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Supplementary Information Supplementary Experimental Powered By Docstoc
					Supplementary Information

Supplementary Experimental Procedures:

Cell lines: MPNST cell lines utilized for our functional studies included the NF1-associated:

ST88-14 (provided by Dr Jonathan Fletcher, Brigham and Women’s Hospital, Boston, MA) and

the sporadic MPNST cell lines: STS26T (provided by Dr Steven Porcelli, Albert Einstein College

of Medicine, Bronx, NY) and MPNST724 (provided by Dr Jonathan Fletcher) and were

propagated and maintained as previously described (Miller SJ, et al. Cancer Res 2006;66:2584-

91). Of note, although STS26T cells exhibit a lower pMET expression in vitro as compared to

ST88 and MPNST724 (but higher the NSC) they were included in our studies as they

reproducibly grow in SCID mice (as do MPNST724 and most importantly they are the only

available MPNST cells that result in lung metastasis after tail vein injection – with the goal of

investigating effect of MET in vivo we did not exclude this cell line. Primary human adult

Schwann cell cultures established from human cauda equina nerves were provided by Dr

Patrick Wood (Miami Project, University of Miami, Miami, FL) and maintained as previously

described (Casella GT, et al. Glia, 2000;30:165–177). Human umbilical vein endothelial cells

(HUVEC; ATCC) and human dermal microvessel endothelial cells (HDMEC; PromoCell,

Heidelberg, Germany) were propagated and maintained as instructed by the respective

sources. The human leiomyosarcoma cell line SKLMS1 (ATCC) and fibrosarcoma cell line

HT1080 (ATCC) were utilized as controls for ELISA experiment.

.

Short tandem repeat (STR) DNA fingerprinting

DNA was extracted from all MPNST cell lines using the Qiagen Blood & Cell Culture DNA Maxi

kit according to the manufacturer's protocol (Qiagen, Valencia, CA). DNA fingerprints were

obtained using the AmpFlSTR Identifier PCR Amplification kit (Applied Biosystems, Foster City,

CA) according to the manufacturer's protocol. The kit amplifies the amelogenin gender-

determining marker and 15 tetranucleotide repeat loci in a single PCR amplification using 33

                                                  1
primers (the extra one is a degenerate primer targeting a mutation at the D8S1179 locus). That

combination of markers is consistent with worldwide database recommendations for identity

testing. Each of the STRs used in this study has a tetranucleotide repeat sequence. Allele calls

were made from peak plots by comparing peaks to known fragment sizes using GeneMapper

4.0 (Applied Biosystems).


Cell growth assays

MTS assays: these were conducted using CellTiter96 Aqueous Non-Radioactive Cell

Proliferation Assay kit (Promega Corp, Madison, WI), per manufacturer's instructions. Drugs

were administered at doses and for intervals as indicated. Absorbance was measured at a

wavelength of 490 nm, and the absorbance values of treated cells are presented as a

percentage of the absorbance of untreated cells. Colony formation assay: one hundred viable

cells per well were plated and allowed to grow in normal medium for 10 days and then stained

for 30 min at room temperature with a 6% glutaraldehyde, 0.5% crystal violet solution. Pictures

were captured digitally and colonies were counted. All experiments were repeated at a minimum

twice for each cell line.



Western Blotting

Briefly, 25-50 μg of proteins extracted from cultured cells or tumor tissues were separated by

SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked and

blotted with relevant antibodies. Horseradish peroxidase–conjugated secondary antibodies were

detected by ECL (Amersham Biosciences). IRdye680- and IRdye800-conjugated secondary

antibodies (Molecular Probes) were detected using Odyssey Imaging (LICOR Biosciences).



