Article Figures & Data
Figures
- Figure 1.
Galardin/GM6001/Ilomastat.
- Figure 2.
Primary tumor growth and metastasis in Galardin-treated mice. Tumor size was monitored weekly by palpation in FVB-PymT mice that were randomly distributed to receive the MMP inhibitor Galardin (n = 26), placebo (n = 26), or no treatment at all (n = 27). Treatment was started at age 41 to 45 d by s.c. insertion of 60-d slow-release tablets containing 150 mg Galardin or placebo. The tablets were left in place until the mice were killed at age 92 to 96 d and analyzed for lung metastasis. The lengths (l) and widths (w) of every tumor in each mouse was used to estimate the total tumor volume: V = ∑(lw2π / 6) as described (22). A, geometric mean of tumor volumes plotted versus age for untreated (○), placebo-treated (▵), and Galardin-treated (▴) mice. B, tumor volumes at sacrifice. Columns, geometric means; bars, 95% confidence interval. C, total metastasis volume was quantified with an unbiased stereologic technique (30) on five to eight evenly spaced H&E-stained sections of the lungs from each mouse. Columns, medians; bars, 95% confidence interval.
- Figure 3.
Increased MMP-2 activity in Galardin-treated tumors. Gelatin zymography of tumor extracts from placebo- and Galardin-treated tumors. The levels of pro-MMP-2 and active MMP-2 are increased in Galardin-treated tumors. Pro-MMP-9 is seen sporadically in tumors from both groups.
- Figure 4.
Collagen deposition in healthy mammary glands and Galardin-treated mammary tumors. The no. 4 (right side) mammary glands were obtained from 92- to 96-day-old FVB-PymT mice that had been randomly distributed to receive the MMP inhibitor Galardin or placebo starting at age 41 to 45 d. The no. 4 mammary gland was also obtained from tumor-free nontransgenic mice matched for strain and age. Sections of the tissue samples were stained with PicroSirius Red to label collagen type I (yellow to red) and III (green) under polarizing microscopy. Sections were counterstained with Weigert's hematoxylin (black nuclei). A and B, no. 4 mammary gland of a tumor-free nontransgenic mouse under bright-field (A) and polarized (B) illumination for comparison. C, C′, C″, representative tumors from placebo-treated mice. D, D′, D″, representative tumors from Galardin-treated mice. The collagen-rich skin is visible in several panels. Bar, 500 μm.
- Figure 5.
Histopathologic progression of Galardin-treated tumors. The no. 4 (right side) mammary glands were obtained from 92- to 96-day-old FVB-PymT mice that had been randomly distributed to receive the MMP inhibitor Galardin (n = 26), placebo (n = 26), or no treatment at all (n = 27), starting at age 41 to 45 d. H&E-stained sections of the tissue samples were scored according to the following defined histopathologic stages (25, 33): normal gland (A), hyperplasia (B), adenoma (C), early carcinoma (D), or late carcinoma (E). Data are presented as superimposed histogram traces indicating the percentage of tissue samples that received tumor grade scores A to E for each of the three groups of mice: untreated (○), placebo-treated (▵), and Galardin-treated (▴). The observed distribution differences were not significant (Kruskal-Wallis test, P = 0.13).
- Figure 6.
Expression of MMP-7, -9, and -10 mRNA in MMTV-PymT-induced mammary cancers and lung metastases. MMP mRNAs were detected by in situ hybridization in primary tumors (A-C) and lung metastases (D) with antisense RNA probes. Expression is seen as silver grains in the bright-field images (row 1) and white reflections in the dark-field images (row 2). Hybridization with a nonoverlapping antisense RNA probe (row 3) and the corresponding sense RNA probe (row 4) on adjacent sections provided positive and negative controls. A, MMP-10 mRNA was infrequent in the primary breast tumors but was occasionally detected quite prominently in narrow bands of connective tissue in areas of compact tumor tissue (arrows). B, MMP-7 mRNA was practically absent in the primary breast tumors and was restricted to occasional cancer cells (arrows) adjacent to necrotic areas (n). C, MMP-9 mRNA was practically absent in the primary breast tumors but was found in scattered stromal cells (arrows) near or adjacent to necrotic areas. D, MMP-9 mRNA was present in scattered cells throughout the entire lung tissue but was not convincingly associated with lung metastases (compare rows 2 and 3 with row 4). Sections were counterstained with H&E. Bars, 100 μm (A and D), 50 μm (C), and 25 μm (B).
Tables
- Table 1.
Galardin treatment of MMTV-PymT transgenic mice
Treatment group Untreated Placebo Galardin No. mice 27 26 26 Age at sacrifice (d) 94.1 ± 1 93.8 ± 1 94.4 ± 1 Tumor incidence at sacrifice (%) 100 100 100 Final primary tumor volume (geometric mean; cm3)* 3.37 3.29 1.69 Final primary tumor volume (95% confidence interval of the geometric mean; cm3) 2.50-4.56 2.45-4.41 1.28-2.23 Dissemination to lymph nodes, brachial + axillary (incidence; %)† 30 (31/105) 29 (30/102) 21 (22/103) Dissemination to brachial lymph nodes (incidence; %)† 25 (13/52) 29 (15/51) 22 (11/51) Dissemination to axillary lymph nodes (incidence; %)† 34 (18/53) 29 (15/51) 21 (11/52) Lung metastasis (incidence; %)‡ 100 (27/27) 92 (24/26) 58 (15/26) Lung metastasis volume (median; mm3)§ 1.39 0.56 0.0030 Lung metastasis volume (95% confidence interval of the median; mm3) 0.23-3.05 0.19-2.34 0-0.048 Lung metastasis volume (geometric mean; mm3)∥ 0.92 0.26 0.0033 NOTE: A cohort of FVB-PymT mice were either left untreated or implanted with placebo or Galardin pellets and monitored weekly for tumor onset and killed at age 92 to 96 d. Primary tumor volume and tumor dissemination to regional lymph nodes and to lungs were assessed at the time of sacrifice.
↵* t test on log-transformed data (placebo versus Galardin): P = 0.0014.
↵† Not significant.
↵‡ Fisher's exact test (placebo versus Galardin): P = 0.009.
↵§ Mann-Whitney U test (placebo versus Galardin): P < 0.0001.
↵∥ The geometric mean is also reported for direct comparison with earlier work (22, 23). To allow logarithmic transformation, metastasis volumes of zero were assigned a value of 10-4 mm3. t test on log-transformed data (placebo versus Galardin): P < 0.0001.
Volume 7, Issue 9
