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Keywords:

  • BONE;
  • BREAST CANCER;
  • ANIMAL MODELS;
  • RODENT;
  • N-METHYL-N-NITROSOUREA

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Current theory on the influence of breast cancer on bone describes metastasis of tumor cells to bone tissue, followed by induction of osteoclasts and bone degradation. Tumor influences on bone health in pre- or nonmetastatic models are unknown. Female rats (n = 48, 52 days old) were injected with N-methyl-N-nitrosourea (MNU) to induce breast cancer. Animals were euthanized 10 weeks later, and tumors were weighed and classified histologically. Right femurs were extracted for testing of bone mineral density (BMD) by dual X-ray absorptiometry (DXA), bone mechanical strength by three-point bending and femoral neck bending tests, and structure by micro–computed tomography (µCT). Of 48 rats, 22 developed one or more tumors in response to MNU injection by 10 weeks. Presence of any tumor predicted significantly poorer bone health in 17 of 28 measures. In tumored versus nontumored animals, BMD was adversely affected by 3%, force at failure of the femoral midshaft by 4%, force at failure of the femoral neck by 12%, and various trabecular structural parameters by 6% to 27% (all p < .05). Similarly, greater tumor burden, represented by total tumor weight, adversely correlated with bone outcomes: r = −0.51 for BMD, −0.42 and −0.35 for femur midshaft force and work at failure, and between 0.36 and 0.59 (absolute values) for trabecular architecture (all p < .05). Presence of MNU-induced tumors and total tumor burden showed a negative association with bone health of the femur in rats in the absence of metastasis. Further study is required to elucidate mechanisms for this association. © 2011 American Society for Bone and Mineral Research.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Breast cancer metastasizes to bone tissue in 70% to 80% of patients.1, 2 Metastasis is thought to occur via a “soil and seed” model,3 in which a malignant tumor cell escapes the basement membrane of breast tissue, circulates, is drawn to invade bone through tissue-specific chemoattractant signals, and finds in bone a favorable environment for growth and proliferation.4, 5 Following invasion of bone, tumors may create osteolytic (bone-degrading) or osteoblastic (bone-forming) lesions, with 80% to 90% of lesions following the osteolytic profile.1 Osteolytic lesions are characterized by a positive-feedback loop of tumor growth leading to additional bone degradation and the release of tumor growth factors and continuing tumor growth.1, 2 Intuitively, osteolytic invasion of bone is highly damaging to bone strength, structure, and density.6–8

This pattern of invasion and ostelytic lesion formation is well established, but current models assume that cross-talk between tumor cells and bone tissues occurs on a local, paracrine, or autocrine level. It is also possible that breast tumors may release bone-damaging signals into the circulation prior to actual metastasis to bone tissue. In this experiment, we tested whether the presence of breast tumors in a nonmetastatic rodent model (induction of breast tumors by N-methyl-N-nitrosourea injection in rats) also may influence bone tissue. Specifically, we characterize bone health by (1) bone mineralization using dual X-ray absorptiometry (DXA), (2) bone structural properties using micro–computed tomography (µCT), and (3) bone strength using mechanical three-point bending and femoral neck tests to failure.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Experimental model

Forty-eight female Sprague-Dawley rats aged 45 days (Harlan-Teklad, Indianapolis, IN, USA) were housed individually at 24°C in wire-bottomed cages over a 12-hour light/dark cycle. At 52 days of age, rats were injected once intraperitoneally with 50 mg/kg of body weight of N-methyl-N-nitrosourea (MNU) administered in a 0.9% NaCl solution to induce tumor formation. The MNU induction model is a standard paradigm for studying breast tumors in rats.9–12 Tumors are induced at rates exceeding 50% and are overwhelmingly malignant but nonmetastatic.13 MNU shows mammary tissue specificity, which is augmented by selective administration to the peritoneal cavity in these animals.9, 14

This study was designed to test multiple hypotheses, including the influence of dietary protein level on MNU-induced tumor incidence and progression.15 Accordingly, animals were randomly assigned to one of two modified AIN-93 semipurified diets16 designed to be equal in all regards except for the ratio of protein to carbohydrate (using a substitution of casein for starch). Additional information on these diets may be found in the supplemental material. All results presented herein have been tested statistically for the influence of diet and the interaction of tumor status with diet assignment. No significant interactions between tumor status and diet assignment were observed (all p > .1), ruling out the possibility that the influence of tumor status is confounded by diet assignment. Accordingly, only the influence of tumors is presented in the main body of this article. The supplemental material provides a comprehensive table of bone outcomes according to diet assignment and tumor status, as well as statistical tests of these effects and their interaction.

