www.impactjournals.

com/oncotarget/ Oncotarget, Advance Publications 2017

NADH autofluorescence, a new metabolic biomarker for cancer

stem cells: Identification of Vitamin C and CAPE as natural
products targeting stemness
Gloria Bonuccelli1, Ernestina Marianna De Francesco1,2, Rianne de Boer1, Herbert
B. Tanowitz3 and Michael P. Lisanti4
1
The Paterson Building, University of Manchester, Withington, M20 4BX, United Kingdom
2
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, 87036, Italy
3
Departments of Pathology and Medicine, Albert Einstein College of Medicine, Bronx, NY, 10461, USA
4
Translational Medicine, School of Environment and Life Sciences, Biomedical Research Centre, University of Salford, Greater
Manchester, M5 4WT, United Kingdom
Correspondence to: Michael P. Lisanti, email: [email protected]
Correspondence to: Gloria Bonuccelli, email: [email protected]
Keywords: cancer stem-like cells; metabolic heterogeneity; metabolic cell fractionation; mitochondria; NADH
Received: January 10, 2017 Accepted: January 25, 2017 Published: February 16, 2017

ABSTRACT
Here, we assembled a broad molecular tool-kit to interrogate the role of
metabolic heterogeneity in the propagation of cancer stem-like cells (CSCs). First, we
subjected MCF7 cells to metabolic fractionation by flow cytometry, using fluorescent
mitochondrial probes to detect PCG1 activity, as well ROS and hydrogen-peroxide
(H2O2) production; NADH levels were also monitored by auto-fluorescence. Then, the
various cell populations were functionally assessed for stem cell activity, using the
mammosphere assay (3D-spheroids). Our results indicate that a sub-population of
MCF7 cells, with increased PGC1 activity, high mitochondrial ROS/H2O2 production
and high NADH levels, all form mammospheres with a higher efficiency. Thus, it
appears that mitochondrial oxidative stress and the anti-oxidant response both
contribute to the promotion of mitochondrial biogenesis and oxidative metabolism in
CSCs. Further validation was provided by using specific inhibitors to target metabolic
processes (the NAD+ salvage pathway, glycolysis, mitochondrial protein synthesis
and OXPHOS), significantly reducing CSC propagation. As a consequence, we have now
identified a variety of clinically-approved drugs (stiripentol), natural products (caffeic
acid phenyl ester (CAPE), ascorbic acid, silibinin) and experimental pharmaceuticals
(actinonin, FK866, 2-DG), that can be used to effectively inhibit CSC activity. We
discuss the use of CAPE (derived from honey-bee propolis) and Vitamin C, as potential
natural therapeutic modalities. In this context, Vitamin C was ~10 times more potent
than 2-DG for the targeting of CSCs. Similarly, stiripentol was between 50 to 100
times more potent than 2-DG.

INTRODUCTION However, the metabolic basis of the CSC phenotype

remains largely unexplored.
Cancer stem-like cells (CSCs) are thought to be Recently, based on unbiased label-free proteomics
the root cause of chemotherapy-resistance and radio- analysis, we proposed that mitochondrial biogenesis may
resistance, ultimately leading to treatment failure in be a key driver of the CSC phenotype [7]. A prediction of
patients with advanced disease [1-3]. They have been this hypothesis is that high mitochondrial mass would be
directly implicated mechanistically in tumor recurrence a metabolic biomarker for CSCs. To test this hypothesis
and metastasis, resulting in poor patient survival [4-6]. experimentally, we used a vital dye, called MitoTracker, to

