European Radiology

, Volume 22, Issue 3, pp 551–558 | Cite as

In vivo imaging of human breast cancer mouse model with high level expression of calcium sensing receptor at 3T

  • Gabriella BaioEmail author
  • Marina Fabbi
  • Laura Emionite
  • Michele Cilli
  • Sandra Salvi
  • Piero Ghedin
  • Sabina Prato
  • Grazia Carbotti
  • Alberto Tagliafico
  • Mauro Truini
  • Carlo Emanuele Neumaier
Molecular Imaging



To demonstrate that manganese can visualise calcium sensing receptor (CaSR)-expressing cells in a human breast cancer murine model, as assessed by clinical 3T magnetic resonance (MR).


Human MDA-MB-231-Luc or MCF7-Luc breast cancer cells were orthotopically grown in NOD/SCID mice to a minimum mass of 5 mm. Mice were evaluated on T1-weighted sequences before and after intravenous injection of MnCl2. To block the CaSR-activated Ca2+ channels, verapamil was injected at the tumour site 5 min before Mn2+ administration. CaSR expression in vivo was studied by immunohistochemistry.


Contrast enhancement was observed at the tumour periphery 10 min after Mn2+ administration, and further increased up to 40 min. In verapamil-treated mice, no contrast enhancement was observed. CaSR was strongly expressed at the tumour periphery.


Manganese enhanced magnetic resonance imaging can visualise CaSR-expressing breast cancer cells in vivo, opening up possibilities for a new MR contrast agent.

Key Points

• Manganese contrast agents helped demonstrate breast cancer cells in an animal model.

• Enhancement was most marked in cells with high calcium sensing receptor expression.

• Manganese uptake was related to the distribution of CaSR within the tumour.

• Manganese MRI may become useful to investigate human breast cancer.


Magnetic Resonance Imaging Manganese chloride Breast cancer Calcium receptor In vivo small animal MRI 



The authors thank Dr. Silvano Ferrini for his helpful discussions and revision of the manuscript.

This study was supported by the Italian Ministry of Health ‘Progetto oncologico di Medicina Molecolare: i tumori femminili’.


