Figure 1.
Efficacy of Ko143+ topotecan combination therapy in Brca1−/−;p53−/− mammary tumors.
A, Experimental outline. Spontaneous mammary tumors from K14cre;Brca1F/F;p53F/F females were orthotopically transplanted into six- to eight-week-old Abcg2−/− syngeneic recipients. When tumors reached a volume of about 200 mm3, animals were either left untreated (control) or injected i.p. with 0.5 mg topotecan per kg body weight on days 0–4 and 14–18 resulting in elevated ABCG2 expression. When tumors doubled in size (∼400 mm3), animals were injected i.p. with either vehicle +0.5 mg topotecan or 10 mg Ko143+0.5 mg topotecan combination therapy per kg body weight on days 0–4 and 14–18. If the tumor volume shrank to less than 50% of the start volume, treatments were stopped until tumors relapsed to 100%. The experiment was terminated when tumors reached a volume of about 1500 mm3. B, Kaplan-Meier (K-M) survival curves of untreated and combination therapy-treated tumor-bearing animals. Eight individual tumors were tested, and per tumor one untreated control (blue line), two vehicle + topotecan- (green line) and two Ko143+ topotecan-treated animals (red line) were included. For the vehicle + topotecan group one tumor did not grow out after transplantation. The P value was calculated using the Log-rank test. C, Waterfall plots (upper panel) showing tumor volume change (%) after 5 days of combination therapy per individual mouse, with relative volumes normalized to the treatment start volume (i.e. about 200 mm3). Box and whiskers plots (lower panel) summarizing the waterfall plot data. Lines represent the median response, while the whiskers show the maximum and minimum values. The P values was calculated using the Mann Whitney test.
Figure 2.
Topotecan response and ABCG2 immunoreactivity of eight individual Brca1−/−;p53−/− mammary tumors.
A, Relative tumor volume (%) of matched control (blue lines) and topotecan-treated (green lines) tumors over time. Each arrow indicates one dosing regimen of i.p. injections of 0.5 mg topotecan per kg body weight on days 0–4 and 14–18. B, Semi-quantification of ABCG2 immunoreactivity. Representative micrographs of four classes of stained tumor cells (0%, 1–10%, 11–50% and more than 50% of counted cells in 10 independent 400x magnification fields are ABCG2-positive) are shown. C, Table indicating ABCG2 immunoreactivity of the spontaneous (spon), untreated control (control) and topotecan-treated samples per individual tumor. P values of the spon - control (0.865) and spon - topotecan (0.020) comparisons were calculated using the Wilcoxon rank-sum test.
Figure 3.
Plasma and tumor topotecan pharmacokinetics (PK).
A, Plasma topotecan PK. Abcg2−/− animals, carrying ABCG2-positive tumor T1 (Fig. 2C), were treated with either vehicle +0.5 mg topotecan (green line) or 10 mg Ko143+0.5 mg topotecan (red line) per kg body weight and plasma was collected at 15, 30, 60, 90 and 120 minutes following i.p. injection. Error bars indicate standard deviations of the mean of at least 3 animals per time point. B, Tumor topotecan PK. The tumors of the animals in A were harvested and homogenized to determine topotecan concentrations at the same time points. Error bars indicate standard deviations of the mean of at least 3 animals per time point. P values were calculated using the unpaired t-test.
Figure 4.
Efficacy of topotecan, irinotecan and EZN-2208 in five ABCG2-positive Brca1−/−;p53−/− mammary tumors.
A, K–M curves showing survival (%) until a tumor volume of about 1500 mm3 was reached after one regimen of five consecutive i.v. injections on days 0, 2, 4, 6 and 8 of saline- (dark blue line, N = 23), topotecan- (light blue line, 4 mg i.v., N = 32), irinotecan- (pink line, 40 mg i.v., N = 28) and EZN-2208-treatments (green line, 10 mg SN38 equivalents i.v., N = 38) per kg body weight. P values were calculated using the Log-rank test. B, Box and whiskers plots indicating per drug the tumor volume change (%) after two weeks of treatment. Lines represent the median response, while the whiskers show the maximum and minimum values; the small circle is an outlier. P values were calculated using the Mann Whitney test. C, Waterfall plots showing tumor volume change (%) after two weeks of treatment per individual mouse, with relative volumes normalized to the treatment start volume.