Migration and Invasion Assays




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BioCoat cell culture inserts and polycarbonate filters with 8-μm pores (Becton Dickinson

Labware) in 24-well tissue culture plates were used for migration assays. Lower chamber

compartments contained DMEM supplemented by 1% bovine serum albumin or 1% fetal bovine

serum as chemoattractants. Cells (5x104) were seeded in the upper compartment and incubated

at 37°C in a humidified atmosphere of 95% air and 5% CO2 for overnight. Invasion assays were

conducted similarly using 24-well BioCoat Matrigel invasion chambers with 8-μm pore size

polycarbonate filters coated with Matrigel (Becton Dickinson Labware). After incubation, filters

were fixed with 4% formaldehyde and stained with 0.2% crystal violet (Baxter Healthcare). Cells

on the upper surface of the filters were removed by wiping with a cotton swab, and migratory

and invasive activities were determined by counting the number of cells per high-power field

(×200) that had migrated to the lower side of the filter.



qRT-PCR

Five µg total RNA was used for cDNA synthesis by RT III kit (Invitrogen, Carlsbad, CA)

according to manufacturers’ instructions. Real time quantitative PCR was performed using PCR

Master Mix (Promega, Medison, WI). The following primers were used: MMP2 forward-5′-

TGGCCCACAACCCATTTCAC-3′ and reverse 5′-TCAATGTACATGGCCTTTCCTTCAC- 3′;

GAPDH, forward 5′-GAGCCACATCGCTCAGAC-3′ and reverse 5′-

CTTCTCATGGTTCACACCC-3′. Gene expression was analyzed using a Mastercycler

Epgradients realplex (Eppendorf, Hamburg Germany). The levels of gene expression were

normalized using GAPDH levels based on the comparative threshold cycle method.



Sequencing of the MET gene

Total genomic DNA was isolated using the QIAamp DNA extraction kit (Qiagen, Venlo, The

Netherlands). Quantification of extracted DNA was performed using the NanoDrop



                                                  3
spectrophotometer (NanoDrop Technologies, Wilmington, DE). Exons 10 and 13–22 of MET

were amplified by polymerase chain reaction (PCR). Primer sequences (forward and reverse,

respectively) and PCR conditions are as follows: MET- Exon10F: -

TTGACTGTGCCTCTGACCTG- and MET- Exon10R: -GCAGAGCTCTGAGTAGAACCA-; MET-

Exon13F: -GACCAAAGTGCTACAACCTG- and MET- Exon13R: -

AGGAGACTTTGACCCAGTGC-; MET- Exon14F: -GTCGTCGATTCTTGTGTGCTG-; and MET-

Exon14R: -CAACAATGTCACAACCCACTG-; MET- Exon15F: -AGCTCTTCCTGTTTCAGTCC-

and MET- Exon15R: -CAAATCCCCACGGATATGAT-; MET- Exon16F: -

CGCAGTGCTAACCAAGTTCT- and MET- Exon16R: -TTTTCCACAAGGGGAAAGTG-; MET-

Exon17F: -AAACCCTCAGGACAAGATGC- and MET- Exon17R: -

GGCCTATTTTGAAGGGATGG-; MET- Exon19F: -TTCTATTTCAGCCACGGGTAA- and MET-

Exon19R: -CTGGAATTGGTGGTGTTGAA-. The purified PCR products were sequenced in

forward and reverse directions using the ABI PRISM BigDye Terminator Cycle Sequencing

Ready Reaction kit (Version 3) and ABI PRISM 3730 Genetic Analyzer (Applied Biosystems,

CA). Chromatograms were analyzed by SeqScape V2.5 and manual review.



Transfection/transduction procedures: siRNAs (20nM pools targeting MET and control non-

targeting constructs [Cat # D-001810-10-05; ON-TARGETplus Non-Targeting Pool Negative

control siRNA with at least four mismatches to any human gene]) were introduced into cells

using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per manufacturer’s instructions. Briefly,

2x105 cells were plated in each well of a six-well plate and incubated overnight. A mixture of

siRNA (20 nM) and Lipofectamine 2000 (10 μl) diluted in Dulbecco's modified Eagle medium

(DMEM) was added for 24 hr, followed by incubation in regular medium. Cells were harvested at

indicated time points for specific experiments. For stable anti-MET lentiviral shRNA transduction

the following two double-stranded oligonucleotide encoding MET shRNA of interest (MET1F:

GTAAAGAGGCACTAGCAAA; MET2F: CAAAGAAGGAAGTGTTTAA; MET3F:

                                                4
AAATAAGAGCTGTGAGAAT; MET4F: GGACAATGATGGCAAGAAA ) were cloned into the

pLVTHM GFP expression vector (Addgene, Cat: 12247) to create expression plasmids.