  1. 1.
    Barrett PQ, Kojima I, Kojima K et al (1986) Short term memory in the calcium messenger system. Evidence for a sustained activation of protein kinase C in adrenal glomerulosa cells. Biochem J 238:905–912PubMedGoogle Scholar
  2. 2.
    Rasmussen H (1986) The calcium messenger system (1). N Engl J Med 314:1094–1101PubMedCrossRefGoogle Scholar
  3. 3.
    Capiod T, Shuba Y, Skryma R et al (2007) Calcium signalling and cancer cell growth. Subcell Biochem 45:405–427PubMedCrossRefGoogle Scholar
  4. 4.
    Brown EM, MacLeod RJ (2001) Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 81:239–297PubMedGoogle Scholar
  5. 5.
    Bikle DD, Oda Y, Xie Z (2004) Calcium and 1,25(OH)2D: interacting drivers of epidermal differentiation. J Steroid Biochem Mol Biol 89–90:355–360PubMedCrossRefGoogle Scholar
  6. 6.
    Heaney RP (2006) Calcium intake and disease prevention. Arq Bras Endocrinol Metabol 50:685–693PubMedCrossRefGoogle Scholar
  7. 7.
    Brown EM (2000) G protein-coupled, extracellular Ca2+ (Ca2+(o))-sensing receptor enables Ca2+(o) to function as a versatile extracellular first messenger. Cell Biochem Biophys 33:63–95PubMedCrossRefGoogle Scholar
  8. 8.
    Garrett JE, Capuano IV, Hammerland LG et al (1995) Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. J Biol Chem 270:12919–12925PubMedCrossRefGoogle Scholar
  9. 9.
    Fukumoto S (2008) Basic and clinical aspects of calcimimetics. Structure and function of calcium-sensing receptor. Clin Calcium 18:32–36PubMedGoogle Scholar
  10. 10.
    Cheng I, Klingensmith ME, Chattopadhyay N et al (1998) Identification and localization of the extracellular calcium-sensing receptor in human breast. J Clin Endocrinol Metab 83:703–707PubMedCrossRefGoogle Scholar
  11. 11.
    Prentince A, Jarjou LMA, Cole TJ et al (1995) Calcium requirements of lactating Gambian mothers, effects of a calcium supplement on breast-milk calcium concentration, maternal bone mineral content and urinary calcium excretion. Am J Clin Nutr 62:58–67Google Scholar
  12. 12.
    Galkin BM, Feig SA, Patchefsky AS et al (1977) Ultrastructure and microanalysis of “benign” and “malignant” breast calcifications. Radiology 124:245–249PubMedGoogle Scholar
  13. 13.
    McGrath CM, Soule HD (1984) Calcium regulation of normal human mammary epithelial cell growth in culture. In Vitro 20:652–662PubMedCrossRefGoogle Scholar
  14. 14.
    Percival RC, Yates AJ, Gray RE et al (1985) Mechanism of malignant hypercalcemia in carcinoma of the breast. Br Med J Clin Res 291:776–779CrossRefGoogle Scholar
  15. 15.
    House MG, Kohlmeier L, Chattopadhyay N et al (1997) Expression of an extracellular calcium-sensing receptor in human and mouse bone marrow cells. J Bone Miner Res 12:1959–1970PubMedCrossRefGoogle Scholar
  16. 16.
    Silver IA, Murrils RJ, Etherington DJ (1988) Microlectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp Cell Res 175:266–276PubMedCrossRefGoogle Scholar
  17. 17.
    Sanders JL, Chattopadhyay N, Kifor O et al (2000) Extracellular calcium-sensing receptor expression and its potential role in regulating parathyroid hormone-related peptide secretion in human breast cancer cell lines. Endocrinology 141:4357–4364PubMedCrossRefGoogle Scholar
  18. 18.
    Coleman RE (1997) Skeletal complications of malignancy. Cancer 80:1588–1594PubMedCrossRefGoogle Scholar
  19. 19.
    Guise TA, Mundy GR (1998) Cancer and bone. Endocr Rev 19:18–54PubMedCrossRefGoogle Scholar
  20. 20.
    Guise TA, Yin JJ, Taylor SD et al (1996) Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest 98:1544–1549PubMedCrossRefGoogle Scholar
  21. 21.
    Nemeth EF, Fox J, Delmar EG et al (1998) Stimulation of parathyroid hormone secretion by a small molecule antagonist of the calcium receptor (Abstract). J Bone Miner Res 23:S156Google Scholar
  22. 22.
    Mihai R, Stevens J, McKinney C et al (2006) Expression of the calcium receptor in human breast cancer—a potential new marker predicting the risk of bone metastases. Eur J Surg Oncol 32:511–515PubMedCrossRefGoogle Scholar
  23. 23.
    Drapeau P, Nachsen DA (1984) Manganese fluxes and manganese-dependent neurotransmitter release in presynaptic nerve endings isolated from rat brain. J Physiol 348:493–510PubMedGoogle Scholar
  24. 24.
    Narita K, Kawaski F, Kita H (1990) Mn and Mg influxes through Ca channels of motor nerve terminals are prevented by verapamil in frogs. Brain Res 510:289–295PubMedCrossRefGoogle Scholar
  25. 25.
    Lin YJ, Koretsky AP (1997) Manganese ion enhances T1-weighted MRI during brain activation: an approach to direct imaging of brain function. Magn Reson Med 38:378–388PubMedCrossRefGoogle Scholar
  26. 26.
    Duong TQ, Silva AC, Lee SP et al (2000) Functional MRI of calcium-dependent synaptic activity: cross correlation with CBF and BOLD measurements. Magn Reson 43:383–392CrossRefGoogle Scholar
  27. 27.
    Hu TC, Pautler RG, MacGowan GA et al (2001) Manganese enhanced MRI of mouse heart during changes in inotropy. Magn Reson Med 46:884–890PubMedCrossRefGoogle Scholar
  28. 28.
    Krombach GA, Saeed M, Higgins CB et al (2004) Contrast-enhanced MR delineation of stunned myocardium with administration of MnCl(2) in rats. Radiology 230:183–190PubMedCrossRefGoogle Scholar
