Breast Cancer Research and Treatment

, Volume 99, Issue 2, pp 163–176

LHRH-conjugated Magnetic Iron Oxide Nanoparticles for Detection of Breast Cancer Metastases

  • Carola Leuschner
  • Challa SSR Kumar
  • William Hansel
  • Wole Soboyejo
  • Jikou Zhou
  • Josef Hormes
Preclinical study
  • 654 Downloads

Summary

Targeted delivery of superparamagnetic iron oxide nanoparticles (SPIONs) could facilitate their accumulation in metastatic cancer cells in peripheral tissues, lymph nodes and bones and enhance the sensitivity of magnetic resonance imaging (MRI). The specificities of luteinizing hormone releasing hormone (LHRH) and luteinizing hormone/chorionic gonadotropin (LH/CG)- bound SPIONs were tested in human breast cancer cells in vitro and were found to be dependent on the receptor expression of the target cells, the time of incubation and showed saturation kinetics. In incubations with MDA-MB-435S.luc cells, the highest iron accumulation was 452.6 pg Fe/cell with LHRH-SPIONs, 203.6 pg Fe/cell with β-CG-SPIONs and 51.3 pg Fe/cell with SPIONs. Incubations at 4 °C resulted in 1.1 pg Fe/cell. Co-incubation with the same ligands (βCG or LHRH) decreased the iron accumulation in each case. LHRH-SPIONs were poorly incorporated by macrophages. Tumors and metastatic cells from breast cancer xenografts were targeted in vivo in a nude mouse model. LHRH-SPION specifically accumulated in cells of human breast cancer xenografts. The amount of LHRH-SPION in the lungs was directly dependent on the number of metastatic cells and amounted to 77.8 pg Fe/metastastic cell. In contrast, unconjugated SPIONs accumulated in the liver, showed poor affinity to the tumor, and were not detectable in metastatic lesions in the lungs. LHRH-SPION accumulated in the cytosolic compartment of the target cells and formed clusters. LHRH-SPIONs did not accumulate in livers of normal mice. In conclusion, LHRH conjugated SPIONs may serve as a contrast agent for MR imaging in vivo and increase the sensitivity for the detection of metastases and disseminated cells in lymph nodes, bones and peripheral organs.

Key words

breast cancer chorionic gonadotropin receptors luteinizing hormone releasing hormone receptors metastases nanoparticles superparamagnetic iron oxide nanoparticles 

Abbreviations

LHRH

luteinizing hormone releasing hormone

CG

chorionic gonadotropin

βCG

fragment of the beta chain of CG from amino acid 81–95

MRI

magnetic resonance imaging

SPION

superparamagnetic iron oxide nanoparticles

CT

computed tomography

PET

positron emission tomography

CHO

Chinese Hamster Ovary Cells

PMA

4α Porbol 12 myristate 13 acetate

s.c.