Lentivirus were generated by cotransfection of the packing plasmid (Addgene, Cat: 12259 and

12260) and the above MET shRNA expression plasmids into 293T cells (these procedures were

conducted at the Cancer Biology Department Core Facility). The STS26T and MPNST724 cell

lines were cultured in six-well plates to which 8 μg/mL polybrene and virally infected supernatant

was added for four hours and after 48 hours selected by FACS sorting for GFP expression.

Knock down of MET was confirmed by western blot.



Gelfoam angiogenesis assay

These experiments were approved by the MD Anderson Cancer Center Institutional Animal Care

and Usage Committee. Gel-foam sponges (Pharmacia & Upjohn, Peapack, NJ) were cut into

approximately 0.5X0.5cm square fragments and saturated overnight in PBS at 4°C. The next

day, the sponges were placed on sterile filter paper to allow excess PBS to be drawn out.

Sponges were incubated with conditioned media collected from MPNST cells. To obtain

conditioned media MPNST cells were incubated in serum free overnight and in the morning

culture media containing HGF was added to the plates for 24h. Next, plates were washed and

fresh serum free culture media (i.e. not containing HGF) was added and collected after 24h.

Cells were counted and no difference in number of cells was found between HGF treated and

untreated cells. The sponges were allowed to sit at room temperature for approximately 1 hour

and then implanted subcutaneously into the flank of SCID mice (n=2/condition). Mice were

euthanized after 14 days and gel-foam sponges were harvested and frozen in OCT (Sakura

Fineter, Torrance, CA). The frozen samples were later sectioned and probed for CD31. To

evaluate the effect of XL184 on cytokine (HGF and/or VEGF) induced angiogenesis gel-foams

(suspended in these cytokines) were subcutaneously implanted and mice were assigned to two

treatment groups (n=3 mice/group): XL184 (30 mg/kg/once daily, per gavage) vs. vehicle

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treatment control. After 10 consecutive treatment days mice were euthanized and gel foam

processed as per above.



In vivo animal models

All animal procedures and care were approved by the MD Anderson Cancer Center Institutional

Animal Care and Usage Committee. Animals received humane care as per the Animal Welfare

Act and the NIH "Guide for the Care and Use of Laboratory Animals." For experiments

evaluating effect of treatment on local tumor growth trypan blue staining confirmed viable

MPNST cells (STS26T and MPNST724; 1-2 x 106/0.1 mL HBSS/mouse) were used. Cell

suspensions were injected subcutaneously into the flank of six week old female hairless SCID

mice (n = 10/arm) and growth was measured twice weekly; after establishment of palpable

lesions (average diameter >5mm) or in a repeated experiment when tumors reached an

average size of 7-9mm in larger dimension mice were assigned to one of the following treatment

groups (only mice with tumors of similar size were included in the study; mice with very large or

small tumors were excluded thus explaining the varied number of mice per experiment): 1)

XL184 (30mg/kg/d, per gavage) or 2) control vehicle only. An experimental lung metastasis

MPNST model was used to evaluate metastases’ growth. STS26T cells (1 x 106/0.1 mL

HBSS/mouse) were injected into the tail vein of female SCID mice. A week after injection (a

time-point by which 95-100% of mice develop established lung metastases) mice were allocated

to treatment groups as per above (n=8/group). Mice were followed for tumor size, well being,

and body weight and sacrificed when control group tumors reached an average of 1.5 cm in

their largest dimension. Tumors were resected, weighed, and frozen or fixed in formalin and

paraffin-embedded for immunohistochemical studies. For lung metastasis studies mice were

followed for body weight and well being and sacrificed after three weeks of treatment. Lungs

were resected, evaluated macroscopically for tumor load and weighed. Similarly, mice were

injected (S.C. or I.V.) STS26T cells stably transduced with NTshRNA, anti-MET construct#1, or

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anti-MET construct#4 (S.C. n=8-10 mice/cell line and I.V. n=5mice/cell line) and mice were

followed for local and metastatic growth.




