Article pubs.acs.org/bc
Quinone Methide Signal Amplification: Covalent Reporter Labeling of Cancer Epitopes using Alkaline Phosphatase Substrates Nathan W. Polaske,* Brian D. Kelly, Julia Ashworth-Sharpe, and Christopher Bieniarz Ventana Medical Systems, Inc., 1910 East Innovation Park Drive, Tucson, Arizona 85755, United States S Supporting Information *
ABSTRACT: Diagnostic assays with the sensitivity required to improve cancer therapeutics depend on the development of new signal amplification technologies. Herein, we report the development and application of a novel amplification system which utilizes latent quinone methides (QMs) activated by alkaline phosphatase (AP) for signal amplification in solid-phase immunohistochemical (IHC) assays. Phosphate-protected QM precursor substrates were prepared and conjugated to either biotin or a fluorophore through an amine-functionalized linker group. Upon reaction with AP, the phosphate group is cleaved, followed by elimination of the leaving group and formation of the highly reactive and short-lived QM. The QMs either react with tissue nucleophiles in close proximity to their site of generation, or are quenched by nucleophiles in the reaction media. The reporter molecules that covalently bind to the tissue were then detected visually by fluorescence microscopy in the case of fluorophore reporters, or brightfield microscopy using diaminobenzidine (DAB) in the case of biotin reporters. With multiple reporters deposited per enzyme, significant signal amplification was observed utilizing QM precursor substrates containing either benzyl difluoro or benzyl monofluoro leaving group functionalities. However, the benzyl monofluoro leaving group gave superior results with respect to both signal intensity and discretion, the latter of which was found to be imperative for use in diagnostic IHC assays.
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INTRODUCTION As we broaden our understanding of cancer and the complexity of the protein and gene expression pathways that define it, the ability to confidently detect cellular markers of low abundance becomes increasingly important for diagnostic purposes. Since its introduction, catalyzed reporter deposition (CARD)1,2 utilizing tyramine-labeled biotin and fluorophores has been an important tool for signal amplification in many immunoassays that aid in the diagnosis of cancer. Also referred to as tyramide signal amplification (TSA), this technique takes advantage of the reaction between horseradish peroxidase (HRP) and tyramine, where in the presence of H2O2, tyramine is converted to a highly reactive and short-lived radical intermediate that reacts preferentially with electron-rich amino acid residues on proteins.3 The covalently bound reporter labels can then be detected either by one of a variety of avidin−biotin−enzyme complex visualization techniques,4 or by fluorescence microscopy.5,6 While initially introduced for use in enzyme-linked immunosorbent assays (ELISA)1 and Western blotting,2 this powerful methodology was later adapted for use in solid-phase immunoassays such as immunohistochemistry (IHC)4−6 and in situ hybridization (ISH)7 on formalin-fixed paraffin embedded (FFPE) tissue. In these applications, where spacial and morphological context is highly valued, the short lifetime of the radical intermediate results in covalent binding of the © XXXX American Chemical Society
tyramide to proteins on tissue in close proximity to the site of generation, giving discrete and specific signal. While CARD broadly defines the use of an analyte-dependent reporter enzyme (ADRE) to catalyze the covalent binding of numerous reporter labels to proteins,1 HRP-based TSA remains the sole methodology described under the CARD umbrella. Herein, we report the development and application of Quinone Methide Signal Amplification (QMSA), an alkaline phosphatase8,9 (AP)-based CARD, which utilizes latent quinone methides10 (QMs) activated by AP for signal amplification in solid-phase IHC assays. Enzyme substrates capable of forming QMs were initially investigated as potential mechanism-based inhibitors of hydrolase-type enzymes.11−18 Later, they were employed as selfimmobilizing probes for enzyme activities, including phosphatases,19−34 sulfatases,35,36 glycosidases,26,37−45 lipases,46 and βlactamases.47 While the majority of these examples involved the detection of enzyme activity in solution or on Western blot, a recent publication from Withers reported fluorescent histological staining on plant tissue utilizing coumarin glycosides modified to generate QMs upon reaction with their cognate endogenous Received: December 1, 2015 Revised: January 4, 2016
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Figure 1. (A) Mechanism of AP-based CARD as applied to IHC. (B) Example of visualization of covalently bound biotin reporters using 3,3′diaminobenzidine (DAB) detection.
