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Cancer Research 67, 10304, November 1, 2007. doi: 10.1158/0008-5472.CAN-07-2483
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Tissue-Engineered Bone Serves as a Target for Metastasis of Human Breast Cancer in a Mouse Model

Jodie E. Moreau1, Kristen Anderson1, Joshua R. Mauney2, Trang Nguyen2, David L. Kaplan2 and Michael Rosenblatt1

1 Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts and 2 Department of Biomedical Engineering, Tufts University, Medford, Massachusetts

Requests for reprints: Michael Rosenblatt, Department of Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. Phone: 617-636-6565; Fax: 617-636-0375; E-mail: michael.rosenblatt{at}tufts.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The high frequency and mortality associated with breast cancer metastasis to bone has motivated efforts to elucidate tumor-stroma interactions in the bone microenvironment contributing to invasion and proliferation of metastatic cells. The development of engineered tissues has prompted the integration of engineered bone scaffolds into animal models as potential targets for metastatic spread. Silk scaffolds were coupled with bone morphogenetic protein-2 (BMP-2), seeded with bone marrow stromal cells (BMSC), and maintained in culture for 7 weeks, 4 weeks, and 1 day before s.c. implant in a mouse model of human breast cancer metastasis from the orthotopic site. Following injection of SUM1315 cells into mouse mammary fat pads, tumor burden of implanted tissues was observed only in 1-day scaffolds. Scaffold development and implantation was then reinitiated to identify the elements of the engineered bone that contribute to metastatic spread. Untreated scaffolds were compared with BMP-2–coupled, BMSC-seeded, or BMP-2/BMSC–combined treatment. Migration of SUM1315 cells was detected in four of four mice bearing scaffolds with BMP-2 treatment and with BMSC treatment, respectively, whereas only one of six mice of the BMP-2/BMSC combination showed evidence of metastatic spread. Histology confirmed active matrix modeling and stromal cell/fibroblast infiltration in scaffolds positive for the presence of metastasis. These results show the first successful integration of engineered tissues in a model system of human breast cancer metastasis. This novel platform now can be used in continued investigation of the bone environment and stem cell contributions to the process of breast cancer metastasis. [Cancer Res 2007;67(21):10304–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer is the leading cause of cancer-related death in American women, accounting for more than 200,000 new cases and 40,000 deaths annually (1). The difficulty in obtaining successful treatment (complete remission) results from the migration of cancer cells away from the primary tumor (2–5). As one of the few cancers that metastasizes to bone, breast cancer cells undergo a complex process involving migration away from the primary colony through the extracellular matrix into the blood or lymph followed by organ-specific adherence and extravasation into the stroma of the new tissue environment (5). The expression of identical and complementary bone proteins expressed on the surface of breast epithelial cells suggests a similarity in breast and bone stromal environments (6). This compatibility implicates a mutual selection of breast cancer and bone, emphasizing the contribution of the microenvironment to attracting, harboring, and sustaining skeletal breast cancer metastases.

To further understand the mechanisms of breast cancer metastasis, a mouse model has been developed that maintains both species- and organ-specific migration from the orthotopic site (7). With a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse as host, implanted native human bone has remained viable and becomes an efficacious target for metastatic spread. The human breast cancer SUM1315 cell line showed the capacity to metastasize following mammary fat pad (MFP) injection, maintaining species specificity by migrating exclusively to the human bone implants and not to the mouse skeleton or peripheral organs (except for late-stage appearance in lung).

Despite the positive migration of the SUM1315 cell line to the implanted human bone fragments, the nature of the bone tissue itself renders it a difficult platform for analysis of invading cells or investigation of the stromal contribution to the metastatic process. Although morphologic analysis and histochemistry can be obtained, genetic analysis of metastatic tissues without contaminating bone tissue has proven a difficult obstacle to overcome. Variability of implanted bone may also affect the consistency of metastatic spread in the model due to differences in donated tissue (age, gender, and disease). Incorporation of a tissue-engineered alternative for native bone has the potential to overcome issues related to native bone in the mouse model.

