Vibrational Imaging of Glucose Uptake Activity in Live Cells and Tissues by Stimulated Raman Scattering

Article abstract of DOI:10.1002/anie.201502543

Glucose is a ubiquitous energy source for most living organisms. Its uptake activity closely reflects cellular metabolic demand in various physiopathological conditions. Extensive efforts have been made to specifically image glucose uptake, such as with positron emission tomography, magnetic resonance imaging, and fluorescence microscopy, but all have limitations. A new platform to visualize glucose uptake activity in live cells and tissues is presented that involves performing stimulated Raman scattering on a novel glucose analogue labeled with a small alkyne moiety. Cancer cells with differing metabolic activities can be distinguished. Heterogeneous uptake patterns are observed with clear cell-cell variations in tumor xenograft tissues, neuronal culture, and mouse brain tissues. By offering the distinct advantage of optical resolution but without the undesirable influence of fluorophores, this method will facilitate the study of energy demands of living systems with subcellular resolution.

Full text of DOI:10.1002/anie.201502543

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201502543
German Edition: DOI: 10.1002/ange.201502543
Glucose Uptake
Vibrational Imaging of Glucose Uptake Activity in Live Cells and
Tissues by Stimulated Raman Scattering**
Fanghao Hu, Zhixing Chen, Luyuan Zhang, Yihui Shen, Lu Wei, and Wei Min*
Abstract: Glucose is a ubiquitous energy source for most living
organisms. Its uptake activity closely reflects cellular metabolic
demand in various physiopathological conditions. Extensive
efforts have been made to specifically image glucose uptake,
such as with positron emission tomography, magnetic reso-
nance imaging, and fluorescence microscopy, but all have
limitations. A new platform to visualize glucose uptake activity
in live cells and tissues is presented that involves performing
stimulated Raman scattering on a novel glucose analogue
labeled with a small alkyne moiety. Cancer cells with differing
Figure 1. Structures of existing d-glucose analogues and synthesis of
metabolic activities can be distinguished. Heterogeneous
uptake patterns are observed with clear cell–cell variations in
tumor xenograft tissues, neuronal culture, and mouse brain
the new 3-O-propargyl-d-glucose. a) [18F]2-Fluoro-2-deoxy-d-glucose
([18F]FDG). b) [11C]3-O-methyl-d-glucose ([11C]3-OMG). c) 2-NBD-2-
deoxy-d-glucose (2-NBDG). d) Synthetic route of 3-OPG 1. a) NaH,
tissues. By offering the distinct advantage of optical resolution
but without the undesirable influence of fluorophores, this
method will facilitate the study of energy demands of living
systems with subcellular resolution.
propargyl bromide, DMF, RT, 12 h, 90%; b) Dowex 50WX8, H2O, 808C,
20 h, 96%.
PET studies to be a key diagnostic marker in many diseases.
More recently, magnetic resonance imaging (MRI) has
achieved glucose imaging in tumors through chemical
exchange saturation transfer and hyperpolarization of 13C-
labeled d-glucose.[2] Unfortunately, both PET and MRI
techniques have limited spatial resolution, and cannot visual-
ize glucose uptake in single cells. Glucose uptake and energy
demands in many crucial physiological (for example, brain
activity) and pathological processes (for example, tumor
development) are intrinsically heterogeneous at the cellular
level.[3] Hence, to visualize glucose uptake activity in single
living cells is indispensable in evaluating the metabolic
demand of individual cells and unveiling the underlying
cell-to-cell variations.
To monitor glucose uptake with cellular resolution,
fluorophore-conjugated glucose analogues have been devel-
oped for fluorescence microscopy. 2-[N-(7-nitrobenz-2-oxa-
1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) (Fig-
ure 1c), which is also a glucose analogue that cannot be
metabolized further in glycolysis, is one of the most popular
probes and has been applied in a variety of cell types.[4]
However, conjugation of glucose with fluorophores, which
are mostly hydrophobic or charged, alters the hydrophilic
property of natural glucose. Indeed, the water solubility of 2-
NBDG is substantially reduced to about 10 mgmLÀ1. More-
over, physical sizes of fluorophores are always larger than
glucose itself, owing to the requirement of extended con-
jugation for visible light absorption. Thus, most fluorescent
glucose analogues, including 2-NBDG, exhibit non-specific
interactions and retentions in cells and tissues,[5] which would
bias the true glucose distribution to varying degrees.
