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Tu et al. J Cancer Metastasis Treat 2018;4:58 I http://dx.doi.org/10.20517/2394-4722.2018.67 Page 3 of 16
[19]
enocarcinoma cell line MDA-MB-231 , and compared cellular and metabolic alterations. The MDA-MB-
231HM.LNm5 cell line is a highly angiogenic and metastatic variant of the MDA-MB-231 cell line derived
by in vivo passaging whereby spontaneous secondary lesions were isolated and expanded ex vivo [20-23] . We
recently demonstrated that the metastatic ability of MDA-MB-231HM.LNm5 line is highly elevated com-
pared to the parental MDA-MB-231 cells. In a metastasis model involving surgical resection of the primary
tumour, NSG immune-deficient mice bearing the HM.LNm5 line exhibited primary tumour recurrence, as
well as significant lung, liver, spleen, lymph and spine metastasis. By comparison, no metastatic lesions were
[23]
detected in secondary organs of MDA-MB-231-innoculated mice .

In this study, the metabolic profiles of MDA-MB-231HM.LNm5 were compared to the parental MDA-
MB-231 cell line using the Extracellular Flux (XF) Analyzer thus enabling simultaneous measurement of the
two major cellular energy-producing pathways, glycolysis and OXPHOS. We then used whole transcriptome
sequencing (RNA-seq) and quantitative real-time PCR (RT-qPCR) to ascertain expression differences in
metabolic genes that were associated with enhanced breast cancer metastatic ability.

METHODS
Cell culture
[19]
The MDA-MB-231 human breast adenocarcinoma cell line was purchased from ATCC . MDA-MB-231HM
cells [20,21] were kindly provided by ZM Shao and ZL Ou (Breast Cancer Institute, Fudan University, Shanghai,
China). MDA-MB-231HM.LNm5 cells were derived as described below. All lines were maintained in phenol
red - containing RPMI1640 (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS),
2 mmol/L L-glutamine, 1% (v/v) non-essential amino acids, 5% (v/v) sodium pyruvate, 100 U/mL penicil-
lin, 15 mmol/L HEPES buffer, and 0.2% (v/v) sodium bicarbonate (Sigma-Aldrich). Cells were maintained at
37 °C in 5% CO and passaged every 4-5 days.
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Generation of reporter gene tagged MDA-MB-231 variants
To facilitate optical imaging of tumors in vivo, both parental MDA-MB-231 and MDA-MB-231HM cells were
transduced with retrovirus encoding tdTomato fluorescent protein, selected with Blasticidin S and bulk sort-
[24]
ed for tdTomato expression by flow cytometry (FACSAria, Beckton Dickinson), as previously described .
Both populations were also transduced with retrovirus encoding Firefly luciferase and selected using pu-
[24]
romycin . Parental MDA-MB-231 cells were additionally transduced with retrovirus encoding enhanced
GFP (encoded by the pFBneoGFP plasmid, a kind gift from Hiroshi Nakagawa, University of Pennsylvania),
selected using G418, and bulk sorted for GFP expression using flow cytometry (FACSAria, Beckton Dickin-
son). MDA-MB-231HM.LNm5 cells were isolated from a spontaneous axillary lymph node metastasis that
developed from a reporter gene tagged MDA-MB-231HM primary inguinal mammary tumour in a BALB/c-
SCID mouse.

Cellular proliferation
5
2
Cells seeded at 10 cells/cm into 24-well plastic plates were allowed to adhere overnight and were then
rendered quiescent by incubation in serum-free medium containing 0.25% (v/v) bovine serum albumin for
24 h before re-exposing to FCS (5%, 10%) for 48 h. Viable cells were identified by the trypan blue (0.06% w/v)
[25]
exclusion and enumerated (blinded) manually using a haemocytometer chamber.
Cellular proliferation using Resazurin (Alamar Blue)
Cell proliferation was also assessed using the Resazurin dye method by measuring reduction of the redox
[26]
dye resazurin to resorufin . Cells were seeded and treated as described in previous section, and were then
incubated with Resazurin reagent containing 1.5% (w/v) Resazurin, 0.25 (w/v) methylene blue, 2.9% (w/v)
potassium hexacyanoferrate (III) and 4.22% (w/v) potassium hexacyanoferrate (II) trihydrate for 2 h at 37 °C
in 5% CO . Resorufin formation was measured fluorometrically (excitation 570 nm; emission 620 nm) using
2
a FlexStationII (Molecular Devices, Sunnyvale, CA, USA). Results are expressed in relative fluorescent units.