Reemerging as a prominent matrix protein for scaffold development, extracted silk fibers from the silkworm Bombyx mori offer a mechanically robust and long-term degradable foundation for tissue engineering applications (8, 9). With the capacity to create various geometries ranging from nanofibers and films to sponges and gels, silk is readily adaptable to correspond to the overall morphology of developing tissue. Spongy scaffolds have been implemented in the in vitro and in vivo development of bone tissue by coupling bone morphogenetic protein-2 (BMP-2) to stimulate human bone marrow stromal cell (BMSC) differentiation along the osteogenic lineage (8, 10).

Incorporating engineered tissue in the model of breast cancer metastasis, we are afforded the potential of a controllable, reproducible, and functional bone implant that may be tailored to the study of the metastatic microenvironment. We hypothesize that tissue-engineered bone scaffolds will serve as target sites for metastatic spread within the mouse model of metastasis. Validation of this model system would offer a unique platform for the dissection of the components of the bone microenvironment contributing to metastatic spread.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Silk fibroin scaffolds. Cocoons of B. mori silkworm were supplied by M. Tsukada (Japan Institute for Sericulture). Cocoons were boiled for 20 min in an aqueous solution of 0.02 mol/L Na2CO3 and rinsed thoroughly with distilled water for sericin protein extraction. The extracted silk fibroin was then dissolved in a 9.3 mol/L solution of lithium bromide at 60°C for 4 h, yielding a 20% (w/v) fibroin solution, which was then dialyzed in distilled water for 2 days and lyophilized. The solid was then dissolved directly in hexafluoroisopropanol and sodium chloride (80 mesh particle size; Sigma-Aldrich) to generate 17 wt% fibroin cylindrical scaffolds, 5 mm diameter and 2 mm height in dimension, with 550- to 600-µm pores.

Coupling BMP-2 to silk fibroin scaffolds. BMP-2 was covalently coupled to autoclaved scaffolds through carbodiimide cross-linking. Exposed carboxyl groups of aspartic and glutamic amino acids on silk were activated with an appropriate volume of 0.5 mg/mL 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.7 mg/mL N-hydroxysuccinimide (NHS) solution (Pierce) in MES buffer (pH 6.0) for 15 min at room temperature. Scaffolds were then rinsed extensively with MES to remove excess EDC/NHS and reacted with an appropriate volume of BMP-2 solution, allotting 20 µg BMP-2 per scaffold for 2 h at room temperature with gentle shaking. Scaffolds were then rinsed with PBS and stored at 4°C until seeding/implant.

BMSC culture expansion. All reagents for cell culture experiments were purchased from Invitrogen Corp., unless otherwise specified. Human BMSCs were isolated, expanded in culture, and stored as described previously (11). Briefly, unprocessed human whole bone marrow aspirates were obtained from donors <25 years of age (Clonetic-Poietics), resuspended in DMEM supplemented with 10% fetal bovine serum (FBS), 0.1 mmol/L nonessential amino acids (NEAA), 100 units/mL penicillin and 100 mg/L streptomycin, and 1 ng/mL basic fibroblast growth factor, and plated at 8 µL aspirate/cm2 in tissue culture polystyrene. Nonadherent hematopoietic cells were removed with the culture medium during medium exchange after 7 days of culture. Culture medium was changed twice weekly thereafter. Primary BMSCs were detached at ~80% confluency using 0.25% trypsin/1 mmol/L EDTA and replated at 5 x 103 cells/cm2. Passage 1 BMSCs near confluency were trypsinized and subsequently frozen in 8% DMSO/10% FBS/DMEM for future use. Passage 1 BMSCs were defrosted, replated at 5 x 103 cells/cm2 (passage 2), and trypsinized near confluency for use in matrix seeding.