G
lucose is the primary energy source for virtually all living
organisms, from bacteria to humans. As a hydrophilic small
molecule, the uptake of glucose is mediated by cell surface
transporters to pass through the otherwise impermeable
hydrophobic plasma membrane, during which the ATP-
independent facilitated diffusion down a glucose concentra-
tion gradient is the major pathway. Since the uptake of
glucose is biologically regulated and closely correlated to
a wide range of metabolic transformations, several prominent
techniques have been developed to selectively image glucose
uptake activity. Among them, radioactive tracers of glucose
analogues (Figure 1a,b) are widely applied in clinical positron
emission tomography (PET) to image glucose uptake activity
in vivo. Although these PET analogues cannot be metabo-
lized like d-glucose in the glycolytic pathway and thus
accumulate inside cells, their net flux across cell membrane
still reflects the total exogenous cellular glucose use when
imaged under steady-state conditions.[1] Indeed, imaging
steady-state glucose uptake level has been shown by extensive
[*] F. Hu, Dr. Z. Chen, Dr. L. Zhang, Y. Shen, L. Wei, Prof. W. Min
Department of Chemistry, Columbia University
New York, NY 10027 (USA)
E-mail: wm2256@columbia.edu
Prof. W. Min
Kavli Institute for Brain Science, Columbia University
New York, NY 10027 (USA)
[**] We thank Y. Shin for providing hippocampal neurons and J. Hirtz for
assistance on mouse brain tissues. W.M. acknowledges support
from the NIH Director’s New Innovator Award, ARO MURI
W911NF-12-1-0594, and a Alfred P. Sloan Research Fellowship.
Herein we report a new modality to visualize glucose
uptake activity at the subcellular level by stimulated Raman
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201502543.
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scattering (SRS) imaging of vibrationally labeled d-glucose,
and demonstrate its utility in live cancer cells, tumor
xenograft tissues, primary neurons, and mouse brain tissues.
We designed, synthesized, and characterized a novel glucose
analogue, named 3-O-propargyl-d-glucose (3-OPG, 1, Fig-
ure 1d). 3-OPG has a minor chemical modification compa-
rable to the PET tracer [11C]3-OMG (Figure 1b) yet avoids
the use of radioactive isotopes and allows for subcellular
optical imaging. Compared with fluorophores, the alkyne tag
ꢀ
(C C) is physically much smaller and has been proven to be
chemically inert and bioorthogonal in cells by numerous click-
chemistry studies.[6] Thus, we expect 3-OPG to not only
preserve the hydrophilic nature of glucose but also prevent its
non-specific interactions in cells and tissues. Detection-wise,
ꢀ
the alkyne tag exhibits a pronounced C C stretching Raman
peak (ca. 2100 cmÀ1) in the cell-silent spectral region.[7] Very
recently, alkyne tags have been coupled to SRS microscopy as
a powerful vibrational label for metabolic imaging in live cells
with high sensitivity, specificity, and biocompatibility.[8]
Therefore, by targeting the unique vibrational signature of
alkyne tags, SRS imaging of 3-OPG uptake could be
a valuable method to study energy demands and metabolic
status in living systems with subcellular resolution and
minimal perturbation.
We first synthesized 3-OPG by conjugating a propargyl
group to glucose 3’ OH group (Figure 1d). Briefly, commer-
cially available 1,2:5,6-di-O-isopropylidene-a-d-glucofura-
nose 2 is reacted with propargyl bromide to selectively
introduce the alkyne moiety onto the 3’ position, which gives
3 in 90% yield. Deprotection of 3 in strong acids affords 3-
OPG 1 with 96% yield. Comparing 3-OPG with 3-OMG
(Figure 1), it can be readily ascertained that their structures
are highly similar except for the replacement of a methyl
group by a propargyl group, which is a common strategy to
introduce the alkyne group and maintain the original
function, such as in propargylcholine[6b] and O-propargylpur-
omycin.[6c]
Figure 2. Uptake of 3-OPG in live mammalian cells. a) Spontaneous
Raman spectra of 25 mm 3-OPG solution in PBS (black) and HeLa
cells incubated with 25 mm 3-OPG for 4 h (red). b) SRS imaging of 3-
OPG uptake (2129 cmÀ1) in live HeLa cells incubated with 8 mm 3-
OPG for 1 hour. The average intracellular concentration is estimated to
be 3–5 mm. Images at 2000 cmÀ1 (off-resonance) and 1655 cmÀ1
(protein amide I) show the same area of cells. The 2129 cmÀ1 color
bars in all images correspond to a linear 3-OPG concentration range
of 0–26 mm, unless otherwise noted. Scale bar: 20 mm.
uptake of 3-OPG. No cytotoxicity of 3-OPG is observed
from cell viability assays (Supporting Information, Figure S2).
