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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 5 of 16

GO:0045820; regulation of glycolytic process GO:0006110) and tricarboxylic acid (TCA) cycle (GO:0006099)
were extracted from the gene ontology consortium website using the AmiGO Gene Ontology browser (http://
[34]
amigo.geneontology.org/amigo) . Genes contained within the mitochondrial respiratory chain complexes
[The Hugo Gene Nomenclature Committee (HGNC) family ID: 639; Complex I GO:006120; complex II
GO006121; complex III GO:006122; complex IV GO:006123; complex V GO:006124] as well as mitochondrial
respiratory chain complex assembly factors (HGNC family ID:645) were extracted from HGNC data base
[35]
(http://www.genenames.org) under the “gene family” browser . Genes with CPM < 1 were excluded from
analysis.

Statistical analyses
All statistical analyses were conducted using Prism v6.0 software (Graph Pad, San Diego, CA, USA). Results
are expressed at mean ± SEM from n independent experiments (performed on separate days on cells from
a different passage) and analysed as grouped data. Cell proliferation data are expressed as a percentage of
unstimulated control cell number. Two-way ANOVA with repeated measures with Bonferroni’s post hoc test
was performed to ascertain statistical significance. For XF analyser profiles and qRT-PCR analysis, signifi-
cance was determined by paired two-tailed Student’s t-test. P < 0.05 was considered a significantly difference
for all analyses.

RESULTS
The MDA-MB-231HM.LNm5 cell line shows slower serum-induced proliferation in vitro than the
parental MDA-MB-231 cell line
MDA-MB-231HM.LNm5 cells exhibit lower migratory and invasive capabilities compared to the parental
[22]
MDA-MB-231 cells, despite enhanced metastatic potential . Here we see decreased MDA-MB-231HM.
LNm5 cell proliferation compared to the parental cells, as measured by either cell enumeration or resazurin
dye reduction. FCS (5% or 10%) increased the number of both parental MDA-MB-231 and MDA-MB-231HM.
LNm5 cells [Figure 1A]. However, the number of cells resulting from 48 h of proliferation was significantly
reduced in MDA-MB-231HM.LNm5 cells compared to parental cells [Figure 1A]. The commonly used Re-
sazurin “proliferation” assay demonstrated a similar percentage increase in MDA-MB-231 cell number at 5%
and 10% FCS [Figure 1B], whereas the FCS response of MDA-MB-231HM.LNm5 cell was barely detectable.
The differences in the outcomes of experiments using the two different methodologies not only illustrate the
limitation of Resazurin use for assessment of cell proliferation, but also emphasize that the indirect measure-
ment of cell number using metabolically converted substrates without independent verification is prone to
generate incorrect conclusions.

The metastatic line MDA-MB-231HM.LNm5 is more metabolically active than the parental MDA-
MB-231 cells line
Although the conversion of resazurin to resorufin is widely used as a “proliferation” assay, an estimate of mi-
tochondrial metabolic activity could be extracted by calculating resorufin production per cell, as resazurin
[36]
undergoes enzymatic reduction in the mitochondria to generate the fluorescent resorufin product . Un-
stimulated MDA-MB-231HM.LNm5 cells have significantly higher basal mitochondrial activity compared to
the parental cells, a difference that was not observed in the presence of serum [Figure 1C]. However, as both
[37]
cytosolic and microsomal enzymes have the ability to reduce resazurin , we sought a more precise method
for quantification of the metabolic changes potentially associated with enhanced metastatic phenotype.

The XF bioanalyser facilitates real time measurement of the two major energy-producing pathways in the
cell, namely glycolysis and OXPHOS. The ECAR, is a measure of glycolysis, and is determined by the net
production and extrusion of protons into the culture medium as a result of the conversion of glucose to py-
ruvate and subsequently to lactate plus H . Simultaneously, OXPHOS is measured by calculating the OCR.
+