Presented at the American Association of Cancer Researchers Annual Meeting, April 4, 2017.
Poster Number: 5432

Characterization of fuel dependencies in multidrug resistant breast cancer cells
Yoonseok Kam, Natalia Romero, Pam Swain and Brian P. Dranka
Cell Analysis Division, Agilent Technologies, Lexington, MA.

AResults• Multidrug resistance (MDR), a common drug resistant mechanism of cancer cells ischaracterized by increased expression of ATP-dependent drug exporting pumps which remove
cytotoxic compounds from the cytosol of resistant cells.

MCF7A

MCF7

MCF7 Dox

2-DGRot/AARot/AA2-DG

BBCBasal glycolysis

Compensatory glycolysis

Figure 1: MCF7 Dox loses
mitochondrial glutamine
dependency. Relative dependency
on glutamine oxidation by MCF7 Dox
was eliminated, while maintaining ahigh glucose dependency andcapacity when the Mito Fuel Flex test
was performed. *MCF7 Dox cells weregenerously provided by Dr. Robert Gillies at H.
Lee Moffitt Cancer Center, Tampa, FL.

Methods
The Seahorse XF Glycolytic Rate Assay is an accurate and reliable method for measuring
glycolysis, providing measurements of glycolytic rates for basal conditions and compensatory
glycolysis following mitochondrial inhibition (Fig. 2A). The calculated rates account forcontribution of CO2 to extracellular acidification derived from mitochondrial/TCA cycle activity
and are directly comparable to orthogonal lactate accumulation data (Fig. 2B).

AMCF7

MCF7 Dox

67.6%

75.1%

Oligomycin

COligomycin

mitoOCR/glycoPER 0.83 ± 0.03 0.54 ± 0.02

Figure 3. Seahorse XF Glycolytic Rate assay results obtained in MCF7 and MCF7 Dox cells. MCF7 and MCF7 DOX
cells were plated the day before the assay (25K cell/well) in a XF96 Cell Culture Microplate. After 24 hr, cells were assayedfor both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in DMEM Base Medium without phenol
red, supplemented with 10 mM glucose, 2 mM glutamine, 1 mM pyruvate, 5 mM HEPES, pH 7.4. (A) Representative
Glycolytic Rate Assay profile obtained for MCF7 or the MDR variant MCF7 Dox. MCF7 DOX showed a higher glycolytic
phenotype as indicated by higher Basal Glycolysis (B), higher Compensatory Glycolysis (C) and lower mitoOCR/glycoPERbasal ratio (D).

MCF7

MCF7 Dox-+-+Figure 5. MDR-mediated drug efflux relies on ATP generated by mitochondria. MCF7 and MCF7 Dox were incubated in thepresence of calcein-AM in DMEM Base Medium without phenol red, supplemented with 10 mM glucose, 2 mM glutamine, 1 mM
pyruvate, 5 mM HEPES, pH 7.4 for 10 min at 37oC with or without pretreatment of 2-deoxy-D-glucose (2-DG, 50 mM) oroligomycin (1 µM) for 10 min. The calcein-AM retained in cells were compared by using the fluorescent images captured (A) andthe whole well fluorescent intensities measured (B) by BioTek® CytationTM 5. (C) Total ATP formation was calculated as the sum ofGlycolytic ATP Rate formation (equivalent to glycoPER) and mitochondrial-derived ATP Rate formation that was estimated fromthe ATP-coupled OCR and assuming a P/O ratio of 2.79 3.

Conclusions• Using the Seahorse XF Glycolytic Rate Assay, basal glycolytic rates and compensatory glycolysis(when mitochondrial ATP production is blocked) are higher in MCF7 Dox cells compared to wild
type cells, confirming the highly glycolytic phenotype of the MCF7 Dox variant.

MCF7

MCF7 Dox

Slope = 1.0
R2 = 0.90

For Research Use Only. Not for use in diagnostic procedures

2-DG

D% PER fromglycolysis

BFigure 2. Seahorse XF Glycolytic Rate Assay. (A) Proton efflux from live cells comprises both glycolytic and mitochondrialderived acidification. Inhibition of mitochondrial function by injecting rotenone & antimycin A (Rot/AA) enables calculation ofmitochondrial-associated acidification. Subtraction of mitochondrial acidification from Total Proton Efflux Rate results in Glycolytic
Proton Efflux Rate. (B) Glycolytic Proton Efflux rate obtained using the Glycolytic Rate Assay shows tight correlation with basal
lactate measured as total lactate accumulated (nmol) in extracellular media during 1 hr (using a fluorescence based lactate kit) andconverted to rate values (pmol/min). Results correspond to pooled rate data obtained using the following cell lines (all in triplicate):
A431, A549, BAEC, C2C12, CD4+ T cells, H9C2, HepG2, MDA-MB-231, neonatal rat ventricular myocytes (NRVM) and rat corticalneurons and in the absence or presence of the mitochondrial pyruvate uptake inhibitor UK5099.

ControlHoechst• MCF7 Dox*, an MDR variant of the MCF7 breast cancer cell line has a highly glycolytic
phenotype, with an increased glucose uptake/consumption rate in vitro and in vivo that
contributes to maintaining the MDR phenotype1. However, recently studies suggest thatmitochondrial modulators have a significant role in avoiding development of MDR phenotypes2.
• We have previously showed using the Seahorse XF Mito Fuel Flex Test that MCF7 and MCF7Dox cells are both highly dependent on glucose, however the MDR cells show significantly lessdependence on glutamine oxidation (Fig. 1).

MCF7 DoxCalcein

Introduction

UK5099-++-++BPTES--+--+

Figure 4. Induced Glycolytic Rates obtainedin MCF7 and MCF7 Dox cells withmitochondrial fuel oxidation inhibitors. TheGlycolytic Rate Assay was performed in MCF7and MCF7 Dox cells after injection of themitochondrial pyruvate carrier (MPC) inhibitor
UK5099 (2 µM), and glutaminase I inhibitor
BPTES (3 µM). Glycolytic rates increased afterinhibition of mitochondrial pyruvate oxidation, inagreement of high glucose dependency
obtained using the Mito Fuel Flex Test.
Additionally, inhibition of glutamine oxidation hasa lower impact in MCF7 Dox compared toMCF7, confirming the lack of dependency ofMCF7 Dox on glutamine oxidation. XF assay
conditions are the same as described in Fig. 3above.

• Addition of the MPC inhibitor, UK5099, induced a metabolic switch to glycolysis, confirming thestrong dependency and low flexibility of both cell variants for glucose as mitochondrial fuel. Inaddition, a further glycolytic shift is induced after combined inhibition of glutamine/pyruvate
oxidation in MCF7 cells that is not observed in MCF7 Dox cells, in agreement with their lack ofdependency for glutamine as a metabolic fuel.
• Calcein-efflux studies strongly suggest that the MDR mechanism relies on mitochondrial
generated ATP for maintaining a chemo-resistant phenotype.
• Fuel dependency characterization may have potential therapeutic relevance to understandtherapy resistance.

References

1. Silva, A.S., et al., Evolutionary approaches to prolong progression-free survival in breast cancer. Cancer Res 2012, 72, 6362-70.
2. Fernandez-Cabezudo, M.J., et al., Deficiency of mitochondrial modulator MCJ promotes chemoresistance in breast cancer. JCIInsight 2016, 1.
3. Mookerjee, S.A., et al., Quantifying Intracellular Rates of Glycolytic and Oxidative ATP Production and Consumption Using
Extracellular Flux Measurements. J Biol Chem 2017.