Animals were followed for 10 weeks, after which they were euthanized and their left femurs extracted, wrapped in saline-soaked gauze, and frozen at −20°C until subsequent testing. Storage in this manner has been shown not to alter the mechanical properties of bone,17 including after thawing and refreezing up to three times.18 Tumors also were excised, weighed, and preserved for pathologic examination as described below. Rats then were inspected grossly for evidence of metastasis. The animal protocol was approved by the University of Illinois Institutional Animal Care and Use Committee (principal investigator, DKL).

Tumor incidence and progression

Beginning 5 weeks after induction, rats were palpated twice weekly for detectable tumors. Palpable tumor incidence and latency to detection were recorded, and orthogonal tumor length and width were measured using calipers. After the rats were euthanized, mammary tumors were excised, weighed immediately, rinsed in saline, fixed in 10% neutral buffered formalin over 24 hours, dehydrated in 75% ethanol, sectioned, and stained with hematoxylin and eosin. Slides were sent to an independent pathology lab (College of Veterinary Medicine, University of Illinois at Urbana-Champaign) for diagnosis of tumors by standard pathologic examination.

Bone mineralization

The 48 rat femurs were thawed to room temperature over 4 hours, and areal bone mineral density (aBMD, g/cm2) was measured by dual X-ray absorptiometry (DXA) using settings for isolated bone from small animals on a Hologic QDR 4500A bone densitometer (software Version 11.2; Hologic, Inc., Waltham, MA, USA). Whole-femur BMD and subregional BMD of uniform small areas at the neck, midshaft, and distal metaphysis and epiphysis of the femur were quantified (Fig. 1).

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Figure 1. Regions of analysis in high-resolution DXA of excised femurs. Subregions were selected to correspond with those tested in mechanical testing and µCT: R1 = femoral neck and head; R2 = midshaft; R3 = metaphysic; R4 = epiphysis and condyles.

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Bone strength

Following bone absorptiometry, femurs were subjected to a three-point bend test to failure using an Instron MINI 44 (Instron, Inc., Grove City, PA, USA) at a rate of 0.6 mm/min and loading span of 15 mm, with the crosshead placed at the midshaft in an anteroposterior direction. Following fracture, digital calipers were used to measure the span of the cross section of the fracture site along anteroposterior and mediolateral axes, as well as the thickness of the cortical shell at four points defined as the midpoints of the cortical rim in the four quadrants of the cross section created by the anteroposterior and mediolateral planes. These parameters were used to approximate the cross-sectional moment of inertia I at the fracture site using an elliptical model according to the methods described by Turner and colleagues19:

  • equation image(1)

where a represents the width of the cross section along the mediolateral axis, b is the width along the anteroposterior axis, and t is the average thickness of the cortical rim at the four sites described earlier. This approximation is less accurate and precise than calculation of I using measured surface area at the fracture site, but any bias introduced by this approximation is expected to be systematic between tumored and nontumored animals. This method therefore should not limit inference, except perhaps by increasing the standard error of corresponding outcomes (which would reduce statistical power and decrease the likelihood of a spuriously positive result).

