A major difference between dyes for in vitro and in vivo use are the wavelengths of emission and excitation. Typically, fluorescent dyes for in vitro applications excite and emit in the visible range, at wavelengths between 400 nm and 600 nm [2]. However, light in the visible range does not have deep tissue penetration because of autofluorescence and the scattering nature of tissue as well as high levels of endogeneous absorbers such as hemoglobin, deoxyhemoglobin, water and lipids [2 – 4]. Therefore, fluorescent probes for in vivo studies commonly excite and emit in the near-infrared (NIR) range of light, at wavelengths between 700 nm and 1000 nm, at which these effects are less pronounced. These dye labeled molecules are also called near-infrared fluorescence (NIRF) imaging probes [2, 4].

Investigating Proteases Involved in Cancer

It is already well established that receptor-binding peptides can serve as carriers for labels and cytotoxic cargo for visualization or treatment of tumors, adopting Paul Ehrlich`s concept of the “magic bullet” [5, 6]. In contrast, protease inhibitors have failed thus far as cancer drugs, although our understanding of the tumor “degradome”, the repertoire of proteases and their natural inhibitors and interaction partners, advanced. Knowing which proteases are active in the tumor micro-environment may render it possible to tackle cancers with the use of protease-activated prodrugs (PAPs) [7]. Dye labeled peptides are used as substrates for assaying the activity of proteases associated with the development and progression of cancer, and some examples are discussed in the following.

Matrix metalloproteinases (MMPs) are a family of related calcium-dependent zinc-containing endopeptidases, subdivided on the basis of substrate selectivity, domain assembly and conservation into collagenases, gelatinases, stromelysins, matrilysins, membrane-type and other MMPs [8]. The family has at least 28 members in vertebrates, with 24 members in mammals and 23 members in human. Although they are indispensable for numerous physiological processes, they also carry out angiogenesis, promote tumor development and thus contribute to the spreading of cancer cells within the body [8].

For measuring the activity of MMPs, a convenient and sensitive approach is Fluorescence (or Förster) Resonance Energy Transfer- or “FRET”-measurement. FRET-substrates typically contain a fluorophore (donor), which is excited with light from an adequate source. The donor fluorescence is absorbed and thus attenuated (quenched) by a second, non-emitting chromophore (quencher), separated from the donor by the cleavage site. Upon proteolytic cleavage, the quenching is relieved and a signal can be measured as fluorescence increase [9 – 11]. As variations on this, the fluorescence of amino acids like tryptophan, contained in the peptide substrate sequence itself can serve as donor, described for example in a report about the optimization of collagenase substrates [12], or, the second chromophore can be fluorescent. Today, a broad range of fluorescent peptide substrates is available as catalog products, with diverging kinetic values for different MMPs.

Cysteine-dependent aspartate specific proteases (caspases) are key players in apoptosis, and their activation is generally considered as the “point of no return” in apoptotic pathways. Excessive apoptosis can lead to neurodegenerative diseases and ischemic injuries, whereas cancer and autoimmune diseases can result from insufficient apoptosis [13, 14]. Fluorescent labeled caspase substrates typically contain four amino acid residues with a stringent preference for an aspartic residue left (towards the N-terminus) and a methylamine right (towards the C-terminus) from the cleavage site, as described for instance for the caspase interleukin-Iβ-converting enzyme (ICE) [15]. However, many variations of this sequence exist in order to increase the specificity for particular caspases, and only a small selection of literature examples can be given here [16 – 18].

Cathepsins are a family of lysosomal proteases, including cysteine, serine and aspartic proteases. They cleave their substrates in a relatively unspecific manner and are involved in numerous physiological and pathological processes, the latter including cancer [19]. Cathepsins play a role in cell invasion and metastasis, and are considered as targets for cancer therapy [20]. A dye labeled peptide was developed for example as a fluorogenic substrate for cathepsin D, which has been implicated in pathological processes such as Alzheimer`s Disease and breast cancer. The substrate is designed for FRET-experiments and resulted from a larger set of synthesized substrate sequences, which were evaluated against the background of enzymatic turnover kinetics [21].