www.impactjournals.com/oncotarget 1 Oncotarget
 stain mitochondria in living cancer cells. This allowed us exogenous fluorescent probes, we propose that NAD(P)
to purify the Mito-high and the Mito-low cell populations H auto-fluorescence could be used as a new metabolic
by flow cytometry. Interestingly, the Mito-high cell biomarker for CSC enrichment (Figure 5A). Importantly,
population showed a clear enrichment of cells with the high levels of NAD(P)H auto-fluorescence are known to
characteristics of CSCs [8-13]. Thus, the use of metabolic be a surrogate marker for mitochondrial power, high
fractionation, using mitochondrial-based probes and OXPHOS capacity and increased ATP production. As
flow cytometry, is a new successful strategy that also such, CSCs may be strictly dependent on NAD(P)H to
provides a functional assay for dissecting how metabolic maintain their enhanced mitochondrial function.
heterogeneity contributes to the CSC phenotype. As further functional validation, we next examined
Here, we have expanded this metabolic fractionation the sensitivity of MCF7 mammosphere formation to
approach to the use of i) other mitochondrial probes (for NAD+ depletion, by employing the well-characterized
monitoring mitochondrial biogenesis, ROS production NAMPT inhibitor, namely FK866 [14]. Figure 5B shows
and hydrogen peroxide) and ii) NADH auto-fluorescence. that mammosphere formation was dramatically inhibited
Our results indicate that increased mitochondrial oxidative by FK866, with an IC-50 of less than 1 nM. Thus, an
stress and high NADH levels are both key characteristics intact NAD+ salvage pathway is strictly required for
of the CSC metabolic phenotype. mammosphere formation, supporting our results using
This strategic approach also allowed us to identify NAD(P)H auto-fluorescence, which enriched CSC activity
several new weak points, such as NAD+ depletion, for by more than 5-fold.
metabolically targeting CSCs. Similarly, as further validation, we also show
that a known selective inhibitor of mitochondrial
RESULTS protein synthesis (the natural antibiotic actinonin; [15]),
efficiently blocked mammosphere formation, with an IC-
50 between 25 and 50 M (Figure 6). This is consistent
NAD(P)H auto-fluorescence as a new metabolic with our observations regarding a significant increase in
biomarker for CSCs mitochondrial biogenesis, as reflected by our studies using
the PGC1-eGFP probe.

Here, we set out to assess the role of cell metabolism Identification of vitamin C as a natural product
in generating molecular heterogeneity or diversity in targeting stemness
cancer cells, with a specific focus on the CSC phenotype.
To address this issue experimentally, we chose to
subject the MCF7 cell line to metabolic fractionation via Since the NAMPT inhibitor FK866 suffers
flow cytometry (FACS). For this purpose, we employed from a number of serious side effects, including
a number of fluorescent probes to specifically monitor
mitochondrial biogenesis (PGC1-eGFP), mitochondrial
ROS production (Dihydro-Rhodamine 123) and
mitochondrial hydrogen-peroxide levels (H2O2; Mito-
PY1). In addition, the levels of NAD(P)H were monitored
by auto-fluorescence, at an emission wavelength of
470-nm. The experimental utility of these probes is
summarized in Table 1.
In this fractionation scheme, cells containing low-
or high-levels of a given vital probe were then analyzed
phenotypically, using the mammosphere assay as a
measure of cancer stem cell activity. A summary of this
systematic approach is briefly presented schematically in
Figure 1.
Interestingly, using this reductionistic experimental
approach, our results directly show that a particular sub-
population of MCF7 cells, with the highest levels of Figure 1: Experimental strategy for examining the
mitochondrial biogenesis, mitochondrial ROS/H2O2, role of metabolic heterogeneity in CSC propagation.
Our overall strategy for investigating the role of mitochondrial
and NADH levels, have the highest capacity to efficiently
metabolism in CSCs, using specific fluorescent probes and
generate mammospheres (Figures 2-5). metabolic fractionation by flow cytometry (FACS) is outlined.
As NAD(P)H auto-fluorescence is an intrinsic Specific metabolic inhibitors were also employed to directly
property of cells and does not require the use of any validate our observations; see Table 1.

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 thrombocytopenia, lymphopenia and anemia, as well as 50 near 10 mM (Figure 7A). Similarly, we show that
severe nausea and mild fatigue [16], we sought to identify two other natural products that function as effective
other potentially less toxic metabolic approaches for glycolysis inhibitors, also inhibited mammosphere
targeting CSCs. formation. More specifically, vitamin C (ascorbic acid),
For this purpose, we examined the activity of a which induces oxidative stress and inhibits the activity of
number of clinically-approved drugs, natural products, as GAPDH (a key glycolytic enzyme) [17], also inhibited
well as experimental pharmaceuticals. Since glycolysis mammosphere formation, with an IC-50 of 1 mM (Figure
is especially critical for maintaining the TCA cycle, 7B). Therefore, vitamin C was ~10 times more potent than
OXPHOS and overall mitochondrial function, we next 2-DG at targeting CSC propagation. Interestingly, silibinin
assessed the effects of known glycolytic inhibitors. (the major active constituent of silymarin, an extract of
Interestingly, our results directly show that the milk thistle seeds) [18], which specifically functions as
classical glycolysis inhibitor, namely 2-deoxy-glucose an inhibitor of glucose uptake, blocked mammosphere
(2-DG), inhibits mammosphere formation, with an IC- formation, with an IC-50 between 200 and 400 M