A force-displacement curve was created and processed using Matlab Version 7.6.0 (Mathworks, Natick, MA, USA) to obtain the following biomechanical properties: Ultimate force Fu, the maximum force observed during bend testing, representing strength of the bone; stiffness S, the slope of the force-displacement curve prior to yield or the force produced per unit of displacement; and work to failure U, energy absorbed by the bone prior to fracture. Intrinsic biomechanical parameters of bone were calculated by normalizing Fu, S, and U to the dimensions of the bone. These parameters are ultimate stress σu or intrinsic strength, Young's modulus E or intrinsic stiffness, and modulus of toughness uT or intrinsic work to failure. Formulas for these calculations are shown in Eqs. (1) through (4).19

  • equation image(2)
  • equation image(3)
  • equation image(4)

Damage induced by three-point bend testing is localized to the femoral shaft, leaving the femoral neck intact.19 Following bend testing, the proximal end of the femur was mounted vertically in a chuck for mechanical testing of the femoral neck to failure. Load was applied at 0.6 mm/min at the superior edge of the femoral head in a direction parallel to the shaft of the femur. Resulting force-displacement curves were integrated for work to failure, and force at catastrophic fracture was recorded. Because of the irregular and nonuniform nature of the femoral neck test, no attempt was made to normalize femoral neck data to the size and form of the neck.19

Bone structure

The distal half of the femur was scanned using micro–computed tomography (µCT) in order to determine additional mineralization parameters and structural indices of the trabecular bone. The distal femur was scanned using a Skyscan 1172 (Skyscan, Aartselaar, Belgium) and acquisition software Version 1.5. Projection radiographs were reconstructed using NRecon software (Skyscan). Reconstructed voxels of 15.89 µm3 were obtained using the following scanner parameters: 1-mm Al filter to reduce beam hardening from the cortical shell, 74-kV source voltage, 100-mA source current, and five-frame averaging. Two regions of interest within reconstructed image sets were defined to isolate trabecular bone. These regions consisted of the first fifteen 15.89-µm slices (0.24 mm total) distal to the distal-most edge of the epiphyseal plate and the first fifty 15.89-µm slices (0.79 mm total) proximal to the proximal-most point of the epiphyseal plate. The innermost border of the cortical shell was hand traced by the same investigator for all image sets, and all data outside this border were excluded such that only the trabecular bone was analyzed. Data in the reconstructed images were binarized such that bone appeared as white and all background and soft tissue as black with no intermediate gray-scale values. The volume of bone was measured and expressed as a percentage of the total space (soft tissue plus bone) as an additional measure of bone mineralization, and the structural model index (SMI) and model-derived trabecular thickness, number, and spacing20, 21 were quantified using Skyscan CT-analyzer software. The SMI reflects whether trabeculae are more rodlike or platelike and has been correlated with bone strength independently.21, 22

Statistical analysis

Initial statistical models covaried for experimental diet assignment and the interaction of diet with tumor status. Where no interaction or diet effects were observed, these terms were dropped from the model, allowing a direct comparison with independent-samples t tests. A multivariate ANOVA also was performed to test whether tumor status influenced all bone outcomes collectively, accounting for intercorrelations among outcomes. This analysis tests the more global hypothesis that tumor status affects “bone health” rather than any one particular bone health index.23

In order to test whether increasing total tumor burden was predictive of poorer bone health, the effect of total tumor weight for each animal on bone outcomes was assessed using ordinary-least-squares regression. All statistics were performed using SPSS Version 18 (SPSS, Inc., Chicago, IL, USA). For all hypothesis tests, α = 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

By week 10, 22 of 48 rats had palpable tumors. After the rats were euthanized, tumors were not seen in rats without previously palpable tumors. Mean tumor-free body weight was 238.9 ± 14.8 g, with no difference between tumored and nontumored animals (p > .2). In all statistical models, the interaction of assigned diet with tumor status (defined first by whether or not any tumor was present in the animal and in subsequent models by total tumor weight in each animal) did not affect outcome variables (all p > .2); therefore, the diet × tumor interaction term was dropped from final statistical models.

Multiple tumors were observed in 10 animals, two tumors were found in 8 animals, and three in 2 animals, making 34 tumors in all. Tumors were overwhelmingly diagnosed as low-grade adenocarcinomas by histology. Two tumors were classified as benign, five as high-grade adenocarcinoma, and the remainder as low-grade adenocarcinoma. No signs of metastasis were observed in any animal, including in µCT imaging of bone regions of interest. Because of the low numbers of benign and high-grade tumors, we were unable to determine the differential influences of these tumors statistically. All tumors therefore are treated as equivalent in the analysis.