Cellular distribution of 3-OPG can be visualized with high
sensitivity and speed by SRS imaging of the alkyne Raman
peak at 2129 cmÀ1 in HeLa cells incubated with 8 mm 3-OPG
for 1 hour (Figure 2b). The corresponding off-resonance
image at 2000 cmÀ1 confirms the background-free nature of
SRS. As a reference, SRS image at 1655 cmÀ1 shows cellular
protein distribution by targeting at protein amide I band.
To quantify the intracellular uptake level, we correlated
SRS intensity at 2129 cmÀ1 with known concentrations of 3-
OPG solutions under identical conditions, which gives
a detection sensitivity of 1.4 mm with 30 ms acquisition time
per pixel and circa 26 s per frame (Supporting Information,
Figure S3). For higher signal-to-noise ratio and more accurate
quantifications, we chose 25 mm 3-OPG as incubation con-
centration for further biochemistry characterizations.
To validate that 3-OPG is transported into cells through
glucose transporters, several control experiments were per-
formed. First, uptake of 3-OPG was competed using d-
glucose at different concentrations (Figure 3). As the con-
centration of d-glucose increases, cellular uptake of 3-OPG
decreases accordingly, reaching 78% with 10 mm d-glucose
and 53% with 50 mm d-glucose, consistent with the higher
transporter affinity of d-glucose (Figure 3a–c). In contrast,
with 50 mm l-glucose, which is not recognized by glucose
transporters for its opposite chirality, 3-OPG uptake level is
unaffected (Figure 3d).
Spontaneous Raman spectrum of 3-OPG is then charac-
terized with a sharp and strong peak at 2129 cmÀ1 in the cell-
silent region (Figure 2a, black), indicative of the Raman-
ꢀ
active mode of C C stretching. Negligible cellular back-
ground in 1800–2400 cmÀ1 is confirmed by both spontaneous
Raman spectrum and SRS image at 2129 cmÀ1 of blank HeLa
cells (Supporting Information, Figure S1). Intrinsic Raman
À
À
peaks of glucose, such as those from C C and C O bond
vibrations, are also observed in the fingerprint region from
1000–1800 cmÀ1 (Figure 2a, black). Note that these peaks are
not only much weaker than the alkyne peak but also
overlapping with the cellular background (Figure 2a, red),
which makes label-free Raman imaging of glucose in cells
rather challenging.[9] Thus, introducing an exogenous alkyne
tag not only promotes the sensitivity but also endows imaging
specificity in the otherwise crowded cellular background
without the need of spectral unmixing.
The uptake of 3-OPG was then tested in mammalian cell
cultures. A distinct peak (2129 cmÀ1) emerges after incubating
HeLa cells with 25 mm 3-OPG for 4 h (Figure 2a, red), which
exactly matches with that in the phosphate-buffered saline
(PBS) solution spectrum of 3-OPG, confirming cellular
Four different transporter inhibition and stimulation
assays were then conducted. Using RNA interference to
suppress the expression of GLUT1 transporter, HeLa cells
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transport kinetics for different glucose analogues.[10] Low-
ering to room temperature slows down 3-OPG uptake, as
expected. Linear fitting of 3-OPG uptake at early times also
reveals a circa threefold decrease in initial transport rate from
378C to room temperature. Quantitatively, by plotting initial
rates of 3-OPG uptake against different incubation concen-
trations (Supporting Information, Figure S7), its Km and Vmax
parameters are extracted in HeLa cells where GLUT1 is the
most abundant isoform.[11] The fitted values of Km = 26 Æ
4 mm and Vmax = 0.71 Æ 0.05 mm minÀ1 of 3-OPG uptake turn
out to be very close to that for 3-OMG uptake by human
GLUT1 expressed in Xenopus oocytes (Km = 13–20 mm and
V
max = 0.74 mm minÀ1 assuming 106 oocytes per L).[12] Com-
petition experiments using 3-OMG show a moderate reduc-
tion to 70% in 3-OPG uptake (Supporting Information,
Figure S8), which is again in agreement with their similar Km.
Furthermore, important insights can be gained by simul-
taneously imaging cellular uptake of 3-OPG and 2-NBDG by
the same set of cells (Supporting Information, Figure S9).
