Metabolic discovery platform:“Seahorse-Bravo”
Gestin M1, Lacoste E1, Vikstrom S2, Schultz L2, Giampaolo G2, Arnaud L2, Iochem C2, Desmoulins S2,Faustin B1,Obre E1 and Rossignol R1

www.agilent.com/chem/discoverxf

1) CELLOMET (ADERA/University of Bordeaux) – email: emilie.obre@cellomet.com
2) Agilent Technologies – email : lesley.schultz@agilent.com

www.cellomet.com

INTRODUCTION
The regulation of energy metabolism is very complex and involves a large number of signaling pathways andmolecular effectors. Recent development in biology revealed that cancer cells can rewire most metabolic pathways,
revealing novel means of bioenergetic regulation (Figure 1). For instance, it was shown that C-MYC oncogene controls
glutamine-supported cellular respiration and lipid synthesis or that glycolysis bottlenecking by PKM2 promotes denovo serine biosynthesis. With the recently ascertained role of mitochondria in cancer biology, the search for novel
regulators of mitochondrial respiration was performed using genetic means, as CRISPR-CAS9 or shRNA libraries, or
pharmacological means such as drug-libraries. There is a growing need in academia and industry to disposetechnology solutions enabling the discovery of mitochondrial respiration regulators (genes or compounds) using
standardized and reproducible high-precision methods. The aim of this unprecedented CELLOMET-Agilent
collaboration is to evaluate the quality and the efficiency of coupling the Agilent Seahorse XF96 Analyzer
extracellular flux analyzer with the Agilent Bravo Automated Liquid Handling Platform.

Agilent Bravo

For each objective we seeded 30.000 cells of each of the two lung cancer celllines (A549 and H460) in each well of a 96-well plate. Respiration wasmeasured using the Agilent Seahorse XF96 Analyzer at the CELLOMET
bioenergetic investigation in Bordeaux (France). A mitostress kit was used inthe cartridge plate to measure basal, minimal and uncoupled respiration rate.

Objective 1 : Decipher the metabolic profile
of two cancer cell lines of variablebioenergetics. ( Seahorse analysis)

Agilent Seahorse
XF96 Analyzer

VS

Objective 2 : Compare the performance ofthe Bravo Platform against human pipetting(Evaluation of the Z prim factor).

 Automatedpipetting platform

Mitochondrial
physiology

 Metabolism
analyzers

Metabolicremodeling

FIGURE 1

Bravo platform

Objective 3 : Identification of bioenergetic
modulators adapted to the metabolic
profile of the cancer cells(Determination of theIC50 ofmetformin)

Z prim evaluation : The precision and reproducibility of our procedures can beevaluated by assessing the Z prim factor, which is a measure used for theGO/NO GO decision when performing high throughput screens of drugs orsiRNA libraries. When precised, the cells were incubated with a cocktail ofmitochondrial inhibitors for two hours. After this incubation time, a Seahorse
test was performed at the CELLOMET facility.

HumanIC50 of metformin test : The performance of the Bravo automate in the dilutionof compounds was tested by the evaluation of the IC50 of metformin, acomplex I inhibitor approved in the clinics for the treatment of diabetes.
Metformin efficacy was tested on the cell lines of varying bioenergetics in cotreatment with doxorubicin, a valid chemotherapeutic drug (1µM). Different
concentrations of metformin (0µM to 4mM) were tested, during 24h. To
complete the results obtained on IC50 measurement with the Agilent Seahorse
XF96 Analyzer, we performed a measure of respiratory chain complex I
spectrophotometric activity using CELLOMET standardized protocol.

Bioenergetic
therapy

We brought the precision of theBravo platform to the highthroughput efficiency of theAgilent Seahorse XF96 Analyzer
and CELLOMET’s know-how.

Experts in the functional andmolecular investigation of cellular
metabolism

Agilent Seahorse XF96Analyzer

Objective 2 : Bravo platform versus human pipetting: Where’s the difference?
(Evaluation of Z prim factor.)

Objective 1 : Bioenergetic profiling of two lung cancer cell lines
Seahorse
analysisallowed to stratify twocell lines according totheirmetabolism.
A549 is oxidative andH460
is
more
glycolytic.

The coefficient of variation is a useful statistical index for comparing thedegree of variation from one data series to another.
Interpretation : 0<CV<4,9% : excellent, 5%<CV<9,9%: very good 10%<CV<14,9%: good,15%<CV<24,9%: acceptable, 25%<CV :marginal
coefficient of variation (CV)

A549

H460

Routine Respiration

6,68,1Non phosphorylating respiration

4,6

3,2Maximal respiration

9,37

Non-mitochondrial respiration

3,9

4,8

The coefficient ofvariation is very goodin each cell line

The Z’ factor is a statistic tool that permit to evaluate the precision of pipetting. The Z’ robust factor isused for the high- throughput screening assay.
Interpretation of Z’ factor : Z’ = 1 : Ideal; 0,5<Z’< 1 : an excellent assay; 0<Z’<0,5 : a marginal assay

 Agilent Seahorse XF96
Analyzer can stratify cancer
cells according to theirbioenergetics

A549
Z’Z’ Robust

Bravo
0,700,86

Human
0,50,65

H460
Z’Z’ Robust

Bravo
0,730,89

Human
0,220,12Z’ factor was calculated from the data of respiration using OXPHOS inhibitors as positive controls

In A549 cells as in H460 cells, the Z’ factor and the Z’ robust factor was better (close to 1) with Bravo platform
than human pipetting.

CELLOMET
Agilent
Collaboration

 The Bravo platform has a much better precision ofpipetting than a trained qualified engineer

Objective 3 : Secondary drug screening using a phenotypic read-out (mitochondrial respiration) and hit validation by targeted CELLOMET assay

No significant cancer-killing effect of metformin was found on
H460 cells of glycolytic profile.

14016120

1412100

1080

860

640
202002000

400006000

1816100
OCR
Complex I activity
Log. (OCR)

4[Metformin, µM]

The Bravo automate combined to Agilent Seahorse
XF96 Analyzer allowed to find one drug, metformin,that specifically targets cancer cells with oxidativebioenergetic profile

120Log. (Complex Iactivity)

1480

1210608406Complex I activity

18

OCR (% of control)

160

Complex I activity

In A549 cells with Bravo pipetting, the IC50 determination curve
showed a sigmoid profile that could not be established with humanpipetting.

MOA study by CELLOMET confirmed that Metfomin inhibits Complex I in A549 cells

OCR (pmol/min))(% of control)

We hypothesized that metformin could alter specifically theoxidative lung cancer cells (A549) based on the metabolic profiling
analysis (objective 1).

OCR
Complex I activity
Log. (OCR)

4202001000

2000

3000

4000

Log. (Complex Iactivity)

05000[Metformin, µM]

 With the Bravo platform the determination of the IC50 is more precise thanhuman pipetting and IC50 determination more precise.

 CELLOMET targeted enzymatic assay provide a MOA for
metformin anti-cancer effect.

CONCLUSION
Combining the Bravo Automated Liquid Handling Platform and the Agilent Seahorse XF96 Analyzer provides an accurate bioenergetic platform with excellent
characteristics in terms of reproducibility and efficiency. Such platform can be used to study cellular energetics in a large number of contexts and to discoverdrug or gene modulations that impact mitochondrial respiration and cellular glycolysis. The Seahorse-Bravo equipment was validated by CELLOMET experts
and a proof-of-concept experiment presented here showed the differences between A549 and H460 cells as well as their sensitivity to mitochondrial
respiration by metformin.

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