Figure 2: Mitochondrial biogenesis correlates with stemness. PGC-1 is a key regulator of energy metabolism, since it stimulates
mitochondrial biogenesis. Thus, we generated a stable MCF7 cell line transfected with the promoter of the murine PGC-1 gene linked to
eGFP, as a surrogate marker of mitochondrial biogenesis. Therefore, using this reporter system, increased GFP fluorescence is a predictor
of increased PGC-1 levels and/or activity. MCF7-PGC-1-eGFP cells were first subjected to metabolic fractionation via flow cytometry
(FACS) to isolate the GFP-high and GFP-low cell populations. These two cell populations were then immediately plated in low-attachment
cell culture plates, to assess their capacity for mammosphere formation. Importantly, GFP-high cells showed an ~1.5-fold increase in their
capacity for mammosphere formation, relative to GFP-low cells. These results suggest that there is a clear correlation between increased
PGC-1 activity and stemness in cancer cells. GFP-high = highest or top 10% GFP intensity; GFP-low = lowest or bottom 10% GFP
intensity. Bar graphs show the mean SEM, t-test, two-tailed test. *p < 0.05. N = 3 independent experiments. A representative FACS tracing
is presented in panel A. and quantitation of mammosphere formation is shown in panel B.

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 Figure 3: Increased mitochondrial ROS production contributes to stemness. MCF7 cells were first live-stained with a
fluorescent probe [dihydro-rhodamine 123 (DHR)] to monitor mitochondrial ROS production. Then, DHR-stained MCF7 cells were
subjected to metabolic fractionation by flow cytometry (FACS), to isolate the DHR-high, -low and -negative cell populations. Each cell
population was then plated in low-attachment cell culture plates, to assess their capacity for mammosphere formation. Note that the DHR-
high cell population showed a 2.3-fold increase in mammosphere formation, relative to DHR-negative cells. Therefore, high mitochondrial
ROS production directly correlates with CSC activity. DHR-high = highest or top 10% intensity; DHR-low = lowest or bottom 10%
intensity; DHR-negative = lowest possible intensity (0 to 4%). Bar graphs show the mean SEM, t-test, two-tailed test. *p < 0.05, ***p
< 0.0001. N = 3 independent experiments. A representative FACS tracing is presented in panel A. and quantitation of mammosphere
formation is shown in panel B.

Figure 4: Increased mitochondrial hydrogen peroxide (H2O2) production contributes to stemness. MCF7 cells were first
live-stained with a fluorescent probe [Mitochondria peroxy yellow 1 (MPY1)] to monitor mitochondrial H2O2 production. Then, MPY1-
stained MCF7 cells were subjected to metabolic fractionation by flow cytometry (FACS), to isolate the MPY1-high, -low and -negative
cell populations. Each cell population was then plated in low-attachment cell culture plates, to assess their capacity for mammosphere
formation. Note that the MPY1-high cell population showed a 2.1-fold increase in mammosphere formation, relative to MPY1-negative
cells. Therefore, high mitochondrial H2O2 production directly correlates with CSC activity. MPY1-high = highest or top 5% intensity;
MPY1-low = lowest or bottom 5% intensity; MPY1-negative = lowest possible intensity (0 to 2%). Bar graph shows the mean SEM, t-test,
two-tailed test. ***p < 0.0001. N = 3 independent experiments. A representative FACS tracing is presented in panel A. and quantitation of
mammosphere formation is shown in panel B.

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 (Figure 7C). Finally, the FDA-approved drug stiripentol, CAPE quantitatively inhibits the mitochondrial oxygen
which behaves as an LDHA inhibitor [19], blocked consumption rate (OCR) and, in turn, induces the onset of
mammosphere formation with an IC-50 between 100 and aerobic glycolysis (ECAR).
200 M (Figure 7D). Thus, stiripentol was the most potent Next, we tested the effects of CAPE on the ability
glycolysis inhibitor that we identified for targeting CSCs of MCF7 cells to form 3D mammospheres, under
and it was 50 to 100 times more potent than 2-DG. anchorage independent growth conditions. Interestingly,
Figure 11A illustrates that CAPE significantly reduces
Identification of CAPE as a natural product that mammosphere formation, with an IC-50 of approximately
metabolically targets mitochondria and CSC 2.5 M. However, it was less effective on adherent bulk
activity cancer cells, with an IC-50 of 10 M (Figure 11B). As
such, CAPE showed a 4-fold selectivity for targeting CSC
propagation, as compared with adherent MCF7 monolayer
Honey-bee propolis (a.k.a., bee glue) is a cells. In addition, at a concentration of 25 M, CAPE
resinous-like substance that bees manufacture as a sealant was twice as toxic for MCF7 cancer cells, as compared
for filling unwanted crevices in their hive [20]. Propolis with normal fibroblasts. At this concentration, ~60% of
has a strong history of medicinal use, dating back more normal fibroblasts survived, while >70% of cancer cells
than 2,000 years [20-22]. In fact, caffeic acid phenyl ester were eliminated. Thus, CAPE shows a clear selectivity
(CAPE), a key component of honey-bee propolis, has for targeting CSCs and adherent cancer cells, relative to
potent anti-cancer properties [22]. However, CAPEs exact normal fibroblasts.
mechanism of action remains largely unknown. Often CSCs are associated with an epithelial
Because of it aromatic ring structure (Figure 8), we mesenchymal transition (EMT) phenotype, resulting
speculated that CAPE might function as a potent inhibitor in an increased capacity for cell migration [23]. As a
of oxidative mitochondrial metabolism. To test this consequence, we also tested the effects of CAPE on MCF7
hypothesis, we examined its metabolic effects on MCF7 cell migration, using a standard scratch assay. In this
cell in culture, using the Seahorse XFe96 Metabolic Flux assay system, cell migration is monitored via wound
Analyzer. Indeed, Figures 9 and 10 directly show that