While 2-NBDG displays a heterogeneous pattern with
stronger signal enriched in cellular structures surrounding
the nucleus, 3-OPG is distributed more homogeneously
throughout the cells including inside the nucleus. Such
differential contrast between 3-OPG and 2-NBDG is likely
due to non-specific retention of 2-NBDG in extra-nuclear
organelles (such as endoplasmic reticulum) and its low
permeability across nuclear membrane caused by the strong
hydrophobicity and/or large size of the NBD fluorophores.[5]
Hence we have obtained direct imaging evidence that 3-OPG
could be a more truthful probe to report the genuine glucose
uptake and distribution inside cells.
Figure 3. Glucose transporter dependence of 3-OPG uptake in mam-
malian cells. Representative SRS images of live HeLa cells incubated
with 3-OPG in the presence of a) 0 mm d-glucose, b) 10 mm d-
glucose, c) 50 mm d-glucose, and d) 50 mm l-glucose. e,f) HeLa cells
were transfected with 10 nm negative control siRNA (e) or GLUT1
siRNA (f) before incubation with 3-OPG. g,h) HeLa cells were incu-
bated with 3-OPG in the presence of DMSO vehicle (g) or 10 mm
cytochalasin B (h). Scale bar: 20 mm. i) Relative SRS intensity of 3-OPG
uptake in HeLa cells under the conditions above. Data are shown as
mean+standard deviation (SD; nꢁ3 replicates for each group). *
p<0.05; *** p<0.001 by Student’s t-test. p<0.05 is considered
statistically significant.
Tumors are well known to have aberrant metabolism with
upregulated glucose uptake. Cancer cells of different pheno-
types also have varied glucose uptake levels, which could
serve as a marker for tumor malignancy and metastatic
potential.[2a] We thus compare glucose uptake activity
between HeLa, a human cervical cancer cell line and U-87
MG, a human primary glioblastoma cell line, using 3-OPG as
imaging probe (Supporting Information, Figure S10). As the
highest-grade glioma (WHO grade IV), glioblastoma is the
most aggressive form of malignant primary brain tumor.
Incubated with 3-OPG, U-87 MG cells display a much higher
alkyne peak than that of HeLa cells in Raman spectra,
suggesting a more pronounced glucose uptake activity. SRS
images further show the whole intracellular distribution of 3-
OPG in both cancer cells. Consistent with the spectroscopic
results, U-87 MG cells indeed exhibit a brighter intracellular
signal than HeLa cells, confirming much increased glucose
uptake activity. Quantitatively, 3-OPG uptake in U-87 MG
cells is over 1.7 times higher than that of HeLa cells. Thus SRS
imaging of 3-OPG uptake could be a useful technique to
distinguish cancer cells with varied metabolic characteristics.
Uptake of 3-OPG is further visualized in mouse subcuta-
neous tumor xenograft model. Tumor regions with distinct
glucose uptake levels are observed (Figure 4). In outer region
of the tumor, cells undergo fast proliferation and hence
uptake glucose significantly (Figure 4a). In contrast, toward
the center of tumor, cells display a small and broken
morphology (as shown in the amide channel at 1655 cmÀ1),
transfected with 10 nm GLUT1 siRNA for 48 h show a much
reduced uptake of 3-OPG (54%) compared to the control
groups (Figure 3e,f). Moreover, HeLa cells incubated with
10 mm cytochalasin B, a potent inhibitor of glucose carriers,
display a drastic reduction to 35% in 3-OPG uptake (Fig-
ure 3g,h). Efflux experiments of 3-OPG-loaded HeLa cells
were also performed with a rapid decay after 30 min and the
addition of cytochalasin B significantly inhibits the efflux of 3-
OPG (Supporting Information, Figure S4), indicating the
transport of 3-OPG in both directions is carrier-mediated. We
further studied the effect of insulin stimulation on 3-OPG
uptake by GLUT4 in rat muscle cells. With 1 mgmLÀ1 insulin
for 24 h, which stimulates GLUT4 translocation to plasma
membrane, a circa 30% increase in 3-OPG uptake is observed
in fused L6 cells (Supporting Information, Figure S5), sug-
gesting GLUT4-mediated uptake of 3-OPG. Taken together,
these results further prove the glucose transporter depend-
ence of 3-OPG uptake and its role as a d-glucose analogue to
mammalian cells.
Uptake kinetics of 3-OPG were also examined (Support-
ing Information, Figures S6 and S7). At 378C, buildup of 3-
OPG inside HeLa cells reaches a plateau within 30 min,
indicating fast uptake of 3-OPG at physiological temperature
and allowing steady-state imaging. This equilibrium time is
consistent with the reported size and weight dependence of
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Figure 5. SRS imaging of 3-OPG uptake in neuronal systems. a) SRS
images of cultured mouse primary hippocampal neurons incubated
with 3-OPG. Scale bar: 20 mm. b) SRS images of mouse brain tissue
slice cultured ex vivo with 3-OPG. Significant uptake of 3-OPG is seen
at the granule cell layer of dentate gyrus. Scale bar: 40 mm.
