Supplementary material

Preclinical activity of the liposomal cisplatin Lipoplatin in ovarian cancer

1Naike Casagrande, 1Marta Celegato, 1Cinzia Borghese, 1Maurizio Mongiat,

1,2 Alfonso Colombatti, 1*Donatella Aldinucci

Supplementary methods

Cyt-c release.Cyt-c release was assessed, with minor modifications, as explained elsewhere, (14). Briefly, cells were permeabilized with 100 µg/mL digitonin (Sigma) and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) solution for 20 min at room temperature. After washing twice with PBS (to eliminate Cyt-c in the cytoplasm), cells were incubated 15 min with labeling medium (2% FBS, 0.2% sodium azide, 0.5% TritonX-100 in PBS) to permeabilize mitochondrial membranes, then residual Cyt-c was detected using 1 µg/mL of the mouse anti-Cytochromec antibody (Becton-Dickinson [BD] Pharmingen, San Jose, CA) followed by PE-conjugated goat anti-mouse IgG (BD). Histograms represent the Cyt-C in the mitochondria before and after Lipoplatin treatment(1).

Tumor xenograft experiments. All the in vivo studies were approved by the Institutional Ethics Committee.

Six-week-old female athymic nu/nu (nude) mice were purchased from Charles River (Lecco, Italy) and 2.7x106 OVCAR5 cells suspended in 0.1 mL of matrigel (1:3 in PBS) were inoculated in the right flank of each mouse. When tumors reached ~42 mm3 in volume, mice were divided randomly into four groups of 5 mice each and were treated twice a week with intraperitoneal (IP) injection of drug-free vehicle (PBS), 3 mg/kg or of 6 mg/kgCisplatin. Mice were weighed during the treatment.

Supplementary Figure Legends

Figure S1

Lipoplatin activity in A2780 and A2780cis cells. (A) FACS analysis of cells after 72 h incubation with different concentrations of Lipoplatin and double stained with Annexin-V-FITC and PI. Values in the bar graph represent the mean ± SEM of three different experiments. *** P<0.001; ****P<0.0001 drug vs.medium. (B) Analysis of caspase-3 activation after incubating cells with Lipoplatin (30 μM) for 24 and 48 h. Cells were harvested, washed and resuspended in complete medium supplemented with FLICA for 1 hour at 37°C, then washed again and analyzed by flow cytometry. Dotted lines indicate background fluorescence of cells. X- and Y-axes indicate the logarithms of the relative fluorescence intensity and relative cell number, respectively. (C) DNA fragmentation (Apo-Direct) was assessed by flow cytometry after treatment for 72 h with Lipoplatin (30 μM). FACS histograms are representative of one of three different experiments. (D) ROS production: cells were treated with Lipoplatin (30 μM) for 72 h and the bar graphs represent the percentage of ROS as the mean ± SEM of three different experiments. (E) Representative FACS dot plots of one of three independent experiments showing ROS generation.

Figure S2

Cisplatin activity in A2780, A2780cis and OVCAR5 cells. (A) FACS analysis of cells after 72 h incubation with different concentrations of Cisplatin and double stained with Annexin-V-FITC and PI. Values in the bar graph represent the mean ± SEM of three different experiments. ** P<0.01; *** P<0.001; ****P<0.0001 drug vs.medium. (B) Analysis of caspase-3 activation after incubating cells with Cisplatin (30 μM) for 24 and 48 h. Cells were harvested, washed and resuspended in complete medium supplemented with FLICA for 1 hour at 37°C, then washed again and analyzed by flow cytometry. Dotted lines indicate background fluorescence of cells. X- and Y-axes indicate the logarithms of the relative fluorescence intensity and relative cell number, respectively. (C) DNA fragmentation (Apo-Direct) was assessed by flow cytometry after treatment for 72 h with Cisplatin (30 μM). FACS histograms are representative of one of three different experiments.

(D) ROS production: cells were treated with Cisplatin (30 μM) for 72 h and the bar graphs represent the percentage of ROS as the mean ± SEM of three different experiments. (E) Representative FACS dot plots of one of three independent experiments showing ROS generation.

Figure S3.Cisplatin activity in SKOV3 spheroids and OVCAR5 tumor xenograft. (A)SKOV3 single pre-formed spheroids were cultured for 3 days in the presence of increasing concentrations of Cisplatin (12.5-50 μM). Responses were evaluated by spheroid volume measurements. The percentages of spheroid volume reduction by Cisplatintreatment are reported in the representative phase contrast microphotographs (original magnification 4x).(B) In vivo anticancer activity of Cisplatin(OVCAR5 xenograft). Tumor volumes were measured in female athymic nude mice after IP injection of either drug-free medium or containing 3 and 6 mg/Kg of Cisplatin, two times a week using a caliper for 42 days.Each value represents the mean ± SEM of 5 (or less when died) animals per group. (C) Histograms represent the mice weight measured at the end of the treatment (day 42).Each value represents the mean ± SEM of 5 animals treated with drug-free medium or Cisplatin (3mg/Kg) and the weight of the only mouse survived after Cisplatin (6 mg/Kg) treatment.** P<0.01; *** P<0.001; ****P<0.0001drug vs. medium.

Supplementary references

Reference List

1.Campos CB, Paim BA, Cosso RG, Castilho RF, Rottenberg H, Vercesi AE. Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane. Cytometry A 2006;69:515-23.