Download Supplemental Materials

Li et al, “Axl as a Potential Therapeutic Target in Cancer: Role of Axl in Tumor Growth, Metastasis and
Angiogenesis”
Supplemental Materials
Materials & Methods
Antibodies. Antibodies were obtained from the following suppliers: Mouse monoclonal
antibody (mAb) against human Axl (clone 6C8, Abnova, Taiwan), phospho-Akt (Ser
473) mouse mAb and Akt polyclonal antibody (Cell Signaling), mouse mAb against
human Gas6 (R&D System). ELISA kit for phospho-Axl was purchased from R&D
System.
Cell lines and culture. Human carcinoma cell lines were from ATCC. Primary human
umbilical vein endothelial cells (HUVECs) and pulmonary artery smooth muscle cells
(PASMCs) were from Cambrex (Walkersville, MD) and cultured following vendor’s
recommendations.
siRNA transfection. siRNA for Axl and luciferase control (GL2) were purchased from
Dharmacon. The other Axl siRNAs (Axl2 and 4) based on previously published sequence
(Holland, 2005) were synthesized in house. For siRNA transfection, 3X105 cells/well (6well plate) HUVECs were transfected with 50 pmol siRNAs using lipofectamine
RNAiMax (Invitrogen), and Axl knockdown was quantified 24 to 96 hours post
transfection by FACS.
Generating inducible AxlshRNA cell lines. The shRNA vector (pHUSH-GW)
comprises an shRNA expression shuttle plasmid and a retroviral vector backbone that
contains a TetR-IRES-Puro cassette to enable tetracycline-regulated shRNA expression.
293GP2 cells were transfected using lipofectamine2000 reagent (Invitrogen). 48 hours
Li et al, “Axl as a Potential Therapeutic Target in Cancer: Role of Axl in Tumor Growth, Metastasis and
Angiogenesis”
post transfection, infectious supernatant was collected, filtered and was then incubated
with targeting cancer cell lines for 24 hours. Cells resistant to puromycin treatment were
pooled to generate stable cell lines that express AxlshRNA.
Proliferation assay. Cells were seeded at 5000 cells/well in 96-well plates. Axl shRNA
expression was induced by treatment of cell with doxycycline for 72 hours. Cell
proliferation was measured using CellTiter-Glo Luminescent Cell Viability Assay
(Promega) following manufacturer’s instructions.
Migration assay. AxlshRNA cell lines were cultured with or without doxycycline for 48
hrs. 10,000 cells were seeded in 50 l serum free medium on the top well of the transwell
plate (BD Bioscience). Migration was induced by adding 200 l medium with 5% FBS in
the bottom well for 16 hrs. Cells were fixed with MeOH and then stained with YOPRO.
Migration was quantified by measuring fluorescence at 485/535 nm.
Co-culture. PASMCs (2.4 x 105 cells /well) were seeded into collagen-coated 12-well
plates one day before adding HUVECs. Early-passage HUVECs (p<5) were transfected
with 50pmol siRNA, and 24-hr post transfection cells were trypsinized and 4x104 cells
were placed onto the monolayer of PASMCs. Cells were co-cultured for 3 days and were
characterized by staining with FITC conjugated anti-CD31 antibodies.
Tubes were
viewed and photographed under ImageXpressMICRO imaging system. Tubes were defined
by the tube width between 7 to 40 m.
The total tube length was analyzed by
MetaXpress software.
Microarray. HUVECs were transfected with Axl or control siRNA. 72-hour post
transfection, RNA was extracted using RNeasy Plus mini kit (Qiagen).
mRNA
expression profiling of HUVECs was carried out on Affymetrix GeneChip Human
Li et al, “Axl as a Potential Therapeutic Target in Cancer: Role of Axl in Tumor Growth, Metastasis and
Angiogenesis”
Genome U133 Plus 2.0 Array per the manufacturer's protocol. HUVECs were transfected
with Axl or control siRNA. 72-hour post transfection, RNA was extracted using RNeasy
Plus mini kit (Qiagen). mRNA expression profiling of HUVECs was carried out on
Affymetrix GeneChip Human Genome U133 Plus 2.0 Array per the manufacturer's
protocol. Signal intensity values from Microarray Analysis Suite version 5 were utilized
with a scaling factor of 500 for data analysis. Student's t-tests were performed on the logtransformed data to detect differentially expressed genes with false discovery rate cutoff
values of 0.05. The following probe sets were chosen to represent the expression of the
transcripts: 204439_at for IFI44L, 204904_at for GJA4, 202686_s_at for AXL,
203153_at for IFIT1, 203889_at for SCG5, 219837_s_at for CYTL1, 203716_s_at for
DPP4, 202196_s_at for DKK3, 209757_s_at for MYCN, 217028_at for CXCR4,
214438_at for HLX1, 202192_s_at for GAS7, 205659_at for HDAC9, 204947_at for
E2F1, 206942_s_at for PMCH, and 211148_s_at for ANGPT2 (Ang-2).
