Tumour samples and cell culture
Patients were diagnosed with TSC according to clinical criteria42
and enrolled in NHLBI Institutional Review Board–approved protocol 00-H-0051. Written informed consent was obtained from all patients. Skin samples from TSC patients were bisected to use one portion for histology and the other for cell culture. To isolate fibroblasts-like cells, the skin biopsies from TSC patients were cut into small pieces and placed in DMEM with 10% FBS in culture dishes. Medium was changed twice weekly until the fibroblasts migrated out to cover the dishes. Cells were harvested for serial passage and cryopreservation43
. Keratinocytes were isolated from foreskins of unidentified normal neonates (provided by Dr. Jonathan Vogel, NCI) and grown using standard methods44
. Briefly, tissues were treated overnight with dispase (Becton Dickinson Labware, Bedford, MA) at 4°C. Epidermal sheets were separated from dermal sheets and digested with 0.05% trypsin 0.53 mM EDTA (Invitrogen, Gaithersburg, MD) at 37°C for 20 min. Cells were collected and placed on tissue culture dishes in keratinocyte serum-free media (Invitrogen) supplemented with bovine pituitary extract and recombinant epidermal growth factor.
Cell treatment and protein measurement
Cells from TSC patients were seeded into 60-mm dishes at 5 × 105
cells in DMEM with 10% FBS and treated with or without 2 nM rapamycin (EMD Biosciences, Inc. La Jolla, CA) in serum-free DMEM for 24 hours. Cells were lysed in protein extraction buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 20 mM NaF, 2.5 mM Na2
, 1 mM β-glycerophosphate, 1 mM benzamidine, 10 mM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride] for quantification of TSC2 and pS6 by western blot as described26,37
. Briefly, total proteins were separated in 10% (wt/vol) polyacrylamide gels and transferred to 0.45-μm Invitrolon PVDF membranes (Invitrogen) before immunoblotting with anti-TSC2 (C-20, Santa Cruz), anti-phospho-S6 ribosomal protein (Ser-235/236), anti-S6 ribosomal protein antibodies (Cell Signaling), or β-actin (Sigma-Aldrich), horseradish peroxidase-conjugated secondary antibodies (GE Healthcare), and a SuperSignal West Pico chemiluminescence detection kit (Pierce).
Cell viability assay
Cells from TSC patients were seeded in 96-well plates (2000 cells per well) with 10% FBS/DMEM and treated with rapamycin (0. 0.2, 2, 20 nM) for 3 days. Cell viability was assessed using a MTT kit (CellTiter 96® Non-Radioactive Cell Proliferation Assay, Promega, Madison, WI).
Creation of composites
Three-dimensional in vitro
composites were prepared for grafting using established methods45
, modified as described. Briefly, TSC2-null cells or TSC normal fibroblasts (passage 3 to 5) were mixed with 1 mg/ml of rat tail collagen type 1 (BD Biosciences, Bedford, MA) in 10% FBS/DMEM, and added to 6-well Transwell plates (Corning Incorporated, Corning, NY) at 0.5 × 106
cells per well. The dermal constructs were grown in 10% FBS/DMEM for 3 days before addition of 106
foreskin keratinocytes on top. The dermal-epidermal composites were incubated for 2 days submerged in DMEM and Ham’s F12 (3:1)(GIBCO/Invitrogen, Grand Island, NY) containing 0.1% FBS, after which the composites were brought to the air-liquid interface and grown for another 2 days in DMEM and Ham’s F12 (1:1) containing 1% FBS.
Female 6-8 week old Cr:NIH(S)-nu/nu mice (FCRDC, Frederick, MD) were anesthetized using inhalant anaesthesia with isoflurane (2-4%). The grafting area on the mouse back was washed with povidine and 70% ethanol and excised using scissors. Composites were placed on the graft bed, covered with sterile petrolatum gauze, and secured with bandages. The bandages were changed at 2 weeks and removed after 4 weeks.
Mice grafted with composites were treated with rapamycin (2 mg/kg) or vehicle (0.9% NaCl, 5% Polyethylene glycol and 5% Tween-80) every other day by intraperitoneal injection, as used in other mouse models46,47
. Mice were treated for 12 weeks starting 5 weeks after grafting. All mouse experiments were performed in accordance with relevant guidelines and regulations following protocol approval by the USUHS Institutional Animal Care and Use Committee.
Immunohistochemistry and quantification
Paraffin sections were deparaffinized and boiled in 10 mM sodium citrate buffer (pH 6.0) for 20 minutes. Frozen sections were fixed in acetone at −20°C for 10 minutes. All sections were stained for cellular markers using specific antibodies and Vectastain ABC-AP kit or Elite ABC kit with Vector Red or DAB substrate, respectively (Vector Laboratories, Burlingame, CA). Numbers of positive cells, staining intensities, and areas were quantified using an Olympus BX40 microscope (Olympus, Melville, NY) and Openlab 4.0 software (Improvision, Lexington, MA). Paraffin sections were stained for COX-IV (1:500, 3E11, Cell Signaling), Nestin (1:30000, 10C2, Millipore), Versican (1:1000, V0/V1 Neo, Thermo scientific), keratin 15 (1:5000, PCK-153P, Covance), keratin 75 (1:1000, K6hf, Progen Biotechnik GmbH), pS6 (1:200, #2211, Cell Signaling), CD68 (1:50, KP1, DakoCytomation), mouse CD31 (1:30, ab28364, Abcam) respectively. Frozen sections were stained for HLA-ABC (1:20, W6/32, Serotec), Ki-67 (1:250, SP6, Thermo Scientific) and F4/80 (1:1000, Cl:A3-1, Abcam).
