Table 1.
Tumor growth and metastases in 4T1 breast carcinoma-bearing mice.
Fig 1.
Representative sections from orthotopic 4T1 tumors and nodular metastatic deposits.
Tumor cells invading the surrounding mammary parenchyma (A—C), muscle fibers (D) and adjoining skin (E–F) show the highly invasive capacity of 4T1 breast tumors. Examples of metastatic lesions were observed in the lungs (G), mesentery (H), pancreas (I) lymph nodes (J), or salivary gland (K). MP, mammary parenchyma; MF, muscle fibers; S, skin; LP, lung parenchyma; LN, lymph node; MES, mesentery; PC, pancreas; SG, salivary gland; * indicates tumor areas. All images present original magnification x200, except upper left and inset images, x50.
Fig 2.
Fitting mathematical growth models to tumor experimental data as a function of inoculated cell density.
Exponential and Gompertz models were fitted to the population’s tumor growth curves (A) and compared using the Akaike’s information criteria (AIC) and the extra sum-of-squares F test (B). Specific growth rates (SGR) and doubling times of each group were determined from the mathematical equations of the best fit (B). Dark symbols represent experimental mean tumor volumes. The solid line represents the best fit for each group with a 95% confidence interval, light lines.
Fig 3.
Effect of cell density and tumor mean volume on viable rim area and vascular density.
Quantitative analysis of viable rim area (A) and vascular density (B) was assessed in tumors with mean volumes of 100–200 mm3 and > 250 mm3, based on H&E or CD31 immunostaining in carcinoma sections derived from 500, 2000, 5 x 104 and 1 x 106 cancer cells, original magnification x400 (C). Data represent the mean ± SEM of 3–6 independent sections. ns, p > 0.05 two-way ANOVA with Tukey’s multiple comparisons test.
Fig 4.
Therapeutic activity of Caelyx® in 4T1 breast tumor-bearing mice.
Balb/c mice implanted with 500 4T1 cancer cells in the mammary fat pad were treated weekly with Caelyx®, at 5 mg doxorubicin/kg body weight for 5 weeks (indicated by grey arrows). An additional control group included non-treated mice (injected with saline). Individual tumor growth curves (A) and survival curves (B) are illustrated. H&E staining, original magnification x200 (C), quantification of viable rim area (D), CD31 immunostaining, original magnification x400 (E), and quantification of vascular density (F), on representative sections from 4T1 primary tumors, following treatment either with Caelyx® or saline. MF, muscle fibers; S, skin; MP, mammary parenchyma; * indicates tumor areas. Data represents individual relative tumor volumes and median survival curves of saline- (n = 14) and Caelyx®-treated (n = 8) mice. Viable rim area and vascular density represent mean ± SEM of 3 independent sections. ns, p > 0.05 two-tailed nonparametric Mann-Whitney test.
Table 2.
Incidence of metastatic lesions in 4T1 breast carcinoma-bearing mice.
Fig 5.
Metastatic burden in the lungs following treatment with Caelyx®.
Metastatic burden was determined upon organ weight for individual mice treated either with Caelyx® or saline, and further normalized for the whole body weight. Data represent individual (dots) or mean ± SEM of control (n = 13) and Caelyx® (n = 6) mice. ns, p > 0.05 two-tailed nonparametric Mann-Whitney test.
Fig 6.
Effect of Caelyx® in the spleen and kidneys of mice bearing 4T1 tumors.
Relative weight (A) and representative H&E images, original magnification x50 (left) or x200 (right) (B) of spleens from naive mice and mice treated with Caelyx® or saline (control). Images illustrate the red pulp markedly expanded by numerous hematopoietic cells (black arrowheads), including megakaryocytes (black arrows), and myeloid precursor cells, particularly in the controls. Red circles represent white pulp areas with lymphoid nodules. The relative weight of kidneys (C) from Caelyx®- or saline-treated control mice was also analyzed. ***, p < 0.001, nonparametric one-way ANOVA with Dunn’s multiple comparisons test; **, p < 0.01, two-tailed nonparametric Mann-Whitney test.