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CDy1. CDy1 was a generous gift from Dr. YT Chang, generous gift from Dr. Andrew Kung, Lurie family Imaging
[22]
Laboratory of Bioimaging Probe Development, Singapore Center, Dana-Farber Cancer Institute, MA) according to
Bioimaging Consortium Agency for Science, Technology, an established protocol. Imaging-ready UP-LN1 F cells
[14]
and Research, Singapore through Dr. Gi-Min Lai (Wan- were harvested and subcutaneously injected into the left
Fan Hospital, Taipei, China). The cells were harvested by flank for NOD/SCID mice (3 × 10 cells/mouse; 5 mice/
5
trypsin treatment, washed with PBS, and re-suspended in group). Tumor-bearing mice were then subdivided into
PBS. The cells were fixed with 4% paraformaldehyde for control and WA-treated groups (10 mg/kg intraperitoneal
10 min, permeabilized with 0.1% Triton X-100/PBS for 10 injection [i.p.], 3 times a week). For intravenous (i.v.) tumor
min, and blocked with 2% bovine serum albumin/PBS for 1 injection, 5.5 × 10 cells/mouse were injected, followed by
5
h. After incubation in dark at room temperature for 15 min, WA i.p injection as described for subcutaneous (s.c.) tumor
the cells were rinsed with PBS. The fluorescence images injected animals. For either the s.c. or i.v.-tumor injection
of the cells were acquired using fluorescence microscope group, WA treatment was initiated 2 weeks after tumor
(Nikon, Lewisville, TX, USA). injection into the animals. Tumor burden was then non-
invasively assessed based on bioluminescence intensity for
In vitro cell migration and invasion assays 6 weeks using IVIS200 system (Caliper Life Sciences Inc.,
Hopkinton, MA, USA). Tumor autopsies were obtained at
A Boyden chamber system was used to measure the the end of the experimental period by humanely sacrificing
invasive ability of UP-LN1 cells. Briefly, UP-LN1 P, A, and the animals for pathological and immunohistological
F cells were harvested, washed with PBS, and re-suspended analyses. All experiments were conducted in accordance
in a serum-free RPMI medium (5 × 10 cells/200 µL) in with the Guide for the Care and Use of Laboratory Animals
4
the presence or absence of WA. The cells were then seeded of the National Health Research Institutes of Taiwan and
into the upper chambers of Matrigel-coated filter inserts. A following the Institutional Animal Care and Use Committee
serum-containing RPMI-1640 medium (500 µL) was added protocol authorized by Taipei Medical University.
to the lower chambers. After incubating for 24 h at 37 °C,
filter inserts were removed from the wells, the cells that Histology and immunohistochemical staining
had invaded were stained with PI, and fluorescence images
were taken. The number of invaded cells was determined Tumor tissues were fixed in 10% formalin and embedded
using Analytical Imaging Station Software Package in paraffin. Serial sections of the embedded specimens
(Imaging Research, ON, Canada). The migration assay was were deparaffinized and then rehydrated in a gradient
performed accordingly but with 8-µm pore polycarbonate fashion and stained with hematoxylin and eosin. For
filters, which were not coated with Matrigel. immunohistochemical staining, the deparaffinized slides
were subjected to antigen retrieval and probed with anti-
Western blotting CXCR4 (1:100), anti-caspase-3 (1:200), anti-PARP (1:100)
antibodies, or isotype IgG control. Slides were washed
UP-LN1 P, A, and F cells lysates were prepared using and incubated with biotinylated link universal antiserum,
ReadyPrep Protein Extraction Kit (Bio-Rad, Hercules, followed by horseradish peroxidase-streptavidin conjugate
CA, USA) according to the instructions provided. Total (LSAB 1 kit). The slides were rinsed, and the color was
cell lysates (50 µg) were separated electrophoretically by developed using 3,3-diaminobenzidine hydrochloride as a
a 10% polyacrylamide SDS-PAGE gel and transferred to a chromogen. Finally, sections were rinsed in distilled water,
polyvinylidene fluoride membrane using the BioRad Mini counterstained with Mayer’s hematoxylin, and mounted
Protean transfer system. The blots were then blocked with with DPX mounting medium for evaluation. Pictures were
5% skim milk in PBST for 1 h and probed with primary captured with a Photometrics CoolSnap CF color camera
antibodies overnight at 4 °C. All primary antibodies were (Nikon, Lewisville, TX, USA).
purchased from cell signaling unless otherwise specified.
The membranes were sequentially detected with an Statistical analysis
appropriate peroxidase-conjugated secondary antibody
incubated at room temperature for 1 h. Blots were washed Each experiment was performed in triplicate. The results
3 times with PBS. Signals were then detected using the were expressed as means ± standard deviation. The
enhanced chemiluminescence detection system and the significant difference between control and experimental
BioSpectrum Imaging System (UVP, Upland, CA, USA). groups was analyzed using t-test (*P < 0.05; **P < 0.01).

In vivo evaluation of WA-mediated anti-UP-LN1 RESULTS
F cell effects
F subset of UP-LN1 cells are enriched with
All animal studies were performed strictly under the cancer stem cells
animal experimentation protocols approved by Taipei
Medical University. UP-LN1 F cells were first modified to We utilized the SP method to compare and analyze the
express dual reporter system, FUW-Luc-mCherry-Puro (a percentage of F cells in the UP-LN1 cell line. In the

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Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ Issue 1 ¦ January 15, 2016 ¦