Using boronolectin in MALDI-MS imaging for the histological analysis of cancer tissue expressing the sialyl Lewis X antigen

Article abstract of DOI:10.1039/c1cc11814e

Certain carbohydrate-based biomarkers are known to correlate with cancer formation and progression. By targeting sialyl Lewis X, we have developed the first boronolectin-MS tag conjugate, which allows for MALDI-based imaging of cancer based on its cell su

Full text of DOI:10.1039/c1cc11814e

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Using boronolectin in MALDI-MS imaging for the histological analysis
of cancer tissue expressing the sialyl Lewis X antigenw
Chaofeng Dai,a Lisa H. Cazares,b Lifang Wang,a Yong Chu,ac Siming L. Wang,a
Dean A. Troyer,b O. John Semmes,b Richard R. Drake*b and Binghe Wang*a
Received 30th March 2011, Accepted 4th August 2011
DOI: 10.1039/c1cc11814e
Certain carbohydrate-based biomarkers are known to correlate
with cancer formation and progression. By targeting sialyl Lewis
X, we have developed the first boronolectin–MS tag conjugate,
which allows for MALDI-based imaging of cancer based on its
cell surface carbohydrate.
biomarkers prior to analysis, and ion desorption can be
targeted to specific ‘‘points’’ in a grid pattern and the data
rasterized. The resulting spectra can then be used to generate
two-dimensional molecular maps of hundreds of biomolecules
directly from the surface of a tissue section. These molecular
maps display the relative abundance and spatial distribution
of these molecules. MALDI tissue profiling has the power to
link the molecular detail of mass spectrometry with molecular
histology, generating mass spectra correlated to known locations
within a thin tissue section.15 We and others have recently
demonstrated the potential of MALDI-IMS to clinical histo-
pathology applications.15–18
Biomarker-based histological work is known to save lives, help
the formulation of therapeutic intervention strategies, and allow
for improved prognosis.1,2 Among all the known biomarkers,
cancer cell surface carbohydrate antigens play a very important
role, and most clinically measured cancer biomarkers are
glycoproteins.3 Cell surface carbohydrate structures as part
of glycosylated proteins, peptides, and lipids are characteristic
signatures of different cell types4–8 and are associated with
many forms of cancer.9,10 For example, the sialyl Lewis X
(sLex) antigen is being assessed in many cancers; serum sLex
and the cytokeratin 19 fragment are said to be predictive
factors for recurrence in patients with stage I non-small
cell lung cancer;11 and sLex plus CA 15.3 levels in serum of
breast cancer patients were reported to be more effective than
CA 15.3 plus CEA in diagnosing cancer.12 Furthermore, the
combination of sLex and sLea expression is known to be
correlated with the adhesion of urothelial cancer cells to
activated endothelium.13 Detection of changes in the expression
of these cell surface carbohydrates is clearly very important in
cancer histological work.
A recently developed variant of MALDI-IMS, termed
Targeted Imaging Mass Spectrometry (TIMS) or TAMSIM
for Targeted multiplex Mass Spectrometry Imaging, first
described by Thiery et al.,19 allows for the targeted analysis
and spatial visualization of a molecule of interest directly from
tissue sections by the use of laser-reactive photo-cleavable
molecular tags attached to affinity molecules.20,21 The bond
conjugating the mass tag to the affinity molecule is photo-
cleavable so that exposure to the UV laser in a MALDI mass
spectrometer releases the tag without the need for matrix
assistance. The released tag is readily detected. Changing the
mass of the tags also allows for multiplexed detection of
different molecules simultaneously within the same tissue,
and sections prepared by standard methods (fixed or frozen)
can be used for TIMS so that existing pathology workflows are
the same.
In histological work, fluorescent and/or color staining agents
are most commonly used. However, such approaches suffer
from difficulties in multiplexing due to spectral resolution/overlap
issues and in quantitation. A novel but maturing technology,
MALDI imaging mass spectrometry (MALDI-IMS)14,15
allows for direct examination of tissue biopsies without
the need for micro-dissection and solubilization of tissue
We have had a long-standing interest in the development of
boronic acid-based ‘‘receptors’’ (named boronolectins)10 that
can recognize carbohydrate biomarkers. In one study, a
bisboronic acid sensor for sLex was developed.22,23 We envisioned
that conjugation of this boronolectin 1 with a trityl-based tag24–29
would allow matrix-free MALDI-IMS analysis of cancer
tissues with a high level of sLex (Scheme 1). Because the trityl
cation is very stable, MALDI laser can ionize compounds similar
to 2 by removing the thiol linkage to give carbocation 3.
Therefore, this boronolectin was conjugated to a trityl-based
MS tag 15 for imaging applications (Scheme 2). Herein we
describe the first example of conjugating a carbohydrate
biomarker-targeting small molecule with a matrix-free MALDI
mass spectrometric tag for MALDI-IMS work by tracking
cancer cell surface sLex expression.
a Department of Chemistry and Center for Diagnostics and Therapeutics,
