Breast Cancer Research and Treatment

, Volume 122, Issue 1, pp 87–94

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

  • Fangbing Liu
  • Preeti Misra
  • Elaine P. Lunsford
  • Joanne T. Vannah
  • Yuxia Liu
  • Robert E. Lenkinski
  • John V. Frangioni
Preclinical study

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.

Keywords

Breast cancer Microcalcifications BMP-2 Animal models Micro-SPECT/CT Micro-CT 

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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Copyright information

© Springer Science+Business Media, LLC. 2009

Authors and Affiliations

  • Fangbing Liu
    • 1
  • Preeti Misra
    • 1
  • Elaine P. Lunsford
    • 2
  • Joanne T. Vannah
    • 2
  • Yuxia Liu
    • 2
  • Robert E. Lenkinski
    • 3
  • John V. Frangioni
    • 1
    • 3
  1. 1.Division of Hematology/Oncology, Department of MedicineBeth Israel Deaconess Medical CenterBostonUSA
  2. 2.Longwood Small Animal Imaging FacilityBeth Israel Deaconess Medical CenterBostonUSA
  3. 3.Department of RadiologyBeth Israel Deaconess Medical Center (BIDMC)BostonUSA

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