Figure 5: Increased NAD(P)H levels directly correlate with stemness. A. NAD(P)H auto-fluorescence. NAD(P)H levels are
known to directly correlate with mitochondrial oxidative capacity (OXPHOS), as well as anti-oxidant capacity. NAD(P)H generates a
significant amount of cellular auto-fluorescence and is detectable by its emission peak at 470-nm. To examine the possible contribution of
NAD(P)H to stemness, MCF7 cells were subjected to metabolic fractionation by flow cytometry (FACS), to isolate the NAD(P)H-high,
-low and -negative cell populations. Each cell population was then plated in low-attachment cell culture plates, to assess their capacity for
mammosphere formation. Note that the NAD(P)H-high cell population showed a 5.4-fold increase in mammosphere formation, relative to
the NAD(P)H-negative cells. Therefore, high cellular NAD(P)H levels directly correlate with CSC activity. NAD(P)H-high = highest top
10% intensity; NAD(P)H-low = lowest bottom 10% intensity; NAD(P)H-negative = lowest possible intensity (no auto-fluorescence). B.
Effects of the inhibitor FK866. To induce cellular depletion of NAD(P)H, we used a known inhibitor of the NAD+ salvage pathway. Note
that treatment with FK866 dose-dependently inhibited mammosphere formation, with an IC-50 of less than 1 nM. Bar graphs are shown as
the mean SEM, t-test, two-tailed test. ***p < 0.0001. N = 3 independent experiments.

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 closure. Interestingly, Figure 12 shows that treatment
with CAPE (25 M for 24 hours) significantly reduced
wound closure by ~70%.
Thus, we conclude that CAPE functions as
a natural mitochondrial OXPHOS inhibitor, that
preferentially targets the CSC sub-population. This could
explain CAPEs known anti-cancer properties.

DISCUSSION

Here, we interrogated how metabolic heterogeneity

plays a key role in the generation of CSCs. More
specifically, we employed metabolic fractionation
by flow cytometry, to assess the potential role of i)
mitochondrial biogenesis, ii) oxidative stress (ROS and
H2O2 production) and iii) anti-oxidants, such as NAD(P)
Figure 6: Actinonin, a naturally-occurring
H, in CSC propagation (Figure 13). Our data directly
inhibitor of mitochondrial protein synthesis, blocks
shows that a small fraction of the total cell population,
mammosphere formation. Actinonin is a naturally-
characterized by increased PGC1 activity, high
occurring antibiotic. However, actinonin also functions as an
inhibitor of mitochondrial protein translation, by targeting a mitochondrial ROS/H2O2 and high NADH levels, has the
human mitochondrial protein, known as peptide deformylase ability to survive and grow under anchorage-independent
(abbreviated as PDF). PDF is required to remove the formyl- conditions, driving mammosphere formation. In order to
group from the initiator methionine of mitochondrial proteins carefully validate our observation, we used a battery of
synthesized on mitochondrial ribosomes. Note that actinonin selective metabolic inhibitors to target specific energetic
inhibited mammosphere formation, with an IC-50 between 25 pathways, significantly reducing and/or eliminating CSC
and 50 M. The bar graph shows the mean SEM, t-test, two- propagation. Thus, we have now identified a number
tailed test. ***p < 0.0001. N = 3 independent experiments.