SUM1315 cell culture. The human breast cancer epithelial cell line SUM1315, expressing green fluorescent protein and luciferase, was maintained in subculture 2 weeks before MFP inoculations, with medium composed of F-12 medium with L-glutamine supplement, 5% FBS, 50 units/mL penicillin, 50 µg/mL streptomycin, 5 µg/mL insulin, and 10 ng/mL epidermal growth factor (Sigma-Aldrich).

Silk scaffold cell seeding. Scaffolds were incubated overnight at 4°C in the culture medium used in BMSC expansion. Scaffolds were transferred to the bottom of individual 50 mL centrifuge tubes. Passage 2 BMSC were prepared in a suspension of 15 x 106 cells/mL in culture medium, applying 100 µL of suspension gently to each side of the cylindrical scaffold. Scaffolds were then incubated in the cell suspension for 30 min in a humidified incubator to facilitate primary cell seeding (cap loosened to allow gas exchange) with gentle agitation of the scaffold/solution at 15-min intervals. After primary seeding, an additional 4 mL of BMSC culture medium were added per tube and the tube was loosely recapped and stored at 37°C, 5% CO2 overnight. For extended in vitro culture, scaffolds were transferred to individual wells of a 12-well culture plate and supplemented with osteogenic differentiation medium composed of DMEM, with 10% FBS, 0.1 mmol/L NEAA, 100 units/mL penicillin/streptomycin, 100 nmol/L dexamethasone, 0.05 mmol/L ascorbic acid-2-phosphate, and 10 mmol/L ß-glycerophosphate.

Surgical implantation. All surgical procedures were approved through the Tufts University Department of Laboratory Animal Management (protocol 53-06) before experimentation. Human bone obtained from femurs removed from total hip replacement surgeries was cut into cylindrical fragments of ~5 mm diameter x 1 cm length dimension. Anesthesia of NOD/SCID mice 6 to 8 weeks of age was induced using an i.p. injection of 2,2,2-tribromoethanol (Sigma-Aldrich). The mouse was then shaved and the area was cleaned with 7.5% povidone-iodine solution. Engineered bone scaffolds and native human bone fragments were implanted s.c. over the respective left and right shoulders of the mouse through a dorsal incision of ~7 mm. Incisions were closed using a one-layer closure using skin clips. Animals were monitored daily over the course of 10 days, at which time skin clips were removed.

MFP injection of SUM1315 in NOD/SCID mice. One month following bone and scaffold implantation, mouse MFP was inoculated with 0.5 million cells/20 µL Matrigel (BD Biosciences) per fat pad by noninvasive injection between the fourth and fifth nipple of the mouse using a Hamilton syringe (Hamilton Co.). Animals were anesthetized during the procedure using isoflurane inhalation as means of restraint.

Luminescent imaging. Primary tumors and metastases were detected via luminescent imaging of the SUM1315 cells using the Xenogen 2000 and IVIS software. Mice received i.p. injections of luciferin (100 µL of 1 mg/mL; Molecular Probes). After 10 min, animals were anesthetized (isoflurane inhalation) for imaging. At the conclusion of study, mice were first imaged and then euthanized (CO2 inhalation). Implanted bone and scaffolds were removed for imaging and detection of metastasis. All images were standardized through IVIS software to pixels/second/cm2.

Histology. Implants bearing metastases and corresponding primary tumors were preserved in 10% formalin for histologic preparation. The day following harvest, cassettes bearing native bone samples were decalcified in an 8% formic acid solution for 48 h. Following decalcification, all samples were embedded in paraffin blocks and slides were prepared for each sample in quadruplicate. Cross-sections (7 µm) were cut from each block, stained with each of H&E, Masson's Trichrome, von Kossa, or antibody to cytokeratin 5/6 (Ventana Medical Systems), and mounted for light microscope visualization at x20 and x60 magnification (BX41, Olympus). Digital imaging incorporated a MacroFire color video camera and image capture software Picture Frame v.2.2 (Optronics).

