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Tafur et al. J Cancer Metastasis Treat 2018;5:xx I http://dx.doi.org/10.20517/2394-4722.2018.102 Page 3 of 14

However, different isoforms have different affinities for lactate and pyruvate, which influences whether they
act predominantly as cellular exporters or importers of lactate and H+. MCT1 is a ubiquitous lactate/H+
symporter that is involved in both import and export of lactate, whereas MCT4 is primarily involved in lactate/
H+ export. Both MCT1 and MCT4 are differentially expressed in several cancers, including breast cancer .
[21]
Additional important players in lactate shuttling between cancer cells are the lactate dehydrogenases A and
B (LDHA and LDHB) that catalyze the reversible conversion of pyruvate to lactate, alter the NAD+/NADH
ratio and thus glycolytic flux, and have also been implicated in EMT and metastasis .
[22]
Previous studies have identified the cell-surface G-protein-coupled receptor, GPR81, as an endogenous lactate
receptor. GPR81 family of receptors consists of three highly homologous members, GPR109a, GPR109b and
GPR81, which are regulated by the specific agonists, 3-hydroxybutyrate, 3-hydroxyoctanoate, and lactate,
respectively . GPR81 has been mainly studied in adipocytes, in which the receptor is coupled to Gi/q.
[23]
GPR81 activation by lactate decreases cAMP production, which ultimately reduces lipolysis . Recently
[24]
GPR81 has been shown to be upregulated in several cancers (pancreas, colon, liver, breast, cervix) and its
expression levels associated with tumor growth and metastasis [25-29] . In this current study, we examined
GPR81 expression in the context of EMT in breast cancer. We provide evidence that GPR81 promotes
lactate transport by MCT1 in hormone-positive breast cancer cells and its expression is crucial for their cell
proliferation and survival.

METHODS
Cell culture
BT-474 and MCF-7 cell lines were obtained from the American Type Culture Collection (Manassas, VA).
EMT in MCF-7 and BT-747 cells was induced using prolonged mammosphere culture method as reported
previously . BT-474 and MCF-7 and their corresponding EMT-derived BT-474-M and MCF-7-M cells were
[7,8]
grown in DMEM/F-12 containing 10% heat inactivated FBS (Gibco, Grand Island, NY) and 1 × MycoZap TM
Plus-CL antibiotic (Lonza, Walkersville, MD). BT-474 and MCF-7 cells were additionally supplemented with
1 × Insulin-Transferrin-Selenium solution (Gibco, Grand Island, NY). All cells were incubated at 5% CO2
and at 37 °C.

3-dimensional culture
TM
TM
TM
Corning Matrigel GFR (Fisher Scientific) or Matrigel (Corning) was used as culture matrix following
a modified version of the previously described protocol for 3-dimensional on-top assay . Twenty-four-well
[30]
TM
plates were coated with 200ul of the mix of 1:1 Matrigel /Physiological modified medium supplemented
with 17 µmol/L Insulin-Transferrin-Selenium solution (Gibco, Grand Island, NY) and kept at 37 °C for 30
min to allow the Matrigel to solidify. All cell lines were suspended in physiological modified medium
TM
supplemented with 1× insulin and seeded into the coated twenty-four-plates. Epithelial and mesenchymal
cells were seeded in 250 µL at 100,000 cells/well and 20,000 cells/well, respectively, and incubated at 37 °C for
30 min to allow cell attachment to the Matrigel. All wells were overlaid with 250 µL of physiological modified
TM
medium containing 4% Matrigel . Culture medium was replaced with fresh medium daily and cells were
used for each assay after seven days, unless otherwise indicated.

Physiological modified medium and drugs
RPMI 1640 Medium Modified without L-glutamine, amino acids and glucose (powder, R9010-02; US
Biological, Swampscott, MA) was supplemented with the following: from Sigma (St. Louis, MO) - 1X MEM
amino acid mixture (minus glutamine) (M5550), 1× Lipid Mixture 1 (L0288), 14.3 mmol/L sodium bicarbonate
powder (S5761); from ThermoFisher/Gibco, (Waltham, MA) -1× non-essential amino acid mixture (11140),
10% heat-inactivated FBS (16000069) and 15 mmol/L HEPES (15630-106); from (Lonza, Walkersville, MD)
- 1× MycoZap Plus-CL antibiotic.
TM