Scheme 1. Synthesis of Biotin-Labeled Quinone Methide Precursors 1 and 2a
a
Reagents and conditions: (a) diethyl chlorophosphate, TEA, EtOAc; (b) Deoxo-Fluor, CH2Cl2; (c) Pd/C, H2, EtOAc; (d) N-Boc-6-aminohexanoic acid, EDAC, DMAP, DMF; (e) TMSBr, CH2Cl2; (f) biotin NHS ester, TEA, DMF; (g) NaBH4, THF, MeOH.
glycosidase.44 This report demonstrated the potential of QMs for the covalent labeling of solid-phase proteins with minimal diffusion from the site of generation, which is imperative for solid-phase immunoassays and an important feature of the current TSA technology. A key limitation of the Withers probe is the requirement for each reporter molecule to be modified synthetically to contain a QM precursor functionality. In addition to adding significant cost and complexity, the necessity for modification may render many reporters unusable. We proposed a single QM precursor scaffold containing an amine-functionalized linker group allowing for simple conjugation to nearly any reporter molecule. Given our primary application of CARD for solid-phase immunoassays such as IHC on FFPE tissue,48 the use of the phosphate group as the enzyme-
cleavable recognition head was a logical choice due to the ubiquity of its cognate enzyme AP in current immunoassays.49 The application of AP-based CARD for IHC begins with the incubation of a primary antibody (Ab) that recognizes the antigen of interest, followed by incubation with a secondary Ab:AP complex that binds the primary Ab by typical antispecies Ab binding (Figure 1). The reporter-labeled QM precursor is then applied, resulting in the recognition and hydrolysis of the phosphate group by AP, elimination of the leaving group (fluoride), and finally the formation of QMs. These QMs either react with immobilized nucleophiles in close proximity to the site of generation, or are quenched by nucleophiles in the reaction media. The reporter molecules that covalently bound to the tissue are then detected by fluorescence microscopy in the case of B
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Figure 2. AP-based CARD IHC (BCL6 on FFPE tonsil tissue) using biotinylated difluoro QM precursor 1 (A thru F) and biotinylated monofluoro QM precursor 2 (G thru L) followed by DAB detection. Conditions for 1 (A thru F): 20 μM 1 in TRIS buffer with varying pH: (A) DAB control; (B) pH = 7.0; (C) pH = 8.0; (D) pH = 9.0; (E) pH = 10.0; (F) pH = 11.0. Conditions for 2 (G thru L): 250 nM 2 in TRIS buffer with varying pH: (G) DAB control; (H) pH = 7.0; (I) pH = 8.0; (J) pH = 9.0; (K) pH = 10.0; (L) pH = 11.0.
Figure 3. (A) Fluorescent IHC staining (Ki67 on FFPE tonsil tissue) with the monofluoro QMP-Cy5 conjugate. The blue box marks an area of positive signal, while the red box marks an area of background signal. The graph compares the positive and background signals, along with the signal to background ratio, at different QMP-Cy5 staining concentrations. Conditions: 5 μM monofluoro QMP-Cy5 in TRIS buffer pH 10. (B) Fluorescent duplex IHC staining (Ki67 and E-cadherin on FFPE breast tissue). Ki67 (nuclear stain, false-colored red) was stained using QMSA and AP substrate QMP-Cy5; E-cadherin (membrane stain, false-colored green) was stained using TSA and the HRP substrate tyramine-TAMRA.