Table 1 summarizes outcomes for all measured outcomes. We observed differences in bone health in both group comparisons of tumored versus nontumored animals and decreasing bone health proportional to the tumor burden, defined by total tumor weight (Table 2). These relationships are illustrated for selected bone outcomes in Figs. 2 through 5.

Table 1. Bone Health Parameters in Animals With and Without Breast Tumors 10 Weeks Following Intraperitoneal Injection With MNU (mean ± SD)a
 NontumoredTumored% Differencep Valueb
  • a

    No evidence of metastasis was observed nor expected in the N-methyl-N-nitrosourea model.

  • b

    p Values derived from independent-samples t tests.

Areal bone mineral density (BMD)
 Total BMD, mg/cm2210.1 ± 7.7202.8 ± 8.9−3%.004
 Femoral neck BMD219.5 ± 13.6214.1 ± 13.6−2%.177
 Midshaft BMD179.4 ± 5.5177.8 ± 6.8−1%.377
 Metaphysis BMD179.9 ± 9.1171.8 ± 9.6−5%.005
 Epiphysis BMD242.3 ± 9.3231.5 ± 11.8−4%<.001
Femoral midshaft geometry
 Mediolateral thickness, mm3.67 ± 0.153.57 ± 0.13−3%.011
 Anteroposterior thickness, mm2.96 ± 0.12.91 ± 0.08−2%.061
 Average cortical thickness, mm0.63 ± 0.0300.61 ± 0.02−3%.012
 CSMI4.1 ± 0.53.76 ± 0.39−8%.010
Femoral midshaft biomechanical properties, bending test
 Ultimate force, N115 ± 7110 ± 7−4%.024
 Stiffness, N/mm280 ± 25275 ± 26−2%.444
 Work to failure, mJ46.8 ± 8.844.0 ± 8.5−6%.266
 Stress at failure, MPa156 ± 9160 ± 73%.057
 Young's modulus, GPa4.84 ± 0.575.16 ± 0.596%.062
 Modulus of toughness, MPa5.00 ± 0.814.94 ± 0.81−1%.797
Femoral neck biomechanical properties
 Force at failure, N90 ± 1379 ± 17−12%.019
 Stiffness, N/mm224 ± 58197 ± 57−12%.116
 Work to failure, mJ43 ± 1834 ± 11−21%.066
Epiphyseal microarchitecture
 Bone volume, %36.0 ± 2.831.5 ± 2.4−13%<.001
 Structural model index1.24 ± 0.111.41 ± 0.1114%<.001
 Trabecular thickness7.60 ± 0.377.08 ± 0.34−7%<.001
 Trabecular number0.047 ± 0.0020.044 ± 0.003−6%<.001
 Trabecular spacing14.5 ± 0.414.7 ± 0.61%.179
Metaphyseal microarchitecture
 Bone volume, %26.6 ± 5.619.4 ± 4.2−27%<.001
 Metaphyseal structural model index1.43 ± 0.241.75 ± 0.1922%<.001
 Metaphyseal trabecular thickness6.7 ± 0.46.4 ± 0.4−4%.004
 Metaphyseal trabecular number0.039 ± 0.0070.030 ± 0.006−23%<.001
 Metaphyseal trabecular spacing18.0 ± 3.622.6 ± 4.026%<.001
Table 2. Correlation of Bone Health Measures With Tumor Burden, Represented by Total Tumor Weight, in All Animals (n = 48) and in Animals With One or More Tumors (n = 22)
 All animalsTumored only
  • a

    p < .01.

  • b

    p < .05.

