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Keywords:

  • bone metastasis;
  • breast cancer;
  • green fluorescent protein;
  • bisphosphonate

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A very common metastatic site for human breast cancer is bone. The traditional bone metastasis model requires human MDA-MB-231 breast carcinoma cell inoculation into the left heart ventricle of nude mice. MDA-MB-231 cells usually develop osteolytic lesions 3–4 weeks after intracardiac inoculation in these animals. Here, we report a new approach to study the formation of bone metastasis in animals using breast carcinoma cells expressing the bioluminescent jellyfish protein (green fluorescent protein [GFP]). We first established a subclone of MDA-MB-231 cells by repeated in vivo passages in bone using the heart injection model. On stable transfection of this subclone with an expression vector for GFP and subsequent inoculation of GFP-expressing tumor cells (B02/GFP.2) in the mouse tail vein, B02/GFP.2 cells displayed a unique predilection for dissemination to bone. Externally fluorescence imaging of live animals allowed the detection of fluorescent bone metastases approximately 1 week before the occurrence of radiologically distinctive osteolytic lesions. The number, size, and intensity of fluorescent bone metastases increased progressively with time and was indicative of breast cancer cell progression within bone. Histological examination of fluorescent long bones from B02/GFP.2-bearing mice revealed the occurrence of profound bone destruction. Treatment of B02/GFP.2-bearing mice with the bisphosphonate zoledronic acid markedly inhibited the progression of established osteolytic lesions and the expansion of breast cancer cells within bone. Overall, this new bone metastasis model of breast cancer combining both fluorescence imaging and radiography should provide an invaluable tool to study the effectiveness of pharmaceutical agents that could suppress cancer colonization in bone.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BREAST CANCER is one of the most common carcinomas in humans that metastasizes to bone.(1) Bone metastases in breast cancer patients are associated frequently with extensive bone destruction, leading to pathological fractures and hypercalcemia.(1) A major impediment to understand the pathogenesis of bone metastases has been the lack of appropriate animal models. Most of the studies of metastasis in animals have used tumor cell inoculation into the tail vein. In this animal model, tumor cells rapidly arrest in the lungs because this organ is the first to be encountered after an intravenous injection.(2) This accumulation of tumor cells in the lungs does not simply result from a mechanical trapping of the cells because of capillary lumen size restrictions, they also specifically attach to the endothelium of pulmonary capillaries to form tumor colonies.(3) For example, the analysis of real-time tumor cell trafficking using positron emission tomography shows that lung-metastatic B16BL6 melanoma cells accumulate in lungs after intravenous injection in animals whereas, under similar experimental conditions, liver-metastatic RAW117 lymphoma cells are released rapidly from lungs and subsequently accumulate in the liver of these animals.(4) Nevertheless, other routes of tumor cell inoculation have been used to bypass the pulmonary vasculature. For bone metastasis formation in animals, tumor cells have been injected into the medullary cavity of long bones,(5) into the abdominal aorta,(6) or into the left heart ventricle.(7, 8) The heart injection model closely mimics the development of osteolytic lesions observed in breast cancer patients(8) and has been extremely useful to dissect the pathogenesis of bone metastases induced by intracardiac injection of human MDA-MB-231 breast carcinoma cells in animals.(9) Mechanisms of bone metastasis formation involve the stimulation of osteoclastic bone resorption by MDA-MB-231 tumor products (parathyroid hormone-related protein [PTHrP], cytokines) and the growth of MDA-MB-231 tumor cells in the bone environment in response to bone-derived growth factors released from resorbed bone.(9) The net result is a vicious circle in which MDA-MB-231 tumor cells stimulate bone resorption and resorbed bone promotes MDA-MB-231 tumor growth. However, from a technical point of view, the heart injection model does not produce bone metastases at 100% because of its requirement of technical skillfulness.(10) In addition, although radiography is a well-suited noninvasive imaging method to monitor the appearance of osteolytic lesions, it allows the detection of bone metastases at a late stage when bone destruction is occurring. Thus, alternative detection methods are required to detect early events during the formation of bone metastases.

