorubicin (B), vorinostat (SAHA; D) and DMSO control (F) treatment. Coronal [¹⁸F]FDG maximum intensity projections (MIP) and fused PET/MR images from E. multilocularis metacestode infected gerbils are depicted in the (G and H). Arrows indicate the positions of the [¹⁸F]FDG uptake in the metacestode tissue.

Figure 3. The weights of E. multilocularis metacestodes tissues isolated from infected Meriones unguiculatus

Metacestodes were exposed in vitro to the cytostatic drugs docetaxel (10 μM, 5 μM, 2 μM), doxorubicin (4.5, 3 and 1.5 μg/ml), navelbine (4.5, 3, 1.5 and 0.75 μg/ml), paclitaxel (10 μM, 5 μM and 2 μM), vorinostat (SAHA) (10, 7.5, 2 and 1 μg/ml) and DMSO (0.1%, 0.05%, solvent control, CTRL) at the indicated concentrations for 7 days, subsequently metacestodes rested in drug-free media for another 7 days, and then the drug-exposed metacestodes were injected into the peritoneum of M. unguiculatus. At 4 and 5 months post infection, the grown metacestode tissues were collected from M. unguiculatus and weighted. The drug concentration groups at which E. multilocularis metacestodes tissues were exposed to the cytostatic drugs are merged. The Figure shows the treatment groups, the mean metacestode tissue weights and the 95% confidence intervals. No significant differences in weights were observable between the treatment groups.

Figure 4. The in vitro “budding” of vesicles from E. multilocularis metacestode tissues after drug-exposure

The “budding” of E. multilocularis vesicles from in vitro cultured metacestodes tissue previously exposed in vitro to cytostatic drugs was evaluated during 14 weeks post drug exposure. The number of vesicles produced in culture was scored, i.e. Score 0 = no vesicle, Score 1 = very few vesicles (1–5), Score 2 = few vesicles (6–10), Score 3 = vesicles (11–20), Score 4 = vesicles (21–30), Score 5 = vesicles (>31). The production scores 1–5 of E. multilocularis metacestodes tissues are shown.

Figure 5. The in vitro production of vesicles from E. multilocularis metacestode tissues after in vitro drug-exposure

In vitro cultured E. multilocularis metacestode tissue blocks (1 cm³) were exposed to 10 μM, 5 μM, 2 μM of docetaxel, to 4.5 μg/ml, 3 μg/ml and 1.5 μg/ml of doxorubicin, to 4.5 μg/ml, 3 μg/ml, 1.5 μg/ml and 0.75 μg/ml of navelbine, to 10 μM, 5 μM, and 2 μM, paclitaxel, to 10 μg/ml, 7.5 μg/ml, 2 μg/ml and 1 μg/ml of vorinostat (SAHA), and to DMSO (solvent control) at the indicated concentrations. The effects of these cytostatic compounds on the in vitro production by E. multilocularis vesicles was scored and studied for 14 weeks. The number of vesicles produced in culture was scored (Figure 5), i.e. Score 0 = no vesicle, Score 1 = very few vesicles (1–5), Score 2 = few vesicles (6–10), Score 3 = vesicles (11–20), Score 4 = vesicles (21–30), Score 5 = vesicles (>31).