Generation of anti-Axl monoclonal antibodies. Five Balb/c mice (Charles River
Laboratories) were hyperimmunized with recombinant his6-tagged human Axl expressed
in 293 human fetal kidney cells (Genentech, Inc., South San Francisco, CA), prepared in
Ribi adjuvant (Ribi Immunochem Research, Inc., Hamilton, MO). B-cells from the five
mice, demonstrating high anti-Axl antibody titers, were fused with mouse myeloma cells
(X63.Ag8.653; ATCC, Rockville, MD) using a modified protocol analogous to one
previously described (Hongo et al, 1995; Kohler & Milstein, 1975). After 10-14 days, the
supernatants were harvested and screened for Axl binding specificity by direct enzymelinked immunosorbent assay (ELISA) and for their ability to inhibit Gas6-dependent
proliferation of Baf3-Axl cells. Four positive clones, showing the highest immunobinding
Li et al, “Axl as a Potential Therapeutic Target in Cancer: Role of Axl in Tumor Growth, Metastasis and
Angiogenesis”
and ability to inhibit Baf3-Axl proliferation after the second round of subcloning by
limiting dilution, were expanded for large scale in vitro production of MAb. Conditioned
supernatants were pooled and purified by Protein A affinity chromatography (Pharmacia,
Uppsala, Sweden) as previously described (Hogo et al, 1995)). The purified antibody
preparations were sterile filtered and stored at 4°C in phosphate buffered saline (PBS).
ELISA: All ELISA base assay were performed by standard assay. Briefly, goat antihuman IgG Fc coated plates were blocking with 0.5% BSA, PBS, 0.05% Tween 20
(PBST). For cross reaction assay, coated plates were incubated with human Axl.Fc,
mouse Axl.Fc or human Mer.Fc, Tyro-3.Fc for 1 hr at room temperature, washed four
times in PBST, incubated with anti-Axl mAbs and HRP-conjugated anti-mouse Ig. For
binding assay, coated plates were incubated with human Axl.Fc for 1 hr at room
temperature, washed four times in PBST, incubated with rmGas 6 and anti-Axl mAbs for
1 hr at room temperature. Plates were washed four times in PBST, incubated with
biotinylated anti-mGas6 and strepavidin-HRP. Secreted Ang-2 and DKK3 was measured
using R&D ELISA kit, according to the manufacturers instruction. Details are available
on request.
Fluorescence-activated cell sorting (FACS). Axl expression on cell surface was
determined by FACS using standard techniques. Briefly, cells were harvested, stained
with anti-Axl mAb (12A11, 10g/ml) for 30 mins on ice, washed twice in PBS, and then
staining with PE-conjugated second antibody. To determine antibodies blocking of Gas6
binding to Axl on cell surface, cells were harvested, stained with anti-Axl mAbs for 30mins and incubated with rmGas6 for 30-mins on ice. They were washed twice in PBS,
and stained with biotinylated anti-Gas6 and PE-conjugated strepavidin.
Samples were
Li et al, “Axl as a Potential Therapeutic Target in Cancer: Role of Axl in Tumor Growth, Metastasis and
Angiogenesis”
analyzed on BD FACScalibur Flow Cytometer.
Xenograft experiments. For the A549 & H1299AxlshRNA model, 5X106 cells were
implanted into right flank of nude mice. Mice were randomized (n=10 for each group),
and fed with drinking water containing either 5% sucrose or 1 mg/ml doxycycline. For
MDA-MB-231AxlshRNA model, 107 cells in matrigel were implanted in SCID mice.
When the tumor sized reached 200 mm3, mice were randomized into Dox treated or
untreated groups (n=10 for each group). Tumor volume was measured twice weekly for
30 to 40 days.
For antibody efficacy studies, 5X106 A549 cells were implanted SQ in nude mice.