The bioluminescent jellyfish protein (green fluorescent protein [GFP]) expression in eukaryotic cells recently has been described as a convenient marker of transgene expression.(11) The use of GFP-expressing Chinese hamster ovary (CHO) cells,(12, 13) melanoma cells,(14, 15) and lung and prostate carcinoma cells(16, 17) enables the detailed imaging of tumor growth and metastasis formation in live tissues. In this respect, Yang et al.(15, 16) recently have shown the usefulness of whole-body noninvasive optical imaging in live mice to detect the colonization of bone by GFP-expressing B16F0-GFP melanoma and H460 lung cancer cells. Here, we report a new approach to study the formation of metastatic carcinoma of the breast to bone using GFP-expressing human MDA-MB-231 breast cancer cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Tumor cell lines

Human MDA-MB-231 breast carcinoma cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). CHO dhfr+ tumor cells were provided by Dr. Nelly Kieffer (Laboratoire Franco-Luxembourgeois de Recherche Biomédicale, Luxembourg). Tumor cell lines were cultured routinely in RPMI-1640 (Life Technologies, Cergy-Pontoise, France) supplemented with 10% (vol/vol) fetal bovine serum (Bio-Media, Boussens, France) and 1% (vol/vol) penicillin/streptomycin (Life Technologies) at 37°C in a 5% CO2 incubator.

Animals

All procedures were performed on 4-week-old female Balb/c nu/nu mice (Harlan, Gana, France). Studies involving animals, including housing and care, method of euthanasia, and experimental protocols were conducted in accordance with a code of practice established by the Experimentation Review Board from the Faculty of Medicine Laënnec. These studies were inspected routinely by an attending veterinarian to insure continued compliance with the proposed protocols.

Establishment of a human breast carcinoma cell line highly metastatic to bone in vivo

Anesthetized mice were inoculated with a tumor cell suspension (105 cells in 0.1 ml of phosphate-buffered saline [PBS]) of MDA-MB-231 cells into the left heart ventricle as described by Sasaki et al.(8) Twenty-one days after tumor cell inoculation, mice were examined by radiography for the presence of osteolytic lesions. Radiographs were taken after anesthetized mice were laid on their back against X-OMAT AR2 films (Kodak, Rochester, NY, USA) and exposed to an X-ray at 35 kV for 5 s using a cabinet X-ray system (Model 43855A; Faxitron X-Ray Corp., Wheeling, IL, USA). At death, on day 21, metastatic hindlimbs were collected. Tumor cells were harvested from metastatic bone specimens and maintained in culture until confluence, at which time they were again inoculated into the left heart ventricle of nude mice. After six in vivo passages in bone, a subclone (MDA-MB-231/B02) was selected for its unique predilection to metastasize to bone when injected into the tail vein of nude mice.(18)

Transfection and selection of stable MDA-MB-231/B02 and CHO cell clones expressing GFP

The expression vector for GFP (pEGFP-N1; Clontech, Palo Alto, CA, USA) was provided by Dr. François Bourre (UMR 5533, Pessac, France). MDA-MB-231/B02 and CHO dhfr+ cells were transfected using TransFast (Promega, Charbonnières, France). Transfected cells were maintained for 2 days in complete RPMI medium and then harvested by trypsin/EDTA treatment and subcultured at a ratio of 1:30 into selective RPMI medium containing 1 mg/ml of G418 (Promega). After 2 weeks, single clones were isolated using cloning cylinders (PolyLabo, Strasbourg, France). Positive clones were selected by flow cytometry (FACScan; Becton Dickinson, San Diego, CA, USA) and the brightest fluorescent clones were maintained subsequently in culture in the presence of 0.25 mg/ml of G418. Four MDA-MB-231/B02 and CHO dhfr+ clones expressing GFP were selected. Subsequent analyses were performed using clone 2 of GFP-transfected MDA-MB-231/B02 cells (B02/GFP.2) and clone 1 of GFP-transfected CHO dhfr+ cells (CHO/GFP.1).