Figure 7: Glycolysis inhibitors also block mammosphere formation. Here, we examined if 4 distinct glycolysis inhibitors have
the capacity to block mammosphere formation: A. 2-deoxy-glucose (2-DG); B. vitamin C (ascorbic acid); C. silibinin; and D. stiripentol.
Note that the two most potent glycolysis inhibitors that we identified were silibinin and stiripentol. Silibinin, which functionally blocks
glucose uptake, inhibited mammosphere formation, with an IC-50 between 200 and 400 M. Similarly, the FDA-approved drug stiripentol,
which behaves as an LDHA inhibitor, blocked mammosphere formation with an IC-50 between 100 and 200 M. Bar graphs are shown as
the mean SEM, t-test, two-tailed test. **p < 0.005, ***p < 0.0001. N = 3 independent experiments.

www.impactjournals.com/oncotarget 6 Oncotarget
 of clinically-approved drugs, natural substances and
experimental drugs that can be used to effectively inhibit
CSC activity. We highlight the utility of certain natural
products, such as Silibinin, Vitamin C and CAPE, that
could be used to therapeutically target CSCs. Silibinin
is the major active component of silymarin, which is an
extract prepared from milk thistle seeds.

NADH auto-fluorescence: A new biomarker for

CSCs
Figure 8: Chemical structure of caffeic acid phenyl
ester (CAPE), a key component of honey-bee propolis. NADH fluorescence is the dominant form of auto-
The presence of two aromatic rings in CAPE conveys significant fluorescence observed in living cells [24,25]. Interestingly,
hydrophobicity upon the molecule, which could increase previous studies have shown that normal intestinal
its affinity for cellular membranes, such the mitochondrial crypt stem cells have the highest levels NADH auto-
membrane. fluorescence [26], relative to other intestinal cell types.

Figure 10: Treatment with CAPE increases glycolysis.

Figure 9: Treatment with CAPE lowers mitochondrial The metabolic ECAR profile of MCF7 cells grown in monolayers,
respiration. The metabolic OCR profile of MCF7 cells after 72 hours of treatment with CAPE, was examined using
grown in monolayers, after 72 hours of treatment with CAPE, the Seahorse XFe96 analyser (Top panel, combination of three
was examined using the Seahorse XFe96 analyser (Top panel, experiments). The bar graph, in the Lower panel, shows that
representative experiment). The bar graph, in the Lower glycolysis (measured after addition of D-glucose) is increased
panel, shows that the all the parameters relative to the oxygen after treatment with CAPE at the concentrations of 10 M
consumption (OCR), are significantly decreased by both and 25 M, as compared to the vehicle-treated control cells
concentration of CAPE, 10 M and 25 M, as compared to the (DMSO). Data are presented in the graph as combination of N =
vehicle-treated control cells (DMSO). The bar graph shows the 3 independent experiments. Bar graphs are shown as the mean
mean SEM, t-test, two-tailed test. **p < 0.005, ***p < 0.0001. SEM, t-test, two-tailed test. ***p < 0.0001. N = 3 independent
N = 3 independent experiments. experiments.

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 So, high NADH is a property that is conserved between Moreover, it also behaves as an inhibitor of glycolysis, by
normal and cancerous stem cells. Previous studies have targeting the activity of GAPDH, a key glycolytic enzyme.
also shown that when non-CSCs and CSCs are both fed However, its effects on CSC activity was not previously
mitochondrial fuels (such as L-lactate or ketone bodies), evaluated. Here, we show that Vitamin C can also be used
that CSCs quantitatively produce more NADH in response to target the CSC population, as it is an inhibitor of energy
to this stimulus [27]. Similarly, Heeschen and colleagues metabolism that feeds into the mitochondrial TCA cycle
have previously shown that FAD auto-fluorescence is and OXPHOS. Similar results were also obtained with 3
also an excellent biomarker for CSCs [28]; however, they other glycolysis inhibitors, namely 2-DG, silibinin and
did not examine the utility of NADH as a biomarker for stiripentol. Importantly, stiripentol is a clinically-approved
CSCs in their studies [29]. Interestingly, high levels of drug, but its use is mainly restricted to the treatment of
NADH auto-fluorescence are a known to be a surrogate epileptic seizures in children, and not for cancer therapy
marker for mitochondrial power, high OXPHOS [19]. Thus, Vitamin C may prove to be promising agent
capacity and increased ATP production. Thus, CSCs for new clinical trials, aimed at testing its ability to
may be strictly dependent on NADH to maintain their reduce CSC activity in cancer patients, as an add-on to
enhanced mitochondrial function. In accordance with this more conventional therapies, to prevent tumor recurrence,
hypothesis, NAD+ depletion, using the NAMPT inhibitor further disease progression and metastasis. Interestingly,
FK866, potently blocked mammosphere formation, with a breast cancer based clinical study has already shown
an IC-50 of less than 1 nM. that the use of Vitamin C, concurrent with or within
6 months of chemotherapy, significantly reduces both
Vitamin C: Targeting metabolism and glycolysis tumor recurrence and patient mortality [31,32]. However,
in CSCs the mechanism underlying its potential clinical benefit
remained obscure. Similarly, Vitamin C treatment inhibits
tumor growth in murine animal models in vivo [33].
The Noble Prize winner, Linus Pauling, was among
the first to describe and clinically test the efficacy of CAPE: Targeting mitochondrial OXPHOS in
Vitamin C, as a relatively non-toxic anti-cancer agent
CSCs
[30]. More recently, Lew Cantleys group has revisited
the mechanism(s) by which Vitamin C targets cancer cells
[17]. Interestingly, they showed that Vitamin C has two CAPE is another natural product that we identified
mechanisms of action. First, it is a potent pro-oxidant, that which mechanistically targets oxidative mitochondrial
actively depletes the reduced glutathione pool, leading metabolism and, as a consequence, CSC activity. We
to cellular oxidative stress and apoptosis in cancer cells. directly show, using the Seahorse metabolic flux analyzer,

