Bone metastasis in a novel breast cancer mouse model containing
human breast and human bone
Tian-Song Xia1* · Guo-Zhu Wang1* · Qiang Ding1 · Xiao-An Liu1 · Wen-Bin
Zhou1 · Li-Jun Ling1 · Yi-Fen Zhang2 · Xiao-Ming Zha1 · Qing Du3 · Xiao-Jian Ni1 · Jue
Wang1 · Su-Yu Miao1 · Shui Wang1
1
Department of Breast Surgery, the First Affiliated Hospital of Nanjing Medical
University
2
Department of Pathology, Nanjing Drum Tower Hospital, Nanjing University Medical
School
3
Jiangsu Province Academy of Clinical Medicine, Institute of Tumor Biology
* Contributed equally
Requests for reprints: Prof. Shui Wang. Department of Breast Surgery, the First Affiliated
Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China.
Phone: 86-25-83737472; Fax: 86-25-83724440; Email: ws0801@hotmail.com
Suppl. 1 Material and method
1.1 Transfection of green fluorescent protein
Breast cancer cells were grown in a six-well cell culture cluster. When grown to about
80% confluence, the culture medium was removed and 2 ml of plenti-GFP lentivirus
(kindly provided by Dr. Sun) combined with 12 μl of the Polybrene (Sigma, St Louis,
MO, USA) was added. After incubation for 4 h, 2 ml of the culture medium was added.
After 24 h, the mixed medium was replaced by the fresh culture medium for further
culturing and passaging.
1.2 Experimental animal
Three- to Four-week-old female severe combined immunodeficiency (SCID, C
B-17IcrCrl-scid-bgBR) mice were purchased from Model Animal Research Center of
Nanjing University (MARC, Nanjing, Jiangsu Province, China). SCID mice were kept
under specific pathogen free (SPF), temperature-controlled-air condition (temperature of
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20-24℃, humidity of 40-70%). Cages, bedding, and drinking water were autoclaved and
changed regularly. Food was sterilized by irradiation. The mice were maintained in a
daily cycle of 12 hour period of light and darkness.
1.3 Micro array gene-expression profiling
RNA quality was assessed by Nanodrop ND-1000 and RNA integrity was assessed using
standard denaturing agarose gel electrophoresis. The Human 12 x 135K Gene Expression
Array was manufactured by Roche NimbleGen. About 45033 genes are collected from
the authoritative data source including NCBI. Double-strand cDNA (ds-cDNA) was
synthesized from 5 μg of total RNA using an Invitrogen SuperScript ds-cDNA synthesis
kit in the presence of 100 pmol oligo dT primers. ds-cDNA was cleaned and labeled in
accordance with the Nimblegen Gene Expression Analysis protocol (Nimblegen Systems,
Inc., USA). Briefly, ds-cDNA was incubated with 4μg RNase A at 37℃ for 10 min and
cleaned using phenol: chloroform: isoamyl alcohol, followed by ice-cold absolute ethanol
precipitation. The purified cDNA was quantified using a nanodrop ND-1000. For Cy3
labeling of cDNA, the Nimblegen One-Color DNA labeling kit was used according to the
manufacturer's guideline detailed in the Gene Expression Analysis protocol (Nimblegen
Systems, Inc., Madison, WI, USA). One μg ds-cDNA was incubated for 10 min at 98℃
with 1 OD of Cy3-9mer primer. Then, 100 pmol of deoxynucleoside triphosphates and
100U of the Klenow fragment (New England Biolabs, USA) were added and the mix
incubated at 37℃ for 2 hours. The reaction was stopped by adding 0.1 volume of 0.5 M
EDTA, and the labeled ds-cDNA was purified by isopropanol / ethanol precipitation.
Microarrays were hybridized at 42℃ during 16 to 20h with 4μg of Cy3 labelled ds-cDNA
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in Nimblegen hybridization buffer/ hybridization component A in a hybridization
chamber (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA).
Following hybridization, washing was performed using the Nimblegen Wash Buffer kit
(Nimblegen Systems, Inc., Madison, WI, USA). After being washed in an ozone-free
environment, the slides were scanned using the Axon GenePix 4000B microarray scanner.
Slides were scanned at 5 μm/pixel resolution using an Axon GenePix 4000 B scanner
(Molecular Devices Corporation) piloted by GenePix Pro 6.0 software (Axon). Scanned
images (TIFF format) were then imported into NimbleScan software (version 2.5) for
grid alignment and expression data analysis. Expression data were normalized through
quantile normalization and the Robust Multichip Average (RMA) algorithm included in
the NimbleScan software. The Probe level files and Gene level files were generated after
normalization. All gene level files were imported into Agilent GeneSpring Software
(version 11.0) for further analysis. Differentially expressed genes were identified through
fold change filtering. Hierarchical clustering was performed using the Agilent
GeneSpring GX software (version 11.0).
1.4 Micro array microRNA-expression profiling
The 5th generation of miRCURYTM LNA Array (v.14.0) (Exiqon) contains more than
1891 capture probes, covering all human, mouse and rat microRNAs annotated in
miRBase 14.0, as well as all viral microRNAs related to these species. In addition, this
array contains capture probes for 385 new miRPlus™ human microRNAs. Total RNA
was isolated using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to
manufacturer’s instructions, which efficiently recovered all RNA species, including
miRNAs. RNA quality and quantity was measured by using nanodrop spectrophotometer
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(ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel
electrophoresis. After RNA isolation from the samples, the miRCURY™ Hy3™/Hy5™
Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the
manufacturer’s guideline for miRNA labelling. One microgram of each sample was
3'-end-labeled with Hy3TM fluorescent label, using T4 RNA ligase by the following
procedure: RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP
(Exiqon). The mixture was incubated for 30 min at 37℃, and was terminated by
incubation for 5 min at 95℃. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label
(Hy3TM), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture. The
labeling reaction was incubated for 1 h at 16℃, and terminated by incubation for 15 min
at 65℃. After stopping the labeling procedure, the Hy3TM-labeled samples were
hybridized on the miRCURYTM LNA Array (v.14.0) (Exiqon) according to array manual.
The total 25 μL mixture from Hy3TM-labeled samples with 25 μL hybridization buffer
were first denatured for 2 min at 95℃, incubated on ice for 2 min and then hybridized to
the microarray for 16–20 h at 56℃ in a 12-Bay Hybridization Systems (Hybridization
System - Nimblegen Systems, Inc., Madison, WI, USA), which provides an active mixing
action and constant incubation temperature to improve hybridization uniformity and
enhance signal. Following hybridization, the slides were achieved, washed several times
using Wash buffer kit (Exiqon), and finally dried by centrifugation for 5 min at 400 rpm.
Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon
Instruments, Foster City, CA). Scanned images were then imported into GenePix Pro 6.0
software (Axon) for grid alignment and data extraction. Replicated miRNAs were
averaged and miRNAs that intensities > 50 in all samples were chosen for calculating
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normalization factor. Expressed data were normalized using the Median normalization.
After normalization, differentially expressed miRNAs with statistically significance were
identified through Volacano Plot filtering. Hierarchical clustering was performed using
MEV software (v4.6, TIGR).
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