Fluorescence spectroscopy: An early detection tool for cancer.

Millions of people are afflicted with cancer each year. 2016 statistics for the USA alone show over half a million deaths attributed to cancer 1. We have come a long way from our early understanding of cancer, and now have highly sophisticated and personalized treatments available in the 21st century. One of the major challenges facing effective treatment is detection time. Cancer detected early has a higher chance of being cured compared to a late prognosis in most cases. Modern medicine uses several tests such as MRI and CT scans, ultrasound, pathology reports etc. to gather as much information as possible. According to American Cancer Society, it takes a minimum of a few weeks from diagnosis to starting treatment. This article proposes a novel mechanism that uses fluorescence spectroscopy as a rapid, early detection tool for most solid tumor carcinomas.

Fluorescence is a form of luminescence, and as such occurs when a substance absorbs light resulting in its electrons being excited, which return to ground state by emitting a photon. This property is exhibited by compounds called fluorophores. Sir John Frederick William Herschel first reported the phenomenon from a quinine solution in 1845. Since then, the field has rapidly advanced, and is now used extensively in biomedical sciences to study molecular interactions in real time. One extensively used technique from the fluorescence toolbox is Forster Resonance Energy Transfer (FRET). Discovered and readily applied in 1950’s, it has become really popular in the last two decades. Briefly, FRET results in a non-radiative energy transfer from one fluorophore to another when the two are within 10 nm of each other. The excited fluorophore transferring its energy is called the “donor” and the fluorophore accepting the energy is called the “acceptor”. Several well characterized FRET pairs are available today in the market for various biomedical assays. The advantage of using fluorescence is the sensitivity to detect even single molecule interactions in real time. This characteristic can be used to our advantage to develop a tool to detect one of the several biomarkers associated with cancer, such as Matrix Metalloproteases (MMP’s).

Matrix metalloproteases are a family of zinc dependent peptidases. So far, over 26 MMP’s have been identified. They are collectively involved in degrading the extracellular matrix for tissue turnover and growth, and are normally found in nominal amounts in healthy adults. However, their levels increase significantly during cancer leading to increased degradation of the extracellular matrix, thus furthering extravasation8. Of these MMP’s, Matrix metalloproteinase-9 (MMP-9) has been implicated to have elevated levels in several solid state cancers, including bladder, breast, prostate, kidney, liver etc. There is also a correlation shown between the levels of plasma and urine MMP-9 levels and progression of cancer in cases of bladder, gastric, colon and hepatocellular carcinoma. Combining this information with the sensitivity of fluorescent detection, it is possible to come up with a FRET pair that can accurately predict increased levels of MMP-9 in patients, thereby serving as an early detection tool that is also very rapid.

Challenges: 1: The FRET pair has to be recognized and cleaved exclusively by MMP-9. 2: Additionally the reaction should be finished within a suitable time frame. 3: Finally, the fluorescent signal from the pair should be clear of any background noise from biological components.

Approach:

Specificity: Kridel et al. developed a peptide sequence Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2 that is recognized and cleaved exclusively by MMP-914. The peptide is small, and provides lysine residues to establish a strong bond between the FRET pair and peptide and recognizes only MMP-9 out of the entire MMP family. The small size ensures that the fluorophores are within the specified distance of 10nm of each other. Since the FRET pair is attached to a peptide that is recognized and cleaved exclusively by MMP-9, the rate of changes in the emission of FRET would be a direct consequence of enzyme activity. In other words, a higher concentration of MMP-9 would result in rapid cleavage of the peptide compared to a lower concentration. If the FRET probe concentration was standardized, it would be very easy to establish a standard curve of FRET changes directly corresponding to plasma MMP-9 levels.

The FRET pair: In addition to the fluorophores being within 10nms of each other, a successful FRET pair also requires a good overlap between the absorption spectra of the donor and the emission spectra of the acceptor. This ensures optimum energy transfer and a good signal from the acceptor. As the peptide with the FRET pair is cleaved by the enzyme, the distance between the donor and acceptor fluorophore will increase, leading to a gradual decline in transfer efficiency to the acceptor, and an increased signal from the donor fluorophore. We selected TAMRA as the donor and HiLyte647 as the acceptor for our FRET pair (Figure 1). Both fluorophores are commercially available, well characterized, have a good overlap between their absorption and emission spectra, good quantum yield, and have an emission signal from 500nm-600nm, thus avoiding fluorescence noise from the biological background.

Figure 1: A representation of the FRET pair (5/6 TAMRA and HiLyte 647) attached to the lysine residues of peptide sequence synthesized by Kridel et. al

Figure 2: Fluorescence spectra of FRET pair in the presence and absence of 2.5nM of MMP-9 enzyme. The sample incubated with the enzyme shows a significant increase in donor intensity after 3 hours (green and red) whereas the sample with no enzyme shows no changes in its emission spectra (black).

Proof of concept: Two cuvettes measuring 1cm x 1cm were prepared with equal concentrations of the FRET probe (Anaspec, NJ) dissolved in phosphate buffer saline (PBS). 2.5 nM of MMP-9 enzyme (Calbiochem, CA) was added to one cuvette and the other was used as control with no enzyme. Both cuvettes were incubated at 370C for 3 hours and their fluorescence measured at regular intervals. The excitation was 490nm to selectively excite only the donor fluorophore to observe energy transfer to acceptor. At the end of 3 hours, the FRET pair incubated with 2.5 nM of MMP-9 showed a significant decrease in energy transfer and an increase in donor intensity; whereas the control showed no changes in its emission spectra (Figure 2).

The future: Rich et al. demonstrated a mechanism for signal enhancement while further characterizing micro transponders a.k.a. RFID p-chip. These chips already serve as platforms for quantification of low abundance biomolecules in nucleic acid based immunoassays16. Coating such chips with a standardized FRET probe (Figure 3) can result in a precise, rapid and potentially early warning system for most cancers using MMP-9 enzyme levels as a biomarker.

Figure 3: A schematic representation of RFID chips developed (500 µM) for the FRET probe. 1:photocells, 2:antenna, 3:memory, 4:logic, 5:registration marks.

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