Figure 11: CAPE potently blocks mammosphere formation. A. 3D-sphere formation. MCF7 cells were plated in low-attachment
cell culture plates (6-well format), to assess the ability of CAPE to inhibit mammosphere formation. Note that CAPE treatment was
sufficient to significantly reduce mammosphere formation, with an IC-50 of approximately 2.5 M. B. Cell viability. The effects of CAPE
on the viability of adherent cells (MCF7 cells and hTERT-BJ1 fibroblasts) was assessed in parallel, using the SRB assay, in a 96-well
format. Note that CAPE significantly reduces the viability of MCF7 cells, with an IC-50 of 10 M. Similar results were obtained with
hTERT-BJ1 cells. Cell viability was assessed after 72 hours of treatment. Bar graphs are shown as the mean SEM, t-test, two-tailed test.
*p < 0.05, ***p < 0.0001. N = 3 independent experiments.

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 that CAPE quantitatively reduces mitochondrial oxygen MATERIALS AND METHODS
consumption (OCR), while inducing a reactive increase
in glycolysis (ECAR). As such, it potently inhibits
mammosphere formation with an IC-50 of ~2.5 M. Materials
Similarly, it also significantly inhibits cell migration.
Therefore, further metabolic studies using CAPE and
other natural components of honey-bee propolis may be MCF7 cells were originally purchased from the
warranted. ATCC. The lentiviral vector encoding the promoter of
the murine PGC1 gene, linked to the eGFP reporter,
and containing a puromycin-resistance cassette, was
CONCLUSIONS custom synthesized by GenecopoeiaTM (Maryland,
USA; mPGC1-eGFP-Puro-R). This construct was
In summary, we have discovered that NAD(P)
then used to stably transduce MCF7 cells. Media for
H auto-fluorescence and several mitochondrial-based
cell culture was DMEM (D6546, Sigma-Aldrich). Cell
fluorescent probes can all be employed to enrich for a
culture media (DMEM/F12) for spheroid culture was
population of cells with the characteristics of CSCs. In
purchased from Life Technologies. Caffeic acid phenetyl
accordance with these observations, we also demonstrate
ester (CAPE), sulforhodamine B (SRB), the glycolytic
that 7 different inhibitors of key energetic pathways can
inhibitor 2-Deoxy-D-Glucose (2-DG), L-ascorbic acid,
be used to effectively block CSC propagation, including
mitochondria peroxy yellow 1 (Mito-PY1), dihydro-
three natural products (silibinin, ascorbic acid and CAPE).
rhodamine 123, and FK866 hydrochloride hydrate, were
Future studies will be necessary to test their potential for
all purchased from Sigma-Aldrich.
clinical benefit in cancer patients.
Generation of stably-transduced MCF7 cells

MCF7 cells were transduced with a lentiviral vector

encoding the following reporter plasmid: mPGC1-eGFP-

Figure 12: CAPE decreases the migratory capacity of Figure 13: Mitochondrial oxidative stress drives
MCF7 cells. MCF7 cell migration was monitored using an mitochondrial biogenesis and the anti-oxidant
in vitro scratch assay. Images were acquired with the IncuCyte response, leading to increased mitochondrial
Zoom instrument at 0 and 24 hours. The Upper panel of the metabolism and stemness. We summarize here our overall
figure shows representative images. MCF7 were treated with or findings schematically, with a focus on how mitochondrial
without CAPE, at a concentration of 25 uM. Note that CAPE- ROS and H2O2 production may be a trigger, for driving
treated MCF7 cells migrated slower, as compared to vehicle- mitochondrial oxidative stress, ultimately leading to increased
treated MCF7 cells. In the Lower panel, quantification of cell mitochondrial oxidative metabolism in CSCs. Pharmaceuticals,
migration is presented as % wound-closure. Bar graphs are chemical inhibitors, or natural products that we have identified
shown as the mean SEM, t-test, two-tailed test. ***p < 0.0001. to target CSCs, which interfere with a specific metabolic process
Scale bar = 250 m. N = 3 independent experiments. or function, are shown in BLUE.

