Supplementary Materials and Methods
Cell lines and cell culture. The cell lines SKBR3, MCF7, MDA MB231, Me180,
NCI-N87, and Calu3 were all obtained from American Type Culture Collection. The
human breast cancer cell line BT474 M1 was a subclone of BT474, from Dr. Dihua Yu
(UT MDACC, Houston, TX). The human melanoma cell line, A375m was obtained
from Dr. Isaiah J. Fidler (UT MDACC, Houston, TX). The cells were maintained in
DMEM or RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine
serum, plus 2mM L-glutamine and 1mM antibiotics.
Cell lines were validated by STR DNA fingerprinting using the AmpFℓSTR
Identifiler kit according to manufacturer instructions (Applied Biosystems). The
STR profiles were compared to known ATCC fingerprints (ATCC.org), to the Cell
Line Integrated Molecular Authentication database (CLIMA) version 0.1.200808
(http://bioinformatics.istge.it/clima/) (Nucleic Acids Research 37:D925-D932 PMCID:
PMC2686526) and to the MD Anderson fingerprint database. The STR profiles
matched known DNA fingerprints or were unique.
Internalization and competitive inhibition analysis. Cells treated with
immunotoxins or rGel were subjected to immunofluorescent staining with anti-rGel
antibody (FITC-conjugated secondary antibody). Nuclei were counterstained with
propidium iodine. Visualization of immunofluorescence was performed with a
confocal laser scanning microscope Zeiss LSM510 (Carl Zeiss). The internalization
rate was calculated using Zeiss LSM Image Brower software (Version 3.5.0.223)
assuming that the nuclear area per cell did not change in the experiment. Relative
Fluorescence = (mean value of green fluorescence × fluorescence area)/ nuclear area.
For competitive inhibition assays, BT474 M1 cells were treated with the mixture
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containing 20nM of each scFv/rGel with 0, 5, 20 or 100nM of Her2/neu ECD.
Thereafter, the treated cells were chased the immunotoxin internalization on the
relative green fluorescence. The mixtures after treatment were subjected to Her2/neu
immunoprecipitation, and immunoblotted by tracking the scFv/rGel amount.
Cytotoxicity of scFv/rGel and competitive assay. Log-phase cells were seeded
(~5×103/well) in flat-bottomed 96-well microtiter plates and allowed to attach
overnight. Immunotoxins were diluted in culture medium and added to the wells in
3-fold serial dilutions. Cells were incubated for 72 h. The remaining adherent cells
were stained with crystal violet (0.5% in 20% methanol) and solubilized with
Sorensor’s Buffer [0.1M sodium citrate (pH4.2) in 50% ethanol]. Finally, the
absorbance was measured at 595nm. Percent control refers to the percentage of viable
cells in treated wells compared with that of control (untreated cells).
The competitive cytotoxicity was performed on SKBR3 and BT474 M1 cancer cell
line based on two strategies: Strategy 1, each scFv/rGel protein (fixed concentration
as 20nM) was admixed with 0, 10, 20, 50 or 100nM Her2/neu ECD at 37℃ for 1h,
and then added to the 96-well plate pre-seeded with cancer cells. For Strategy 2, cancer
cells were pretreated with a fixed concentration of Her2/neu ECD (20nM) at 37℃ for
1h and then cells were treated with various concentrations of each scFv/rGel in
triplicate wells. For each strategy, the cells treated with each scFv/rGel were used as
positive control, and with Her2/neu ECD as negative control. After 72h treatment, the
cell viability was then assessed as crystal violet staining method.
Western blot analysis of apoptosis and autophagy. BT474 M1 cells treated with
100nM immunotoxins were pelleted and lysed. Proteins from each cell lysates were
analyzed by western blot with antibodies that recognized poly ADP-ribose
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polymerase (PARP), β-actin, high mobility group box 1 (HMGB1) (Santa Cruz
Biotechnology) and microtubule-associated protein 1 light chain 3 (MAP LC3) (Novus
Biological). For HMGB1 release assay, the medium was harvested, concentrated and
further analyzed by western blot.
Shed Her2/neu level detection. The competitive ELISA was applied to quantitate
soluble shed Her2/neu antigen in the cell culture medium and in serum from
tumor-bearing mice. The medium samples were tested at the original dilution, and the
serum samples were tested at an initial dilution of 1:60. All assays were performed
described below: 96-well plates were coated with 100µL of Her2/neu ECD at
0.2µg/mL per well for 2h and then blocked overnight at 4℃ with 200 µL blocking
solution. After TBST washes, the plates were incubated with a mixture of either
Her2/neu ECD standard dilution samples (0.01-1 µg/mL) or diluted test samples, with
0.02 µg/mL anti-Her2/neu Herceptin antibody for 1h. The plates were washed and
incubated with anti-human IgG HRP for 1h at 1: 5000 dilution. The plates were
finally added with ABTS substrate buffer, and incubated for 15min. Optical density
was measured at 405nm with a microplate reader. The shed Her2/neu level was
calculated based on the subtraction values of positive samples in negative control.