Experimental design. Initial studies were designed to determine if engineered bone would serve as a target for metastasis for human breast cancer cells originating at the orthotopic site. After seeding with BMSC, scaffolds were cultured in vitro for 7 weeks, 4 weeks, and 1 day, respectively, in osteogenic differentiation medium (n = 12 per group) before implantation in mice. Injections of SUM1315 cells into mouse MFP were given 4 weeks following bone implant. Animal health was monitored weekly for 3 months following MFP injection, with final imaging and sacrifice for assessment of metastatic spread.

Subsequent experiments were aimed at identifying the components of bone that contribute to the metastatic spread of SUM1315. A total of eight scaffolds were prepared for each of four treatment groups: BMP-2 treatment with BMSC seeding (BMP/BMSC), BMP-2 coupling alone (BMP), BMSC seeding alone (BMSC), and untreated scaffold. Groups incorporating BMSC were seeded 1 day before implant. All scaffolds were maintained in osteogenic differentiation medium overnight. MFP injections of SUM1315 cells were given 4 weeks following bone implant. Animal health was monitored weekly for 3 months following MFP injection, with final imaging and sacrifice for assessment of metastatic spread.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Luminescent images of bone and scaffold explants 3 months after primary tumor inoculation indicate the presence of metastatic breast cancer to tissue-engineered bone scaffolds (Fig. 1 ). One-day scaffolds and native bone controls supported metastatic spread of SUM1315 in two of six surviving animals. Signal intensity relating to the tumor burden in bone and scaffolds was quantified at 7.91 x 10–5 ± 9.75 x 10–5 pixels/second and 3.97 x 10–5 ± 4.07 x 10–5 pixels/second, respectively. In comparison, all 4-week and 7-week explants were negative for metastatic spread (images not shown). The progression of scaffold remodeling is evident in the analysis of histologic sections, with the increase in both collagen and calcium deposition directly correlating to the length of time in culture before implant (Fig. 1B).


Figure 1
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  		Figure 1. Luminescent and histologic images of scaffold explants. A, composite luminescent image of 1-d explants with metastatic spread of SUM1315 from the orthotopic site-engineered bone scaffolds (left) and native bone (right). B, scaffold cross-sections of BMSC stained with von Kossa and trichrome, respectively. Visible increases of calcium (brown, left column) and collagen type I (blue, right column) are evident with increasing in vitro culture before implant. In all panels, the remaining silk scaffold stains pink and can be identified as the framework of a porous cross-section. D0, 1 d; W4, 4 wk; W7, 7 wk. Bar, 250 µm.

 
In the determination of components contributing to metastatic spread, all scaffold treatments supported metastasis of SUM1315 as confirmed by luminescent imaging and histology (Figs. 2 and 3 , respectively). BMP and BMSC scaffold groups had the highest incidence of metastatic spread, specifically four of four surviving animals in each group, whereas BMP/BMSC scaffolds supported metastasis in one of six. Signal intensity relating to the tumor burden was quantified at 1.18 x 10–5 ± 8.1 x 10–4 pixels/second for all bone explants bearing SUM1315 spread and 8.36 x 10–4 ± 2.92 x 10–4 pixels/second for all scaffolds. Of note, one animal in the BMSC scaffold group supported metastatic spread on the scaffold alone, as the native bone did not register a luminescent signal. Luminescent images of BMP explants were not obtained due to a mechanical malfunction, requiring histologic processing to identify metastatic spread. Untreated explants and respective native bone controls showed no evidence of metastasis in any of six surviving animals at the end of study. Xenogen images of the lungs and the site of scaffold/bone explant of mice bearing metastases were negative for luminescent signal, indicating that peripheral spread to other organs was not observed.


Figure 2
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  		Figure 2. Luminescent and histologic images of scaffold explants. A, composite luminescent image of scaffolds from BMSC and BMSC/BMP treatment groups bearing metastatic spread-engineered bone (left) and native bone (right). B, scaffold cross-sections of labeling cytokeratin 5 and 6 and collagen type I, respectively. The brown stain (left column) confirms the presence of invading SUM1315 cells at the scaffold perimeter. Bar, 100 µm. The predominance of stromal cells and fibroblasts within the scaffold has produced abundant collagen type I visible in the trichrome images (right column). Bar, 250 µm.