Areal bone mineral density (BMD)
 Total BMD, mg/cm2−0.51a−0.55a
 Femoral neck BMD−0.43a−0.59a
 Midshaft BMD−0.06−0.07
 Metaphysis BMD−0.46a−0.48b
 Epiphysis BMD−0.49a−0.49b
Femoral midshaft geometry
 Mediolateral thickness, mm−0.42a−0.48b
 Anteroposterior thickness, mm−0.23−0.19
 Average cortical thickness, mm−0.30b−0.24
 CSMI−0.34b−0.34
Femoral midshaft fragility
 Ultimate force, N−0.42a−0.47b
 Stiffness, N/mm−0.29b−0.38
 Work to failure, mJ−0.35b−0.47b
 Stress at failure, MPa0.130.01
 Young's modulus, GPa0.11−0.05
 Modulus of toughness, MPa−0.27−0.44b
 Force at failure, N−0.17−0.01
 Stiffness, N/mm−0.26−0.27
 Work to failure, mJ−.120.05
Epiphyseal microarchitecture
 Bone volume, %−0.58a−0.63a
 Structural model index0.52a0.45b
 Trabecular thickness−0.45a−0.36
 Trabecular number−0.53a−0.50b
 Trabecular spacing0.36b0.37
Metaphyseal microarchitecture
 Bone volume, %−0.56a−0.66a
 Structural model index0.47a0.45b
 Trabecular thickness−0.59a−0.72a
 Trabecular number−0.52a−0.53b
 Trabecular spacing0.45a0.36
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Figure 2. Estimated cross-sectional moment of inertia of the femoral midshaft in 48 rats with and without breast tumor in response to MNU injection. aIndependent-samples t test, tumored versus nontumored animals. bOrdinary-least-squares regression, response to tumor burden of the animal, represented by total tumor weight. Dotted lines depict the 95% confidence interval of the regression line.

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thumbnail image

Figure 3. Force at failure of the femoral midshaft in 48 rats with and without breast tumor in response to MNU injection. aIndependent-samples t test, tumored versus nontumored animals. bOrdinary-least-squares regression, response to tumor burden of the animal, represented by total tumor weight. Dotted lines depict the 95% confidence interval of the regression line.

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thumbnail image

Figure 4. Trabecular volume fraction of the epiphysis in 48 rats with and without breast tumor in response to MNU injection. aIndependent-samples t test, tumored versus nontumored animals. bOrdinary-least-squares regression, response to tumor burden of the animal, represented by total tumor weight. Dotted lines depict the 95% confidence interval of the regression line.

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thumbnail image

Figure 5. Trabecular number in the metaphysis of 48 rats with and without breast tumor in response to MNU injection. aIndependent-samples t test, tumored versus nontumored animals. bOrdinary-least-squares regression, response to tumor burden of the animal, represented by total tumor weight. Dotted lines depict the 95% confidence interval of the regression line.

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Finally, a multivariate analysis of all outcomes collectively was performed to account for intercorrelations in outcome variables by testing the effect of tumor status on BMD of all measured regions, all final parameters from three-point bending and femoral neck bending tests, and µCT structural parameters. This test also indicated a negative effect of tumor status on global bone health (p = .027).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

We observed a consistent, significant negative association of tumor status with several alternative measures of bone mineralization, structure, and strength at multiple sites of the femur. These associations persist in multivariate analysis, suggesting a global poor quality of bone in tumored versus nontumored animals. Because tumor incidence cannot be assigned randomly using this model, we cannot definitively conclude that tumors caused reductions in bone health. An alternative explanation would be that some unidentified factor present prior to injection causes both poor bone quality and increased susceptibility to tumor initiation in response to MNU. However, the strongly inverse dose-response relationship between total tumor burden (tumor weight) and bone health strongly implicates a direct negative effect of nonmetastatic, MNU-induced breast tumors on bone health in rats (Figs. 2 through 5).

In contrast, we observed no adverse effect but a nonsignificant trend for increased stress at failure and Young's modulus in the presence of tumors. These parameters are measurements of the intrinsic fragility of bone or fragility after adjustment for bone geometry. It is probable that this observation is related to a relatively larger adverse effect on the cross-sectional moment of inertia (−8%) compared with breaking force (−4%) and stiffness (−2%). Accordingly, we suspect that tumor status decreased the denominators of Eqs. (2) and (3) more so than the numerators, resulting in a (non-significant) elevation.

