Molecular imaging have provided great hope for tiny tumors detection and staging upon acquisition of molecular and physiological information[1-2]. The anti-Stokes mechanism of the upconversion luminescence probes offers greater penetration of tissue by the NIR excitation and eliminates the interference of endogenous fluorescence, giving rise to an enhanced signal-to-noise ratio (SNR)[3-6]. Moreover, with the incorporation of gadolinium in the nanoparticle matrix, dual-modality imaging probes can be constructed with integrated magnetic and upconversion properties[7-10]. The culmination of these physical attributes provides unique opportunities in developing molecular probes for bioimaging, including the analysis of the tumor[11-14], blood pool[15], and lymph nodes[16]. Urokinase plasminogenactivator (uPA) is a serine protease that interacts with its receptor, uPAR, to regulate multiple pathways involved in matrix degradation, cell motility, metastasis, and angiogenesis[17]. Urokinase plasminogen activator
receptor (uPAR), a cellular receptor that is highly expressed in
pancreatic cancer cells and tumor stromal cells, is an excellent surface
molecule for receptor-targeted imaging[18-20]. Studies have demonstrated that a high level of uPAR expression in tumor cells correlates with aggressive tumor types, tumor metastasis, and poorer prognosis[21,22]. Furthermore, in the majority of normal tissues or organs, the level of uPAR is very low or undetectable[23]. uPAR-targeted upconversion
nanoparticles change the traditional single and down conversion
luminescent imaging mode, as near-infrared excitation can better
penetrate tissue than visible light while giving rise to greatly reduced
background noise.
The uPAR-targeted probe is designed and prepared by conjugating
amino-terminal fragment of the receptor binding domain of human
urokinase plasminogen activator to the surface of upconversion
nanoparticles. The orthotopic human pancreatic cancer xenograft model
was established by a surgical procedure. A stably luciferase-expressing
SW1990 cell line, which is a typical pancreatic cancer cell line, was
used to enable constant monitoring of tumor development. Ten days after
surgical implantation, the nude mice were screened by bioluminescence
imaging to verify successful inoculation. Only mice exhibiting
comparable bioluminescence intensities were chosen for parallel studies.
Targeted nanoparticles are able to specifically bind to uPAR-expressing
tumor cells. The primary tumor and adjacent lymphatic metastasis site
were clearly differentiated by upconversion luminescence imaging after
the uPAR-specific probe was delivered through tail vein injection into
tumor-bearing nude mice. Target specificity of nanoparticles is further
confirmed by a clinical MRI scanner at a field strength of 3 Tesla.
Furthermore, mice administered targeted nanoparticles exhibit lower
uptake of the particles in the liver and lung compared to those
receiving non-targeted upconversion nanoparticles.
Lymphatic status is a very important factor for the prognosis of cancer patients. With respect to the detection of LN metastasis, no imaging modality consistently achieves both high sensitivity and high specificity. In contrast, the sensitivity and specificity achieved by using the current UCNP-based probes are apparently superior to FDG-PET and other techniques for differentiating lymphatic metastasis from the primary cancer. In fact, MR imaging of lymph nodes enhanced by ultrasmall superparamagnetic iron oxide nanoparticles was reported quite some time ago. The mechanism largely relies on populating differences of macrophages between benign and malignant nodes, which makes detection of micrometatasis in LNs difficult. However, the current study provides a different mechanism for imaging only lymph nodes positive for metastases through active targeting of the malignant nodes via specific antibody antigen recognition. Although optical imaging offers an ultrasensitive tool for tumor detection, the limited tissue penetrationis widely accepted to be a flaw of optical imaging. However, the optical signal does not always need to penetrate through the whole abdominal wall to be detected during surgical lymphadenectomy.
Our results revealed that the probe could be useful for not only tiny tumor lesion diagnosis but also for lymphatic metastasis detections, indicating potential clinical applications in the early pancreatic cancer diagnosis and lymph node status evaluation.
References
1. Weissleder, R. Molecular Imaging in Cancer. Science 2006,312, 1168–1171.
2. Weissleder, R.; Pittet, M. J. Imaging in the Era of MolecularOncology. Nature 2008, 452, 580–589.
3. Zhou, J.; Liu, Z.; Li, F. Y. Upconversion Nanophosphors forSmall-Animal Imaging. Chem. Soc. Rev. 2012, 41, 1323–1349.
4. Liu, C.; Hou, Y.; Gao, M. Y. Are Rare-Earth NanoparticlesSuitable for in Vivo Applications? Adv. Mater. 2014, 26,6922–6932
5. Wang, F.; Liu, X. G. Recent Advances in the Chemistry of Lanthanide-Doped Upconversion Nanocrystals. Chem.Soc. Rev. 2009, 38, 976–989.
6. Wu, S. W.; Han, G.; Milliron, D. J.; Aloni, S.; Altoe, V.; Talapin,D. V.; Cohen, B. E.; Schuck, P. J. Non-blinking andPhotostable Upconverted Luminescence from Single Lanthanide-Doped Nanocrystals. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 10917–10921.
7. Park, Y. I.; Kim, J. H.; Lee, K. T.; Jeon, K. S.; Bin Na, H.; Yu, J. H.;Kim, H. M.; Lee, N.; Choi, S. H.; Baik, S. I.; et al. Nonblinkingand Nonbleaching Upconverting Nanoparticles asan Optical Imaging Nanoprobe and T1 Magnetic ResonanceImaging Contrast Agent. Adv. Mater. 2009, 21,4467–4471.
