Drug-resistant metastatic tumors as well as side effects are major limitations of conventional cancer therapy such as irradiation or chemotherapy [4
], which necessitate the search for novel tumor-targeting agents. Specific targeting of tumors, compared with earlier less specific approaches, is increasingly significant in oncology either for diagnostic or therapeutic purposes [10
]. Our previous report has demonstrated the monitoring of tumor progression and gene therapy using noninvasive planar γ
-camera imaging in a lung metastases model with murine fibrosarcoma cells, NG4TL4-tk, stably expressing HSV1-tk [9
]. Following the previous work, in the current research, we are attempting to develop a new targeting agent which can label the same tumor cells in vivo without the need of the expression of an exogenous marker gene in tumor cells. Using the peptide phage display system on NG4TL4-tk cell line, a new tumor-binding peptide, TK4, was identified. TK4-phage was confirmed for the binding affinity to NG4TL4-tk cells in vitro with different methods including plaque-forming assay, flow cytometry, IF microscopy, and phage ELISA (). IV administration of TK4-phage to mice bearing NG4TL4-tk tumors through tail vein showed significantly higher retention of phages in tumor than in other organs (). As competitive synthetic TK4 peptide was premixed with TK4-phage, the phage titer eluted from tumors reduced to a level comparable to the control tissue. These results confirmed that TK4-phage maintained its tumor-targeting ability in vivo and that the binding is dependent on the displayed surface TK4 peptide.
These results recommended the potential of TK4 peptide as an in vivo tumor-targeting agent. Radio- and fluorescent-labeled TK4 ([131
I]-FITC-TK4) was thus synthesized and systemically administrated in tumor-bearing mice. Although a strong and unspecific distribution of TK4 signal was observed throughout the body 1
hr after injection, most signals diminished rapidly at 4
hr, while the tumor retained a comparable intensity, resulting in a high tumor-to-organ ratio ( and ). This observation would suggest that most signals accumulated in normal tissues were nonspecific. The uptake of TK4 was higher in tumor than in normal organs except kidney, liver, and intestine at 4
hr, and the tumor-to-muscle ratio of %
ID/g was 5.73 (). The weak accumulation of radioactivity in collateral tissues made the diagnostic imaging of tumor more viable. The tumor site can be detected in a whole-body imaging using planar γ
-camera at 4
hr postinjection (), and the FRI confirmed a much stronger FITC signal accumulated in tumors than in surrounding muscles (). All together, these results suggest that TK4 peptide is able to target and accumulate in fibrosarcoma tumors in vivo.
The difference in biodistributions between the phage particles and synthetic TK4 peptide was noticed (Figures and ). The phage particles showed an efficient binding only in tumor, while synthetic peptide had a high retention also in liver and kidney. Such discrepancy may have resulted from the different methodologies applied. Phage particles were recovered from tissues of sacrificed animal in a few minutes after administration, which may prevent the further accumulation of phages in excretion route like liver, intestine, or kidney. In addition, the whole body perfusion in sacrificed mice may have helped to wash off the unbound phages. To optimize the use of TK4 as an efficient tumor-imaging peptide; however, the substantial accumulation of radioactivity in kidney and liver should be reduced. Small changes in peptide sequence, linkers, chelator, and isotope can dramatically affect the biodistribution [18
]. For example, a high radioactivity retention in liver and intestine could result from the hepatobiliary elimination. Strategies including glycosylation or conjugation with hydrophilic amino acid have been developed to reduce the liver uptake of RGD peptides (for a recent review, see [20
]). Renal excretion is a preferable elimination pathway of radiopharmaceuticals and/or their metabolites from the body. Nevertheless, high kidney retention may obscure tumor imaging and result in potential radiation nephrotoxicity, and thus limit the use of radiolabelled peptides in medical applications [21
]. Current methods for kidney protection include coadministration of basic amino acids, the bovine gelatin-containing solution Gelofusine or albumin fragments, which interfere with the tubular reabsorption pathway of peptides (for a recent review, see [21
]). These and other researches have led to developments of more ideal peptide probes for tumor imaging and therapy, and the knowledge can be expanded to further modification of TK4.
Our current research showed that TK4 is homologous to a FN3 domain in a novel protein similar to vertebrate fibronectin family and that the TK4 binding on cancer cells can be inhibited by fibronectin (), suggesting that TK4 may have targeted tumors via fibronectin-binding site(s). Fibronectin is a glycoprotein which serves as one of the major fractions of ECM. It contains an array of repeating domains (FN1, 2, and 3) that mediate interactions between cells, other ECM components (e.g., collagen), and other fibronectin molecules [15
]. The tumor-targeting motif RGD, which serves as a common binding site for a variety of integrins, has been originally derived from the tenth FN3 repeat in fibronectin [15
]. Besides RGD, other peptide sequences have been exploited from FN domains with cell-binding activity such as the essential α4 β1
adhesion motif LDV [23
], the 25-mer endothelial cell adhesion peptide CS1 [24
], and the mesenchymal stem cell-adhesive peptide ALNGR that interacts with integrin β1
]. These studies would suggest that due to the pivotal role of fibronectin in the complex ECM architecture, novel binding motif residing in FN sequences or their homologous domains, like TK4, may be still discovered. We have recently found that TK4 binds efficiently to human lung cancer cell lines and that the binding can be also inhibited by fibronectin pretreatment (data not shown). Thus one of our future works will be to evaluate the potential of TK4 as a multitumor-targeting probe. Identifying the cell surface receptor(s) of TK4 on tumor cells, certainly, is crucial and should be performed in the future.
In conclusion, our data presented here showed that TK4, a novel peptide isolated from phage display library, showed a homology to FN3 domain and can target fibrosarcoma tumor cells in vivo after systemic administration. TK4 holds promise as a lead structure useful for selective delivery of therapeutics or diagnostic probe to fibrosarcoma or other tumors.