Molecular Image of Superparamagnetic Iron Oxide Nanopariticle Labeled with hATF in Colon Tumor Models_Journal of Biomedical Engineering

Authors：

ZHANGShu ¹ , WANGLei ² , CHENLu ¹ , XUHuayan ³ , WUQiang ¹ , BIFeng ¹ , GAOFabao ² ,  XUFeng ¹

1. Oncology center, West China Hospital, Sichuan University, Chengdu 610041, China;
2. Molecular Image Institute, West China Hospital, Sichuan University, Chengdu 610041, China;
3. Department of Radiology, West China Hospital, Sichuan University, Chengdu 610041, China;

Corresponding author：

XUFeng, Email: fengxuster@gmail.com

Keywords：

urokinase plasminogen activator receptor; molecular image; magnetic resonance imaging; colon cancer

DOI：

10.7507/1001-5515.20150189

Video：

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Urokinase plasminogen activator receptor (uPAR) is a membrane protein which is attached to the cellular external membrane. The uPAR expression can be observed both in tumor cells and in tumor-associated stromal cells. Thus, in the present study, the human amino-terminal fragment (hATF), as a targeting element to uPAR, is used to conjugate to the surface of superparamagnetic iron nanoparticle (SPIO). Flowcytometry was used to examine the uPAR expression in different tumor cell lines. The specificity of hATF-SPIO was verified by Prussian blue stain and cell phantom test. The imaging properties of hATF-SPIO were confirmed in vivo magnetic resonance imaging (MRI) of uPAR-elevated colon tumor. Finally, the distribution of hATF-SPIO in tumor tissue was confirmed by pathological staining. Results showed that the three cells in which we screened, presented different expression characteristics, i.e., Hela cells strongly expressed uPAR, HT29 cells moderately expressed uPAR, but Lovo cells didn't express uPAR. In vitro, after incubating with Hela cells, hATF-SPIO could specifically combined to and be subsequently internalized by uPAR positive cells, which could be observed via Prussian blue staining. Meanwhile T2WI signal intensity of Hela cells, after incubation with targeted probe, significantly decreased, and otherwise no obvious changes in Lovo cells both by Prussian blue staining and MRI scans. In vivo, hATF-SPIO could be systematically delivered to HT29 xenograft and accumulated in the tumor tissue which was confirmed by Prussian Blue stain compared to Lovo xenografts. Twenty-four hours after injection of targeting probe, the signal intensity of HT29 xenografts was lower than Lovo ones which was statistically significant. This targeting nanoparticles enabled not only in vitro specifically combining to uPAR positive cells but also in vivo imaging of uPAR moderately elevated colon cancer lesions.

Citation： ZHANGShu, WANGLei, CHENLu, XUHuayan, WUQiang, BIFeng, GAOFabao, XUFeng. Molecular Image of Superparamagnetic Iron Oxide Nanopariticle Labeled with hATF in Colon Tumor Models. Journal of Biomedical Engineering, 2015, 32(5): 1067-1074. doi: 10.7507/1001-5515.20150189 Copy

