Yonsei Med J.  2017 Jan;58(1):51-58. 10.3349/ymj.2017.58.1.51.

The Effectiveness of Ferritin as a Contrast Agent for Cell Tracking MRI in Mouse Cancer Models

• Lee CW
• Choi SI
• Lee SJ
• Oh YT
• Park G
• Park NY
• Yoon KA
• Kim S
• Kim D
• Kim YH
• Suh JS

• KMID: 2374188
• DOI: http://doi.org/10.3349/ymj.2017.58.1.51

Abstract

PURPOSE
We aimed to investigate the effectiveness of ferritin as a contrast agent and a potential reporter gene for tracking tumor cells or macrophages in mouse cancer models.
MATERIALS AND METHODS
Adenoviral human ferritin heavy chain (Ad-hFTH) was administrated to orthotopic glioma models and subcutaneous colon cancer mouse models using U87MG and HCT116 cells, respectively. Brain MR images were acquired before and daily for up to 6 days after the intracranial injection of Ad-hFTH. In the HCT116 tumor model, MR examinations were performed before and at 6, 24, and 48 h after intratumoral injection of Ad-hFTH, as well as before and every two days after intravenous injection of ferritin-labeled macrophages. The contrast effect of ferritin in vitro was measured by MR imaging of cell pellets. MRI examinations using a 7T MR scanner comprised a T1-weighted (T1w) spin-echo sequence, T2-weighted (T2w) relaxation enhancement sequence, and T2*-weighted (T2*w) fast low angle shot sequence.
RESULTS
Cell pellet imaging of Ad-hFTH in vitro showed a strong negatively enhanced contrast in T2w and T2*w images, presenting with darker signal intensity in high concentrations of Fe. T2w images of glioma and subcutaneous HCT116 tumor models showed a dark signal intensity around or within the Ad-hFTH tumor, which was distinct with time and apparent in T2*w images. After injection of ferritin-labeled macrophages, negative contrast enhancement was identified within the tumor.
CONCLUSION
Ferritin could be a good candidate as an endogenous MR contrast agent and a potential reporter gene that is capable of maintaining cell labeling stability and cellular safety.

Keyword

Ferritin; cell tracking; MR imaging; reporter gene

MeSH Terms

Animals
Brain Neoplasms/*diagnostic imaging/pathology
Cell Line, Tumor
Cell Tracking/*methods
Colonic Neoplasms/*diagnostic imaging/pathology
*Contrast Media/administration & dosage
Disease Models, Animal
Female
*Ferritins/administration & dosage
Genes, Reporter
Glioma/*diagnostic imaging/pathology
Humans
Injections, Intravenous
Macrophages
Magnetic Resonance Imaging/*methods
Male
Mice
Neoplasm Transplantation
Skin Neoplasms/*diagnostic imaging/pathology
Time Factors
Contrast Media
Ferritins

Figure

• 

  Fig. 1 Adenoviral construct for hFTH expression. (A) Schematic diagram of hFTH expressing an adenoviral vector with the Ad5 backbone and E1/E3 deletion containing the CMV promoter. The adenoviral vector was designed so that GFP expression was able to reflect adenoviral transduction. (B) GFP-positive cells indicate hFTH expression at 48 h after adenovirus infection in U87MG cells. CMV, promoter cytomegalovirus; ITR, inverted terminal repeat sequences; GFP, green fluorescence protein; HC, heavy chain; Ad, adenoviral; hFTH, human ferritin heavy chain.

• 

  Fig. 2 Ferritin-induced magnetic resonance (MR) contrast effect in vitro in U87MG cells. To prepare cell pellets for MR imaging, U87MG cells (3×106) were infected with Mock and Ad-hFTH (2×108 PFU) for 48 h under complete culture conditions. FeCl3 was added at indicated concentrations during infection. After incubation, all cells were pelleted by centrifugation and washed with PBS three times, followed by MRI measurements of cell pellets transferred to strip tubes. Experimental detail is described in the Materials and Methods. T1w, T1-weighted; T2w, T2-weighted; T2*w, T2*-weighted; Ad-hFTH, adenoviral human ferritin heavy chain; PBS, phosphate-buffered saline; NT, no treatment.