In situ alkaline phosphatase activity assay
Frozen sections were fixed in acetone for 10 minutes and incubated with the pre-equilibration buffer (100 mM NaCl, 50 mM MgCl2, 100 mM Tris-HCl, pH 9.5, 0.1% Tween-20) for 15 minutes at room temperature. BM Purple AP substrate (Roche, Indianapolis, IN) was applied for 2 hours. The reaction was stopped with 20 mM EDTA in PBS.
Laser microdissection and DNA isolation
Cryosections were placed on Leica slides (Leica Microsystems, Bannockburn, IL) for membrane-based laser microdissection. Cells from dermal sheath/dermal papilla, follicular epithelium, dermis, and interfollicular epidermis were microdissected using the LEICA AS LMD laser dissection microscope. DNA was isolated using PicoPure DNA Isolation kit (Molecular Devices, Sunnyvale, CA).
TSC2 sequence analysis
DNA isolated from TSC2-null fibroblast-like cells was sequenced for TSC2 mutations by Athena Diagnostics (Worcester, MA). Part of exon 10 of TSC2 was amplified using AmpliTaq gold DNA polymerase (Applied Biosystems) and PCR primers 5′TGGTGTCCTATGAGATCGTCC3′ and 5′ AAGGAGCCGTTCGATGTT3′ (for 1074G>A) or 5′AAGCAGCTCTGACCCTGTGT 3′ and 5′ CACTGCGAATCACCAGAGAA 3′ (for 1058_1059delTC). The PCR product was purified by QIAquick Gel Extraction kit (QIAGEN) and sequenced using 3130xl Genetic Analyzer (Applied Biosystems). To confirm DNA mutations using restricted enzyme digestion, PCR products were amplified using primers of 5′TGGTGTCCTATGAGATCGTCC3′ and 5′AAGGAGCCGTTCGATGATGTT3′. The products were digested with BsmA1 or SacI for detecting 1074G>A or 1058_1059delTC, respectively, and separated by electrophoresis in 10% Novex TBE (Tris-Borate-EDTA) polyacrylamide gels (Invitrogen). Part of exon 36 was amplified and sequenced using primers of 5′CAATGAGCATGGCTCCTACA3′ and 5′GGCACCTCCTGATTACTCCA 3′. The mutation of 4830G>A in exon 36 was confirmed by PCR amplification using primers of 5′TCATCGAGCTGAAGGACTGC3′ and 5′AGGCCGTACCTTGCATGAT3′ followed by restricted enzyme digestion with BslI and electrophoresis in 10% TBE gels.
Loss of heterozygosity analysis
Genomic DNA was isolated from TSC patient cells using DNeasy Blood & Tissue kit (QIAGEN) and amplified by PCR with primers flanking microsatellite loci D16S291, D16S521 and D16S663 on chromosome 16p1348
. One primer of each pair was fluorescently labeled with 6-FAM during synthesis (Invitrogen, Carlsbad, CA). PCR products were denatured in formamide containing GeneScan-500 (Rox) size standards, and separated on the Genetic Analyzer 3100 (PE Biosystems, Norwalk, CT) capillary electrophoresis system.
Y chromosome fluorescence in situ hybridization
The presence of male-derived human cells in xenografts was analyzed using the Vysis CEP Y (DYZ1) SpectrumOrange probe (Abbot Laboratories, Des Plaines IL) according to the manufacturer’s protocol. Briefly, 8-μm cryosections were air-dried for 20 min before incubating in 2 X SSC at 37°C for 20 min. Following sequential dehydration in ethanol, sections were treated with 10 mM HCl plus 0.006% pepsin at 37°C for 5 min and washed twice in PBS before dehydrating and air drying. Sections were denatured in 70% formamide, 2X SSC at 73°C for 5 min and dehydrated before hybridizing overnight with probe mixture (7 μl of CEP hybridization buffer, 2 μl of water and 1 μl of probe) at 42°C. The sample was washed twice at 68°C with 2X SSC, 0.1% NP-40 and counterstained by applying 10 μl of DAPI (Vector Laboratories) on each target area.
Gene Array Analysis
Gene array data deposited in the Gene Expression Omnibus (GEO) database26
(GEO accession GSE9715 and GEO dataset GDS3281), was analyzed for levels of genes in TSC angiofibroma cells that were twofold or more those in TSC normal fibroblasts. Affymetrix gene probes that were assigned an “absent” call in all samples were excluded from the list, and significance was defined as p<0.05 using a two-tailed t-test. Genes overexpressed by TSC angiofibroma cells were intersected with the human homologues of dermal papilla genes listed in Table S6 in Rendl et al35
and Table S1 in Driskell et al36
Means and standard errors (SE) of data were analyzed by two-way analysis of variance with interaction between two factors. One factor is treatment group (vehicle and rapamycin) and another factor is cell source (normal and tumour). If significant, a multiple pairwise comparison procedure by Tukey-Kramer was used for assessing the difference of one group from others. Significance was defined as p<0.05, and all statistical tests were two-sided. PC SAS version 9.2 software was used for statistical analysis.