1.	SIEGEL R, MA Jiemin, ZOU Zhaohui, et al. Cancer statistics, 2014[J]. CA Cancer J Clin, 2014, 64(1):9-29.
2.	ITTRICH H, PELDSCHUS K, PELDSCHUS K, et al. Superparamagnetic iron oxide nanoparticles in biomedicine:applications and developments in diagnostics and therapy[J]. Rofo, 2013, 185(12):1149-1166.
3.	LEE J H, HUH Y M, JUN Y W, et al. Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging[J]. Nat Med, 2007, 13(1):95-99.
4.	KAIDA S, CABRAL H, KUMAGAI M, et al. Visible drug delivery by supramolecular nanocarriers directing to sngle-patformed diagnosis and therapy of pancreatic tumor model[J]. Cancer Res, 2010, 70(18):7031-7041.
5.	HADJIPANAYIS C G, MACHAIDZE R, KALUZOVA M A, et al. EGFRvIII antibody-conjugated iron oxide nanoparticles for magnetic resonance imaging-guided convection-enhanced delivery and targeted therapy of glioblastoma[J]. Cancer Res, 2010, 70(15):6303-6312.
6.	YANG L, MAO Hui, WANG Y A, et al. Single chain epidermal growth factor receptor antibody conjugated nanoparticles for in vivo tumor targeting and imaging[J]. Small, 2009, 5(2):235-243.
7.	SUN C, LEE J S H, ZHANG M. Magnetic nanoparticles in MR imaging and drug delivery[J]. Advanced Drug Delivery Reviews, 2008, 60(11):1252-1265.
8.	PYKE C, RALFKIAER E, RONNE E, et al. Immunohistochemical detection of the receptor for urokinase plasminogen activator in human colon cancer[J]. Histopathology, 1994, 24(2):131-138.
9.	PYKE C, GRAEM N, RALFKIAER E, et al. Receptor for urokinase is present in tumor-associated macrophages in ductal breast carcinoma[J]. Cancer Res, 1993, 53(8):1911-1915.
10.	SMITH H W, MARSHALL C J. Regulation of cell signalling by uPAR[J]. Nat Rev Mol Cell Biol, 2010, 11(1):23-36.
11.	YANG L, MAO Hui, CAO Zehong, et al. Molecular imaging of pancreatic cancer in an animal model using targeted multifunctional nanoparticles[J]. Gastroenterology, 2009, 136(5):1514-1525.
12.	YANG L, SAJJA H K, CAO Zehong, et al. uPAR-targeted optical imaging contrasts as theranostic agents for tumor margin detection[J]. Theranostics, 2014, 4(1):106-118.
13.	LEBEAU A M, DURISETI S, MURPHY ST, et al. Targeting uPAR with antagonistic recombinant human antibodies in aggressive breast cancer[J]. Cancer Res, 2013, 73(7):2070-2081.
14.	ABDALLA MO, KARNA P, SAJJA HK, et al. Enhanced noscapine delivery using uPAR-targeted optical-MR imaging trackable nanoparticles for prostate cancer therapy[J]. J Control Release, 2011, 149(3):314-322.
15.	ILLEMANN M, BIRD N, MAJEED A, et al. Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases[J]. Int J Cancer, 2009, 125(6):1494-1496.
16.	APPELLA E, ROBINSON E A, ULLRICH S J, et al. The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases[J]. J Biol Chem, 1987, 262(10):4437-4440.
17.	BARINKA C, PARRY G, CALLAHAN J, et al. Structural basis of interaction between urokinase-type plasminogen activator and its receptor[J]. J Mol Biol, 2006, 363(2):482-495.
18.	MAGDOLEN V, RETTENBERGER P, KOPPITZ M, et al. Systematic mutational analysis of the receptor-binding region of the human urokinase-type plasminogen activator[J]. Eur J Biochem, 1996, 237(3):743-751.
19.	PLOUG M, ELLIS V, DANO K. Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor[J].Biochemistry, 1994, 33(30):8991-8997.
20.	温志鹏,刘海燕,文明,等.SPIO标记的c-erbB2反义探针用于活体荷瘤裸鼠MRI的最佳扫描浓度.南方医科大学学报,2013,33(4):496-501.
21.	PERSSON M, MADSEN J, OSTERGAARD S, et al. Quantitative PET of human urokinase-type plasminogen activator receptor with ⁶⁴Cu-DOTA-AE105:implications for visualizing cancer invasion[J].J Nucl Med, 2012, 53(1):138-145.
22.	MAEDA H. Tumor-selective delivery of macromolecular drugs via the EPR effect:background and future prospects[J]. Bioconjug Chem, 2010, 21(5):797-802..
23.	HUH Y M, JUN Y W, SONG H T, et al. In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals[J]. J Am Chem Soc, 2005, 127(35):12387-12391.
24.	MEDAROVA Z, PHAM W, KIM Y, et al. In vivo imaging of tumor response to therapy using a dual-modality imaging strategy[J]. Int J Cancer, 2006, 118(11):2796-2802.
25.	KRISHNAMACHARY B, BERG-DIXON S, KELLY B, et al. Regulation of colon carcinoma cell invasion by hypoxia-inducible factor 1[J]. Cancer Res, 2003, 63(5):1138-1143.
26.	LESTER R D, JO M, MONTEL V, et al. uPAR induces epithelial-mesenchymal transition in hypoxic breast cancer cells[J]. J Cell Biol, 2007, 178(3):425-436.
27.	LIU Gang, GAO Jinhao, AI Hua, et al. Applications and potential toxicity of magnetic iron oxide nanoparticles[J]. Small, 2013, 9(9-10):1533-1545.
28.	HUANG Gang, CHEN Huabing, DONG Ying, et al. Superparamagnetic iron oxide nanoparticles:amplifying ROS stress to improve anticancer drug efficacy[J]. Theranostics, 2013, 3(2):116-126.
29.	YU Miao, HUANG Shaohui, YU K J, et al. Dextran and polymer polyethylene glycol (PEG) coating reduce both 5 and 30 nm iron oxide nanoparticle cytotoxicity in 2D and 3D cell culture[J]. Int J Mol Sci, 2012, 13(5):5554-5570.
30.	PERSSON M, RASMUSSEN P, MADSEN J, et al. New peptide receptor radionuclide therapy of invasive cancer cells:in vivo studies using 177Lu-DOTA-AE105 targeting uPAR in human colorectal cancer xenografts[J]. Nucl Med Biol, 2012, 39(7):962-969.
31.	LI Zibo, NIU Gang, WANG Hui, et al. Imaging of urokinase-type plasminogen activator receptor expression using a ⁶⁴Cu-labeled linear peptide antagonist by microPET[J]. Clin Cancer Res, 2008, 14(15):4758-4766.
32.	KOROSOGLOU G, WEISS R G, KEDZIOREK D A, et al. Noninvasive detection of macrophage-rich atherosclerotic plaque in hyperlipidemic rabbits using "positive contrast" magnetic resonance imaging[J]. J Am Coll Cardiol, 2008, 52(6):483-491.
33.	ROBSON M D, BYDDER G M. Clinical ultrashort echo time imaging of bone and other connective tissues[J]. NMR Biomed, 2006, 19(7):765-780.
34.	ZHANG Longjiang, ZHONG Xiaodong, WANG Liya, et al. T(1)-Weighted ultrashort echo time method for positive contrast imaging of magnetic nanoparticles and cancer cells bound with the targeted nanoparticles[J]. J Magn Resone Imaging, 2011, 33(1):194-202.