fluorophore reporters (Figure 1A), or by one of a variety of brightfield microscopy visualization techniques such as 3,3′-
diaminobenzidine (DAB) in the case of hapten reporters (Figure 1B). C
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RESULTS AND DISCUSSION Design, Synthesis, and Evaluation of QM Precursors. In evaluating our QM precursors for IHC staining, we found the
observed compared to the DAB control sample when on-slide concentrations of 1 greater than 20 μM were utilized. However, increased diffusion of signal away from the cell nuclei was also observed in all cases, resulting in considerable off-target staining. We speculated that the diffusion of signal afforded by QM precursor 1 arose from a combination of two kinetic factors: (1) the rate of leaving group elimination and subsequent QM formation after the cleavage of the phosphate group, and (2) the rate of QM quenching, either by a nucleophile on the tissue or in the reaction media. In the case of the difluoro QM precursor 1, the geminal fluorine atom provides stabilization that may decelerate both factors, resulting in unacceptable diffusion from the target site. We theorized that by increasing the pH of the reaction media, the rates of both QM formation and quenching may be accelerated. For (1), a more alkaline pH would increase the population of deprotonated phenol after phosphate cleavage, therefore encouraging leaving group elimination and QM formation. For (2), increased pH would raise the population of available quenchers from both the water and the buffer (TRIS), effectively decreasing the distance the QMs would be able to travel. To test the effect of pH on signal diffusion, difluoro QM precursor 1 was deposited as before at various pH levels within the working range for AP (7−11 with two shoulders of maximal activity at pH = 8.5 and 11).51 The nuclear marker BCL6 on FFPE tonsil tissue was again chosen as the model. Positive staining was observed across the entire pH range, although a balance between overall signal and diffusion was seen (Figure 2A−F). As expected, the diffusion consistently decreased with increasing pH. At a pH of 7, almost no on-target staining was observed with a nearly homogeneous signal seen across the entire tissue section. As pH rose, the off-target staining gradually decreased, although overall signal diffusion and off-target staining remained significant even at a pH of 11. The level of amplification increased only as the pH rose to 8.5 and then decreased gradually over the rest of the range. This effect may be due to a combination of enzyme activity and the population of QM quenchers in the reaction media. At pH levels well below the optimal AP reaction conditions (i.e., 7.0), the activity of AP is depressed, resulting in low signal. As pH rises (8.0) but remains near the pKa of TRIS (8.1), the activity of AP increases at a faster rate than the population of quenchers in the reaction media, leading to a significant increase in signal. Raising the pH further (9−10) increased the quencher population while decreasing AP activity, resulting in a decrease of signal. Although AP has maximal activity at pH 11 in TRIS, we observed a slight decrease in staining intensity most likely due to the excessively high population of quenchers that could not be overcome by the increased AP activity (raising the pH from 8.5 to 11 increases [TRIS base] ∼50%, with AP activity increasing by only ∼20%). Unfortunately, even at the upper end of the pH range, the diffusion from the difluoro QM precursor 1 was still too great to be of practical use for IHC. We hypothesized that by utilizing the less stable monofluoro QM precursor 2, diffusion may be reduced for reasons described above. IHC staining was first carried out under typical AP IHC condition (pH 8.5 in TRIS buffer) across a wide concentration range again using the nuclear marker BCL6 on FFPE tonsil tissue to determine the optimal concentration of monofluoro QM precursor 2. Surprisingly, a much lower concentration of monofluoro QM precursor 2 (250 nM) was required to give the desired level of amplification when compared to difluoro QM precursor 1 (20 μM). In addition, overall diffusion was greatly reduced compared to difluoro QM
Figure 4. Trapping of the QM intermediate 10 from monofluoro QM precursor 2 by TRIS. (A) HPLC chromatogram of 2 in TRIS pH 10 at t = 0; (B) HPLC chromatogram of 2 in TRIS pH 10 at t = 10 min; (C) HPLC chromatogram of the reaction of 2 with AP in TRIS pH 10 at t = 10 min.
identity of the leaving group to be of paramount importance. We prepared two phosphate-protected ortho-QM precursors based on 4-nitrosalicylaldehyde starting material containing benzyl difluoro and benzyl monofluoro functionalities (Scheme 1). Both compounds utilize fluoride as the leaving group, but the reactivity of the QM derived from difluoro 1 should be considerably lower than monofluoro 2 due to the electronic stabilization offered by the geminal fluorine atom. In fact, previous reports have suggested QMs derived from some monofluoro precursors may be excessively reactive as probes of enzyme activity, resulting in active site labeling and subsequent enzyme inhibition.18,19,44 Therefore, we first evaluated the difluoro QM precursor 1 for IHC staining performance on FFPE tissue using the B-cell lymphoma marker BCL6 on tonsil as a model system.50 The biotin-labeled difluoro QM precursor 1 successfully bound to the sample as evidenced by subsequent biotin visualization using DAB detection. In addition, significant amplification of signal was D
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suggesting that the adduct 11 was formed from the attack of the 1° amine of TRIS on the QM 10, rather than direct displacement of the fluoride of 2 (Figure 4B). These results strongly support the proposed mechanism of QM-mediated staining and implicates TRIS base as the primary source of quenchers under the IHC staining conditions.