Georgia State University, Atlanta, Georgia 30302-4098, USA.
E-mail: wang@gsu.edu; Tel: +1 404-413-5544; +1 404-413-5888
b Department of Microbiology and Molecular Cell Biology and The
Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia
Medical School, Norfolk, Virginia, 23507, USA.
E-mail: drakerr@evms.edu
c Department of Medicinal Chemistry, School of Pharmacy, Fudan
University, No.826, Zhangheng Rd, Shanghai, 201203, China
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc11814e
c
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We use click chemistry for side chain modification in order to
allow for future diversity generation and conjugation to various
molecules. The final compound (16) was obtained following
alkylation and amidation reactions. The trityl tag was synthe-
sized following similar literature procedures (see ESIw for
details) and its MALDI MS in the absence of any matrix
showed formation of an intense peak corresponding to the
trityl tag (Fig. 1a, top panel).24–29
The boronolectin–trityl reporter conjugate (16) was then
tested with fresh-frozen renal tissues containing both tumor
and normal regions. The same peak for the trityl tag (Fig. 1a,
bottom panel) was observed and monitored for imaging
studies. In Fig. 1b, a picture of the renal tissue slide is shown,
with immunostaining of the sLex (mouse monoclonal antibody,
GenWay Biotech San Diego, CA) expressing regions coincident
with the tumor area (dark stained bottom tip area; confirmed
by a pathologist).
Scheme 1 The general concept of boronolectin–MS tag conjugation
for imaging applications.
An adjacent tissue slice (7 mm) was placed on a conductive
slide and incubated with the sLex–trityl probe in 100% methanol
(2 ng mlÀ1) in a humid chamber overnight at 4 1C. Slides were
washed for 5 min in PBS followed by brief water wash to
remove any unbound material. The slides were placed in a
desiccator for 20 min and analyzed directly by MALDI-TOF
(no matrix is added) in the reflectron mode using a laser raster
width of 200 mm.
Only in the region of tumor with sLex expression was there
binding of the sLex–trityl conjugate, as shown in red pixel
intensities (Fig. 1b). These red pixels correlate to the MALDI
peaks in the spectra shown in the Fig. 1a panel and the color
intensity is related to peak abundance as shown in the expression
scale. Peaks were obtained in the absence of any chemical
matrix. Addition of external sLex clearly attenuated the ability
of the conjugate to bind to the tissue (Fig. S1, ESIw section).
This same probe also has shown the ability to bind to alcohol
fixed renal tissues, as shown in Fig. 1b. This opens the possibility
for probing tumor tissue microarrays prepared from alcohol
or formalin fixed tissues. Thus, it is clear that MALDI-IMS
results tracked with the immunostaining results and tumor
tissue location, showing feasibility of using a boronolectin–MS
tag conjugate for MALDI-IMS work. This represents the first
Scheme 2 (a) Propargyl bromide, K2CO3, CH3CN, reflux 24 h,
quantitative; (b) 2 N NaOH, CH3OH, reflux 3 h, 84%; (c) CH2Cl2,
EDCI, HOBT, DMAP, 88%; (d) TFA, CH2Cl2, 82%; (e) CuI,
DIPEA, DMSO, 80 1C, microwave, 55%; (f) K2CO3, CH3CN, 79%;
(g) TFA, CH2Cl2, 73%; (h) Et3N, DMF, CH2Cl2, 36%.
Scheme 2 shows a general flowchart for the synthesis of the
boronolectin–MS tag conjugate. Specifically, compound 9 can
be synthesized following procedures similar to the synthesis of
the parent boronolectin without the side arm.23 Subsequent
Cu-mediated Huisgen cycloaddition30–33 allowed for the
installation of a side chain functional group for conjugation.
Fig. 1 (a) MALDI-IMS trityl peak obtained using frozen kidney tissue cut and stored at À80 1C. Other peaks track to the RCC (renal cell
carcinomas). (b) MALDI-IMS (left) and immunostaining (middle) images of kidney tissue described in (a). A pathologist confirmed that
immunostaining and MALDI-IMS-boronolectin signal results overlap in the tumor region, and not in normal cell areas. The third panel (right)
shows boronolectin staining of a Sakura/UMFix alcohol fixed renal tumor tissue (top).
c
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example of using small molecules for targeting carbohydrate-
based cancer biomarkers in MALDI-IMS work.
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In conclusion, using a sLex-recognizing boronolectin, we
were able to develop a mass spectrometric probe for histological
work on cancer tissues expressing the target carbohydrate
using MALDI-IMS. Compared with protein-based targeting
molecules, small molecule boronolectins have the advantage of
excellent stability, easy storage, well-defined conjugation
chemistry, and compatibility with organic solvents in sample
preparation and tissue handling. The availability of additional
boronolectins will allow for the development of a ‘‘toolbox’’
for MALDI-IMS of cancer based on carbohydrate biomarkers.
We gratefully acknowledge the financial support of this work
by the National Institutes of Health (GM084933 (BW) and
GM086925 (BW); CA137704/CA135087 (RRD); CA085067
(OJS)) and Department of Defense W81XWH-10-1-0136 (RRD).
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10340 Chem. Commun., 2011, 47, 10338–10340
This journal is The Royal Society of Chemistry 2011