Cell Culture

4T1 cells were from the Chinese Academy of Sciences Cell Bank of Type Culture Collection (Shanghai, China) and cultured in monolayers in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 µg/ml penicillin and 100 µg/ml streptomycin. MDA-MB-231 cells were purchased from American Tissue Culture Collection (No. HTB-26; ATCC, Manassas, VA) and maintained in Leibovitz’s L-15 medium with 10% FBS, 100 µg/ml penicillin and 100 µg/ml streptomycin (all from Invitrogen). Both cells were maintained in a humidified incubator at 37°C 5% CO₂ atmosphere.

Primary Cell Culture

Fresh breast tumors were obtained from patients undergoing surgery with written informed consent. Specimens of infiltrating ductal breast carcinoma without chemotherapy were selected for cell culture as describe by Speirs et al. [21]. The cells were cultured in HuMEC Ready Medium (Invitrogen) supplemented with 2% FBS in a humidified incubator at 37°C 5% CO₂ atmosphere.

Animals

Female BALB/c and BALB/c nu/nu mice (18–22 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China; Animal certificate: No. SCXK [Hu] 2007–0005). Animals were housed in a standard polypropylene cage containing sterile bedding under a controlled condition of temperature (23±2°C), humidity (50±5%), and light (10 and 14 h of light and dark) in Zhejiang University Laboratory Animal Center (No. SYXK [Zhe] 2007–0029).

4T1 Syngeneic Xenograft Model

The animal model is based on the previous report [22]. Each mouse was inoculated with 5×10⁴ viable 4T1 cells s.c. in the second right mammary fat pad area. Mice were randomly assigned to Sch B or control group the next day after inoculation. Each mouse was gavaged with either Sch B (100, 30, or 10 mg/kg body weight) or vehicle (0.5% paraxamer, Sigma) every day for a total of 7 doses. To investigate the effect of Sch B on 4T1 metastasis, we used 3 protocols. First, mice were gavaged with Sch B (100 mg/kg body weight), and tumors grew uninterruptedly. Second, mice were dosed with Sch B (100 mg/kg body weight) and then primary tumors were resected on day 10 as previously described [23]. Third, mice were administered with three doses of Sch B (100, 30, or 10 mg/kg body weight) and primary tumors were excised on day 10. The tumor volume was measured by vernier calipers twice or thrice weekly and calculated by an ellipse volume formula V= (L×W²)×0.5, where L is length and W is width. Lung metastases were scored by counting the macroscopic metastatic nodules based on the previous reports [24], [25], and the average number of lung metastases per mouse was determined as an indicator of general cancer disseminations [23], [26].

Delayed Treatment Model

Primary tumors were resected on day 10, and Sch B (100 mg/kg body weight) or vehicle was gavaged on day 13–19. All animals were sacrificed on day 30 for data collection.

Tail Vein Injection Model

This in vivo model is based on the previous report [27]. Before inoculation with 4T1 cells, each mouse was gavaged with Sch B (30, 100 mg/kg) or vehicle (0.5% paraxamer) for 3 consecutive days. Each mouse was then inoculated with 2×10⁵ cells intravenously via tail vein injection, followed by Sch B or vehicle treatment for the next 7 consecutive days. All mice were sacrificed on day 18.

MDA-MB-231 in vivo Animal Model

Each mouse was inoculated with 5×10⁶ MDA-MB-231 cells s.c. in the second right mammary fat pad area. Mice were randomly assigned to Sch B or control group the next day after inoculation. Each mouse was gavaged with either Sch B (100 mg/kg body weight) or vehicle (0.5% paraxamer, Sigma) for the next 7 consecutive days with a total of 7 doses. Mice were sacrificed on day 30 after inoculation.

Criterion to Assess the Mouse Moribund Status

A moribund mouse is defined as one which exhibits more than one of the following six clinical signs: inability to eat or drink; severe lethargy (reluctance to move when gently prodded with forceps); severe balance or gait disturbance, according to the NIH criterion [28].

Histological Analyses

Tumor tissue was fixed, embedded, and sliced into 5 µm thick sections. Paraffin sections were stained with hematoxylin and eosin (H&E) (Sigma). For immunohistochemistry, immunostaining of E-cadherin and vimentin was carried out using a standard protocol [29]. The H&E sections were evaluated and photographed with a Nikon Eclipse 50i microscope controlled by the NIS-Elements software (version 3.22). The relative area of metastases was measured using the ImageJ 1.45S software (NIH) [30]. Lung metastasis was quantified by counting the total tissue area per lung section (D1) and metastasis present in the same area (D2). The metastatic index was calculated by the ratio D2/D1 [31]. For bone metastasis, leg bones (femora and tibiae) were fixed in 10% neutral buffered formalin, decalcified in 1% HCl for 24 hours, and embedded in paraffin. Sections were stained with H&E and were examined microscopically. The incidence of bone metastasis was reflected by the visible osteolysis [32].