In vivo efficacy studies. Balb/c nude mice were received implants of slow release
estrogen pellets (0.72mg β-estradiol; Innovative Research of America) and, on the
second day, s.c. inoculated with 1×107 BT474 M1 cells in the right flank. After one
week when the tumors reached (~150-200 mm3), animals were treated (i.v. tail vein)
with rGel as control, or different scFv/rGel fusion proteins (24mg/kg), every other day
for six times. Animals were monitored, and tumors were measured for an additional 40
days.
Fluorescence dye labeling. MH3-B1/rGel and B1D3/rGel fusion proteins were
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directly labeled with infrared dye reagent IRDye 800CW according to Protein Labeling
Manual. Briefly, 0.05mg IRDye 800 CW Reactive Dye (Li-Cor Biosciences) and 1mg
scFv/rGel (MH3-B1/rGel or B1D3/rGel) were mixed to a final volume of 1mL,
incubated for 2 h at 4℃, protected from light. After reaction, the dye labeled scFv/rGel
was purified using a disposable PD-10 desalting column (GE Healthcare). The
scFv/rGel conjugates were tested for affinity and cytotoxicity in vitro, and stored at 4℃
protected from light.
Tissue distribution study. After inoculation of mice with BT474 M1 tumor cells
and allowing the tumors to establish, tumor-bearing mice were injected (i.v. tail vein)
with 1.5 nM IRDye800 labeled immunotoxins (MH3-B1/rGel or B1D3/rGel). For
optical imaging, an IVIS imaging system 200 series (Xenogen Corp.,) was used with
ICG filter sets (excitation/emission, 710-760/810-875 nm). The field of view was 25cm
in diameter. The fluency rate for near-infrared fluorescence excitation light was
2mW/cm2. The camera settings included maximum gain, 2 × 2 binning, 640 × 480 pixel
resolution, and an exposure time of 1 second.
Whole body images were obtained at 4, 24, 48 and 72 h after injection. 24h and 72h
after fluorescent agent injection, groups of mice were sacrificed and organs were
excised for quantitative optical imaging. For quantitative comparison, ratios of
fluorescent intensities in regions of interest (ROIs) corresponding to the tumor and
normal tissue regions were determined. Statistical analysis of the tissue-to-muscle ratio
(TMR) was performed with fluorescence images obtained at various time points. In this
study, TMR was calculated as follows: TMR = fluorescence intensity in tissue region/
fluorescence intensity in muscle region.
Co-immunoprecipitation assay. 72 h after i.v. injection of MH3-B1/rGel and
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B1D3/rGel, the mice were sacrificed and the liver samples were collected. The liver
was miced and then homogenized in a dounce apparatus. After centrifugation, the
supernatants were quantified, and 500 μg supernatants were subjected to Her2/neu
immunoprecipitation, and further immunoblotted using anti-rGel antibody to detect
intact scFv/rGel fusion protein.
In situ immunofluorescent detection. Liver samples from the mice after the
treatment of rGel, MH3-B1/rGel or B1D3/rGel were collected and frozen for section
slides. The slides were fixed in 3.7% paraformaldehyde, permeabilized with PBS
containing 0.2% Triton X-100, and blocked in 5% nonfat milk in PBS. After incubation
with anti-rGel antibody (followed by FITC-conjugated secondary antibody), and
anti-human Her2/neu antibody (followed by phycoerythrin (PE)-conjugated secondary
antibody), the slides were mounted for the immunofluorescence observation under a
Nikon Eclipse TS-100 fluorescence microscope (Nikon).
Liver toxicity study. Hepatotoxicity was investigated at 72 h after MH3-B1/rGel
and B1D3/rGel injections by measuring the enzymatic activities of serum Alanine
Transaminase (ALT), Aspartate Transaminase (AST) and lactate dehydrogenase (LDH)
using an enzymatic kit (Roche) on an autoanalyzer (Cobas Integra 400/800, Roche).
For the histologic examination of hepatotoxicity, mice were anesthetized 72h after
immunotoxin administration, and liver organs were perfused with PBS, resected and
paraffin-embedded section were stained with hematoxylin and eosin.
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