Signal Amplification for Imaging Mass Cytometry

Article abstract of DOI:10.1021/acs.bioconjchem.9b00559

An enzyme-catalyzed reporter deposition stain has been developed for Imaging Mass Cytometry (IMC). The reagent consists of an alkaline phosphatase substrate tethered to a tellurophene which serves as reporter group for mass cytometry. Upon phosphate hydro

Full text of DOI:10.1021/acs.bioconjchem.9b00559

Article
pubs.acs.org/bc
Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
Signal Amplification for Imaging Mass Cytometry
†
‡
†,§
Rahul Rana, Rodolfo F. Gomez-Biagi, Jay Bassan, and Mark Nitz*,†
́
†Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada
‡SPARC BioCentre−The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, 686 Bay Street, 21st
Floor, Toronto, ON M5G 0A4, Canada
§BIMDAQ Ltd, 9 Lessness Avenue, Bexleyheath DA7 5SH, United Kingdom
S
* Supporting Information
ABSTRACT: An enzyme-catalyzed reporter deposition stain has
been developed for Imaging Mass Cytometry (IMC). The
reagent consists of an alkaline phosphatase substrate tethered to a
tellurophene which serves as reporter group for mass cytometry.
Upon phosphate hydrolysis, a quinone methide is released which
covalently labels local nucleophiles. This strategy is a useful
complement to heavy isotope antibody conjugates as it facilitates
signal amplification for low-abundance biomarker detection. The workflow is conveniently integrated with standard IMC
antibody staining to allow multiparametric antigen detection.
ass cytometry (MC) has enabled highly multiparametric
single-cell analysis (>40 parameters/cell) which has
MC experiments, and this approach shows promise with
primary antibody conjugates.17 Here we sought to introduce
an IMC-compatible enzyme amplification strategy which
would improve the sensitivity of IMC while not compromising
mass channels commonly used with antibody conjugates. An
enzyme amplification strategy would be compatible with
standard secondary immunohistochemistry (IHC) antibody
protocols, allowing direct translation and comparison between
existing analyses and IMC analysis. In addition, primary
antibodies not bearing a mass tag can readily be incorporated
into characterized commercial MaxPar libraries.
AP-conjugates are ubiquitous in immunoassays with
numerous well-characterized synthetic substrates.18,19 Latent
quinone methide (QM) derivatives, developed originally as AP
covalent inhibitors,20,21 have been used to covalently label
phosphatases in solution for visualization.22−26 Kinetic analysis
and product analysis of QMs generated enzymatically suggest
these products diffuse away from the enzymes active site and
label local nucleophiles.16,27 In the case of an AP antibody
conjugate, the QM released will be immobilized by local
nucleophiles, in close proximity to the site of QM generation
(Scheme 1). Provided adequate turnover can be realized, a
significant degree of signal amplification with minimal diffusion
can be achieved. This improves both the limit of detection and
dynamic range of signal. Thus, we developed a latent-QM AP
substrate bearing a tellurophene (QMTe) as a universal
reagent for use with AP-conjugates in IMC.
M
yielded insight into disease and therapy.1,2 MC uses heavy
isotope mass tags tethered to bioaffinity reagents to identify
biomarkers. The heavy isotopes are quantified through single-
cell inductively coupled plasma mass spectrometry (ICP-MS)
analysis in a workflow analogous to flow cytometry. The mass
resolution of the ICP-MS and the large number of available
heavy isotopes allow highly parametrized experiments with
minimal complications due to signal overlap.3 Similarly,
imaging mass cytometry (IMC) couples these high-dimen-
sional measurements with 2-D resolution via laser ablation of
immunohistochemical and immunocytochemical sections.4
IMC has been successfully used to study immunophenotypic
states,5 study drug distribution in tissues,6 and quantify mRNA
transcripts.7
At present, the catalogue of MC- and IMC-compatible stains
consists of antibodies conjugated with metal-chelating
polymers which bind, with high affinity, the lanthanide series
as well as indium, yttrium, palladium, and bismuth.8−10 Beyond
these bioaffinity reagents, small-molecule MC-compatible
stains have been validated for multiple applications. For
example, DNA cycle studies have been performed using 5-
iodo-2′-deoxyuridine (IdU), and palladium and platinum
complexes have been used as cell barcoding and live dead