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 Puro-R. Briefly, the lentiviral vector was transfected into trypsinized, resuspended in PBS and were separated by
the 293Ta packaging cells, following the manufacturers flow cytometry. Based on their fluorescence intensity, the
instructions. Two days post-transfection, the cell culture MCF7 were fractionated and sorted into three distinct cell
supernatant was collected and centrifuged and used to populations: very low fluorescent cells, low 5% and top
infect MCF7 cells. After lentiviral transduction, MCF7 5% GFP cells. The cells were immediately plated under
cells were selected for 12 days in puromycin (2 g/ml). non-adherent conditions at the density of five thousands
cells in each well of a six wells-plate to evaluate their
FACScan analysis and sorting mammosphere-forming ability.

NAD(P)H measurements and treatment with

MCF7 cells were subjected to metabolic
fractionation using either the BD FACSAriaTM III and FK866
BD InfluxTM cells sorters. Data were analyzed using
FlowJo software (Tree Star, Ashland, OR). Different cell To optically monitor the levels of mitochondrial
populations were isolated and subjected to mammosphere OXPHOS in MCF7 cells, it is possible to measure NADH
analysis. auto-fluorescence by FACS analysis [24,25]. After UV
excitation, NADH emits auto-fluorescence at 470-nm.
Mammosphere formation (3D-Spheroid culture) So, we used the fluorescence properties of NADH to sort
MCF7 cells into three distinct populations, based on their
cellular auto-fluorescence.
A single cell suspension was prepared using
Afterwards, we tested their mammosphere-forming
enzymatic (1x Trypsin-EDTA, Sigma Aldrich, #T3924),
ability. Furthermore, we investigated the effect of
and manual disaggregation (25 gauge needle) was
FK866, a potent inhibitor of the NAD+ salvage pathway
used to create a single cell suspension. Five thousand
biosynthesis, on the mammosphere-forming ability of
cells were plated with in mammosphere medium
the sorted cell populations. As it is difficult to distinguish
(DMEM-F12/B27/20ng/ml EGF/PenStrep), under non-
between NADH and NADPH auto-fluorescence (both emit
adherent conditions, in six wells plates coated with
at 470-nm), the values observed represent the sum of these
2-hydroxyethylmethacrylate (poly-HEMA, Sigma,
two molecules: NAD(P)H auto-fluorescence. In contrast,
#P3932) [34]. Cells were grown for 5 days and maintained
FAD emits auto-fluorescence at 525-nm. Thus, NAD(P)H
in a humidified incubator at 37C at an atmospheric
and FAD auto-fluorescence are distinct.
pressure in 5% (v/v) carbon dioxide/air. After five days
of culture, the number of spheres with diameter >50 m
were counted. Metabolic flux analysis

Mitochondrial ROS production Extracellular acidification rates (ECAR) and

oxygen consumption rates (OCR) were analysed using
the Seahorse XFe96 bioenergetic analyzer (Seahorse
To measure mitochondrial ROS production, we used
Bioscience, MA, USA). MCF7 cells were maintained
a well-known fluorogenic vital probe, namely dihydro-
in DMEM supplemented with 10% FBS (fetal bovine
rhodamine 123 (Cat. No. D1054). For this purpose,
serum), 2 mM GlutaMAX, and 1% Pen- Strep. Fifteen
samples containing 5 x 106 MCF7 cells (volume 500 l)
thousand MCF7 cells were seeded per well, into XFe96-
were incubated for 10 min at 37oC, together with dihydro-
well cell culture plates, and incubated at 37C in a 5%
rhodamine 123 at 10 M. Then, cold PBS was added and
CO2 humidified atmosphere. After 72 hours, MCF7
the samples were FACS sorted based on the intensity of
cells were washed in pre-warmed XF assay media, as
the fluorochrome. The three cell populations obtained by
previously described. ECAR and OCR measurements
sorting were seeded under non-adherent conditions and
were normalized for cell protein content, by the SRB
tested for their mammosphere- forming ability.
colorimetric assay. Data sets were analyzed using XFe-96
software and Excel software.
Mitochondrial hydrogen peroxide production
Cell migration: in vitro scratch assay
For the detection of mitochondrial hydrogen
peroxide production, we used Mito-PY1 as a vital
MCF7 cells were seeded in 12 wells-plates to create
fluorescent dye (Cat. No. SML0734). Live cells under
a confluent monolayer. Cell monolayers were then scraped
adherent conditions were labelled with Mito-PY1
to create a scratch with a p-200 pipet tip. Plates were
(10 M) in a PBS solution. After 45 min, Mito-PY1
incubated in the IncuCyteTM Zoom (Essen Bioscience) in
was removed and replaced with fresh warm PBS. The
5% CO2 at 370C for 24 hours. Complete culture medium
washes were repeated one more time and the cells were

