Pamela Swain1, Natalia Romero1, Yoonseok Kam1, Genevieve Van de Bittner2,Mahmoud ElAzzouny3, Brian P. Dranka1

Differential use of lactate for mitochondria respiration by NSCLC cells

1Cell Analysis Division, Agilent Technologies, Lexington, MA; 2Agilent Research

AACR 2019
POSTER #810

Laboratories and 3Mass Spectrometry Division, Agilent Technologies, Santa Clara, CAResults and Discussion

Introduction• Identification of metabolic liabilities in cancer cells
through metabolic phenotypic screening provides
opportunity for novel therapeutic approaches.
• Non-small cell lung cancer (NSCLC) is typically driven byoncogenic mutations in either KRAS or EGFR. However,
the impact of these mutations on cellular metabolic
phenotype is not well-studied.
• There is emerging evidence to suggest that lactate is amajor fuel for cancer cell energy metabolism, especiallyin the glucose-limited tumor microenvironment.
• In this study, we evaluated metabolic phenotypes ofKRAS- and EGFR-mutated NSCLC cells and thecontribution of lactate in their bioenergetic metabolism
by measuring the real-time ATP production rates andstable isotope incorporation in the TCA cycle using theAgilent Seahorse XF Analyzer and the Agilent LC/Q-TOF
Technology respectively.
3 6 % N o n o n c o g e n ic
25 %KRAS

21%

EGFR

8 % ALK
3 % ERBB2
2 % BRAF
5 % E tc (< 1 % )Percent data from
Kris, MG JAMA 2014

Figure 1: Frequency of
oncogenic drivers inadenocarcinoma of thelung detected by the Lung
Cancer Mutation
Consortium.

Results and Discussion

EGFR- and KRAS-mutated NSCLC cells show distinct
bioenergetic phenotypes
AB

Conclusions

Inhibition of MPC, but not MCT1 suppressesmitochondrial use of lactate in PC9AID

Inhibitor Treatment Target

ARC AR-C155858 (10µM) MCT1AZD AZD3965 (25nM)
MCT1
UK UK5099 (2µM)
MPCB• Recent reports strongly suggest that some cancer cells
preferentially use lactate as a mitochondrial fuelsource. We identified that EGFR-mutated PC9 cells but
not A549 cells (KRAS mutant) use lactate to increase
mitochondrial ATP production regardless of glucose
availability.

EGFR

KRAS

Figure 3: (A) Metabolic Profile of 4 cellular models of NSCLC instandard assay medium (XF RPMI containing 10 mM Glucose,
1 mM Pyruvate, 2 mM Glutamine, pH 7.4). (B) Percent of total
ATP production rate from glycolysis and oxidative
phosphorylation.

Mitochondrial fuel utilization varies between EGFRand KRAS-mutated NSCLC cells
A

PC9 cells (EGFR mut.)

B

A549 cells (KRAS mut.)

Figure 6: (A) Mitochondrial ATP production rates in PC9 cells
treated with MPC inhibitor, UK5099 (UK) or with MCT1 inhibitor,
AZD3965 (AZD) or AR-C155858 (ARC) for 30 min prior toaddition of 10 mM lactate in the presence of 5mM glucose.
(B) Scheme of selected lactate metabolic reactions.

Metabolite tracing data confirms lactate uptake into
the cell and incorporation in TCA cycle intermediates
mainly thru MPC
BAID

Inhibitor Treatment Target

ARC AR-C155858 (10µM) MCT1
UK UK5099 (2µM)
MPC

Experimental

Kinetic profile of oxygen consumption rate (OCR) andextracellular acidification rate (ECAR) measurement. The
injection of oligomycin and rotenone plus antimycin A enables
calculation of mitochondrial and glycolytic ATP production(Romero, et. al., Agilent White Paper, 2018 ).

Stable isotope labeling using U-13C lactate: Cells wereincubated with 5 mM glucose, 10 mM 13C3-lactate +/MPC or MCT inhibitors for 2hr at 37oC. Samples weresnap frozen, extracted with ice cold 8:1:1 methanol:
water:chloroform and supernatant was injected aftercentrifugation. Metabolite samples were analyzed using a6546 LC/Q TOF coupled to an Agilent 1290 Infinity II LC.
Chromatographic separation was performed on anAgilent InfinityLab Poroshell 120 HILIC-Z, 2.1 × 100 mm,
2.7 μm with a HILIC-Z UHPLC Guard column. Data wereacquired and analyzed using MassHunter Acquisition
software and Agilent Vistaflux.

Figure 4: Metabolic profile of ATP production rate in thepresence of multiple fuels. (A) PC9 cells can utilize lactate as
mitochondrial fuel. (B) A549 cells do not use lactate.

EGFR-mutated NSCLC cells utilize lactate as
mitochondrial fuel regardless of glucose availabilityA

PC9 cells (EGFR mut.)B• PC9 cells labeled with13C3-lactate showed theincorporation of lactate carbons into TCA cycle
metabolites that was blocked in the presence of theMPC inhibitor UK5099. These results correlate with theobserved increase in mitochondrial ATP production
rate in the presence of lactate using the XF ATP Rate
Assay, which was inhibited by pretreatment withUK5099.
• MCT1 inhibition did not block lactate uptake, although
MCT1 is known to be used both for import and exportof lactate. The presence of other MCT isoform was not
excluded and further studies are required.
• These results suggest NSCLC variants have adopted
different metabolic phenotypes depending on theoncogenic background. This variation in thebioenergetic phenotype can be a critical factor for acancer therapy design targeting metabolic vulnerability.

The Agilent Seahorse XF Real-Time ATP Rate Assay wasused to simultaneously measure the two main
bioenergetic pathways
Figure 2.
Representative
scheme ofAgilent Seahorse
XF Real-time ATPRate Assay.

• We observed that the two KRAS-mutated NSCLC celllines (A549 and H460) are relatively more reliant on
glycolysis for ATP production than EGFR-mutated celllines (H1975 and PC9).

References

A549 cells (KRAS mut.)

Romero, N., et al. Quantifying cellular ATP production rate using
Agilent Seahorse XF Technology. Agilent White Paper (2018)(http://www.agilent.com/chem/realtimeatp)
Kerr, EM., et al. Metabolic rewiring in mutant Kras lung cancer.
FEBS J. 285 (1), 28-41 (2018)

Faubert, B., et al. Lactate Metabolism in Human Lung Tumors.
Cell 171, 358-371 (2017).
Kris, MG., et al. Using multiplexed assays of oncogenic drivers inlung cancers to select targeted drugs. JAMA. 311(19),1998–2006(2014)

Figure 5: Profile of ATP production rate from glycolysis andmitochondrial respiration +/- lactate and different glucose
concentrations in (A) PC9 cells (B) A549 cells.

Figure 7: (A) Stable isotope labeling of TCA cycle metabolites
using 13C3-lactate in PC9 cells (n=3 replicates). (B) Subset of theXF mitoATP production rate which correlates to the metabolic
flux data.

For Research Use Only. Not for use in diagnostic
procedures.