It is well known that tumor metastasis to bone degrades bone in humans and in multiple animal models.1, 6–8, 24 Within the positive-feedback loop of metastatic bone disease, initiation of osteoclast activity by interleukin 8 (IL-8),1, 25 parathyroid hormone–related peptide (PTHrP),4, 26, 27 and receptor activator of NF-κB ligand (RANKL)1, 28 is implicated. In turn, osteoclast activity releases growth factors in bone that promote tumor proliferation and growth, including transforming growth factor β,29, 30 bone morphogenic proteins,31, 32 and others.1, 4 Our data imply that some cross-talk between tumor and bone tissues may exist even in the absence of metastasis. This study was not designed to assess tumor signaling molecules in circulation, and mechanistic explanations can only be speculative. It is feasible, however, that IL-8, PTHrP, and other tumor cytokines may be increased in the circulation and affect bone metabolism remotely.33 It is further possible that such a mechanism might increase the susceptibility of bone tissue to invasion by tumor cells, thereby priming bone tissue for metastasis.

As might be expected, insults to bone health in these rats were not as prominent as those observed in metastatic osteolytic bone lesions. Kurth and colleagues8 observed a 9% decline in BMD measured by DXA and a 35% or greater decline in mechanical strength of rat femurs 28 days after implantation with Carcinosarcoma 256 malignant breast cancer cells compared with sham implant controls. In a comparable article,34 Kurth and Müller observed a 30% reduction in percent bone volume of the trabecular compartment using µCT, as well as a 24% reduction in trabecular number, a 10% decrease in trabecular thickness, and a doubling of trabecular spacing. Arrington and colleagues6 assessed bone at 3, 6, and 9 months following injection of breast cancer cells into femurs of nude mice. They observed progressive deterioration of BMD, strength, and structure of bone from 3 to 9 weeks after injection, with an approximate 21% difference in BMD, 86% to 94% difference in torsion strength, and 89% to 126% difference in µCT structural parameters of the metaphysis at final testing between pairs of tumored and nontumored femurs from the same animals. In contrast, 10 weeks after injection of MNU, we observed a 1% to 5% detriment to BMD, a 14% to 21% detriment to strength, and a 6% to 14% detriment to structure at the epiphysis and a 22% to 27% detriment to structure at the metaphysis.

MNU induction is a well-accepted rodent breast cancer model,12, 14, 35 but it is not without limitations.11, 12 Thompson and Singh9 describe a course of MNU-induced tumors that is analogous to human breast cancer, although markedly more rapid. They also note, however, that the cellular processes underlying chemically induced cancer models are not well understood at this time. It is possible that our observations represent an idiosyncratic biologic effect of MNU-induced tumors. This possibility can be ruled out only with investigation into alternate cancer models. A second limitation of this experiment is the absence of a non-MNU-exposed control group. Our use of MNU-exposed controls allows us to infer that differences between groups are attributable to tumors, not protocol; however, it is theoretically possible that MNU exposure potentiated (or mitigated) the effect of tumors. We are aware of no influence of MNU per se on bone tissue in the available literature, and our mechanical strength parameters appear to be within the ranges reported for various rat strains reported by Turner and colleagues.19

In summary, we observed moderate and statistically significant degradation of bone mineralization, strength, and structure in the femurs of rats developing breast tumors in response to MNU injection relative to injected rats without tumors. These changes occurred in the absence of metastasis to bone tissue over a 10-week period from injection. Bone health was reduced in proportion to the total tumor burden, quantified as combined weight of all tumors in each animal. Further study is required to identify what, if any, circulating tumor signals might explain these data. If replicated and generalizable, it is possible that these mechanisms may have some influence over the poorer bone health observed in nonmetastatic clinical breast cancer36, 37; however, this observation is presently attributed to documented side effects of current breast tumor pharmaceutical therapies.36, 38

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Funding for this study was provided by the Illinois Council on Food and Agricultural Research.

MPT performed DXA, bending, and µCT tests of femurs and statistical analysis and interpretation and created the original draft of this article. RJV and CJM contributed to the design and execution of the experiment, oversaw the handling and use of all animals, and made substantive revisions to this article. AJWJ contributed to the design of the experiment and oversaw bending µCT testing and contributed substantive revisions to this article. EME and DKL contributed to the design and execution of the experiment and made substantive revisions to this article.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Additional Supporting information may be found in the online version of this article.

FilenameFormatSizeDescription
jbmr_277_sm_suppMat.doc76KSupplementary Material

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