Page 59 - Read Online

Page 4 of 14 Anand et al. J Cancer Metastasis Treat 2019;5:6 I http://dx.doi.org/10.20517/2394-4722.2018.98

Elmer Inc., respectively, and cultured as per the instructions provided by the source/vendor.

4T1 murine breast tumor model
6
6
Murine 4T1 cells (0.5 × 10 ) and 4T1-Red-Fluc cells (0.005 × 10 ) were resuspended in the growth medium
and injected into breast fat pads of female nude mice. Visible/palpable tumors developed in 3-5 days post
injection. All experimental procedures involving mice were preapproved by the Institutional Animal Care
and Use Committee of the Cleveland Clinic.

Pretreatment with CPBN
CPBN (Sigma Aldrich, 600 mg/kg/day) was dissolved in 5% gum arabic-sodium citrate solution (vehicle)
and was given by oral route (gavage) once daily for 3 days. Control mice received vehicle solution only. On
day 4, ALA was administered through intraperitoneal route (IP; 200 mg/kg in phosphate buffered saline)
for 4 h and the mice were either exposed to red light (633 nm) for PDT, or euthanized for tumor harvest
and histological and immunohistochemical analyses [23,24] . The dose of oral CPBN chosen for our study
(600 mg/kg/day for three days) was based upon 80% of the maximum tolerated dose determined in nude
[33]
mice by Kolinsky et al. , maximum tolerated dose 700 mg/kg/day when given for 7 days. Note that dose
translation between CPBN (Xeloda®) doses used in humans [34,35] and biologically equivalent doses in mice is
not straightforward, and typically involves body surface normalization methods [36,37] .

®
Imaging of tumor growth/regression and metastases using in vivo imaging system (IVIS
spectrum)
4T1 tumor bearing mice were injected with D-luciferin (IP; 150 mg/kg; Perkin Elmer Inc.) and imaged
under continuous isoflurane anesthesia using the in vivo imaging system (IVIS spectrum; Perkin Elmer
Inc.) following the manufacturer’s instructions. Bioluminescence imaging signals were recorded as
2
bioluminescence units (BLUs, photons/s/cm /sr), and used to analyze tumor growth/regression and
[25]
metastases to distant sites using Live Imaging Software . Digital images were captured 5 min after
D-luciferin injection, along with a radiance calibration standard scale, at each weekly time point during
tumor growth/regression experiments. Tumor-associated signals (emitted light units) from each region of
interest (ROI) were normalized per this radiance standard scale.

Treatment with PDT
Mice were anesthetized using intraperitoneal ketamine and xylazine. Anesthetized mice with 4T1 tumors
2
(± CPBN pretreatment and after 4 h of ALA), were exposed to 100 J/cm of 633 nm light using a LumaCare®
xenon source (LumaCare). The light source was calibrated using a FieldMate® laser power meter (Coherent).
Mice were either imaged at different time points to analyze the therapeutic response and metastases to
distant sites or sacrificed and tumors harvested at 24 h post PDT for histological and immunohistochemical
analyses [23,24] .

Imaging of PpIX in 4T1 tumors by confocal microscopy
Tumor cryosections (10 mm) were placed on glass slides, briefly air dried, mounted under coverslips
with Vectashield (Vector Laboratories), and viewed on a confocal microscope (Leica Microsystems; 40´
magnification; excitation at 635 nm and image collection at 650-780 nm). Quantitation of relative PpIX levels
from digital photomicrographs was performed using IP Lab software [23,24] .

Histology, immunohistochemistry and cell death analyses
The 4T1 tumors and distant metastatic sites were harvested at different time points, fixed in formalin,
paraffin-embedded and sectioned (5 mm) following a standard protocol. Hematoxylin and eosin staining,
immunofluorescence staining, and cell death analysis [terminal-deoxynucleoitidyl transferase dUTP nick
end labeling (TUNEL)] were performed as described [23,24] .