Figure 1.
Cmpd-1 treatment timeline of prevention and intervention studies in xenograft and syngeneic mouse models.
Figure 2.
Prevention studies with Cmpd-1 and its effect on primary tumor growth in the MDA-MB-231 human breast cancer xenograft model.
(A) Three groups of mice inoculated with cells at day 0 were treated with vehicle (n = 10) or either of the low dose (25 mg/kg twice daily; n = 10) or two-tier (50 mg/kg for initial 10 days and then reduced to low dose; n = 10) Cmpd-1 regimens from day 2 until the end point of the experiment. There was a significant dose-dependent effect of Cmpd-1 on the inhibition of primary tumor growth (vehicle vs. low dose p = 0.0018; vehicle vs. two-tier p<0.0001; ANOVA and Bonferroni post-test). Tumor measurements plotted are expressed as the mean ± S.E.M. (B) Mice treated with low or two-tier Cmpd-1 regimen showed prolonged survival compared to vehicle control (p = 0.0066 and p<0.0001, respectively; Kaplan-Meier survival).
Figure 3.
Prevention treatment with Cmpd-1 and its effect on osteolytic damage in the tibiae bones.
(A) Three groups of mice were inoculated with MDA-MB-231 cells intracardially at day 0 and were treated with vehicle (n = 10) or either of the low dose (25 mg/kg twice daily; n = 10) or two-tier (50 mg/kg for initial 17 days and then reduced to low dose; n = 10) Cmpd-1 regimens from day 2 until the end point of the experiment. A fourth group of mice (n = 10) received weekly treatment of Zometa for 3 weeks beginning on day 3. The long bones were analyzed by high resolution X-ray analysis and the degree of osteolytic damage was scored on a semi-quantitative scale, with a score of 0 representing no visible damage and a score of 4 representing severe damage. The percentage of mice without (A) initial osteolytic damage (Cmpd-1 low p = 0.027, two-tier p = 0.0037) or (B) substantial osteolytic damage (Cmpd-1 low p = NS, two-tier dose p = 0.017) of the proximal tibia was significantly lower when treated with Cmpd-1 compared to vehicle-treated mice (Kaplan-Meier analysis). (C) Examples of osteolytic lesions (shown using black arrowheads) in long bones following the treatment with vehicle alone, Zometa or Cmpd-1 given under either the low dose or two-tier regimens are illustrated in the panel and the lesion score is displayed on respective image.
Figure 4.
Prevention treatment with Cmpd-1 and its effect on primary tumor growth in a syngeneic mouse model of breast cancer.
Three groups of mice were inoculated with 4T1.2 cells into mammary fat pad at day 0 and were treated with vehicle (n = 8) or either of the low (25 mg/kg twice daily; n = 8) or high (50 mg/kg; n = 8) Cmpd-1 regimens from day 2 until the end point of the experiment. The primary tumor growth was significantly delayed in mice treated with low or high dose of Cmpd-1 compared with vehicle-treated mice (p = 0.0216 and p<0.0001, respectively; RM-ANOVA with Bonferroni post-test). Each time point represents tumor measurements as mean ± S.E.M.
Figure 5.
Metastatic burden in the lungs (A) and brain (B) of mice treated with vehicle or Cmpd-1 (low or two-tier regimens) following intra-cardiac inoculation of MDA-MB-231 cells, as assessed by qPCR for the human Alu repeat sequence on DNA extracted from whole organs.
Mouse only controls were included in the qPCR analysis and all mouse only samples were negative for human DNA. Mean values are indicated. (C) Percentage survival of intra-cardially inoculated mice. Cmpd-1 did not prolong overall survival of the mice treated with Cmpd-1 (Kaplan-Meier survival). (D) Metastatic burden in the lungs of mice treated with vehicle or Cmpd-1 (low or two-tier regimens) following mammary fat pad inoculation of spontaneously metastasizing 4T1.2 cells. Tumor burden in lungs was assessed by luminescence intensity of ex-vivo lung imaging using IVIS at the termination of the experiment. Cmpd-1 showed no inhibitory effect on metastatic tumor burden on soft tissues in both xenograft and syngeneic mice models (Mann-Whitney test). (E) Representative images of lung macrometastasis in mice inoculated with 4T1.2-Luc and treated placebo/low dose/high dose.
Figure 6.
Intervention treatment with Cmpd-1.
Mice were inoculated with MDA-MB-231 cells at day 0 and randomized to receive either vehicle (n = 7) or high Cmpd-1 regimen (50 mg/kg; n = 8) from day 27 continued until harvest (day 43). Each time point of tumor measurements plotted on the graph represents the mean ± S.E.M. Cmpd-1 inhibits established tumors in high dose treated mice at the largest tumor size (p<0.01 by student-t test, p = NS by RM-ANOVA).
Figure 7.
Concentration of Cmpd-1 in serum collected at the time of culling plotted against the time since last dose of Cmpd-1.
Figure 8.
(A) MMP13 immunofluorescence images of the clinical breast tumors from Patients 1 and 2. Immunofluorescence staining was performed on FFPE sections for MMP13 using a sheep polyclonal MMP-13 antibody as indicated by green fluorescence, whereas nuclei are stained red with propidium iodide. Parallel sections from the same tissue blocks were probed with control sheep serum to monitor for non-immune background (lack of green signal displayed as an inset in each panel). (B) Representative images of MMP13 immunofluorescence (green) from the syngeneic (top panel) and xenograft (bottom panel) mouse tumors. (C) MMP13 immunofluorescence (red) in human breast to bone metastasis. Dashed box in upper panels represents area of magnification in lower panels. Multinucleated (DAPI; blue) osteoclasts (TRAcP; green) were identified at the tumor-bone interface (represented by dashed line in the lower panels).
Table 1.
Clinicopathologic details of breast cancer patients.
Figure 9.
Expression of Ki67 in the experimental mice tumors from syngeneic (top panel) and xenograft (bottom panel) models treated with either vehicle, low and high dose of Cmpd-1.