When the tumor size reached 100 mm3, mice were randomized and divided into 4 groups
(n=10 for each group), and treated with Anti-Axl or control antibodies at 30 mg/kg (IP),
twice weekly, for 40 days. Tumor volume was measured twice weekly for 43 days.
For PD studies, mice were treated with antibodies at 30 mg/kg (IP) for 0, 24, 48
and 72 hours. At each time point, tumors were excised and cell lysates generated for
Western blotting analysis.
Orthotopic metastasis model. The breast cancer cell line MDA-MB-231 was
sequentially infected with AxlshRNA and then dsRedLuc to generate a stable cell line
that expresses both Dox-inducible AxlshRNA and the firefly luciferase reporter gene. 105
cells were implanted into mammary fat pads of SCID mice. Mice were divided into Doxtreated and untreated groups (n=5 for each group) the day after implantation
(experiment1), or after removal of the primary tumors (experiment 2). When the primary
tumor size reached 1000 mm3, the tumors were excised. Lungs were harvested for
Li et al, “Axl as a Potential Therapeutic Target in Cancer: Role of Axl in Tumor Growth, Metastasis and
Angiogenesis”
bioluminescence imaging and H & E staining 5 weeks after removal of the primary
tumors.
Bioluminescence imaging
Mice were injected intraperitoneally (i.p.) with 200 µL of 25 mg/ml D-luciferin
(Invitrogen, Carlsbad, CA) in PBS and were anesthetized during imaging using isoflurane
(Henry Schein, Sparks, NV) via nose cone. Bioluminescence images were acquired using
a cooled intensified charge-coupled device camera fixed to a light-tight imaging chamber
(Stanford Photonics, Palo Alto, CA). Image acquisition times were typically less than 5
minutes, and were processed by co-registering a reference image with the
bioluminescence data image. For ex-vivo imaging, lungs were harvested, rinsed in PBS
and bathed in 100 µl of 25 mg/ml D-luciferin sodium salt.
References:
Hongo JS, Mora-Worms M, Lucas C, and Fendly BM. (1995) Development and characterization
of murine monoclonal antibodies to the latency associated peptide of transforming growth factor
ß1. Hybridoma14:253-260.
Kohler G and Milstein C. (1975) Continuous cultures of fused cells secreting antibody of
predefined specificity. Nature 256:495-497.
Li et al, “Axl as a Potential Therapeutic Target in Cancer: Role of Axl in Tumor Growth, Metastasis and
Angiogenesis”
Figure legends
Figure 1. Axl knockdown by siRNA in HUVECs. HUVECs were transfected with
Luciferase control (GL2) siRNA or with Axl specific (D1-4, Axl2 & 4) siRNAs. 48-hr
post transfection Axl expression on cell surface was analyzed by FACS. A. FACS
profiles of Axl expression. Bar graph represents quantification of FACS. B. siRNA
sequences. C. Effect of Axl knockdown on HUVEC growth. Cell viability was
determined by CellTiter Glo Assay 72-hour post transfection.
Figure 2. Knockdown of DKK3 or Ang2 by RNAi does not affect Axl protein levels
in HUVECs. HUVECs were transfected with control (GL2), Axl, Dkk3 or Ang-2
siRNAs. 48-hrs post transfection, protein levels of Axl were analyzed by FACS and
Li et al, “Axl as a Potential Therapeutic Target in Cancer: Role of Axl in Tumor Growth, Metastasis and
Angiogenesis”
protein levels of DKK3 and Ang-2 in the medium were determined by ELISA. Expressed
as fold changes over control.
Figure 3. Cell-based assay for screening of Axl monoclonal antibodies (mAbs). A.
Stable expression of Axl in Baf3 cells (Western blotting). B. Gas6-dependent growth of
Baf3Axl cells. C. Inhibition of Gas6-dependent growth of Baf3Axl cells by AxlFc. D.
Screen for Axl mAbs that block Gas6-dependent Baf3Axl growth. Baf3Axl cells were
grown in the presence of 200 ng/ml Gas6 (C & D) with indicated dose of AxlFc (C), or
hybridoma supernatants (D) for 72 hrs. Cell viability was determined by CellTiter Glo.
Error bars represent standard deviation (n=8).
Figure 4. Cross reactivity of Axl mAbs. Upper panels: Axl mAbs do not cross react
with murine Axl. Lower panels: Axl mAbs do not cross react with Mer and Tyro-3.
ELISA performed using human AxlFc, murine AxlFc, and human MerFc and Tyro-3Fc
as antigens for antibody binding.