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A dose- and time-controllable syngeneic animal model of breast cancer microcalcification

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Abstract

The development of novel diagnostic agents for the detection of breast cancer microcalcifications requires a reliable animal model. Based on previous work from our group, we hypothesized that a single systemic injection of recombinant bone morphogenetic protein-2 (rBMP-2) could be used to create such a model. The cDNA encoding mature human BMP-2 was expressed in BL21(DE3) bacteria, purified to homogeneity, and refolded as a dimer. Bioactivity was confirmed using a C2C12 alkaline phosphatase assay. rBMP-2 was radiolabeled with 99mTc, and its biodistribution and clearance were quantified after both intravenous (IV) and intraperitoneal (IP) injection. Fischer 344 rats bearing syngeneic R3230 breast tumors received a single intraperitoneal injection of rBMP-2 at a specified dose. Tumor microcalcification was quantified over time using micro–single photon emission computed tomography (SPECT) and microcomputed tomography (CT). rBMP-2 could be expressed in E. coli at high levels, isolated at >95% purity, and refolded to a bioactive dimer. Beta-phase half-life was 30.5 min after IV administration and 47.6 min after IP administration. Renal excretion was the primary mode of clearance. A single IP injection of ≥50 μg rBMP-2 when tumors were not yet palpable resulted in dose-dependent microcalcification in 8 of 8 R3230 tumors. No calcification was found in control tumors or in normal tissues and organs of animals injected with rBMP-2. Tumor calcification increased progressively between weeks 2 and 4 post-rBMP-2 injection. A single IP injection of rBMP-2 in rats bearing a syngeneic breast cancer will produce dose-dependent and time-dependent microcalcifications. This animal model lays the foundation for the development of novel diagnostic radiotracers for breast cancer.

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Abbreviations

%ID:

Percent injected dose

%ID/g:

Percent injected dose per gram of tissue

aa:

Amino acids

ALP:

Alkaline phosphatase

BMP-2:

Bone morphogenetic protein-2

CT:

Computed tomography

CV:

Column volumes

DCE:

Dynamic contrast-enhanced

DMEM:

Dulbecco’s modified Eagle’s medium

DMSO:

Dimethyl sulfoxide

FBS:

Fetal bovine serum

Gnd-HCl:

Guanidinium hydrochloride

HA:

Hydroxyapatite

H&E:

Hematoxylin and eosin

IP:

Intraperitoneal

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

IV:

Intravenous

MDP:

Methylene diphosphonate

MIP:

Maximal intensity projection

MRI:

Magnetic resonance imaging

NHS:

N-hydroxysuccinimide

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PEM:

Positron emission mammography

PET:

Positron emission tomography

PPV:

Positive predictive value

rBMP-2:

Recombinant bone morphogenetic protein-2

SPECT:

Single-photon emission computed tomography

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Acknowledgments

We thank Barbara L. Clough and Lorissa A. Moffitt for editing. This work was funded by NIH grant R01-CA-115296, NCRR shared instrumentation grant S10-RR-023010, and grants from the Lewis Family Fund and Ellison Foundation.

Competing interests:

Fangbing Liu: None.

Preeti Misra: None.

Elaine Lunsford: None

Joanne T. Vannah: None

Yuxia Liu: None

Robert E. Lenkinski: None

John V. Frangioni: None

Author Contributions

Fangbing Liu: Preparation of all reagents. Conducted all experiments. Wrote first draft of manuscript. Edited final draft of manuscript.

Preeti Misra: 99mTc-radiolabeling and purification of BMP-2. In vivo biodistribution and clearance experiments. Reviewed final draft of manuscript.

Elaine Lunsford: Micro-CT and micro-SPECT experiments and quantitative analysis of data. Reviewed final draft of manuscript.

Joanne T. Vannah: Micro-CT and micro-SPECT experiments and quantitative analysis of data. Reviewed final draft of manuscript.

Yuxia Liu: Micro-CT and micro-SPECT experiments and quantitative analysis of data. Reviewed final draft of manuscript.

Robert E. Lenkinski: Design of study. Review and interpretation of all primary data. Edited final draft of manuscript.

John V. Frangioni: Design of study. Review and interpretation of all primary data. Wrote final draft of manuscript.

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Authors and Affiliations

  1. Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, 02215, USA

    Fangbing Liu, Preeti Misra & John V. Frangioni

  2. Longwood Small Animal Imaging Facility, Beth Israel Deaconess Medical Center, Boston, MA, 02215, USA

    Elaine P. Lunsford, Joanne T. Vannah & Yuxia Liu

  3. Department of Radiology, Beth Israel Deaconess Medical Center (BIDMC), Room SL-B05, 330 Brookline Avenue, Boston, MA, 02215, USA

    Robert E. Lenkinski & John V. Frangioni

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  1. Fangbing Liu
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Correspondence to John V. Frangioni.

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Liu, F., Misra, P., Lunsford, E.P. et al. A dose- and time-controllable syngeneic animal model of breast cancer microcalcification. Breast Cancer Res Treat 122, 87–94 (2010). https://doi.org/10.1007/s10549-009-0535-6

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  • DOI: https://doi.org/10.1007/s10549-009-0535-6

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