Cell Toxicity of Sch B on 4T1 Cells

2×10³ cells were seeded per well in 96-well plates. Sch B (5, 10, 20, 40, 80, 160 µg/ml) was added and cultured for 60 hours, cell viability was estimated by trypan blue exclusion. Six wells were measured for each concentration. Half inhibitory concentration (IC₅₀) was determined.

Effect of Sch B on TGF-β Induced EMT

The murine 4T1 cells and human primary breast cancer cells were pretreated with Sch B (5 µg/ml) for 24 hours, then TGF-β (5 ng/ml) was added and cultured for another 36 hours. The cell lysates were used in the western blotting analysis as previously described [33]. Fluorescence immunostaining was performed according to our previous reported method [34].

Cell Migration and Invasion Assays

For migration assays, 2×10⁵ cells were plated in six-well plates. After pretreatment of Sch B (5 µg/ml) for 24 hours, the FBS concentration of the culture medium was changed to 2% and a wound was incised as previously described [35]. Then TGF-β (5 ng/ml) was added and cultured for another 24 hours, photographs were taken by phase-contrast microscopy. Transwell (Corning Costar, Cambridge, MA) with a filter coated with Matrigel (BD Biosciences) was used for invasion assays. 4×10³ cells in serum-free RPMI-1640 were seeded into the 24-well transwell upper chamber, the lower chambers were filled with RPMI-1640 supplemented with 10% FBS as a chemo-attractant. After 12 hours pretreatment of Sch B (5 µg/ml) in both upper chamber and lower chamber, TGF-β (5 ng/ml) was added and treated for another 12 hours. At the end of the assays, the filter side of the upper chamber was cleansed with a cotton swab and the filter was stained for one hour with crystal violet (Sigma) in 2% ethanol and then rinsed in water. Then, cells on the filter were counted in at least 10 fields under the view of a phase-contrast microscope.

Statistical Analysis

Data were expressed as the Mean ± SD. Mouse survival was evaluated by Kaplan–Meier analysis and the log rank test. Chi-square analysis was used for bone metastasis comparison. Student t tests were used for the comparison between two mean values. P values less than 0.05 were considered statistically significant.

Sch B Significantly Increases Survival Time of Mice Via Inhibiting 4T1 Lung Metastasis

To exclude the effect of the primary tumor on mice survival time, we removed primary tumor by surgical resection on day 10 after 4T1 inoculation. Then two experiments with different purposes were performed on these mice. Experiment 1 was to observe the lung metastasis. The mice were sacrificed on day 30 after inoculation and lung metastasis was examined. Results showed that Sch B significantly inhibited 4T1 lung metastasis (Fig. 2 A & B & Fig. S1). Experiment 2 was to observe survival time. Fig. 2C demonstrated that mice receiving Sch B lived significantly longer than control and this effect was dose dependent (Fig. 3A). The growth curves of mice and weight of tumors were comparable between Sch B and control group (Fig. 2D&E, Fig. 3B&C).

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Figure 2

Sch B reduces lung metastasis and prolongs mice survival time.

5×10⁴ viable 4T1 cells were inoculated s.c. in the second right mammary fat pad area to establish the spontaneously metastatic model. Mice were treated with Sch B (100 mg/kg body weight) or vehicle every day for a total of 7 doses. Primary tumors were resected on day 10. Two thirds of the mice (n=20 for each group) were sacrificed on day 30 and one third of the mice (n=10 for each group) were for the survival experiment. (A) Numbers of visible 4T1 lung metastasis. (B) Lung metastatic index calculated by H&E staining. (C) Mouse survival time. (D) Mouse body weight. (E) Tumor weight resected on day 10. *, P<0.05, **, P<0.01.

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Figure 3

Sch B prolongs mouse survival time in dose-dependent manner.

Mice (n=8 for each group) receiving Sch B (100, 30, 10 mg/kg body weight) or vehicle every day for total 7 doses, and primary tumors were resected on day 10. (A) Mouse survival time. (B) Mouse body weight. (C) Tumor weight resected on day 10. *, P<0.05, **, P<0.01, Sch B versus control group.

It is noted that 10% of mice receiving Sch B (100 mg/kg body weight, Fig. 2C&3A) survived for 3 months, after which the experiment was terminated. This phenomenon was observed in three independent experiments. These mice were executed and examined pathologically. There were no visible lung metastasis in these mice (data not shown). It has been demonstrated by Pulaski and Demaria [37], [38] that metastasis of 4T1 syngeneic tumor could occur at very early stage (e.g., within the first week after s.c. inoculation), and our study suggests that Sch B could interfere with early metastasis. Consistently, we showed that Sch B could attenuate 4T1 local invasion in vivo and inhibit EMT in vitro (see below). The results suggest that early surgery (e.g., surgery done within 10 days after inoculation) combined with Sch B treatment may completely block the in vivo metastasis of 4T1 cells.

Sch B Attenuates 4T1 Metastasis to Bone

The 4T1 mouse model is commonly used as a model of mammary cancer metastasis to bone [32], [39]. Inhibiting mammary tumor metastasis to the lung may divert tumor cells to metastasize to alternative organs such as bone [32]. In order to rule out this possibility, we investigated if Sch B would divert 4T1 to bone metastasis. Our data demonstrated that Sch B significantly reduced 4T1 metastasis to bone (Figure 4), as reflected by the severity of osteolysis.