stains.11,12 We have introduced tellurophenes as versatile
functionalities to incorporate into small-molecule MC-
compatible reagents.13 Using tellurophenes, probes have
been developed to follow tumor hypoxia,14 protein synthesis,15
and cellular senescence.16
Polaske et al. reported the use of o-monofluoromethyl- and
o-difluoromethyl-substituted fluorescent AP substrates as QM
precursors for IHC.28 While both reagents were able to detect
While the benefits of highly parametrized assays are evident,
MC and IMC are less sensitive than fluorescence, and methods
to improve the detection of low-abundance antigens would
expand the scope of their applications. Recently, nanoparticle
reagents have been introduced to improve the sensitivity of
Received: August 19, 2019
Revised: October 9, 2019
© XXXX American Chemical Society
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DOI: 10.1021/acs.bioconjchem.9b00559
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Article
a
Scheme 1. Mechanism of Amplified Labelling by QMTe
Scheme 2. Synthetic Preparation of QMTe
antigens via an AP secondary antibody, the difluoromethyl
analogue exhibited higher nonspecific staining. This was
attributed to the longer half-life of the difluoromethylphenol,
which allows diffusion from the site of QM generation
compromising resolution. The o-monofluoromethyl QM
precursors gave better resolution, but o-monofluoromethyl is
known to be chemically less stable as it is prone to solvolysis
and undesirable side reactions with thiols and other
nucleophilic buffer components.29 Furthermore, from a
synthetic perspective, the o-monofluoromethyl group is
susceptible to displacement by the o-phosphate during final
deprotection, resulting in a cyclic phosphodiester byproduct
and reduced yields. To circumvent these issues, we prepared p-
phosphobenzylic fluoride scaffold, a latent trapping group that
undergoes a 1,6-elimination mechanism.30,31
a
Reagents and conditions: (a) NBS, AgNO3, acetone, rt, quantitative;
(b) 4-pentynoic acid, CuCl, NH2OH·HCl, 30% aq. BuNH2, 0 °C,
70%; (c) i. TBAF, (1.0 M in THF), THF 50 °C, ii. Te0, NaBH4,
EtOH/H2O, 60 °C, 63%; (d) N-hydroxysuccinimide, T3P (50% wt.
EtOAc), DIPEA, THF, 0 °C, 82%; (e) N-Boc- 2,2′-(ethylenedioxy)-
diethylamine, EDC, HOBt, DMF, rt, 84%; (f) diethylchlorophos-
phate, NEt3, DCM, 0 °C, 66%; (g) XtalFluor-E, NEt3.HF, DCM, −78
°C, 84%; (h) i. TMSBr, DCM 0 °C, ii. 4, NEt3, DMF, rt (18%).
The tellurophene mass reporter group was synthesized
according to previous reports with minor modifications
(Scheme 2).16 After Cadiot−Chodkiewicz coupling of 1 with
4-pentynoic acid, diyne 2 was deprotected with tetrabutylam-
monium fluoride. The crude mixture was treated immediately
with a solution of sodium hydrogen telluride generated from
excess sodium borohydride and tellurium metal. The major
isolated product, 3-(tellurophen-2-yl)propanoic acid (3), was
activated to prepare the corresponding N-hydroxysuccinimide
ester 4. The AP substrate coupling partner was synthesized in
four steps. Briefly, N-Boc-2-(2-aminoethoxy)ethylamine was
coupled to 4-hydroxymandelic acid to afford amide 5. The
phosphate recognition head was then installed on the phenol
using diethylchlorophosphate, yielding intermediate 6. We
screened several fluorination conditions and found XtalFluor-E
gave satisfactory yields of 7. Trimethylsilyl bromide was used
to cleave the phosphodiethyl ester and N-Boc groups, followed
by coupling with 4. QMTe was purified and obtained in 18%
over two steps.
Prior to developing staining protocols, we investigated the
turnover of QMTe by AP. Time course inhibition studies using
p-nitrophenolphosphate to monitor AP activity showed that
increasing concentrations of QMTe (0 to 1 mM) gave no
significant AP inhibition over 1 h incubations with near
complete probe hydrolysis (Supporting Information Figure
S1). These results suggested significant amplification could be
achieved as both the QMTe concentrations and incubation
period were greater than those used in comparable IHC
staining protocols.28 To gain further insight into the fate of the
quinone methide, AP-catalyzed turnover of QMTe was carried
out in TRIS buffer, and the products were analyzed by LC-MS
(Supporting Information Figure S2). The major product peak
observed was consistent with a QM-TRIS buffer adduct
suggesting the dephosphorylated product leaves the enzyme
active site and reacts with local nucleophiles.
