Supplementary Material and Methods
B-cell isolation and cell culture
All patients studied here provided written informed consent according to the Declaration
of Helsinki to the Mayo Clinic Institutional Review Board, which approved these studies.
Primary CLL B-cells or normal B-cells were purified from blood samples of previously
untreated CLL patients or healthy individuals (apheresis cones)1, using the RosetteSep
B-cell enrichment kit (StemCell Technologies). The typical purification range of
CD5+/CD19+ CLL B-cells was >95–99% as determined by flow cytromeric analysis2, 3.
CLL B-cells were cultured for optimum viability in serum-free AIM-V (Gibco) medium.
Primary bone marrow stromal cells (BMSCs) were isolated from the bone biopsy
materials and were maintained in vitro as previously described4. A breast cancer cell
line MDA-MB-231 was purchased from the American Type Culture Collection and grown
in Dulbecco’s modified essential medium/F-12 (Invitrogen) supplemented with 10% fetal
bovine serum (FBS) (Invitrogen), as needed.
Reagents
High-affinity Axl inhibitor, TP-09034,
5
and FGFR inhibitor, TKI-2586,
7
were kind gifts
from Tolero Pharmaceuticals and Novartis Pharmaceuticals, respectively. Ibrutinib (PCI32765) was purchased from Selleck Chemical4. Goat F(ab’)2 anti-human IgM was
obtained from Southern Biotech. All the antibodies were purchased from Cell Signaling
Technologies with the exception of the phosphotyrosine antibody 4G10 (Millipore),
actin, Axl and FGFR3 antibodies (Santa Cruz Biotechnologies), B-cell lymphoma 2
1
antibody (Bcl-2; BD Transduction), fluorescein isothiocyanate (FITC)–conjugated
secondary antibody to rabbit IgG (R&D Systems), fluorescence-conjugated antibodies
to CD5 and CD19 (BD Biosciences). Alexa Fluor-488 (green) anti-mouse and Alexa
Fluor-546 (red) anti-rabbit secondary antibodies were purchased from Molecular
Probes. FITC-conjugated annexin V and Lipofectamine 2000 transfection reagent were
purchased from Invitrogen. Propidium iodide (PI) and other chemicals were obtained
from Sigma or Bio-Rad. Axl- and FGFR3-specific smart pool siRNA and control
scrambled (sc)-siRNA were purchased from Santa Cruz Biotechnologies and
Dharmacon, respectively. Amaxa human B-cell nucleofactor kit was obtained from
Lonza. Recombinant(r) ligands, growth arrest specific gene6 (Gas6), bFGF and a
neutralizing antibody to bFGF were purchased from R&D Systems.
Treatment of cells with specific ligands or neutralizing antibody
Purified CLL B-cells or MDA-MB-231 cells were treated with r-bFGF (100ng/ml) for 0, 5
and10 min or neutralizing antibody to bFGF (10µg/ml) for 24 hours and cell lysates were
prepared to detect P-FGFRs by Western blot analysis. In separate experiments, MDAMB-231 cells were serum starved for 24 hours and treated with r-Gas6 (200ng/ml) for 0,
5, 10 and 20 min. Cell lysates were prepared to detect phospho-Axl (Y702) and
phospho-FGFRs by Western blot analysis. Additionally, purified CLL B-cells in serum
free AIM-V were treated with Gas6 (200ng/ml) for 0, 5 and 10 min. Cell lysates were
prepared to detect phospho-Axl by immunoprecipitation/Western blot analysis using
anti-Axl and anti-P-Tyr (4G10) antibodies.
2
Drug treatment, apoptosis induction assay, flow cytometric analysis and
preparation of cell lysates
CLL B-cells (2.0x106 cells/mL) were treated with increasing doses (0.1–5.0μM) of TKI258 for 24–72 hours or left untreated (DMSO control) and apoptosis induction was
determined by flow cytometry after staining the cells with annexin V-FITC/PI as
described earlier2, 3. For the detection of surface FGFR3 expression, normal B- or CLL
B-cells were stained with fluorescein conjugated-antibodies to CD19 and CD5 followed
by addition of an antibody to FGFR3 (Cell signaling). Cells were washed, followed by
addition of FITC-conjugated anti–rabbit IgG and subjected to flow cytometric analysis
for the detection of FGFR3 expression. A two sided student’s t-test was used for
statistical analysis. Whole cell lysates were prepared after treating purified CLL B-cells
(4.0x106/mL) with DMSO or TKI-258 (2.5μM) for 48 hours or TP-0903 (0.1μM) for 16–20
hours as described earlier2-4.