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Figure 4

Sch B inhibits bone metastasis of 4T1 cells.

Bone metastasis of 4T1 in mice demonstrated in Figure 2 were evaluated using H&E staining as described in Materials and Methods. The incidence of bone metastasis was reflected by the visible osteolysis. (A) Representative photo of bone metastasis. Osteolysis was marked by arrow. (B) Representative photo of bones with no osteolysis. (C) Incidence of bone metastasis (n=20) compared using chi-square analysis. *, P<0.05, Sch B versus control group.

Sch B Inhibits Local Invasion of 4T1 Cells

Although the above results indicated that Sch B could inhibit 4T1 lung and bone metastasis, it was not known how Sch B interfered with 4T1 metastasis. Since cancer metastasis is a complex process involving local invasion, intravasation, survival in the bloodstream, extravasation and colonization at a secondary site, it would be important to identify at which step Sch B interferes with 4T1 metastasis. We thought that the model via tail vein injection of cancer cells may help to tell if Sch B disturbs metastasis at step of the local invasion or not. If Sch B does not inhibit lung metastasis of 4T1 cells injected via tail vein, it should interfere with 4T1 metastasis at the step of local invasion. The results showed that Sch B had no significant inhibitory effects on lung metastasis of 4T1 cells via tail vein injection (Fig. 5A).

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Figure 5

Sch B does not inhibit lung metastasis of 4T1 cells using a tail vein injection or a delayed treatment model.

(A) Tail vein injection model: mice (n=10 for each group) were gavaged with Sch B (daily doses: 30 or 100 mg/kg body weight or vehicle) for 3 days prior to 4T1 cell inoculation. Then, each mouse was inoculated with 2×10⁵ 4T1 cells via tail vein injection, followed by treatment with Sch B (daily doses: gavaged with 30 or 100 mg/kg body weight or vehicle) for the next consecutive 7 days and sacrificed on day 18. (B) Delayed treatment model. Mice (n=10 for each group) were inoculated with 5×10⁴ 4T1 cells s.c. into the second right mammary fat pad area, and the primary tumors were resected on day 10. Sch B (100 mg/kg body weight) or vehicle was given every day from day 13–19 for a total of 7 doses, and mice were sacrificed on day 30. The lung metastasis was scored by macroscopic metastatic nodule as described in Materials and Methods. *, P<0.05, Sch B versus control group.

In order to further confirm if Sch B affect 4T1 metastasis at the step of early stage, we performed an experiment of delayed treatment with Sch B in mice with pre-existing tumors. The results showed that delayed treatment with Sch B had no significant effect on 4T1 metastasis (Fig. 5B).

Based on the above results, we performed the histopathological examination. As shown in Fig.6A, tumor cells had invaded deeply into the muscles in most of the primary tumors in control group (13 out of 20), while in Sch B group, most tumors showed a clear borderline between the region of tumor cells and muscles (14 out of 20). In addition, cells in the tumor margin in control group were spindle-shaped, more like mesenchymal cells, whereas in Sch B group, tumor cells kept “cobblestone” morphology, more like epithelial cells. These observations suggested that Sch B might inhibit epithelial mesynchymal transition (EMT).

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Figure 6

Sch B inhibits local invasion of 4T1 cells in vivo.

Mice (n=20 for each group) were treated with Sch B (100 mg/kg body weight) or vehicle intragastrically daily for 7 days. (A) Primary tumors and surrounding tissues were resected on day 15 for hematoxylin-eosin stain (upper, ×40, lower, ×100). (B) immunostaining of E-cadherin and vimentin.(×100).

Besides morphological changes, EMT is also accompanied with expressional changes of some biomarkers, such as E-cadherin, vimentin, etc [6], [40], [41]. When cells are under this transition, E-cadherin would be downregulated and vimentin upregulated. We examined the expressions of E-cadherin and vimentin in tumors. Fig.6B showed tumor cells in Sch B group had a higher E-cadherin expression (an epithelial biomarker) but lower vimentin expression (a mesenchymal biomarker) than those in control, suggesting that Sch B inhibited 4T1 metastasis likely via targeting EMT.

Apart from 4T1 model, we also investigated the effect of Sch B on in vivo metastasis of human breast cancer cell line MDA-MB-231 (Fig. S2). 30 days after inoculation, mice were sacrificed. We found that there were neither macroscopic metastatic nodules in lung nor bone metastasis in control and Sch B treated mice. The reports on metastatic ability of this cell line from different laboratories seems mixed: while several groups reported a poor metastatic ability of this cell [42], [43], [44], others [45],via vigorous selection, acquired several subclones (bone-seeking MDA-231BO and brain-seeking MDA-231BR) with high metastatic ability. Nevertheless, despite no obvious metastasis, we noticed that, similar to 4T1 cells, MDA-MB-231 cells invaded deeper into the muscles in most of the primary tumors in control than in Sch B treated group.