www.impactjournals.com/oncotarget 10 Oncotarget
 containing 10% FBS, -/+ CAPE 25 M was used. The 5. Martinez-Outschoorn UE, Peiris-Pags M, Pestell RG,
images were acquired and scanned over a period of 24 Sotgia F, Lisanti MP. Cancer metabolism: a therapeutic
hours. Values for migration were obtained after scanning perspective. Nat Rev Clin Oncol. 2017; 14: 11-31.
the whole wells (with a 4X objective) and represent the 6. Peiris-Pags M, Martinez-Outschoorn UE, Pestell RG,
average of three independent experiments. The rate of Sotgia F, Lisanti MP. Cancer stem cell metabolism. Breast
the migration was evaluated using Image-J software, to Cancer Res. 2016; 18: 55.
measure wound closure. 7. Lamb R, Harrison H, Hulit J, Smith DL, Lisanti MP, Sotgia
F. Mitochondria as new therapeutic targets for eradicating
Statistics cancer stem cells: Quantitative proteomics and functional
validation via MCT1/2 inhibition. Oncotarget. 2014; 5:
All data are presented as the mean SEM. The 11029-37. doi: 10.18632/oncotarget.2789.
Students t-test was used. P < 0.05 was considered 8. De Luca A, Fiorillo M, Peiris-Pags M, Ozsvari B,
statistically significant. Smith DL, Sanchez-Alvarez R, Martinez-Outschoorn
UE, Cappello AR, Pezzi V, Lisanti MP, Sotgia F.
ACKNOWLEDGMENTS Mitochondrial biogenesis is required for the anchorage-
independent survival and propagation of stem-like cancer
Dr. Ernestina M. De Francesco was supported by cells. Oncotarget. 2015; 6: 14777-95. doi: 10.18632/
an iCARE fellowship from the Associazione Italiana per oncotarget.4401.
la Ricerca sul Cancro (AIRC) cofunded by Marie Curie 9. Farnie G, Sotgia F, Lisanti MP. High mitochondrial mass
Actions. The Lisanti Laboratory is currently supported identifies a sub-population of stem-like cancer cells that
by private donations, and by funds from the Healthy Life are chemo-resistant. Oncotarget. 2015; 6 :30472-86. doi:
Foundation (HLF) and the University of Salford. 10.18632/oncotarget.5401.
10. Lamb R, Bonuccelli G, Ozsvri B, Peiris-Pags M, Fiorillo
CONFLICTS OF INTEREST M, Smith DL, Bevilacqua G, Mazzanti CM, McDonnell LA,
Naccarato AG, Chiu M, Wynne L, Martinez-Outschoorn
There is no conflict of interest. UE, et al. Mitochondrial mass, a new metabolic biomarker
for stem-like cancer cells: Understanding WNT/FGF-driven
Author contributions anabolic signaling. Oncotarget. 2015; 6: 30453-71. doi:
10.18632/oncotarget.5852.
11. Fiorillo M, Lamb R, Tanowitz HB, Mutti L, Krstic-
Professor Lisanti conceived and initiated this Demonacos M, Cappello AR, Martinez-Outschoorn UE,
collaborative project. All the experiments in this paper Sotgia F, Lisanti MP. Repurposing atovaquone: targeting
were designed and performed by Gloria Bonuccelli. mitochondrial complex III and OXPHOS to eradicate
Experiments on CAPE were jointly performed by Gloria cancer stem cells. Oncotarget. 2016; 7: 34084-99. doi:
Bonuccelli and Rianne de Boer. Gloria Bonuccelli 10.18632/oncotarget.9122.
analyzed all the data and generated the final figures
12. Fiorillo M, Lamb R, Tanowitz HB, Cappello AR, Martinez-
and tables, and she wrote significant portions of the
Outschoorn UE, Sotgia F, Lisanti MP. Bedaquiline, an
manuscript. Professor Lisanti, Ernestina M. De Francesco
FDA-approved antibiotic, inhibits mitochondrial function
and Herbert B. Tanowitz contributed to the writing and the
and potently blocks the proliferative expansion of stem-like
editing of the manuscript. Professor Lisanti generated the
cancer cells (CSCs). Aging (Albany NY). 2016; 8: 1593-
schematic summary diagram.
607. doi: 10.18632/aging.100983.
13. Lamb R, Ozsvari B, Lisanti CL, Tanowitz HB, Howell A,
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