Page 268 - Read Online

Ansari et al. J Cancer Metastasis Treat 2019;5:20 I http://dx.doi.org/10.20517/2394-4722.2018.68 Page 3 of 14

METHODS
Patient consent and tissue processing
De-identified archival and fresh tumor tissue samples were collected from consented patients undergoing
resection of primary breast or breast to brain metastases, in accordance with a City of Hope Institutional
Review Board (IRB)-approved protocol (#05091).

RNA isolation and sequencing
The flash frozen patient tissue samples were subjected to total RNA preparation using an RNeasy Mini Kit
(Qiagen), according to the manufacturer’s instructions, eluted in 50 μL of RNase/DNase-free water, and
the initial concentration and purity assessed on a NanoDrop ND-1000 spectrophotometer (NanoDrop
Technologies, Wilmington, DE). Prior to sequencing, RNA quality was assessed by microfluidic capillary
electrophoresis using an Agilent 2100 Bioanalyzer and the RNA 6000 Nano Chip kit (Agilent Technologies,
Santa Clara, CA). Sequencing libraries were prepared with the TruSeq RNA Sample Prep Kit V2 (Illumina,
San Diego, CA), according to the manufacturer’s protocol with minor modifications. Briefly, ribosomal
RNA was removed from 500 ng of total RNA using a RiboZero kit (Illumina) and the resulting RNA was
ethanol precipitated. Pellets were re-suspended in 17 μL of Elute/Prime/Fragment Mix (Illumina) and
first-strand cDNA synthesis performed using DNA polymerase I and RNase H. cDNA was end repaired,
3’ end adenylated, and universal adapter ligated followed by 10 cycles of PCR using Illumina PCR Primer
Cocktail and Phusion DNA polymerase (Illumina). Libraries were purified with Agencourt AMPure XP
beads, validated with the Agilent 2100 Bioanalyzer, and quantified with Qubit (Life Technologies). Libraries
were sequenced on an Illumina Hiseq 2500 with single-end 40-bp reads. Raw sequences were aligned to
the human genome assembly (version 19, GRCh37.p13) using Tophat v2 and RefSeq gene expression levels
were counted using HTseq-count. The genes expression counts were normalized using the trimmed mean of
M-values method implemented in the Bioconductor package “edgeR.” The differential expression analysis,
clustering analysis, and pathway analysis were conducted using the DAVID online annotation tool and
Ingenuity Pathway Analysis (IPA) to determine tissue-specific gene signatures and signaling pathways. The
gene expression data of candidate genes was confirmed by real-time PCR.

Breast cancer brain metastasis cell cultures
HER2+ tumor samples were acquired from patients undergoing resection of the breast to brain metastases
in accordance with a City of Hope IRB-approved protocol (IRB #05091). A portion of each specimen was
cultured in DMEM-F12 (Life Technologies) supplemented with 10% fetal bovine serum, 1% glutamax, and
1% antibiotic-antimycotic(Life Technologies) in collagen-coated T75 flasks (Life Technologies) to derive low-
passage primary cell lines COH-BBM1 (BBM1) and COH-BBM2 (BBM2).

High throughput screening of therapeutic candidates
The Lopac 1280 compound library (Sigma), DiscoveryProbe Neuronal Signaling Library (ApexBio),
and clinical drugs targeting primary breast cancer and CNS tumors (Cayman Chemical) were obtained
commercially. All the compounds of the two libraries are in preclinical or clinical candidates. Over 50%
compounds of the library target neural disorders. The compounds were dissolved in DMSO at concentrations
of 100 μmol/L. For the initial screening, BBM1 cells were grown in 96-well plates (10000/well) and treated
with the compounds at a final concentration of 1 μmol/L (n = 3 per treatment). Control cells were treated
with DMSO only. The viability of the cells was measured at 48 and 72 h post‐treatment by using a CellTiter‐
Glo® Luminescent Cell Viability Assay kit (Promega). The compounds that suppressed BBM1 cell viability
by at least 70% compared to the control were selected for secondary screening in both BBM1 and BBM2 cells
lines. The compounds that showed consistent toxicity of both BBM1 and BBM2 cells over a period of 10 days
were assessed for toxicity against BBM1 cells but not astrocytes.

To analyze cell type-specific toxicity, human astrocytes and BBM1 cells were grown overnight prior to
treatment with 1 μmol/L of the active compounds (n = 6). Control cells were treated with DMSO only. The