Tumor xenograft and bone metastasis models and fluorescence imaging

For tumor xenograft formation, mice were inoculated subcutaneously with tumor cell suspensions (106 cells in 0.1 ml of PBS) of MDA-MB-231 or B02/GFP.2 cells into the right flank. Tumor size was assessed twice weekly by measurement of the length (L) and width (W) using a vernier caliper. Tumor volume (TV; expressed in mm3) was calculated using the following equation: TV = (L × W2)/2. For bone metastasis formation, anesthetized mice were inoculated intravenously with tumor cell suspensions (5 × 105 cells in 0.1 ml of PBS) of B02/GFP.2 or CHO/GFP.1 cells into the tail vein. Radiographs were monitored twice weekly as mentioned previously, and bone metastases were enumerated on each radiograph. All radiographs were evaluated independently by three examiners without knowledge of the experimental groups. In case of disagreement between examiners, radiographs were reviewed, and a consensus opinion was obtained. The area of osteolytic lesions was measured using a computerized image analysis system (Visiolab 2000; Biocom, Paris, France) and results were expressed in square millimeters. Tumor-bearing mice analyzed by radiography also were examined by noninvasive, whole-body fluorescence imaging using a fluorescence scanning system (Fluorimager; Molecular Dynamics, Sunnyvale, CA, USA). Then, scanned images were analyzed using computerized image analysis system ImageQuant (Molecular Dynamics). At death, autopsy was performed on all mice, and lungs and bones were collected for further fluorescence and histological analyses.

Bisphosphonate treatment

The bisphosphonate zoledronic acid [1-hydroxy-2-(1H-imidazole-1-yl)ethylidene-bisphosphonic acid] was obtained in the form of its hydrated disodium salt from Novartis Pharm AG (Basel, Switzerland). Zoledronic acid was dissolved in water and stored at 4°C. For bisphosphonate treatment, zoledronic acid was diluted in PBS and the pH was adjusted to 7.4. Animals were inoculated intravenously with B02/GFP.2 cells (day 0) and examined for the presence of bone metastases at day 18 using both fluorescence imaging and radiography. One hundred percent of the animals developed fluorescent bone metastases at day 18. Then, animals were divided into two groups. One group of mice received PBS (vehicle) and another group received zoledronic acid (3 μg/mouse per day) subcutaneously once a day from day 18 to 29. At the end of the experiments, animals were examined again by fluorescence imaging and radiography.

Histological analysis of lung and bone tissues

Formalin-fixed, paraffin-embedded lung tissues were cut using a semimotorized rotary microtome (RM2145; Leica, Rueil-Malmaison, France). Five-micrometer lung tissue sections were subsequently stained with hematoxylin, phloxin, and safran. Hindlimbs from animals were fixed with 80% (vol/vol) alcohol, dehydrated, and embedded in methylmethacrylate. Seven-micrometer sections of undecalcified long bones then were cut with a microtome (Polycut E; Reichert-Jung, Heidelberg, Germany) and stained with Goldner's trichrome. Histological analysis was performed on longitudinal medial sections of tibial metaphysis using computerized image analysis system Visiolab 2000 (Biocom).

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Isolation of a GFP-expressing human breast carcinoma cell line highly metastatic to bone in vivo

Human MDA-MB-231 breast carcinoma cells develop radiologically distinctive osteolytic lesions 3-4 weeks after cell inoculation into the left heart ventricle of nude mice.8-10) However, from a technical point of view, the heart injection model does not produce bone metastases at 100% due to its requirement of technical skillfulness.(10) For the purpose of this study, a subclone of MDA-MB-231 breast carcinoma cells MDA-MB-231/B02 was first established by repeated in vivo passages in bone using the heart injection model(18) and was subsequently transfected for GFP expression (this study). The selected clone (B02/GFP.2) remained stable in the absence of selective agents after numerous passages in vitro (Fig. 1A, left panel) and had a strikingly bright GFP fluorescence (Fig. 1A, right panel). Stable high-level expression of GFP in B02/GFP.2 cells also was investigated after subcutaneous growth in nude mice. Four weeks after tumor cell inoculation, mice had B02/GFP.2 tumor xenografts (Fig. 1B, left panel) that were strongly fluorescent in live animals (Fig. 1B, middle panel) and ex vivo after resection (Fig. 1B, right panel), showing stable high-level GFP expression in vivo during tumor growth. The growth of B02/GFP.2 cells in vivo was similar to that of MDA-MB-231 cells (data not shown). No spontaneous GFP fluorescent metastases were found in systemic organs using this tumor xenograft model (results not shown). The observation that a fluorescence scanning system allowed the external imaging of B02/GFP.2 tumor xenografts in nude mice was in agreement with previous findings showing that GFP-expressing B16F0 tumors can be viewed externally in intact animals using a transilluminated epifluorescence microscope.(15)