• 

  Fig. 3 Serial magnetic resonance imaging (MRI) of ferritin as a reporter in a glioma model with Ad-hFTH. (A) Experimental scheme using U87MG glioma model. (B) From serial MRI of mice after injection of Mock and Ad-hFTH (2×108 PFU), T2w and T2*w images of mouse brain were acquired for six days. Dark signal intensity at the periphery of the Ad-hFTH-treated tumor appeared at day 1 post-injection, which was distinct with time, and more apparent in T2*w images. T2*w axial images show a hypo-signal peripheral rim (white circles) around the tumor three days after injection of Ad-hFTH. There was no signal change around the Mock-treated tumor, with the exception of linear low signal intensity suggestive of the injection site (white dotted circles). I.C., Intracranial injection; Ad-hFTH, adenoviral human ferritin heavy chain; T2w, T2-weighted, T2*w, T2*-weighted.

• 

  Fig. 4 Magnetic resonance imaging (MRI) of ferritin in a HCT116 subcutaneous tumor model. Ad-FTH (5×108 PFU) was intratumorally injected into the tumor mass at right flank. Mock viruses were also intratumorally injected into the other side of Ad-FTH-treated tumor mass as indicated with gray arrows. To observe the contrast change inside the tumor mass, a series of T2-weighted MR images were acquired. The dark curvilinear signal intensity appeared at 6 h and 24 h after injection (white arrows) disappeared two days post-injection. Ad-FTH, adenoviral human ferritin heavy chain.

• 

  Fig. 5 Tracking of macrophages labeled with ferritin in a colon cancer mouse model using MRI. (A) The macrophage pellets labeled with equine ferritin showed a strong negative enhanced contrast in the conventional T2w images. To prepare macrophage pellets, RAW 264.7 cells were incubated with culture medium containing equine ferritin (5 mg/mL) for 24 h, and then cells were centrifuged in order to collect them at the bottom of tube. (B) Experimental scheme of macrophage tracking in a colon cancer mouse model using HCT116 cells. RAW264.7 macrophage cells labeled with equine ferritin were administrated intravenously (I.V.) after 14 days after subcutaneous (S.C.) inoculation of HCT116 colon cancer cells. MR monitoring was performed every two days after acquiring pre-imaging. (C) In T2w axial images, the dark spot inside the tumor mass (white dotted circle) could be observed four days after the injection of macrophages labeled with ferritin. (D) Prussian blue staining of the tumor mass showed scattered purple dots suggesting iron uptake in the tumor mass. The staining method is described in the Materials and Methods. T2w, T2-weighted.

Reference

1. Brader P, Serganova I, Blasberg RG. Noninvasive molecular imaging using reporter genes. J Nucl Med. 2013; 54:167–172. PMID: 23318292.

2. Youn H, Chung JK. Reporter gene imaging. AJR Am J Roentgenol. 2013; 201:W206–W214. PMID: 23883235.

3. Weissleder R, Moore A, Mahmood U, Bhorade R, Benveniste H, Chiocca EA, et al. In vivo magnetic resonance imaging of transgene expression. Nat Med. 2000; 6:351–355. PMID: 10700241.

4. Kang HW, Josephson L, Petrovsky A, Weissleder R, Bogdanov A Jr. Magnetic resonance imaging of inducible E-selectin expression in human endothelial cell culture. Bioconjug Chem. 2002; 13:122–127. PMID: 11792187.

5. Zhao M, Beauregard DA, Loizou L, Davletov B, Brindle KM. Noninvasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent. Nat Med. 2001; 7:1241–1244. PMID: 11689890.

6. Artemov D. Molecular magnetic resonance imaging with targeted contrast agents. J Cell Biochem. 2003; 90:518–524. PMID: 14523986.

7. Huh YM, Jun YW, Song HT, Kim S, Choi JS, Lee JH, et al. In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals. J Am Chem Soc. 2005; 127:12387–12391. PMID: 16131220.

8. Jun YW, Huh YM, Choi JS, Lee JH, Song HT, Kim S, et al. Nanoscale size effect of magnetic nanocrystals and their utilization for cancer diagnosis via magnetic resonance imaging. J Am Chem Soc. 2005; 127:5732–5733. PMID: 15839639.

9. Czernin J, Phelps ME. Positron emission tomography scanning: current and future applications. Annu Rev Med. 2002; 53:89–112. PMID: 11818465.

10. Graves EE, Weissleder R, Ntziachristos V. Fluorescence molecular imaging of small animal tumor models. Curr Mol Med. 2004; 4:419–430. PMID: 15354872.

