Supplementary Methods
Establishment of human TNBC
The breast cancer parental cells including MDA-MB-231 and MDA-MB-436 were obtained
from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified
Eagle’s medium (Life Technologies) supplemented with 10% foetal bovine serum and 1%
penicillin-streptomycin solution (Sigma) 37 °C, 5% CO2. Subconfluent parental cells were
harvested and a single cell suspension with >95% viability (1×106) was injected subcutaneously
into the mammary fat pads of female nude mice (5-8 weeks). Tumours were established (~30
mm3) approximately 4 weeks after tumour cell injection. By approximately 8 weeks post-tumour
cell inoculation when the tumour volumes were allowed to reach ~700 mm3 (1.2 cm of diameter
is the maximum size that the tumours are allowed to attain under institutional guideline of the
University of Florida), tumours were excised, grown in culture and inoculated into the mammary
fat pads of female nude mice. This procedure was repeated several times until similar tumour
volumes (~700 mm3) were obtained with fewer cells (1×105) for shorter time (2 weeks).
MDA-MB-231Br (brain metastatic) was a generous gift from Dr Patricia Steeg (National Cancer
Institute, USA).
Determination of maximum tolerated dose of NCI-41356
A single athymic female nude mice (nu/nu) was given a singly IP injection of 0.8g/kg of NCI41356 (equivalent to 100μM), while a second mouse received a dose of 0.4g/kg and a third
mouse received a single dose of 0.2g/kg. Their weights were observed and recorded every two
days for two weeks.
Immunohsitochemistry detection and histological morphometric analysis
The breast cancer xenograft tissues were fixed in 4% paraformaldehyde. Three serial frozen
sections from each treatment of the breast cancer xenograft tissues were incubated with
antibodies against CRYAB (Abcam), VEGF (Abcam) and von Willbebrand factor (Millipore),
respectively, for 1 hour. Detection was done with an avidin-biotin complex method-based system.
The secondary biotinylated antibodies were from Dako, Inc., and avidin biotin complex were
from Vector laboratories. The sections were counterstained with Gill’s hematoxylin. At ×200, we
used an eyepiece systematic point sampling grid with 100 points and 50 lines to count the
fraction of points overlaying the positive stained structures of the 10 most vascular areas within
the chosen sections. We average this over 10 microscopic fields to obtain a final result as a
percentage of staining.
Supplementary figure legend
Supplementary Figure 1. a. Concentration response curves to the decoy peptides in the U2OS
cells (20k/well) expressing the PathHunter™ cell-based CRYAB/VEGF165 interaction assay.
Relative RUL is expressed as the percentage of the vehicle treatment. b. Synthetic decoy
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peptides corresponded to the five sequences of VEGF165 interacting regions of human CRYAB.
A control “scrambled” (*scr) peptide with the amino acid composition of *157/164.c. All decoy
peptides exhibit similar internalization efficiencies in U2OS cells.
Supplementary Figure 2. a. Schematic of the customized PathHunter™ cell-based
CRYAB/VEGF165 interaction assay. b. Concentration response curves to the three leading
compounds as indicated in the two cell densities (5k/well or 20k/well) of U2OS cells expressing
PathHunter™ cell-based CRYAB/VEGF165 interaction assays.
Supplementary Figure 3. a. The tumour volumes of different in vivo passages of breast cancer
cells. The broken line indicated the maximum size that the tumours are allowed to attain under
institutional guideline of the University of Florida. b. The percentage change of body weight of
normal mice treated with different doses of NCI-41356 for two weeks. c. Western blot analysis
of total protein levels of CRYAB in breast cancer cell lines.
Supplementary Figure 4. a. Annexin V-propidium iodide-positive cells were shown by flow
cytometric analysis (top right, late stage apoptosis; bottom right, early apoptosis). b. Phasecontrast of photographs of third in vivo passaged MDA-MB-231 cells show that NCI-43156
treatment leads to the cells less stellate with lower proportions of lamellipodia, filopodia and
trailing compared with those of vehicle treatment. c. Vimentin mmunostaining was detected in
MDA-MB-231 cells. d. Western blot analysis of total protein levels of vimentin in the MDAMB-231 cells normalized to GAPHD loading control. e. Survival assay of breast cancer cells
were determined by crystal violet staining. f. VEGF165 protein levels in both supernatant and cell
lysates of MCF-7 were measured by ELISA.
Supplementary Figure 5. a. Western blot analysis the effect of NCI-41356 treatment on
CRYAB expression in breast cancer cell lines. b. showing no detectable inhibitory effect on
endothelial cells proliferation in the absence of MDA-MB-231 cell-stimulation. c. IHC staining
for CRYAB, VEGF and von Willebrand factor. d. Three plots demonstrated the relationships
between NCI-43156 treatment and the staining of CRYAB, VEGF or VWF (% of points).
Supplementary Table
NCI#
IC50±SD (μM)
Maximal inhibition at 100μM (%±SD)
MDA-MB-231
MDA-MB-436
MDA-MB-231
MDA-MB-436
41356
24.5±1.4
30.8±3.2
97.6±1.9
90.1±2.2
525117
86.3±2.5
96.5±5.7
75.8±7.2
55.0±9.6
24681
61.9±7.7
77.4±1.1
81.0±0.5
59.2±1.3
52296
93.8±5.2
92.5±2.1
51.5±3.1
49.1±2.8
Supplementary table 1. IC50 of the four SMIs for MDA-MB-231 and MDA-MB-436 cells
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