Figure 4. SRS imaging of 3-OPG uptake in U-87 MG tumor xenograft
tissues. a) Strong uptake of 3-OPG in the proliferating region of tumor.
b) Little uptake is seen in the tumor region with necrotic cell
neural signaling. Intercellularly, significant variation in 3-
OPG uptake is observed among different neurons, with over 2
times difference from cells with low-glucose level to high-
glucose demanding cells (Figure 5a, arrowhead). This obser-
vation from the 3-OPG channel, which is absent from the
corresponding protein amide image, indicates heterogeneous
energy requirement at single neuron level. Going beyond the
neuron cultures, 3-OPG uptake is also visualized in the
hippocampus of mouse brain tissue slice cultured ex vivo.
Pronounced signal is present in both the granule cell layer and
nearby hilar region of dentate gyrus, which is known to exhibit
high metabolic activity[14] (Figure 5b). Thus, 3-OPG is gen-
erally applicable as a vibrational imaging probe for glucose
transport in both tumor and brain study with subcellular
resolution.
In summary, we have designed, synthesized, and charac-
terized a novel glucose analogue with an alkyne vibrational
tag (3-OPG). By coupling SRS microscopy to the alkyne tag,
we have demonstrated vibrational imaging of glucose uptake
activity in living cells and tissues with high sensitivity,
specificity, and biocompatibility. Our method can differen-
tiate cancer cells with varied metabolic activities and reveal
heterogeneous glucose uptake patterns in tumor xenograft
tissues as well as in neuronal culture and mouse brain tissues
at cellular resolution. Previous studies have been reported on
glucose sensing in blood and metabolic incorporation using
isotope-labeled glucose for imaging downstream products
with Raman techniques.[15] However, they are either not in
cellular environment or cannot visualize glucose uptake
activity specifically. If compared to the classic PET tracers,
3-OPG can be synthesized in a highly cost-effective way
without special handling of radioactive materials and endows
optical imaging with subcellular resolution. If compared to
the bulky fluorescent glucose analogues, 3-OPG has a minor
structural modification without adding a significant size and
weight to glucose, rendering better affinity to glucose trans-
porters, less non-specific interaction, and higher translocation
efficiency (for example, crossing the blood–brain barrier).
morphology. The 2129 cmÀ1 color bars in (a) and (b) correspond to
a 3-OPG concentration range of 0–53 mm. c) Sharp contrast of 3-OPG
uptake at the interface of two tumor regions. Scale bar: 40 mm.
suggesting necrosis and diminished cellular activity. Indeed,
little 3-OPG uptake is seen in the SRS image with a signal at
least five times lower than that in the tumor-propagating
region (Figure 4b).
The interface between the two tumor regions can be
clearly distinguished by highlighting the 3-OPG uptake
contrast at the single cell level, providing useful microscopic
insights (Figure 4c). Necrosis is known to be a histological
hallmark of glioblastoma and an important predictor for
tumor prognosis in clinical glioma diagnostics and staging.[13]
We discover that the metabolic contrast from 3-OPG uptake
is much more pronounced than the label-free images of
endogenous lipid (at 2845 cmÀ1) or protein (at 1655 cmÀ1)
distributions. Most notably, a small population of tumor cells
with high glucose uptake activity is identified within the
region of diminished activity (Figure 4c, arrowhead), reflect-
ing cell–cell variation of metabolic status. Therefore, this
showcases the capability of vibrational imaging of glucose
uptake activity using 3-OPG and provides opportunity for
studying intratumoral heterogeneity in energy consumptions
during brain tumor growth with superb contrast and cellular
resolution.
The brain is the central control of activity in higher
organisms and has a high energy demand. Fast and adequate
glucose supply is critical for maintaining the neural activity.
We therefore demonstrate imaging 3-OPG uptake in both
in vitro neuron culture and ex vivo mouse brain tissue
(Figure 5). In cultured mouse primary hippocampal neurons,
not only a high level of 3-OPG is imaged inside the soma, but
also it can be seen clearly in the neuronal processes (Fig-
ure 5a), reflecting energy need in axons and dendrites for
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Keywords: alkyne tags · glucose uptake · imaging agents ·
Raman spectroscopy · SRS microscopy
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Received: March 18, 2015
Revised: April 26, 2015
Published online: July 16, 2015
Angew. Chem. Int. Ed. 2015, 54, 9821 –9825
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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