subcutanously

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References

  1. 1.
    Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ Cancer Statistics, 2005CA Cancer J Clin55:10–30, 2005PubMedCrossRefGoogle Scholar
  2. 2.
    Braun S, Kentenich Ch, Janni W, Hepp F, Waal J de, Wilgeroth F, Sommer H, Pantel K: Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patientsJ Clin Oncol18: 80–86, 2000PubMedGoogle Scholar
  3. 3.
    Gerber B, Krause A, Muller H, Richter D, Reimer T, Makovitzky J, Herrnring C, Jeschke U, Kundt G, Friese K Simultaneous immunohistochemical detection of tumor cells in lymph nodes, bone marrow aspirates in breast cancer and its correlation with other prognostic factorsJ Clin Oncol19: 960–971, 2001PubMedGoogle Scholar
  4. 4.
    Braun S, Cevatli BS, Assemi C, Janni W, Kentenich CR, Schindlbeck C, Rjosk D, Hepp F: Comparative analysis of micrometastasis to the bone marrow, lymph nodes of node-negative breast cancer patients receiving no adjuvant therapyJ Clin Oncol19: 1468–1475, 2001PubMedGoogle Scholar
  5. 5.
    Woelfle U, Cloos J, Sauter G, Riethdorf L, Janicke F, van Diest P, Brakenhoff R, Pantel K: Molecular signature associated with bone marrow micrometastasis in human breast cancerCancer Res 63:5679–5684, 2003PubMedGoogle Scholar
  6. 6.
    Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CR, Gastroph S, Wischnik A, Dimpfl T, Kindermann G, Riethmuller G, Schlimok G: Cytokeratin-positive cells in the bone marrow, survival of patients with stage I, II, or III breast cancerN Engl J Med342:525–533, 2000PubMedCrossRefGoogle Scholar
  7. 7.
    Pantel K, Otte M: Occult micrometastasis: enrichment, identification, characterization of single disseminated tumor cellsSemin Cancer Biol11: 327–337, 2001PubMedCrossRefGoogle Scholar
  8. 8.
    Pantel K, Cote RJ, Fodstadt O: Detection, clinical importance of micrometastatic diseaseJ Natl Cancer Inst91:1113–1124 1999PubMedCrossRefGoogle Scholar
  9. 9.
    Pantel K, Mueller V, Auer M, Nusser N, Harbeck N, Braun S: Detection, clinical implications of early systemic tumor cell dissemination in breast cancerClin Cancer Res9:6326–6334, 2003PubMedGoogle Scholar
  10. 10.
    O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J: Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinomaCell79:315–328, 1994PubMedCrossRefGoogle Scholar
  11. 11.
    Kruger WH, Kroger N, Togel F, Renges H, Badbaran A, Hornung R, Jung R, Gutensohn K, Gieseking F, Janicke F, Zander AR: Disseminated breast cancer cells prior to, after high-dose therapyJ Hematother Stem Cell Res10:681–689, 2001PubMedCrossRefGoogle Scholar
  12. 12.
    Diel IJ, Krempien B, Kaufmann M, Costa SD, Goerner R, von Fournier D, Bastert G: Ergebnisse von Beckenkammbiopsien von 475 Patientinnen mit primaerem und metastasiertem MammakarzinomTumor Diagn Ther13: 85–90, 1992Google Scholar
  13. 13.
    Morikawa K, Walker SM, Nakajima M, Pathak S, Jessup JM, Fidler IJ: Influence of organ environment on the growth selection and metastasis of human colon carcinoma cells in nude miceCancer Res48: 6863–6871, 1988PubMedGoogle Scholar
  14. 14.
    Dearnaley DP, Sloane JP, Ormerod MG, Steele K, Coombes RC, Clink Hmc, Powles TJ, Ford HT, Neville AM: Increased detection of mammary carcinoma cells in marrow smears using antisera to epithelial membrane antigenBr J Cancer44: 85–90, 1981PubMedGoogle Scholar