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Figure FIG. 1. Stable, high-level expression of GFP in human B02/GFP.2 breast carcinoma cells in vitro and in vivo. One subclone of human MDA-MB-231 breast carcinoma cells (MDA-MB-231/B02) was isolated by repeated in vivo passages in bone using the heart injection model. Then, MDA-MB-231/B02 cells were stably transfected with an expression vector for GFP. Positive clones were selected by flow cytometry and the brightest fluorescent clones were maintained by selection with 0.25 mg/ml of G418. (A, left panel) Analysis of B02/GFP.2 cells in culture in the absence of G418 (16th passage); right panel shows flow cytometry analysis of clone B02/GFP.2 versus parental cell line MDA-MB-231/B02. The y axis depicts the number of cells per channel and the x axis depicts the relative fluorescence intensity in arbitrary units (log scale). (B, left panel) representative tumor xenograft 4 weeks after subcutaneous inoculation of B02/GFP.2 cells in a nude mouse; middle panel shows corresponding tumor xenograft visualized after external fluorescence scanning of the same animal; right panel shows corresponding tumor xenograft visualized ex vivo after tumor resection. (C) Fluorescent images and (D) radiographs of the same nude mouse 8, 14, and 29 days after intravenous inoculation of B02/GFP.2 cells. Fluorescent and osteolytic bone metastases are depicted by arrowheads.

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Bone metastases are identified usually by radiography of intact animals.8-10) However, radiographs must be evaluated independently by at least two examiners who are without knowledge of the experimental groups to avoid false positive or false negative results. In this study, we showed that B02/GFP.2 cells displayed a unique predilection for dissemination to bone after intravenous cell inoculation in animals (Figs. 1C and 1D). In addition, the number and area of osteolytic lesions induced by B02/GFP.2 cells were significantly increased compared with that previously reported for MDA-MB-231 cells8-10) (Table 1). More importantly, the occurrence of fluorescent bone metastases in animals closely matched the localization of osteolytic lesions (Figs. 1C and 1D). Therefore, we compared fluorescence imaging and radiography for the detection of bone metastases.

Table Table 1.. Quantitative Assessment of Osteolytic Bone Metastases Induced by MDA-MB-231 and B02/GFP.2 Breast Carcinoma Cells 4 Weeks After Tumor Cell Inoculation in Nude Mice
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Comparison between fluorescence imaging and radiography as noninvasive methods to detect bone metastases induced by B02/GFP.2 cells

Direct comparison between fluorescence and X-ray technologies was possible because all of the animals inoculated with B02/GFP.2 cells developed bone metastases. In this respect, a series of external fluorescent images and radiographs for each animal was taken from day 0 to 29 after tail vein injection of B02/GFP.2 cells in nude mice. The follow-up of 16 animals by both fluorescence imaging and radiography clearly showed that GFP was far more sensitive than X-rays at identifying bone metastases (Fig. 2). For example, at day 18, all of the animals were positive for the presence of fluorescent bone metastases whereas only 50% of these animals had osteolytic lesions (Fig. 2). Overall, fluorescence imaging allowed the detection of bone metastasis formation approximatively 1 week before the occurrence of radiologically distinctive osteolytic lesions. Mechanisms of bone metastasis formation involve the stimulation of osteoclastic bone resorption by tumor products (PTHrP and cytokines) leading to bone destruction.(9) In our opinion, the tracking of B02/GFP.2 cells by fluorescence imaging was far more sensitive and accurate than radiography because the X-ray technology allows the detection of bone metastases only when bone destruction is occurring.(8)

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Figure FIG. 2. Detection of bone metastases by both noninvasive fluorescence imaging and radiography. Mice were inoculated intravenously with B02/GFP.2 breast carcinoma cells into the tail vein. Serial fluorescent images and radiographs were obtained at day 0, 4, 8, 11, 14, 18, 21, 25, and 29. Incidence of bone metastasis formation in nude mice was assessed by fluorescence imaging (open square) and radiography (open circle). The number of metastatic animals was expressed as the percentage (%) of mice (n = 16) inoculated with B02/GFP.2 cells.