11. Bulte JW, Kraitchman DL. Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed. 2004; 17:484–499. PMID: 15526347.

12. Ahn SJ, Koom WS, An CS, Lim JS, Lee SK, Suh JS, et al. Quantitative assessment of tumor responses after radiation therapy in a DLD-1 colon cancer mouse model using serial dynamic contrast-enhanced magnetic resonance imaging. Yonsei Med J. 2012; 53:1147–1153. PMID: 23074115.

13. Lee JH, Huh YM, Jun YW, Seo JW, Jang JT, Song HT, et al. Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging. Nat Med. 2007; 13:95–99. PMID: 17187073.

14. Song HT, Choi JS, Huh YM, Kim S, Jun YW, Suh JS, et al. Surface modulation of magnetic nanocrystals in the development of highly efficient magnetic resonance probes for intracellular labeling. J Am Chem Soc. 2005; 127:9992–9993. PMID: 16011350.

15. Pawelczyk E, Arbab AS, Pandit S, Hu E, Frank JA. Expression of transferrin receptor and ferritin following ferumoxides-protamine sulfate labeling of cells: implications for cellular magnetic resonance imaging. NMR Biomed. 2006; 19:581–592. PMID: 16673357.

16. Gilad AA, Winnard PT Jr, van Zijl PC, Bulte JW. Developing MR reporter genes: promises and pitfalls. NMR Biomed. 2007; 20:275–290. PMID: 17451181.

17. Cohen B, Ziv K, Plaks V, Israely T, Kalchenko V, Harmelin A, et al. MRI detection of transcriptional regulation of gene expression in transgenic mice. Nat Med. 2007; 13:498–503. PMID: 17351627.

18. Genove G, DeMarco U, Xu H, Goins WF, Ahrens ET. A new transgene reporter for in vivo magnetic resonance imaging. Nat Med. 2005; 11:450–454. PMID: 15778721.

19. Uchida M, Terashima M, Cunningham CH, Suzuki Y, Willits DA, Willis AF, et al. A human ferritin iron oxide nano-composite magnetic resonance contrast agent. Magn Reson Med. 2008; 60:1073–1081. PMID: 18956458.

20. Carrondo MA. Ferritins, iron uptake and storage from the bacterioferritin viewpoint. EMBO J. 2003; 22:1959–1968. PMID: 12727864.

21. Gossuin Y, Muller RN, Gillis P. Relaxation induced by ferritin: a better understanding for an improved MRI iron quantification. NMR Biomed. 2004; 17:427–432. PMID: 15526352.

22. Kim HS, Woo J, Lee JH, Joo HJ, Choi Y, Kim H, et al. In vivo tracking of dendritic cell using MRI reporter gene, ferritin. PLoS One. 2015; 10:e0125291. PMID: 25993535.

23. Ghosh SS, Gopinath P, Ramesh A. Adenoviral vectors: a promising tool for gene therapy. Appl Biochem Biotechnol. 2006; 133:9–29. PMID: 16622281.

24. D'souza N, Rossignoli F, Golinelli G, Grisendi G, Spano C, Candini O, et al. Mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies. BMC Med. 2015; 13:186. PMID: 26265166.

25. Corot C, Robert P, Idée JM, Port M. Recent advances in iron oxide nanocrystal technology for medical imaging. Adv Drug Deliv Rev. 2006; 58:1471–1504. PMID: 17116343.

26. Bach-Gansmo T. Ferrimagnetic susceptibility contrast agents. Acta Radiol Suppl. 1993; 387:1–30. PMID: 8390776.

27. Wang YX. Superparamagnetic iron oxide based MRI contrast agents: current status of clinical application. Quant Imaging Med Surg. 2011; 1:35–40. PMID: 23256052.

28. de Rochefort L, Liu T, Kressler B, Liu J, Spincemaille P, Lebon V, et al. Quantitative susceptibility map reconstruction from MR phase data using bayesian regularization: validation and application to brain imaging. Magn Reson Med. 2010; 63:194–206. PMID: 19953507.

29. Eibofner F, Steidle G, Kehlbach R, Bantleon R, Schick F. Positive contrast imaging of iron oxide nanoparticles with susceptibility-weighted imaging. Magn Reson Med. 2010; 64:1027–1038. PMID: 20564596.