1. SIEGEL R, MA Jiemin, ZOU Zhaohui, et al. Cancer statistics, 2014[J]. CA Cancer J Clin, 2014, 64(1):9-29.
2. ITTRICH H, PELDSCHUS K, PELDSCHUS K, et al. Superparamagnetic iron oxide nanoparticles in biomedicine:applications and developments in diagnostics and therapy[J]. Rofo, 2013, 185(12):1149-1166.
3. LEE J H, HUH Y M, JUN Y W, et al. Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging[J]. Nat Med, 2007, 13(1):95-99.
4. KAIDA S, CABRAL H, KUMAGAI M, et al. Visible drug delivery by supramolecular nanocarriers directing to sngle-patformed diagnosis and therapy of pancreatic tumor model[J]. Cancer Res, 2010, 70(18):7031-7041.
5. HADJIPANAYIS C G, MACHAIDZE R, KALUZOVA M A, et al. EGFRvIII antibody-conjugated iron oxide nanoparticles for magnetic resonance imaging-guided convection-enhanced delivery and targeted therapy of glioblastoma[J]. Cancer Res, 2010, 70(15):6303-6312.
6. YANG L, MAO Hui, WANG Y A, et al. Single chain epidermal growth factor receptor antibody conjugated nanoparticles for in vivo tumor targeting and imaging[J]. Small, 2009, 5(2):235-243.
7. SUN C, LEE J S H, ZHANG M. Magnetic nanoparticles in MR imaging and drug delivery[J]. Advanced Drug Delivery Reviews, 2008, 60(11):1252-1265.
8. PYKE C, RALFKIAER E, RONNE E, et al. Immunohistochemical detection of the receptor for urokinase plasminogen activator in human colon cancer[J]. Histopathology, 1994, 24(2):131-138.
9. PYKE C, GRAEM N, RALFKIAER E, et al. Receptor for urokinase is present in tumor-associated macrophages in ductal breast carcinoma[J]. Cancer Res, 1993, 53(8):1911-1915.
10. SMITH H W, MARSHALL C J. Regulation of cell signalling by uPAR[J]. Nat Rev Mol Cell Biol, 2010, 11(1):23-36.
11. YANG L, MAO Hui, CAO Zehong, et al. Molecular imaging of pancreatic cancer in an animal model using targeted multifunctional nanoparticles[J]. Gastroenterology, 2009, 136(5):1514-1525.
12. YANG L, SAJJA H K, CAO Zehong, et al. uPAR-targeted optical imaging contrasts as theranostic agents for tumor margin detection[J]. Theranostics, 2014, 4(1):106-118.
13. LEBEAU A M, DURISETI S, MURPHY ST, et al. Targeting uPAR with antagonistic recombinant human antibodies in aggressive breast cancer[J]. Cancer Res, 2013, 73(7):2070-2081.
14. ABDALLA MO, KARNA P, SAJJA HK, et al. Enhanced noscapine delivery using uPAR-targeted optical-MR imaging trackable nanoparticles for prostate cancer therapy[J]. J Control Release, 2011, 149(3):314-322.
15. ILLEMANN M, BIRD N, MAJEED A, et al. Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases[J]. Int J Cancer, 2009, 125(6):1494-1496.
16. APPELLA E, ROBINSON E A, ULLRICH S J, et al. The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases[J]. J Biol Chem, 1987, 262(10):4437-4440.
17. BARINKA C, PARRY G, CALLAHAN J, et al. Structural basis of interaction between urokinase-type plasminogen activator and its receptor[J]. J Mol Biol, 2006, 363(2):482-495.
18. MAGDOLEN V, RETTENBERGER P, KOPPITZ M, et al. Systematic mutational analysis of the receptor-binding region of the human urokinase-type plasminogen activator[J]. Eur J Biochem, 1996, 237(3):743-751.