  15. 15.
    Tschentscher P, Wagener C, Neumaier M: Sensitive, specific cytokeratin 18 reverse transcriptase-polymerase chain reaction that excludes amplification of processed pseudogenes from contaminating genomic DNAClin Chem43: 2244–2250, 1997PubMedGoogle Scholar
  16. 16.
    Robsen ME, (2004) Offit K. Breast MRI for women with hereditary cancer riskJAMA 292: 1368CrossRefGoogle Scholar
  17. 17.
    Warner E, Plewes DB, Hill KA, Causer PA, Zubovits JT, Jong RA, Cutrara MR, DeBoer G, Yaffe MJ, Messner SJ, Meschino WS, Piron CA, Narod SA: Related articles, links surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examinationJAMA 292(11): 1317–1325, 2004PubMedCrossRefGoogle Scholar
  18. 18.
    Morris EA, Schwartz LH, Dershaw DD: MR imaging of the breast in patients with occult primary breast carcinoma Radiology205: 437–440, 1997PubMedGoogle Scholar
  19. 19.
    Schorn C, Fischer U: MRI of the breast in patients with metastatic disease of unknown primaryEur Radiol9: 470–473, 1999PubMedCrossRefGoogle Scholar
  20. 20.
    Clement O, Siauwe N: Liver imaging with ferrumxodidesTop Magn Res Imaging9: 167–182, 1998Google Scholar
  21. 21.
    Wang YXJ, Hussain SM, Kresting GP: Superparamagnetic iron oxide contrast agents: physicochemical characteristics and applications in MR imagingEur Radiol11:2319–2331, 2001PubMedCrossRefGoogle Scholar
  22. 22.
    Weissleder R, Elizondo G, Wittenberg J, Rabito CA, Bengele HH, Josephson L: Ultrasmall superparamagnetic iron oxide: characterization of a new class of contrast agents for MR imagingRadiology 175:489–493, 1990PubMedGoogle Scholar
  23. 23.
    Seneterre E, Weissleder R, Jaramillo D, Reimer P, Lee AS, Brady TJ, Wittenberg J: Bone marrow: ultrasmall superparamagnetic iron oxide for MR imagingRadiology179: 529–533, 1991PubMedGoogle Scholar
  24. 24.
    Pouliquen D, Lucet I, Chouly C, Perdrisot R, Le Jeune JJ, Jallet P: Related articles, links liver-directed superparamagnetic iron oxide: quantitation of T2 relaxation effects Magn Reson Imaging11: 219–228, 1993PubMedCrossRefGoogle Scholar
  25. 25.
    Weissleder R, Stark DD, Engelstad BL, Bacon BR, Compton CC, White DL, Jacobas P. Lewis J: Superparamagnetic iron oxide: pharmacokinetics and toxicityAm J Roentgenol152:167–173, 1989Google Scholar
  26. 26.
    Bonnemain B, (1998) Superparamagnetic agents in magnetic resonance imaging: physicochemical characteristics and clinical applicationsJ Drug Target 6: 167–174PubMedCrossRefGoogle Scholar
  27. 27.
    Daldrup HE, Link TM, Blasius S, Strozyk A, Konemann S, Jurgens H, Rummeny EJ: Monitoring radiation induced changes in bone marrow histopathology with ultra small superparamagnetic iron oxide (USPIO) enhanced MRIJ Magn Reson Imaging 9: 643–652, 1999PubMedCrossRefGoogle Scholar
  28. 28.
    Van de Berg BC, Lecouvert FE, Kanku JP, Jamart J, Van Beers BE, Maldague B, Malghem J: Ferrumoxides enhanced quantitative magnetic resonance imaging of the normal and abnormal bone marrow. Preliminary assessmentJ Magn Reson Imaging9: 322–328, 1999CrossRefGoogle Scholar
  29. 29.
    Harisinghani MG, Barentsz J, Hahn PF, Deserno WM, Tabatabaei S, Hulsbergen C, Rosette J, Weissleder R: Noninvasive detection of clinically occult lymph node metastases in prostate cancerNew Engl J Med348: 2491–2499, 2003PubMedCrossRefGoogle Scholar
  30. 30.
