Tissue and melanoma cell lines used for the discovery and first validation in this study were described previously (5
For the melanoma second validation set, Optimum cutting temperature (OCT)–embedded frozen clinical specimens were obtained from the Melanoma Informatics, Tissue Resource, and Pathology Core, and the Central Nervous System Tissue Bank at The University of Texas M. D. Anderson Cancer Center under Institutional Review Board-approved protocols. H&E-guided dissection and isolation of DNA from the tumor-enriched isolates has been described previously (12
PCR, sequencing and mutational analysis of melanoma samples
PCR and sequencing was done as previously described (5
). The primary phase mutation screen was analyzed using Consed (43
). Variants were called using Polyphred 6.11 (44
) and DIPDetector, an indel detector for improved sensitivity in finding insertions and deletions. Sequence traces of the secondary screen were analyzed using the Mutation Surveyor software package (SoftGenetics, State College, PA).
To increase our confidence that the mutations in the M. D. Anderson set, for which no matched normal DNA sample was available, did not represent germline polymorphisms, we searched the corresponding exons of ADAMTS18 in a total of 145 DNA samples and detected no abnormalities.
Construction of wild-type and mutant ADAMTS18 expression vector
(NM _199355.2) was cloned by PCR as previously described(5
) using a clone (#30343625) purchased from Open Biosystems with primers in Supplementary Table S5
. The PCR product was cloned into the mammalian expression vector pCDF-MCS2-EF1-Puro™ (Systems Biosciences, Inc., Mountain View, CA) or pCDNA3.1 (−) (Invitrogen) via the XbaI and NotI restriction sites. The G312E, P452S, C638S, Q904X, Q1002X, and P1035S point mutants were made using Phusion PCR for site-directed mutagenesis using the primers listed in Supplementary Table S5
Cell culture and transient expression
Metastatic melanoma tumor lines were maintained as previously described (45
). HEK 293T cells were purchased from ATCC (Manassas, VA) and maintained in complete Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1× non-essential amino acids, 2 mM L-glutamine, and 0.75% sodium bicarbonate. A375 cells were purchased from National Cancer Institute, Division of Cancer Treatment, Developmental Therapeutics Program, Frederick, MD and maintained in RPMI-1640 and supplemented with 10% FBS. Mel-STR cells were described previously(5
). HEK 293T cells were transfected with Lipfoctamine2000 reagent (Invitrogen, Carlsbad, CA) at a 6:1 ratio with DNA (µl:µg) using 3–5 µg of plasmid DNA per T75 flask.
Immunoprecipitation and western blotting
Transfected cells were gently washed 3× in PBS and then lysed using 0.5–1.0 mL 1% NP-40 lysis buffer (1% NP-40, 50mM Tris-HCl pH 7.5, 150mM NaCl, Complete Protease Inhibitor tablet, EDTA-free (Roche, Indianapolis, IN), 1µM sodium orthovanadate, 1 mM sodium fluoride, and 0.1% β-mercaptoethanol) per T-75 flask, for 20 min on ice. Lysed cells were scraped and transferred into a 1.5 mL microcentrifuge tube. Extracts were centrifuged for 10 min at 14,000 rpm at 4°C. 500 µl of supernatant was immunoprecipitated overnight using 20 µL of anti-FLAG (M2) beads (Sigma-Aldrich A2220). Immunoprecipitation of conditioned medium was done as previously described (5
). The immunoprecipitates were washed and subjected to SDS-PAGE and western blotting as previously described (46
). Primary antibodies used in our analysis were anti-FLAG horseradish peroxidase conjugated (Sigma-Aldrich A8592) and anti-alpha-tubulin (Calbiochem-EMD Biosciences, Gibbstown, NJ CP06).
Pooled stable expression
To make lentivirus, pCDF-MCS2-EF1-Puro ADAMTS18 constructs were co-transfected into HEK 293T cells seeded at 1.5×106 per T75 flask with pVSV-G and pFIV-34N (kind gifts from Todd Waldman, Georgetown University) helper plasmids using Lipofectamine 2000 as described by the manufacturer. Virus-containing media was harvested 48–60hr after transfection, filtered, aliquoted and stored at −80°C.
A375 cells were seeded at 1.5 × 106
cells per T75 flask 24 hr prior to infection. Lentivirus for ADAMTS18
(WT, G312E, P452S, C638S, Q904X, Q1002X, and P1035S) or empty vector control were used to infect A375 cells as previously described(47
). Stable expression of ADAMTS18 proteins (WT and mutants) was determined by SDS-PAGE analysis followed by immunoprecipitation and immunoblotting with anti-FLAG and anti-alpha-tubulin to show equivalent expression among pooled clones.
Mel-STR cells were seeded at 1.5×106 per T75 flask 24 hr prior to transfection with ADAMTS18 (WT, G312E, P452S, C638S, Q904X, Q1002X, and P1035S) or empty vector control in pcDNA3.1(−) using Fugene6 (Roche 11814443001) as per manufacturers protocol. Transfected cells were selected using normal complete growth medium supplemented with 300µg/ml G418 and pooled for future experiments. Stable expression of ADAMTS18 proteins (WT and mutants) was determined by SDS-PAGE analysis followed by immunoprecipitation and immunoblotting with anti-FLAG and anti-alpha-tubulin to show equivalent expression among pools.
Constructs for stable depletion of ADAMTS18 were obtained from Open Biosystems (Huntsville, AL) and two were confirmed to efficiently knockdown ADAMTS18 at the message and protein level. Lentiviral stocks were prepared as previously described (5
). Melanoma cell lines (5T, 12T, 85T and A375) were infected with shRNA lentiviruses for each condition (vector and scrambled controls and three independent ADAMTS18
-specific shRNAs whose sequences are presented in Supplementary table S6
). Selection and growth were done as described above. Stably infected pooled clones were tested in functional assays.