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Supplementary Tables

Table S1: MPNST cell lines DNA fingerprinting results*



               AMEL CSF1PO D13S317 D16S539 D5S818 D7S820 TH01 TPOX vWA
  Cell Line


   STS26T
                  X       10,13       9,10        12,13      11,12       8,11     6,9.3     8        17



 MPNST724
                  X        13        11,12        9,11        9,13       9,10       6       8       15,17



  ST88-14        X,Y      9,12         12          13        12,13        8         9     11,12      16


*As per HIPPA regulations and Institutional policy not all markers are provided. Marker selection
followed ATCC database reporting .




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Table S2: Univariable and multivariable Cox proportional hazard models for biomarker

expression correlation with MPNST patients’ disease specific survival (DSS)


                                   1A. Univariable Analysis
Marker                                       HR                                  P value

HGF                    Intensity             1.03             (0.65-1.63)         0.892

                           %                 1.01             (0.99-1.02)         0.471

MET                    Intensity             0.95             (0.54-1.67)         0.863

                           %                 1.05             (0.99-1.11)         0.108

pMET                   Intensity             2.32             (1.20-4.48)         0.012

                           %                 1.04             (1.02-1.06)        0.0004




                                   1B. Multivariable Analysis
Variable                                      HR              95 % CI            p-value

P53                    intensity             2.31             1.19-4.49           0.014


pMET                       %                 1.04             1.01-1.07           0.014


Size                    <10 cm                --                  --                --
                       >=10 cm               2.03             0.75-5.52           0.164


Negative S100        Neg. vs. pos.           1.19             0.45-3.18           0.725
staining




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Supplementary Figures:

Supplementary Figure 1:




Fig S1. MET activation does not impact MPNST cell proliferation. A. No significant effect on

growth of MPNST cells was found after stimulation with HGF (50µg/48h; MTS); B. Similarly, no

effect of HGF on clonogenicity was found; C. WB analysis of shRNA-mediated MET knockdown.

MPNST cells were transduced with lentiviral shRNA constructs targeting MET or non targeting

control. Constructs 1 and 4 resulted in marked MET knockdown in both cell lines and were

selected for further study; constructs 2 and 3 as well as non-targeting shRNA did not affect MET

protein levels. GFP expression in all transduced cell lines (after FACS sorting) confirms a high



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transduction efficiency; D. MET knockdown did not significantly alter MPNST cell growth or

colony-forming efficiency




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Supplementary Figure 2:




Fig S2: Effects of HGF and XL184 on normal human Schwann cells (NHSCs). A. WB

depicting enhanced MET phosphorylation in NHSCs (no constitutive pMET is expressed by

these cells) in response to HGF. XL184 inhibits HGF induced MET phosphorylation; B. No

significant effect on growth of NHSCs was found after stimulation with HGF (50µg/48h; MTS left

graph). Higher XL184 doses (48hr) were needed to inhibit the growth of NHSC as compared to

MPNST cells (right graph); C. HGF enhances NSCs migration and invasion and XL184 (0.5µM)

blocks HGF induced effects.




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Supplementary Figure 3:




Fig S3: Relative MET levels and signaling after MET siRNA knockdown. Protein expression

levels in Fig 3A determined via densitometry




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Supplementary Figure 4:




Fig S4: Impact of XL184 on constitutive MET phosphorylation and expression of XL184

potential kinase targets in MPNST cells. A. Marked dose dependent inhibition of constitutive

MET phosphorylation and its resultant downstream signaling was observed after four hour

incubation of MPNST cells (cultured in regular media without HGF stimulation) with XL184. B.


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WB analyses depicting the expression of several potential XL184 targets in MPNST cell lines.

As shown the MPNST cells evaluated do not express VEGFR2 while endothelial cells do.

Varying levels of KIT, RET and AXL have been found.




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