Labeled RGD Peptides

The arginine-glycine-aspartic acid (RGD) sequence was discovered as a cell attachment site in fibronectin. Nowadays, RGD-peptides have become a popular tool for imaging αvβ3-integrin expressing tumor vasculature [22]. Cyclization is commonly employed to improve the binding properties and stability of RGD peptides [22]. The drug cilengitide, relying on a cyclic RGD peptide structure, was tested for treatment of glioblastoma, although finally did not alter patterns of progression in clinical trials [23].

As a recent development, proprietary Tide Fluor™ dyes are incorporated into cyclic RGD sequences. This group of dyes shows efficient excitation with light from common light sources and exhibits stronger fluorescence and significantly higher photo-stability, than most conventional or proprietary dyes. For example, a Tide Fluor™ labeled, cyclic RGD peptide is available, absorbing and emitting light in the NIR range.

Conclusion

There is an urgent demand for efficient and specific probes for imaging and assaying of molecules in cancer research. Fluorescence technology is applied in a broad range of fields, including culture assays, cell imaging, tissue staining, and in vivo studies. Dye labeled peptides as fluorescent substrates are particular valuable tools for the investigation of protease activity, leading to the formation of tumors or progression of cancer, and hence may represent a key technology for the development of cancer drugs. Numerous fluorescent peptide substrates are described in the literature and available as ready-made products. As a newer development, cyclic RGD peptides labeled with particularly strong emitting and stable dyes may contribute significantly to our understanding of cancer.

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References

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(3) S. Achilefu, Lighting up tumors with receptor-specific optical molecular probes. Technol. Cancer Res. Treat., 3(4), 393-409 (2004)

(4) J. Rao et al., Fluorescence imaging in vivo: recent advances. Curr. Opin. Biotechnol., 18(1), 17-25 (2007)

(5) P. Ehrlich, The collected papers of Paul Ehrlich, F. Himmelweite, M. Marquardt, and H. Dale, Editors. 1956, Pergamon, Elmsford, New York. p. 596-618.

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(7) J. Vandooren et al., Proteases in cancer drug delivery. Adv. Drug Deliv. Rev., 97144-55 (2016)

(8) A. Khalid and M.A. Javaid, Matrix metalloproteinases: new targets in cancer therapy. J. Cancer Sci. Ther., 8(6), 143-153 (2016)

(9) D.M. Bickett et al., A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9). Anal. Biochem., 212(1), 58-64 (1993)

(10) B. Beekman et al., Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay. FEBS Lett., 418(3), 305-9 (1997)

(11) M.O. Palmier and S.R. Van Doren, Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect. Anal. Biochem., 371(1), 43-51 (2007)

(12) J. Berman et al., Rapid optimization of enzyme substrates using defined substrate mixtures. J. Biol. Chem., 267(3), 1434-7 (1992)

(13) S. Shalini et al., Old, new and emerging functions of caspases. Cell. Death Differ., 22(4), 526-39 (2015)

(14) S.M. Man and T.D. Kanneganti, Converging roles of caspases in inflammasome activation, cell death and innate immunity. Nat. Rev. Immunol., 16(1), 7-21 (2016)

(15) N.A. Thornberry et al., A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature, 356(6372), 768-74 (1992)

(16) D.W. Nicholson et al., Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature, 376(6535), 37-43 (1995)

(17) J. Xiang et al., BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA, 93(25), 14559-63 (1996)

(18) P.S. Schwartz and D.J. Waxman, Cyclophosphamide induces caspase 9-dependent apoptosis in 9L tumor cells. Mol. Pharmacol., 60(6), 1268-79 (2001)

(19) S. Conus and H.U. Simon, Cathepsins and their involvement in immune responses. Swiss Med. Wkly., 140w13042 (2010)

(20) T. Nomura and N. Katunuma, Involvement of cathepsins in the invasion, metastasis and proliferation of cancer cells. J. Med. Invest., 52(1-2), 1-9 (2005)

(21) S.V. Gulnik et al., Design of sensitive fluorogenic substrates for human cathepsin D. FEBS Lett., 413(2), 379-84 (1997)

(22) K. Temming et al., RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature. Drug Resist. Updat., 8(6), 381-402 (2005)

(23) G. Eisele et al., Cilengitide treatment of newly diagnosed glioblastoma patients does not alter patterns of progression. J. Neurooncol., 117(1), 141-5 (2014)