To evaluate staining properties of QMTe, commercial
reference standard sections of paraffin-embedded cell line
cores with negative, low, medium, and high expression levels of
PD-L1 (CD274) were stained with a rabbit-anti-PD-L1
primary antibody followed by either a goat-antirabbit-AP and
QMTe or a goat-antirabbit-175Lu MaxPar secondary antibody.
The sections were then analyzed by IMC.
While both methods were unable to discriminate between
negative and low expression PD-L1 cell cores, significant signal
(>2 count average) was observed in these samples with the
QMTe protocol. Control experiments suggest that the signal
observed in the PD-L1(−) sample is due to nonspecific
binding of the GAR-AP conjugate. Experiments are ongoing to
limit this background signal to further improve the antigen
detection limit. However, with the current protocol, clear
discrimination between the low and medium expression levels
were observed using QMTe which could not be discriminated
by current MaxPar reagents (Figure 1). Both detection
antibodies were able to distinguish between medium and
high expression levels, but the absolute values measured from
QMTe are ∼100× higher than those from MaxPar labeling,
indicating appreciable signal amplification. Additionally, the
ratios of PDL1(+++) to PDL(−) signals for both stains are
similar (approximately 40), suggesting QMTe does not
disproportionately label off-target antigens. Taken together,
these data support that QMTe is able to label its target antigen
in a concentration-dependent manner with sensitivity at better
than current secondary detection IMC methods.
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DOI: 10.1021/acs.bioconjchem.9b00559
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Figure 1. IMC images goat anti-rabbit-175Lu(MaxPar) or 125Te (QMTe) channels from PD-L1 negative (−), low (+), medium (++), or high (++
+) slides. Median cellular labeling of segmented cells quantified 175Lu or 125Te counts accordingly. Three independent PD-L1 standard slides were
stained; each represented data points. * = p < 0.05, NS = p > 0.05. Scale bar = 200 μm.
To validate the labeling specificity of QMTe for AP
immunocomplexes, staining in the presence and absence of
the AP-secondary antibody was performed. In this case, a
different tissue source and target antigen were selected to
evaluate the utility of QMTe across varied histological samples
where MaxPar primary stains are not commercially available.
Accordingly, we selected hornerin (HRNR), a member of the
S100 family of extracellular matrix proteins.32 Relative to
normal pancreatic vasculature, HRNR expression is elevated in
pancreatic ductal carcinoma models.33 A pair of PANC-1
xenograft sections from mice were stained with anti-HRNR
primary antibody and a combination of MaxPar 175Lu and AP
goat-antirabbit secondary antibodies or MaxPar 175Lu secon-
dary antibody alone; both sections were stained with QMTe.
Pixelwise quantification showed good positive correlation
between 175Lu and 125Te in the presence of both secondary
antibodies, further confirming that our strategy labels the target
antigen specifically. Low background levels of staining were
observed with QMTe in the absence of the AP-conjugate
secondary, demonstrating selective amplification only in the
presence of AP-conjugate antibodies (Figure 2).
To demonstrate the use of QMTe in a typical IMC
experimental workflow, we analyzed formalin-fixed paraffin-
embedded sections of HCT116 xenograft tumors. Using
QMTe we sought to visualize the localization of carbonic
anhydrase IX (CAIX), a protein known to correlate with
hypoxia in solid tumors, which is found on the border between
necrotic and viable tissue.34,35 The staining intensity using
QMTe and AP-secondary antibody was compared to staining
with the MaxPar 175Lu secondary antibody as described above.
With both secondary detection methods, CAIX staining
displays the expected morphology of localization to hypoxic
tumor regions close to necrosis, but QMTe offered stronger
median signal intensity. Comparing CAIX staining between
protocols, using DNA (193Ir) signal as an internal standard for
tissue thickness, we observed a 100-fold increase in staining
intensity with QMTe over the MaxPar secondary antibody
(Figure 3).
Figure 2. QMTe labeling is specific to AP-conjugates. (Top): two
PANC-1 xenograft sections stained with a rabbit antihornerin primary
antibody. Both sections stained with a goat antirabbit 175Lu (MaxPar)
antibody, but only the left section was stained with an antirabbit-AP.