 

Figure 3
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  		Figure 3. Nonmetastatic BMP/BMSC scaffolds in cross-section with trichrome staining. In contrast to scaffolds positive for metastatic spread, negative groups showed evidence of necrosis with the infiltration of lymphoid cells and the absence of fibroblast/stromal cell infiltration and remodeling. Bar, 500 µm.

 
Stromal cells and fibroblasts were the most abundant cell types present in all scaffolds supporting metastatic spread, actively remodeling the silk matrix, and depositing collagen as observed in trichrome-stained sections for all groups (Fig. 3). Conversely, the BMP/BMSC scaffolds that did not support metastases seemed necrotic with lymphoid cells in place of fibroblasts and stromal cell remodeling. Significant calcium deposition was observed only in the BMP/BMSC explant in regions that previously contained the silk scaffold. Cytokeratin 5/6 immunostaining confirmed the peripheral invasion of SUM1315 into engineered scaffolds, with the absence of positive stain in native bone controls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By integrating research approaches from the disciplines of tissue engineering and cancer biology, we have identified a functional and reproducible engineered bone scaffold capable of supporting the metastatic spread of breast cancer from the orthotopic site. This novel model system holds promise for advancing the study of the contribution of the bone microenvironment to metastasis. Our findings parallel the spontaneous migration of the SUM1315 human breast cancer cell line to native human bone observed in our mouse model of metastasis. This new animal model offers a novel approach to characterizing the metastatic microenvironment of bone in breast cancer and other models of disease.

In the development of a disease model, engineered tissues offer a unique platform that undergo the development and remodeling characteristic of native tissue while permitting environmental control. Both the structure and porosity inherent in the silk scaffold preparation predispose seeded cells to osteogenic differentiation, characterized by calcium deposition and the up-regulation of bone-specific markers (12). Advanced bone development was attained in 7-week cultures, with progressively immature states at 4 weeks and 1 day, respectively. Although 7 weeks in in vitro culture produced the most mature bone environment, the results observed with these scaffolds contradicted our initial assumption that the most developed bone environment would yield the greatest metastatic spread. Instead, the more primitive bone environments were more conducive to breast cancer cell invasion. Previous work by Lyden et al. has implicated stem cells in the environmental preparation and signaling before breast cancer cell invasion in bone, supporting our findings of metastasis in the most actively regenerating scaffold (13). Perhaps the relatively rich proportion of stem cells in immature scaffolds contributes to enhanced metastasis. Characterization of the 1-day scaffolds will be emphasized in future studies to identify the means by which the s.c. environment affects cell differentiation and the subsequent development of a metastatic niche.

In the attempt to identify a specific component responsible for the initiation of metastasis to the engineered scaffold, we instead found three distinct environments capable of supporting SUM1315 cell invasion. Individually treating the scaffolds with either BMP-2 or BMSC induced metastasis at a remarkable frequency, maintaining a primitive yet actively remodeling bone environment. Distant changes in the premetastatic "soil" environment have been implicated in the migration of breast cancer cells to distant sites (13, 14). Given its capacity to recruit a blood supply, the direct addition of BMSC to the silk scaffold may equip the implant with externally derived components that trigger metastasis. A similar metastatic niche may be achieved through BMP-2 scaffold treatment, given its capacity to attract and select stem cells for bone development and maturation (15). The importance of both the balance and timing of environmental cues is reflected by BMP/BMSC scaffolds and their reduced frequency in fostering metastatic spread.

Combining a mouse model of breast cancer metastasis with tissue-engineered bone has enabled us to demonstrate, for the first time, that engineered scaffolds are capable of functioning as homing sites for metastatic spread. This novel platform will enable future study of bone stromal factors essential for or contributing to metastasis. Moreover, our findings suggest that engineered tissues can be integrated as advantageous components in emerging models of disease.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/ 3/07. Revised 8/20/07. Accepted 9/ 7/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
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