  29. 29.
    Pautler RG, Silva AC, Koretsky AP (1998) In vivo neuronal tract tracing using manganese-enhanced magnetic resonance imaging. Magn Reson Med 40:740–748PubMedCrossRefGoogle Scholar
  30. 30.
    Watanabe T, Michaelis T, Frahm J (2001) Mapping of retinal projections in the living rat using high-resolution 3D gradient-echo MRI with Mn2+-induced contrast. Magn Reson Med 46:424–429PubMedCrossRefGoogle Scholar
  31. 31.
    Lin CP, Tseng WY, Cheng HC et al (2001) Validation of diffusion tensor magnetic resonance axonal fiber imaging with registered manganese-enhanced optic tracts. NeuroImage 14:1035–1047PubMedCrossRefGoogle Scholar
  32. 32.
    Allegrini PR, Wiessner C (2003) Three-dimensional MRI of cerebral projections in rat brain in vivo after intracortical injection of MnCl2. NMR Biomed 16:252–256PubMedCrossRefGoogle Scholar
  33. 33.
    Lin Y (1997) MRI of the rat and mouse brain after systemic administration of MnCl2. Thesis, Carnegie Mellon University, Pittsburgh, PAGoogle Scholar
  34. 34.
    Watanabe T, Natt O, Boretius S et al (2002) In vivo 3D MRI staining of mouse brain after subcutaneous application of MnCl2. Magn Resonan Med 48:852–859CrossRefGoogle Scholar
  35. 35.
    Lee JH, Koretsky AP (2004) Manganese enhanced magnetic resonance imaging. Curr Pharm Biotechnol 5:529–537PubMedCrossRefGoogle Scholar
  36. 36.
    Ogan MD, Revel D, Brasch RC (1987) Metalloporphyrin contrast enhancement of tumours in magnetic resonance imaging. A study of human carcinoma, lymphoma, and fibrosarcoma in mice. Invest Radiol 22:822–828PubMedCrossRefGoogle Scholar
  37. 37.
    van Zijl PC, Place DA, Cohen JS et al (1990) Metalloporphyrin magnetic resonance contrast agents. Feasibility of tumour-specific magnetic resonance imaging. Acta Radiol 374:75–79Google Scholar
  38. 38.
    Remy C, Kirchhoff P, Hafner P et al (2007) Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands. Cell Physiol Biochem 19:33–42PubMedCrossRefGoogle Scholar
  39. 39.
    Günzel D, Amasheh S, Pfaffenbach S et al (2009) Claudin-16 affects transcellular Cl-secretion in MDCK cells. J Physiol 587:3777–3793PubMedCrossRefGoogle Scholar
  40. 40.
    Ye C, Rogers K, Bai M et al (1996) Agonists of the Ca(2+)-sensing receptor (CaR) activate nonselective cation channels in HEK293 cells stably transfected with the human CaR. Biochem Biophys Res Commun 226:572–579PubMedCrossRefGoogle Scholar
  41. 41.
    Tsien RW, Hess P, McCleskey EW et al (1987) Calcium channels: mechanisms of selectivity, permeation, and block. Annu Rev Biophys Biophys Chem 16:265–290PubMedCrossRefGoogle Scholar
  42. 42.
    Sanders JL, Chattopadhyay N, Kifor O et al (2000) Extracellular calcium-sensing receptor expression and its potential role in regulating parathyroid hormone-related peptide secretion in human breast cancer cell lines. Endocrinology 141:4357–4363PubMedCrossRefGoogle Scholar
  43. 43.
    VanHouten J, Dann P, McGeoch G et al (2004) The calcium-sensing receptor regulates mammary gland parathyroid hormone-related protein production and calcium transport. J Clin Invest 113:598–608PubMedGoogle Scholar
  44. 44.
    Wolff SD, Balaban RS (1997) Assessing contrast on MR images. Radiology 202:25–29PubMedGoogle Scholar
  45. 45.
    El Hiani Y, Ahidouch Lehen’kyi V, Hague F et al (2009) Extracellular signal-regulated kinases 1 and 2 and TRPC1 channels are required for calcium-sensing receptor-stimulated MCF-7 breast cancer cell proliferation. Cell Physiol Biochem 23:335–346PubMedCrossRefGoogle Scholar
  46. 46.
    Aydar E, Yeo S, Djamgoz M, Palmer C (2009) Abnormal expression, localization and interaction of canonical transient receptor potential ion channels in human breast cancer cell lines and tissues: a potential target for breast cancer diagnosis and therapy. Cancer Cell Int 18:9–23Google Scholar
  47. 47.
    Grünecker B, Kaltwasser SF, Peterse Y, Sämann PG et al (2010) Fractionated manganese injections: effects on MRI contrast enhancement and physiological measures in C57BL/6 mice. NMR Biomed 23:913–921PubMedCrossRefGoogle Scholar
  48. 48.
    Huang C, Hydo LM, Liu S, Miller RT (2009) Activation of choline kinase by extracellular Ca2+ is Ca(2+)-sensing receptor, Galpha12 and Rho-dependent in breast cancer cells. Cell Signal 21:1894–1900PubMedCrossRefGoogle Scholar

Copyright information

© European Society of Radiology 2011

Authors and Affiliations

  • Gabriella Baio
    • 1
    Email author
  • Marina Fabbi
    • 2
  • Laura Emionite
    • 3
  • Michele Cilli
    • 3
  • Sandra Salvi
    • 4
  • Piero Ghedin
    • 5
  • Sabina Prato
    • 5
  • Grazia Carbotti
    • 2
  • Alberto Tagliafico
    • 1
  • Mauro Truini
    • 4
  • Carlo Emanuele Neumaier
    • 1
  1. 1.Department of Diagnostic Imaging, ISTNational Cancer InstituteGenoaItaly
  2. 2.Unit of Immunological Therapy, ISTNational Cancer InstituteGenoaItaly
  3. 3.Animal Facility, ISTNational Cancer InstituteGenoaItaly
  4. 4.Department of Pathology, ISTNational Cancer InstituteGenoaItaly
  5. 5.General Electric, GEMilanoItaly

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