precursor 1, although some diffusion and off-target staining were still evident at pH 8.5 (Figure 2G−L). In an effort to reduce diffusion and off-target staining, we experimented across a wide pH range (7−11) in TRIS buffer. At low pH (7.0−8.0), significant diffusion was seen, creating a stain similar to those observed with the difluoro QM precursor 1. However, diffusion and off-target staining were gradually reduced as pH rose, without an unacceptable decrease in overall signal. At a pH of 10, diffusion and off-target staining were nearly eliminated, producing a stain with a high level of signal along with comparable specificity to the DAB control. Increasing the pH further to 11 resulted in slightly cleaner stains as evidenced by the slightly more vibrant blue hematoxylin counterstain. However, a slight reduction in overall signal was observed at these higher pH levels. Fluorescent IHC Staining. In an effort to expand the utility of the QMSA approach, a fluorescent IHC assay was also developed. We prepared a monofluoro QM precursor-cyanine 5 (QMP-Cy5) conjugate by reaction of compound 9 with Cy5NHS ester and performed the IHC assay as shown in Figure 1A. We chose a pH of 10.0 as it appeared to give the best combination of overall signal intensity and discretion in our previous experiments with compound 2. As the concentration of QMP-Cy5 was increased (>10 μM), the overall signal intensity began to plateau (Figure 3A). However, the staining pattern for individual cells appeared membraneous and not the expected nuclear stain for Ki67 (see Supporting Information). This suggests that the labeling density was increasing to the point that self-quenching of the Cy5 dyes was occurring. Not surprisingly, when higher concentrations of the QMP-Cy5 conjugate were employed, higher background signal was also detected. The background and self-quenching were easily mitigated by lowering the concentration, to give an optimal balance of signal-to-noise at 1−5 μM. Multiplex Fluorescent IHC with QMSA and TSA. A major advantage of expanding the number of enzymes available for CARD is the ability to perform multiplexed detection without enzyme inactivation or antibody elution steps. Assays using multiple rounds of TSA-fluorophores are well reported52 but typically require multiple sequential detection steps and treatments to inactivate (or elute) the HRP between each target detection. This complicates the assay scheme and in some cases exposes the fluorophores to highly oxidative conditions.53 To demonstrate an amplified duplex detection without inactivation steps we choose membrane marker E-cadherin (mouse antibody) combined with nuclear marker Ki67 (rabbit antibody). In this experiment, coincubation of the primary antibodies was followed by simultaneous binding of an anti-rabbit AP conjugate and an anti-mouse HRP conjugate. TSA detection of E-cadherin with tyramide-TAMRA preceded QMSA detection of Ki67 with QM-Cy5 to eliminate the exposure of Cy5 to oxidizing conditions (Figure 3B). The ability to use two orthogonal detection chemistries and enzymes increases efficiency, decreases time, and simplifies the design of the assay. QM Trapping Experiments. To probe the mechanism of staining and quenching, we attempted to trap the QM intermediate formed by monofluoro QM precursor 2 in solution-phase under similar conditions to those utilized in the staining experiments. QM precursor 2 was dissolved in pH 10 TRIS buffer followed by addition of a catalytic amount of AP. The reaction progress was monitored by HPLC-MS. Within 10 min, complete conversion to the TRIS adduct 11 was observed (Figure 4C). In the absence of AP, no reaction took place,
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CONCLUSIONS We have presented the first example of an AP-based CARD methodology applied to IHC utilizing latent QMs that covalently bind to nucleophilic residues on FFPE tissue. Both biotin and fluorophore labeled QM precursors derived from a single monofluoro QM precursor scaffold have been described. As a result, we have expanded on the powerful peroxidase-based CARD technology to include enzymes other than HRP. Due to the highly amplified and specific signal, we envision the QMbased CARD methodology having a significant impact in the diagnostics field. Efforts to further expand on the capabilities of QMSA to include additional enzymes are currently underway in our laboratory.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00652. Synthetic details and characterization data for all new compounds, experimental details for IHC tissue staining, and expanded fluorescent staining data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
All authors are full time employees of Ventana Medical Systems, Inc. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank Dr. Ben Stevens for his guidance in imaging and analysis of the fluorescently stained tissue slides. REFERENCES
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