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The number, size, and intensity of fluorescent bone metastatic lesions increased progressively with time (Figs. 3A, 3C, and 3E), showing progressive invasion of breast carcinoma cells within bone. Discrepancies in the numbers of osteolytic and fluorescent lesions may reflect a higher resolution for radiographs once bone degradation occurred (Fig. 3A vs. Fig. 3B). The approximately 2-fold higher fluorescent areas versus osteolytic lesion areas may indicate the potential for tumor cells to expand inside and outside the bone medullary cavity (Fig. 3C vs. Fig. 3D). The increase of fluorescent intensity may point out that cancer cells were growing at the bone metastatic site (Fig. 3E). Thus, as previously reported for the colonization of bone by GFP-expressing B16F0-GFP melanoma and H460 lung cancer cells,(15, 16) the use of B02/GFP.2 cells allowed the visualization of breast carcinoma progression in bone, even before the occurrence of radiologically distinctive osteolytic lesions.

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Figure FIG. 3. Quantitation of fluorescent metastases and osteolytic lesions. (A) The number, (C) area, and (E) intensity of fluorescent lesions were measured using computerized image analysis system ImageQuant. (B) The number and (D) area of osteolytic lesions on radiographs were quantitated using computerized image analysis system Visiolab 2000. Values are expressed as the mean ± SE (n = 6).

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At autopsy, fluorescence imaging and histological examination of lungs revealed that B02/GFP.2-bearing mice had no pulmonary metastasis (Fig. 4A). In contrast, the intravenous injection of CHO/GFP.1 cells in nude mice induced the formation of lung metastases that were readily detectable by fluorescence imaging (Fig. 4A). However, external fluorescence imaging of CHO/GFP.1-bearing mice did not allow the detection of these pulmonary metastases (not shown). It has been observed previously that the depth to which fluorescent metastases could be imaged depended on their size.(15) It is likely that CHO/GFP.1 pulmonary metastases were too small at the time of death to be imaged externally. The observation that B02/GFP.2 cells did not induce lung metastasis despite intravenous injection in the mouse tail vein confirmed previous findings2-4) showing that after intravenous injection in animals, B16BL6 melanoma cells accumulate in lungs whereas liver-metastatic RAW117 lymphoma cells are released rapidly from lungs and accumulate in the liver. It is most conceivable that B02/GFP.2 cells also were released rapidly from lungs and accumulated in bones. The externally acquired fluorescent images of bone metastases from B02/GFP.2-bearing mice closely matched ex vivo fluorescent images acquired on long bones collected at autopsy on day 29 (Fig. 4B, left panel). More importantly, histological examination of fluorescent long bones from B02/GFP.2-bearing mice revealed the occurrence of profound bone destruction (Fig. 4B, middle panel) when compared with the histology of normal long bones (Fig. 4B, right panel). Thus, there was a close relationship between the external fluorescence imaging of bone metastases and occurrence of osteolytic lesions as judged by histology.

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Figure FIG. 4. Fluorescence imaging and histological examination of lungs and bones colonized by GFP-transfected tumor cells. (A) Fluorescent images and histological sections of lungs from mice inoculated with B02/GFP.2 or CHO/GFP.1 cells. B02/GFP.2 cells did not induce pulmonary metastases in vivo (magnification ×10). In contrast, several metastatic lesions were detected readily in lungs from CHO/GFP.1-bearing mice as judged by both fluorescence imaging and lung histology (magnification ×10). Lung metastases are depicted by arrows. (B) Representative fluorescent image (left panel) and corresponding histology (middle panel) of a tibia from a mouse 29 days after intravenous inoculation with B02/GFP.2 cells. The histology of a normal tibial section is shown for comparison (right panel). Tissue sections were stained with Goldner's trichrome. Mineralized bone is stained in green and cells in red. The middle panel shows that cortical bone was partially destroyed and that most of the trabecular bone disappeared and was replaced by tumor cells that completely filled the bone marrow cavity (magnification ×25).

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Effects of the bisphosphonate zoledronic acid on the progression of established osteolytic lesions and expansion of B02/GFP.2 tumor cells