Reverse Transcription PCR
Total RNA was extracted from pooled clones of melanoma cells A375 stably knocked-down for endogenous ADAMTS18 following the manufacturer’s protocol for RNeasy Mini Kit (Qiagen #74101). Total RNA was eluted in 30 µl DEPC-treated dH20. A total of 1µg of total RNA was used for single strand cDNA synthesis using a SuperScript III First Strand kit (Invitrogen #18080-051). cDNA was amplified using the olido dT20 primer supplied in the kit. To test for loss of ADAMTS18 message we used 1 µl of cDNA in the PCR with either ADAMTS18 primers (forward primer: 5´-accctggtctcagtgttcca-3´ and reverse primer: 5´-tgcaggtctcttccaagtcc-3´), (forward primer: 5´-ccgtttgtggcttgagtatg-3´ and reverse primer: 5´-cggctagaacctggacagaa-3´) or GAPDH primers (forward primer: 5´-tggaaggactcatgaccaca-3´ and reverse primer: 5´-tgctgtagccaaattcgttg-3´). The product was then analyzed on a 1% agarose gel.
To examine growth potential, pooled A375 and Mel-STR ADAMTS18 clones were seeded into 96 well plates at 250 cells per well in either 1%, 2.5% or 10% serum-containing medium and incubated for 13–17 days. Cell numbers were counted every 48 hr by lysing cells in 50 µl 0.2% SDS/well and incubating for 2 hours at 37°C prior to addition of 150 µl/well of SYBR Green I solution (1:750 SYBR Green I (Invitrogen-Molecular Probes) diluted in dH20). Plates were analyzed using a BMG Labtech FLOUstar Optima.
Foci formation assays
A375 and Mel-STR pooled clones were seeded at 500 cells per T25 flask in normal complete serum containing medium and incubated for 8–10 days prior to staining with Hema 3 Stat Pack (Protocol) to visualize foci for counting.
A375 or melanoma cells with stable knock-down of ADAMTS18 were seeded into pre-conditioned migration wells (8.0 µm – BD Biocoat, BD Biosciences) at 10,000 to 30,000 cells per well in serum-free medium in the top chamber and incubated for 16–18 hr with complete serum containing medium in the bottom chamber prior to harvesting. Inserts were fixed and stained using Hema 3 Stat Pack (Protocol). Inserts were analyzed and counted for cells migrated per field view and quantitated using ImageJ (NIH software).
96-well plates were coated with 5 µg/ml laminin-I or 5 µg/ml fibronectin in 200ul 1×PBS and incubated overnight at 4°C. Prior to plating cells the coated wells were washed once in PBS and then blocked with 0.1mg/ml heat-inactivated BSA (dissolved in PBS) for 1 hr at room temperature. Cells were seeded into the plates at 30,000 cells per well and incubated at 37°C for 2 hr with the lid off. Cells were shaken off plate vigorously then washed three times with PBS. Remaining cells were fixed using 4% paraformaldehyde in PBS overnight at 4°C. Plates were then washed three times using ddH20. Attached cells were stained with 0.1% crystal violet (w/v – 20% methanol in PBS) for 30 min at room temperature followed by three washes with dd H20. Dye was solubilized with 0.1N HCl for 10 min at room temperature. Absorbance was measured at 610nm on Molecular Devices SpectraMax and quantitated using Microsoft Excel.
Immunoflescent analysis of pooled clones
A375 pooled ADAMTS18 clones expressing either (WT, G312E, C638S and P1035S) or empty vector were seeded on 8-well chamber slides at a cell density of 50,000 cells per well. Cells were grown for 24 hr prior to fixing and staining. Chamber slides were wased once with 1× PBS followed by fixation with 4% paraformaldhyde (PBS) for 15 min at room temperature. Cells were subsequently washed three times with ice-cold 1× PBS for 5 min per wash. Slides were blocked with 1% BSA (PBS-T) for 30–60 min at room temperature followed by washing with 1× PBS twice. Chamber slides were immunostained with anti-FLAG (rabbit) (Sigma-cat# F7425) in 1% BSA (PBS-T) at a dilution of 1:50 for 18 hr at 4°C. Chamber slides were washed three times with PBS at 5 min per wash followed by incubation with anti-rabbit (Alexa fluor 568-cat# A11036) (Invitrogen, Carlsbad, CA) diluted at 1:200 in 1% BSA (PBS-T) for 2 hr at room temperature. Slides were washed three times in PBS followed by fixation/mounting with DAPI and analyzed on a Zeiss A×10 (Scope.A1) at 40× using SPOT imaging software for image aquistion. Further analysis was done using Adobe Photoshop and ImageJ/NIH software.
Xenograft Studies in Mice
NOD/SCID mice were purchased from Jackson Laboratories. All mice were housed in a pathogen-free facility and were given autoclaved food and water. Mel-STR pooled clones expressing empty vector or with wild-type ADAMTS18 or mutant ADAMTS18 were grown up in T-75 flasks to 70–80% confluency. 1×106 cells were resuspended in 100 µL of sterile 1×PBS and injected subcutaneously into 11 week old male NOD/SCID mice. Mice were monitored bi-weekly and final tumor weights were measured after tumor was excised from euthanized mice at day 22 post-injection. Lungs from each mouse were harvested in 4% paraformaldehyde and embedded in paraffin for H&E staining followed by histological evaluation.
Statistical analyses were performed using the R statistical environment and Microsoft Excel (two-tailed t-test, binomial test).