    Mintorovich J, Shansi K, Eovist: injection, reovist injection, two liver specific contrast agents for MRI. Oncology Supp 3 14: 37–40, 2000Google Scholar
  31. 31.
    Foster-Gareau P, Heyn C, Alejski A, Rutt BK: Imaging single mammalian cells with a 1.5 T clinical MRI scannerMagn Reson Med49: 968–971, 2003PubMedCrossRefGoogle Scholar
  32. 32.
    Hinds KA, Hill JM, Shapiro EM, Laukkanen MO, Silva AC, Combs CA, Varney T R, Balaban RS, Koretsky AP, Dunbar CE: Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cellsBlood102: 867–872, 2003PubMedCrossRefGoogle Scholar
  33. 33.
    Hogemann D, Josephson L, Weissleder R, Basilion JP: Related articles, links improvement of MRI probes to allow efficient detection of gene expressionBioconjug Chem11:941–946, 2000PubMedCrossRefGoogle Scholar
  34. 34.
    Choi H, Choi SR, Zhou R, Kung HF, Chen IW: Iron oxide nanoparticles as magnetic resonance contrast agent for tumor imaging via folate receptor-targeted deliveryAcad Radiol11:996–1004, 2004PubMedCrossRefGoogle Scholar
  35. 35.
    Zhang Y, Kohler N, Zhang M: Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptakeBiomaterials23:1553–1561, 2002PubMedCrossRefGoogle Scholar
  36. 36.
    Lojun S, Bao S, Lei ZM, Rao CV: Presence of functional luteinizing hormone/chorionic gonadotropin receptors in human breast cell lines: implications supporting the premise that CG protects women against breast cancerBiol Reprod57:1202–1210, 1997PubMedCrossRefGoogle Scholar
  37. 37.
    Chatzistamou L, Schally AV, Nagi A, Szepeshazi K, Halmos G: Effective treatment of metastatic MDA-MB-435 human estrogen independent breast carcinomas with a targeted cytotoxic analogue of luteinizing hormone releasing hormone AN-207Clin Cancer Res6:4158–4168, 2000PubMedGoogle Scholar
  38. 38.
    Leuschner C, Enright F, Gawronska B, Hansel W: Membrane disrupting lytic peptide conjugates destroy hormone dependent and independent breast cancer cells in vitro and in vivoBreast Cancer Res Treat78: 17–27, 2003PubMedCrossRefGoogle Scholar
  39. 39.
    Leuschner C, Hansel W: Targeting breast and prostate cancers through their hormone receptorsBiol Reprod73: 255–260, 2005CrossRefGoogle Scholar
  40. 40.
    Morbeck DE, Roche PC, Keutmann HT, McCormick DJ: A receptor binding site identified in the region 81–95 of the beta-subunit of human luteinizing hormone (LH) and chorionic gonadotropin (hCG)Mol Cell Endocrinol97: 173–181, 1993PubMedCrossRefGoogle Scholar
  41. 41.
    Kumar C, Leuschner C, Doomes EE, Henry L, Juban M, Hormes J: Efficacy of lytic peptide bound magnetite nanoparticles in destroying breast cancer cellsJ Nanosci Nanotechnol4:245–249, 2004PubMedCrossRefGoogle Scholar
  42. 42.
    Rubio N, Espana L, Fernandez Y, Blanco J, Sierra A: Metastatic behaviour of human breast carcinomas overexpressing the Bcl-xl Gene: a role in dormancy and organospecificityLab Invest81: 725–734, 2001PubMedCrossRefGoogle Scholar
  43. 43.
    Rubio N, Villacampa MM, Hilali NE, Blanco J: Metastatic burden in nude mice organs measured using prostate tumor PC-3 cells expressing the luciferase gene as a quantifiable tumor cell markerProstate 44:133–143, 2000PubMedCrossRefGoogle Scholar
  44. 44.
    Moyle WR, Capmpbell RK, Myers RV, Bernard MP, Han Y, Wang X: Co-evolution of ligand-receptor pairsNature368: 251–255, 1994PubMedCrossRefGoogle Scholar
  45. 45.
