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Park et al. J Cancer Metastasis Treat 2019;5:17 I http://dx.doi.org/10.20517/2394-4722.2018.84 Page 3 of 12

Metastatic cancer cell, MDA-MB-231LN, is subtype of MDA-MB231 breast cancer cells and it has shown
[14]
enhanced tumor growth and widespread metastasis than parents cell line in xenograft models . We
observed AF1q, a metastasis enhancer, is highly expressed in metastatic cancer cells (MDA-MB-231LN)
than in the primary tumor cells (MDA-MB-231). In this study, we investigated whether AF1q is responsible
in the acquisition of metastatic phenotype using RNA-sequencing (RNA-Seq) and applied the Metacore
direct interactions network building algorithm. Intriguingly, most genes were directly linked with ICAM-1.
Likewise, we identified that ICAM-1 expression is attenuated in metastatic cancer cells compared to primary
cancer cells.

In addition to AF1q oncogenic functions, we demonstrated that intensity of AF1q expression in metastatic
[2]
sites is higher than in primary sites . Thus, similarly to that reported for certain oncogenes (i.e., Myc and
Ras), AF1q has been shown to be endowed with a dual function in malignancy, being a protein apparently
involved in both initiation and promotion of cancer progression through regulation of ICAM-1 expression.
Our results suggest that targeting AF1q is valuable in developing treatments for breast cancer metastasis.

METHODS
Cell lines and cell culture conditions
MDA-MB-231 was purchased from American Type Culture Collection (ATCC). MDA-MB-231-luc-D3H2LN
(MDA-MB-231LN) was purchased from Caliper Life Science. The cells were maintained as a monolayer
culture in DMEM, supplemented with Glutamax and penicillin-streptomycin (Invitrogen). Fetal bovine
serum (FBS) (Thermo Fisher Scientific) was added to the media. Burkitt’s lymphoma cell lines, Ramos,
Akata, Mutu, Raji, Jiyoye, BL-5, BL-7, and BL-8, were maintained in RPMI-1640, supplemented with
Glutamax, penicillin-streptomycin, and 10% FBS (Thermo Fisher Scientific).

Whole blood was collected from healthy volunteers at James Graham Brown Cancer Center, University of
Louisville with donors’ written consent. The CD4-positive and CD8-positive human T cells were purified
from buffy coats via positive selection using a 1:1 mixture of CD4- and CD8- MicroBeads (Miltenyi Biotec)
according to the manufacturer’s protocol. Isolated T cells were maintained in RPMI-1640, supplemented
with 300 IU/mL IL-2 (R&D) and 10% FBS (Thermo Fisher Scientific). All cells were maintained at 37 °C
under a humidified atmosphere of 5% CO .
2
RNA-Seq
Total RNA were isolated from MDA-MB-231 and MDA-MB-231LN cells with the use of the Purelink
RNA min Kit (Thermo Fisher Scientific) in triplicates. Of total RNA, 1 µg was depleted of cytoplasmic and
mitochondrial ribosomal RNA using the Illumina Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/
Rat) (Illumina). The rRNA-depleted RNA was ligated with Illumina adapters and further processed for
sequencing following the Illumina TruSeq Stranded Total RNA library preparation kit (Illumina). All
samples were pooled and a 75-cycle single-end sequence run was performed using the Illumina High Output
Kit v2 on the Illumina NextSeq 500 platform.

RNA-Seq data analysis
RNA-Seq data were mapped using UCSC human genome, hg38, with STAR (version 2.5.2b). The read count
and reads per kilobase of transcript per million mapped reads (RPKM) were calculated on the basis of the
human GRCh38 Ensembl Release 91 gene annotation. To search differentially expressed genes, Cuffdiff
(version 2.2.1) was used. For further analysis, 1485 significantly differentially expressed genes were selected
as following criteria, when the value of averaged RPKM from three replicates is greater than or equal to 1
in at least one of the two samples, when the absolute value of log (fold-change) is greater than or equal to 1,
2
and when false discovery rate is less than or equal to 0.01.