In separate experiments, CLL B-cells (2 x 106 cells/ml) from high-risk CLL patients with
17p-/11q-deletion were treated with increasing doses of TKI-258 (0.25-2.5 µM) in
combination with ibrutinib (1:1 ratio) or TP-0903 (1:0.1 ratio) for 72 hours. Cells were
harvested and apoptosis induction was determined as described above. Combination
effects of the two drugs were analyzed using the CalcuSyn software program, which
uses the method of Chou and Talalay8. A combination index (CI) value of 1 indicates an
additive effect; values >1 indicate an antagonistic effect and values <1 indicate a
synergistic effect of combined treatment.
3
Treatment of CLL B-cells with TKI-258 in co-culture with bone marrow stromal
cells
CLL bone marrow stromal cells (BMSCs) were plated in 24-well tissue-culture plates
(5.0-7.5 x 104 cells/well) and cultured until the cells were ~80% confluent. After washing,
CLL BMSCs were co-cultured with CLL B-cells at a cell density of 2.0 x 106 cells/well in
serum-free AIM-V medium. Cells were subsequently treated with TKI-258 (2.5 µM) or
DMSO. For comparison, CLL B-cells cultured alone were treated similarly with TKI-258
or DMSO. After 72 hours, CLL B-cells and CLL BMSCs were harvested and apoptosis
induction in both the cell types was determined by flow cytometry as described above.
RNA interference and transfection
CLL B-cells (2.5x106 cells/mL) or MDA-MB-231 cells were transfected with 4.0μg of AxlsiRNA or sc-siRNA using Lipofectamine 2000 (Invitrogen). After 48 hours, cell lysates
were prepared for subsequent analyses. In parallel, CLL B-cells (10-12 x106 cells) were
also transfected with 6.0 g of Axl- or FGFR3-specific siRNA or sc-siRNA using Amaxa
human B-cell nucleofactor reagents. Cell lysates were prepared after 48 hours of
transfection followed by Western blot analysis to detect relevant proteins using specific
antibodies.
Immunoprecipitation and Western blot analysis
For the immunoprecipitation (IP) experiments, 0.2–0.3mg of cell lysates was incubated
with 2μg of specific antibody for 4 hours, followed by addition of 30μl of CHIP grade
agarose beads (Cell Signaling), overnight at 4°C. Beads were washed, digested in
4
Laemmli buffer and subjected to Western blot analysis using specific antibodies as
described earlier2.
Confocal microscopy
1.0x105 purified CLL B-cells were immobilized on slides by Cytospin. Cells were fixed in
3% paraformaldehyde (PFA) and permeabilized in 0.05% Triton-X100 for 5 min. After
blocking, cells were incubated with the primary antibodies; mouse anti-Axl and rabbit
anti-FGFR3, followed by addition of appropriate fluorochrome-conjugated secondary
antibodies. Cell were washed, post-fixed in 3% PFA and mounted in Vectashield
(Vector Labs) containing the nuclear stain DAPI. Confocal microscopy was performed
using a Zeiss LSM 780 confocal laser-scanning microscope with a C-Apochromat 100X
NA1.4 oil-immersion lens. Absence of signal crossover was established using singlelabeled samples. Quantitation of Axl and FGFR3 colocalization was performed using
Image-pro premier software (version 9.1: Media Cybernetics) from 10 different fields.
Results are presented as overlap coefficient of colocalization (coloc overlap) where the
value 0 indicates no overlap and 1 represents maximum overlap of the two RTKs.
Reverse transcriptase-polymerase chain reaction and sequencing of the amplified
products
Total cellular RNA was extracted from purified CLL B-cells using PureLink RNA Mini Kit
(Ambion) and 1μg of total RNA from each sample was reverse-transcribed using the
SuperScript® III First Strand Synthesis Kit (Invitrogen) according to the instructions of
the manufacturer.
5
Primer sequences for FGFR3 amplification were adopted from an earlier study9. The
reverse transcribed products were amplified by polymerase chain reaction (PCR) using
Choice Taq Blue Mastermix (Denville Scientific, Inc.). Primers for PCR amplification
used
were:
FGFR3-exon6-forward
primer;
5’-AACTACACCTGCGTCGTGGAG-3’,
FGFR3-exon8-reverse primer; 5’-GGCCTCCACACTCTCACTGATC-3’, FGFR3-exon9reverse primer; 5’-TAGCTCCTTGTCGGTGGTGTTAG-3’, and FGFR3-exon11-reverse
primer; 5’-AGCTCGAGCTCGGAGACATTG-3’. FGFR3 PCR conditions were; 95ºC for 1
min, then 40 cycles of 94ºC for 30 sec, 60C for 30 sec, and 72ºC for 45 sec. Both
FGFR1 and FGFR3 PCR products were gel purified and sequenced at Mayo Clinic core
facility.
All the experiments were repeated thrice and representative data are shown.
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