    Raynal I, Prigent P, Peyramaure S, Najid A, Rebuzzi C, Corot C: Macrophage endocytosis of superparamagnetic iron oxide nanoparticles: mechanisms and comparison of ferumoxides and ferumoxtran-10Invest Radiol39:56–63, 2004PubMedCrossRefGoogle Scholar
  46. 46.
    Leuschner C, Enright FM, Melrose PA, Hansel W: Targeted destruction of androgen-sensitive and insensitive prostate cancer cells and xenografts through luteinizing hormone receptorsProstate46:116–125, 2001PubMedCrossRefGoogle Scholar
  47. 47.
    Leuschner C, Enright F, Gawronska-Kozak B, Hansel W: Human prostate cancer cells and xenografts are targeted and destroyed through luteinizing hormone releasing hormone receptorsProstate 56:239–249, 2003PubMedCrossRefGoogle Scholar
  48. 48.
    Chen DW, Liao MH: Preparation and characterization of YADH-bound magnetic nanoparticlesJ Mol Cat B:Enzym16: 283–291, 2002CrossRefGoogle Scholar
  49. 49.
    Huang SH, Liao MH, Chen DH: Direct binding and characterization of lipase onto magnetic nanoparticlesBiotechnol Prog19: 1095–1100, 2003PubMedCrossRefGoogle Scholar
  50. 50.
    Sonoda N, Katabuchi H, Tashire H, Ohba T, Nishimura R, Minegishi T, Okamura H: Expression of variant luteinizing hormone/chorionic gonadotropin receptors and degradation of chorionic gonadotropin in human chorionic villous macrophagesPlacenta26, 298–307, 2005PubMedCrossRefGoogle Scholar
  51. 51.
    Zhou J, Leuschner C, Kumar C, Hormes FJ, Soboyejo W: Sub-cellular accumulation of magnetic nanoparticles in breast tumors and metastasesBiomaterials27: 2001–2008, 2006PubMedCrossRefGoogle Scholar
  52. 52.
    Leuschner C, Kumar CSSR, Hansel W, Hormes J: Targeting breast cancer cells and their metastses through luteinizing hormone releasing hormone (LHRH) using magnetic nanoparticlesJ Biomed Nanotechnol2: 229–233, 2005CrossRefGoogle Scholar
  53. 53.
    Shapiro EM, Skrtic S, Sharer K, Hill JM, Dunbar CE, Koretsky AP: MRI detection of single particles for cellular imaging. Proc Natl Acad Sci USA101: 10901–10906, 2004PubMedCrossRefGoogle Scholar
  54. 54.
    Billotey C, Wilhelm C, Devaud M, Bacri JC, Bittoun J, Gazeau F: Cell internalization of anionic maghemite nanoparticles: quantitative effect on magnetic resonance imagingMagn Reson Med 49:646–654, 2003PubMedCrossRefGoogle Scholar
  55. 55.
    Moore A, Marecos E, Bogdanov A, Weissleder R: Tumoral distribution of long-circulating dextran coated iron oxide nanoparticles in a rodent modelRadiology214: 568–574, 2000PubMedGoogle Scholar
  56. 56.
    Funovics MA, Kapeller B, Hoeller C, Su HS, Kunstfeld R, Puig S, Macfelda K: MR imaging of the her2/neu and 9.2.27 tumor antigens using immunospecific contrast agentsMagn Reson Imaging22: 843–850, 2004PubMedCrossRefGoogle Scholar
  57. 57.
    Berry CC, Charles S, Wells S, Da.by MJ, Curtis AS: The influence of transferring stabilized magnetic nanoparticles on human dermal fibroblasts in culture. Int J Pharm 269, 211-, 2004; Int J Pharmaceutics, 2004Google Scholar
  58. 58.
    Kircher MF, Mahmood U, King RS, Weissleder R, Josephson L: A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineationCancer Res 63: 8122–8125, 2003PubMedGoogle Scholar
  59. 59.
    Bergey EJ, Levy L, Wang X, Krebs LJ, Lal M, Kim KS, Pakatchi S, Liebow C, Prasad PN: DC magnetic filed induced magnetocytolysis of cancer cells targeted by LH-RH magnetic nanoparticles in vitro Biomed Microdev4: 293–299, 2002CrossRefGoogle Scholar