19. PLOUG M, ELLIS V, DANO K. Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor[J].Biochemistry, 1994, 33(30):8991-8997.
20. 温志鹏,刘海燕,文明,等.SPIO标记的c-erbB2反义探针用于活体荷瘤裸鼠MRI的最佳扫描浓度.南方医科大学学报,2013,33(4):496-501.
21. PERSSON M, MADSEN J, OSTERGAARD S, et al. Quantitative PET of human urokinase-type plasminogen activator receptor with ⁶⁴Cu-DOTA-AE105:implications for visualizing cancer invasion[J].J Nucl Med, 2012, 53(1):138-145.
22. MAEDA H. Tumor-selective delivery of macromolecular drugs via the EPR effect:background and future prospects[J]. Bioconjug Chem, 2010, 21(5):797-802..
23. HUH Y M, JUN Y W, SONG H T, et al. In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals[J]. J Am Chem Soc, 2005, 127(35):12387-12391.
24. MEDAROVA Z, PHAM W, KIM Y, et al. In vivo imaging of tumor response to therapy using a dual-modality imaging strategy[J]. Int J Cancer, 2006, 118(11):2796-2802.
25. KRISHNAMACHARY B, BERG-DIXON S, KELLY B, et al. Regulation of colon carcinoma cell invasion by hypoxia-inducible factor 1[J]. Cancer Res, 2003, 63(5):1138-1143.
26. LESTER R D, JO M, MONTEL V, et al. uPAR induces epithelial-mesenchymal transition in hypoxic breast cancer cells[J]. J Cell Biol, 2007, 178(3):425-436.
27. LIU Gang, GAO Jinhao, AI Hua, et al. Applications and potential toxicity of magnetic iron oxide nanoparticles[J]. Small, 2013, 9(9-10):1533-1545.
28. HUANG Gang, CHEN Huabing, DONG Ying, et al. Superparamagnetic iron oxide nanoparticles:amplifying ROS stress to improve anticancer drug efficacy[J]. Theranostics, 2013, 3(2):116-126.
29. YU Miao, HUANG Shaohui, YU K J, et al. Dextran and polymer polyethylene glycol (PEG) coating reduce both 5 and 30 nm iron oxide nanoparticle cytotoxicity in 2D and 3D cell culture[J]. Int J Mol Sci, 2012, 13(5):5554-5570.
30. PERSSON M, RASMUSSEN P, MADSEN J, et al. New peptide receptor radionuclide therapy of invasive cancer cells:in vivo studies using 177Lu-DOTA-AE105 targeting uPAR in human colorectal cancer xenografts[J]. Nucl Med Biol, 2012, 39(7):962-969.
31. LI Zibo, NIU Gang, WANG Hui, et al. Imaging of urokinase-type plasminogen activator receptor expression using a ⁶⁴Cu-labeled linear peptide antagonist by microPET[J]. Clin Cancer Res, 2008, 14(15):4758-4766.
32. KOROSOGLOU G, WEISS R G, KEDZIOREK D A, et al. Noninvasive detection of macrophage-rich atherosclerotic plaque in hyperlipidemic rabbits using "positive contrast" magnetic resonance imaging[J]. J Am Coll Cardiol, 2008, 52(6):483-491.
33. ROBSON M D, BYDDER G M. Clinical ultrashort echo time imaging of bone and other connective tissues[J]. NMR Biomed, 2006, 19(7):765-780.
34. ZHANG Longjiang, ZHONG Xiaodong, WANG Liya, et al. T(1)-Weighted ultrashort echo time method for positive contrast imaging of magnetic nanoparticles and cancer cells bound with the targeted nanoparticles[J]. J Magn Resone Imaging, 2011, 33(1):194-202.

Journal of Biomedical Engineering

Molecular Image of Superparamagnetic Iron Oxide Nanopariticle Labeled with hATF in Colon Tumor Models

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