8. Hou, Y.; Qiao, R. R.; Fang, F.; Wang, X. X.; Dong, C. Y.; Liu, K.;Liu, C. Y.; Liu, Z. F.; Lei, H.; Wang, F.; et al. NaGdF4Nanoparticle-Based Molecular Probes for Magnetic ResonanceImaging of Intraperitoneal Tumor Xenografts inVivo. ACS Nano 2013, 7, 330–338.
9. Liu, C. Y.; Gao, Z. Y.; Zeng, J. F.; Hou, Y.; Fang, F.; Li, Y. L.; Qiao,R. R.; Shen, L.; Lei, H.; Yang, W. S.; et al. Magnetic/Upconversion Fluorescent NaGdF4:Yb,Er Nanoparticle-Based Dual-Modal Molecular Probes for Imaging TinyTumors in Vivo. ACS Nano 2013, 7, 7227–7240.
10. Chen, G. Y.; Ohulchanskyy, T. Y.; Liu, S.; Law, W. C.; Wu, F.;Swihart, M. T.; Agren, H.; Prasad, P. N. Core/Shell NaGdF4:Nd3þ/NaGdF4 Nanocrystals with Efficient Near-Infrared toNear-Infrared Downconversion Photoluminescence forBioimaging Applications. ACS Nano 2012, 6, 2969–2977.
11. Xiong, L. Q.; Chen, Z. G.; Yu, M. X.; Li, F. Y.; Liu, C.; Huang,C. H. Synthesis, Characterization, and in Vivo TargetedImaging of Amine-Functionalized Rare-Earth Up-convertingNanophosphors. Biomaterials 2009, 30, 5592–5600.
12. Zako, T.; Nagata, H.; Terada, N.; Utsumi, A.; Sakono, M.;Yohda, M.; Ueda, H.; Soga, K.; Maeda, M. Cyclic RGDPeptide-Labeled Upconversion Nanophosphors forTumor Cell-Targeted Imaging. Biochem. Biophys. Res.Commun. 2009, 381, 54–58
13.Park, Y.; Kim, H. M.; Kim, J. H.; Moon, K. C.; Yoo, B.; Lee, K. T.;Lee, N.; Choi, Y.; Park, W.; Ling, D.; et al. Theranostic ProbeBased on Lanthanide-Doped Nanoparticles for Simultaneousin Vivo Dual-Modal Imaging and PhotodynamicTherapy. Adv. Mater. 2012, 24, 5755–5761.
14.Xiong, L. Q.; Chen, Z. G.; Tian, Q. W.; Cao, T. Y.; Xu, C. J.; Li,F. Y. High Contrast Upconversion Luminescence TargetedImaging in Vivo Using Peptide-Labeled Nanophosphors.Anal. Chem. 2009, 81, 8687–8694.
15. Hilderbrand, S. A.; Shao, F. W.; Salthouse, C.; Mahmood, U.;Weissleder, R. Upconverting Luminescent Nanomaterials:Application to in Vivo Bioimaging. Chem. Commun. 2009,4188–4190.
16. Kobayashi, H.; Kosaka, N.; Ogawa, M.; Morgan, N. Y.; Smith,P. D.; Murray, C. B.; Ye, X. C.; Collins, J.; Kumar, G. A.; Bell, H.;et al. In Vivo Multiple Color Lymphatic Imaging UsingUpconverting Nanocrystals. J. Mater. Chem. 2009, 19,6481–6484.
17.Blasi F, Carmeliet P. uPAR: a versatile signalling orchestrator. Nat Rev MolCell Biol. 2002;3(12): 932-43.
18. Yang Lily,
Sajja Hari Krishna, Cao Zehong, Qian Weiping, Bender Laura, Marcus Adam I,
Lipowska Malgorzata, Wood William C, Wang Y Andrew. uPAR-targeted Optical
Imaging Contrasts as Theranostic Agents for Tumor Margin Detection.
Theranostics, 2014, 4(1):106–18.
19. Yang Lily, Peng Xiang-Hong, Wang Y Andrew, Wang
Xiaoxia, Cao Zehong, Ni Chunchun, Karna Prasanthi, Zhang Xinjian, Wood William
C, Gao Xiaohu, Nie Shuming, Mao Hui. Receptor-Targeted Nanoparticles for In
Vivo Imaging of Breast Cancer. Clin Cancer Res Off J Am Assoc Cancer Res, 2009,
15(14):4722–32.
20. Mekkawy Ahmed H, Pourgholami Mohammad H, Morris
David L. Involvement of Urokinase-Type Plasminogen Activator System in Cancer:
An Overview. Med Res Rev, 2014, 34(5):918–56.
21. Hemsen A, Riethdorf L, Brunner N, et al. Comparative evaluation ofurokinase-type plasminogen activator receptor expression in primary breastcarcinomas and on metastatic tumor cells. Int J Cancer. 2003;107(6): 903-9.
22. Kotzsch M, Bernt K, Friedrich K, et al. Prognostic relevance of tumourcell-associated uPAR expression in invasive ductal breast carcinoma.Histopathology. 2010;57(3): 461-71.
23. Solberg H, Ploug M, Hoyer-Hansen G, Nielsen BS, Lund LR. The murinereceptor for urokinase-type plasminogen activator is primarily expressed intissues actively undergoing remodeling. J Histochem Cytochem. 2001;49(2):237-46.