  60. 60.
    Josephson L, Tung CH, Moore A, Weissleder R: Highefficiency intracellular magnetic labeling with novel superparamagnetic-tat peptide conjugatesBioconjugate Chem10: 186–191, 1999CrossRefGoogle Scholar
  61. 61.
    Dodd CH, Hsu HC, Chu WJ, Yang P, Zhang HG, Mountz JD Jr, Zinn K, Forder J, Josephson L, Weissleder R, Mountz JM, Mountz JD: Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivator-peptide-derived superparamagnetic nanoparticlesJ Immunol Methods256:89–105, 2001PubMedCrossRefGoogle Scholar
  62. 62.
    Chouly C, Pouliquen D, Lucet J, Jeune JJ, Jallet P: Development of superparamagnetic nanoparticles for MRI: effect of particle size, charge and surface nature on biodistributionJ Microencapsul 13:245–255, 1996PubMedCrossRefGoogle Scholar
  63. 63.
    Roser M, Fischer D, Kissel T: Surface-modified biodegradable albumin nano- and microspheres. II: effect of surface charges on in vitro phagocytosis and biodistribution in rats Eur J Pharm Biopharm 46:255–263, 1998PubMedCrossRefGoogle Scholar
  64. 64.
    Shieh DB, Chen FY, Su CH, Yeh CS, Wu MT, Wu YN, Tsai CY, Wu DH, Chen DH, Chou CH: Aequeous dispersions of magnetite nanoparticles with NH+ surfaces for magnetic manipulations of biomolecules and MRI contrast agentsBiomaterials26: 7183–7191, 2005PubMedCrossRefGoogle Scholar
  65. 65.
    Pouliquen D, Le Jeune JJ, Perdrisot R, Ermias A, Jallet P: Iron oxide nanoparticles for use as an MRI contrast agent: pharmacokinetics and metabolismMagn Reson Imaging9:275–283, 1991PubMedCrossRefGoogle Scholar
  66. 66.
    Saini S, Stark DD, Hahn PF, Wittenberg J, Brady TJ, Ferrucci JT Jr: Ferrite particles: a superparamagnetic MR contrast agent for the reticuloendothelial system. Radiology. 162:211–216, 1987PubMedGoogle Scholar
  67. 67.
    Tiefenauer LX, Tschirky A, Kuhne G, Andres RY: In vivo evaluation of magnetite nanoparticles for use as a tumor contrast agent in MRIMagn Reson Imaging14:391–402, 1996PubMedCrossRefGoogle Scholar
  68. 68.
    Pineaud F, King D: Bioactivation and cell targeting of semiconductore CdSeJ Am Chem Soc126: 6115–6123, 2004CrossRefGoogle Scholar
  69. 69.
    Weissleder R, Stark DD, Engelstad BL, Bacon BR, Compton CC, White DL, Jacobs P, Lewis J: Superparamagnetic iron oxide: pharmacokinetics and toxicityAJR Am J Roentgenol152:167–173, 1989PubMedGoogle Scholar
  70. 70.
    Moore A, Josephson L, Bhorade RM, Basilion JP, Weissleder R: Human transferrin receptor gene as a marker gene for MR imagingRadiology221:244–250, 2001PubMedCrossRefGoogle Scholar
  71. 71.
    Zhou JK, Meng J, Thieraux C, Leuschner C, Kumar C, Hormes J, Soboyejo WO: LHRH-Functionalized Magnetite Nanoparticles for Breast Cancer Detection and Treatment, American Academy for Nanomedicine, Baltimore MD, 2005Google Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Carola Leuschner
    • 1
    • 5
  • Challa SSR Kumar
    • 2
  • William Hansel
    • 1
  • Wole Soboyejo
    • 3
  • Jikou Zhou
    • 4
  • Josef Hormes
    • 2
  1. 1.Pennington Biomedical Research CenterLSU SystemBaton RougeUSA
  2. 2. Center for Advanced Microsystems and DevicesLouisiana State UniversityBaton RougeUSA
  3. 3.Princeton UniversityPrincetonUSA
  4. 4.Lawrence Livermore National Laboratory LivermoreUSA
  5. 5.Reproductive BiotechnologyPennington Biomedical Research CenterBaton RougeUSA

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