Both sections were exposed to the QMTe probe. Scale bars 200 μm.
(Bottom): bivariate histograms showing pixelwise 175Lu counts
against 125Te counts.
To demonstrate that QMTe is fully compatible with typical
IMC workflows, we had stained the same HCT116 tissue
sections before QMTe labeling with antibodies against
epithelial cell adhesion molecule (EpCAM, 144Nd), pan-keratin
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Figure 3. QMTe labels CAIX without loss of morphological
information. (a) False color IMC image of HCT116 xenograft
tumor section stained for DNA (193Ir, blue) and CAIX (175Lu MaxPar,
red). Necrotic tumor regions are masked in green. (b) Single-cell
segmented quantification of 175Lu labeling of CAIX in four images
using DNA as an internal control for the thickness of tissue. (c)
Similar image to (a), instead showing CAIX as 125Te signal from
QMTe staining. (d) Quantification of QMTe labeling of CAIX in four
images using DNA as an internal control for the thickness of tissue.
Unstained slides for MaxPar were not stained with QMTe and vice
versa and represent background signal. Mean and standard deviations
are shown. *** = p < 0.001, NS = p > 0.05. Scale bars 500 μm.
Figure 4. QMTe is compatible with a typical IMC workflow. Five
channels from a traditional IMC analysis of HCT116 tumor xenograft
are shown: DNA (193Ir), EpCAM (144Nd), pan-keratin (162Dy), Ki67
168Er), and VEGF (163Dy). CAIX is shown by either (a) MaxPar
(
162Dy), Ki67 (168Er), and vascular endothelial growth factor
(VEGF, 163Dy). After QMTe labeling, DNA was also stained
193Ir). Visually comparing antigen labeling and tissue
(
secondary detection (175Lu) or (b) QMTe (125Te). Scale bars 500
μm.
(
morphology, we observed no difference in DNA or antibody
staining between tissue stained with QMTe and tissue stained
with the 175Lu MaxPar secondary antibody, demonstrating that
QMTe staining does not perturb the binding of other
antibodies or the DNA stain. Furthermore, the morphology
of CAIX stained by QMTe is visually comparable to the
morphology of CAIX stained by MaxPar, suggesting that any
loss of spatial precision by signal amplification is below the 1
μm resolution of the IMC instrument (Figure 4).
In conclusion, we report herein a novel organotellurium
reagent for IMC using a secondary detection method. QMTe
seamlessly integrates with the current IMC workflow, allowing
access to mass channels orthogonal to the current inventory of
MC imaging reagents. By using AP turnover, QMTe was
observed to give amplifications of 100-fold relative to
traditional IMC stains. Further increases in signal can be
realized through the generation of isotopically enriched QMTe
reagents and potentially through optimization of the
amplification protocol for specific tissues and the epitope in
question. The improved signals observed may be particularly
useful when quantifying low-abundance antigens. Similar to
any IHC stains, measurements using QMTe should be further
validated by including proper antigen standards during tissue
ablations. Furthermore, QMTe can be employed as a universal
stain with an AP-secondary antibody conjugate to explore new
antigens prior to developing new MaxPar reagents.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconj-
chem.9b00559.
■
S
General experimental details, synthetic procedures,
NMR and MS characterization, enzyme kinetics, LC-
MS traces, slide staining procedures, data acquisition,
and data analysis methods (PDF)
Unprocessed IMC image files have been deposited in
the Open Science Framework, https://osf.io/unhzj/.
AUTHOR INFORMATION
Corresponding Author
*E-mail: mark.nitz@utoronto.ca.
ORCID
Mark Nitz: 0000-0001-8078-2265
Notes
Imaging Mass Cytometry, IMC, and Maxpar are trademarks
and/or registered trademarks of Fluidigm Corporation in the
United States and/or other countries. For Research Use Only.
The authors declare the following competing financial
interest(s): J.B. owns stock in BIMDAQ Ltd. M.N. owns IP
on the use of Tellurium in MC.
■
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ACKNOWLEDGMENTS
■
We thank T. Closson (Fluidigm) for assistance with IMC. We
are grateful to H. Soor (University of Toronto) for LCMS
experiments and to Bradly G. Wouters and Ravi N. Vellanki for
providing mounted tissue samples. This work was supported
by the Natural Sciences and Engineering Research Council,
Ontario Centres of Excellence and Fluidigm Canada.
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