Bisphosphonates are powerful inhibitors of the osteoclast activity and therefore are used in the treatment of patients with osteolytic bone metastases.(10) Zoledronic acid is the most potent bisphosphonate at inhibiting bone resorption in vivo.(19) However, its efficacy in the treatment of osteolytic bone metastases currently is unknown. In this study, the effects of zoledronic acid were tested on the progression of established osteolytic bone metastases. This experiment closely reflects the clinical situation in which bisphosphonate treatment is given to breast cancer patients with bone metastases. Zoledronic acid (3 μg/mouse) was administered subcutaneously daily to mice with established bone metastases from day 18 to 29. The dose of zoledronic acid was chosen based on previous published works showing the efficacy of similar doses of the bisphosphonates risedronate(8) and ibandronate(20) on inhibition of the progression of established osteolytic lesions in animals. Zoledronic acid totally blocked the formation of new osteolytic lesions and the progression of established osteolytic bone metastases, as judged by radiography (Figs. 5A and 5B). These results were in agreement with those previously reported for risedronate and ibandronate.(8, 20) Moreover, quantitative assessment of changes in the number, area, and intensity of fluorescent tumor foci showed that zoledronic acid drastically inhibited the expansion of fluorescent tumor areas and, to a lesser extent, the development of new fluorescent tumor foci (Figs. 5C and 5D). In contrast, zoledronic acid had no statistically significant effect on the fluorescence intensity of tumor foci for this short period of treatment. Overall, these results provided clear evidence that zoledronic acid inhibits tumor progression as well as bone destruction. Using histomorphometric analysis of tibial bones, Sasaki et al.(8) and Yoneda et al.(20) have reported that risedronate and ibandronate also decrease breast cancer burden within bone. It has been suggested that the inhibitory effect of risedronate and ibandronate on tumor burden might be caused by an inhibition of osteoclastic bone resorption which, in turn, decreases the release of bone-derived growth factors required for tumor growth.(8, 20) Such a hypothesis also could apply to zoledronic acid. Alternatively, zoledronic acid may exhibit antitumor activity by directly stimulating apoptosis of breast carcinoma cells within bone because it reduces viability of human breast carcinoma cells in vitro.(21) The use of our bone metastasis model in which fluorescence imaging allowed the analysis of real-time tumor cell progression in live animals should help to resolve these specific questions.

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Figure FIG. 5. Effects of zoledronic acid on the progression of established osteolytic bone metastases and expansion of tumor cells within bone. Zoledronic acid (3 μg/mouse) or the vehicle alone was administered subcutaneously daily to mice with established bone metastases from day 18 to 29. Vehicle- and bisphosphonate-treated mice (8 animals in each group) were analyzed by radiography and fluorescence scanning at day 18 and day 29. (A) Representative radiographs of osteolytic bone metastases of hindlimbs. The left panel shows a radiograph taken at day 18 before the administration of zoledronic acid; the middle panel shows a radiograph taken at day 29 after a 12-day treatment with the vehicle; the right panel shows a radiograph taken at day 29 after a 12-day treatment with zoledronic acid. (B) Number and area of osteolytic lesions quantitated on radiographs at day 18 and day 29. Results were expressed as the mean ± SE; significantly different from the vehicle-treated group, *p < 0.01, Student's t-test. (C) Fluorescent images corresponding to radiographs shown in panel A. (D) Number, area, and intensity of fluorescent lesions at day 18 and day 29. Results were expressed as the mean ± SE; significantly different from the vehicle-treated group, *p < 0.01, Student's t-test; **p = 0.06 and ***p = 0.24.

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In conclusion, these data show that B02/GFP.2 cells have a unique predilection for dissemination to bone in vivo after tumor cell inoculation into the mouse tail vein. The underlying mechanisms responsible for the propensity of B02/GFP.2 cells to metastasize to bone presently are unknown and are the subject of further studies in our laboratory. Because of the high sensitivity of GFP fluorescence, this bone metastasis model of breast cancer should provide an invaluable tool to study the effectiveness of pharmaceutical agents that could suppress cancer colonization in bone.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Annie Desenfant for technical assistance. This work was supported by grants from INSERM (P.C.), the Comité Départemental du Rhône de la Ligue Nationale contre le Cancer (P.C.), ARC (P.C.), and Novartis (P.C.). O.P. and I.P. are recipients of a fellowship from ARC and the Ministère de la Recherche, respectively.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  • 1
    Mundy GR 1999 Bone Remodeling and Its Disorders, 2nd ed, Biddles Ltd., London, UK.
  • 2
    Oku N, Koike C, Sugawara M, Tsukada H, Irimura T, Okada S 1994 Positron emission tomography analysis of metastatic tumor cell trafficking. Cancer Res 54:25732576.
  • 3
    Al-Medhi AB, Tozawa K, Fisher AB, Shientag L, Lee A, Muschel RJ 2000 Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: A new model for metastasis. Nat Med 6:100102.
  • 4
    Koike C, Watanabe M, Oku N, Tsukada H, Irimura T, Okada S 1997 Tumor cells with organ-specific metastatic ability show distinctive trafficking in vivo: Analyses by positron emission tomography and bioimaging. Cancer Res 57:36123619.
  • 5
    Kjonniksen I, Winderen M, Bruland O, Fodstad O 1994 Validity and usefulness of human tumor models established by intratibial cell inoculation in nude rats. Cancer Res 54:17151719.
  • 6
    Powles TJ, Clark SA, Easty DM, Neville AM 1973 The inhibition by aspirin and indomethacin of osteolytic tumor deposits and hypercalcemia in rats with Walker tumor, and its possible application to human breast cancer. Br J Cancer 28:316321.
  • 7
    Arguello F, Baggs RB, Frantz CN 1988 A murine model of experimental metastasis to bone and bone marrow. Cancer Res 48:68766881.
  • 8
    Sasaki A, Boyce BF, Story B, Wright KR, Chapman M, Boyce R, Mundy GR, Yoneda T 1995 Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice. Cancer Res 55:35513557.
  • 9
    Guise T, Mundy GR 1998 Cancer and bone. Endocr Rev 19:1854.
  • 10
    Yoneda T, Michigami T, Yi B, Williams PJ, Niewolna M, Hiraga T 2000 Actions of bisphosphonates on bone metastasis in animal models of breast carcinoma. Cancer 88:29792988.
  • 11
    Misteli T, Spector DL 1997 Applications of green fluorescent protein in cell biology and biotechnology. Nat Biotechnol 15:961964.
  • 12
    Yang M, Chishima T, Wang X, Baranov E, Shimada H, Moossa AR, Hoffman RM 1999 Multi-organ metastatic capability of Chinese hamster ovary cells revealed by green fluorescent protein (GFP) expression. Clin Exp Metastasis 17:417422.
  • 13
    Chishima T, Miyagi Y, Wang X, Yamaoka H, Shimada H, Moossa AR, Hoffman RM 1997 Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression. Cancer Res 57:20422047.
  • 14
    Yang M, Jiang P, An Z, Baranov E, Li L, Hasegawa S, Al-Tuwaijri M, Chishima T, Shimada H, Moossa AR, Hoffman RM 1999 Genetically fluorescent melanoma bone and organ metastasis models. Clin Cancer Res 11:35493559.
  • 15
    Yang M, Baranov E, Jiang P, Sun FX, Li XM, Li L, Hasegawa S, Bouvet M, Al-Tuwaijri M, Chishima T, Shimada H, Moossa AR, Penman S, Hoffman RM 2000 Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc Natl Acad Sci USA 97:12061211.
  • 16
    Yang M, Hasegawa S, Jiang P, Wang X, Tan Y, Chishima T, Shimada H, Moossa AR, Hoffman RM 1998 Widespread skeletal metastatic potential of human lung cancer revealed by green fluorescent protein expression. Cancer Res 58:42174221.
  • 17
    Yang M, Jiang P, Sun FX, Hasegawa S, Baranov E, Chishima T, Shimada H, Moossa AR, Hoffman RM 1999 A fluorescent orthotopic bone metastasis model of human prostate cancer. Cancer Res 59:781786.
  • 18
    Winding B, Misander H, Sveigaard C, Therkildsen B, Jakobsen M, Overgaard T, Oursler MJ, Fogged NT 2000 Human breast cancer cells induce angiogenesis, recruitment, and activation of osteoclasts in osteolytic metastasis. J Cancer Res Clin Oncol 126:631640.
  • 19
    Green JR, Müller K, Jaeggi KA 1994 Preclinical pharmacology of CGP 42′446, a new, potent heterocyclic bisphosphonate compound. J Bone Miner Res 9:745751.
  • 20
    Yoneda T, Sasaki A, Dunstan C, Williams PJ, Bauss F, De Clerck YA, Mundy GR 1997 Inhibition of osteolytic bone metastasis of breast cancer by combined treatment with the bisphosphonate ibandronate and tissue inhibitor of the matrix metalloproteinase-2. J Clin Invest 99:25092517.
  • 21
    Senaratne SG, Pirianov G, Mansi JL, Arnett TR, Colston KW 2000 Bisphosphonates induce apoptosis in human breast cancer cell lines. Br J Cancer 82:14591468.