Primary Human Cells. Stromal cells were isolated from discarded skin tissue fromthe Dermatology Clinic with approval from the InstitutionalReview Board (Stanford University Medical Center). Fat was removedby using a sterile scalpel and forceps; tissue was minced into ~1-mm cubes. Incubation in a six-well dish without medium at37°C for 10 min allowed for adhesion of the tissue to theplate. Fresh media containing DMEM, 10% FBS, and penicillin-streptomycinwere added, and samples were maintained at 37°C and 5% CO2.Media were replaced every 2 days. Outgrowth of spindle-shapedcells was typically apparent after 5–15 days in cultureand had a success rate of ~60%. When the cells were near confluence,they were subcultured with 0.25% trypsin-EDTA. Cultures wereexpanded until sufficient for RNA isolations (typically fourpassages).
Human BCC keratinocyte cultures were derived from fresh skintissue as described (38). A small crosssectional piece of eachsample was cut and fixed in 10% buffered formalin for histologicalconfirmation. The remaining tissue was placed overnight in 5mg/ml dispase (Gibco, Carlsbad, CA) at 4°C. The next day,epidermis was separated from dermis with dissecting forceps,minced by using sterile forceps and scalpel, and incubated in0.05% trypsin-EDTA at 37°C for 15 min, with occasional mixingto disperse cells. After neutralization with HBSS containing15% FBS, cells were spun down at 900 rpm in a Beckman AllegraGR centrifuge for 5 min, then resuspended in Keratinocyte serum-freemedia supplemented with EGF, bovine pituitary extract, and penicillin-streptomycin(Gibco). Cells were plated onto 12-well collagen I-coated plates(BD Biosciences, Franklin Lakes, NJ) and incubated at 37°Cin 5% CO2. Media were replaced every 2 days. Contamination fromfibroblasts or normal keratinocytes was avoided by subjectingthe culture to differential trypsinization and a transient increasein calcium concentration, respectively (39).
Microarray Procedures. Construction of human cDNA microarrays with ~42,000 elements,representing ~24,000 genes, and hybridizations was as described(40). Forty-eight hours before RNA harvest of stromal cultures,cells were washed three times in prewarmed PBS and then maintainedin low serum media containing DMEM and 0.1% FBS. mRNA was harvestedby using the FastTrack kit (Invitrogen, Carlsbad, CA). UniversalHuman Reference RNA (Stratagene, La Jolla, CA) was used as referencefor array experiments.
Arrays were scanned with a GenePix 4000A scanner and imagesanalyzed with GenePix 3.0 (Axon Instruments, Union City, CA).Microarray data were stored in the Stanford Microarray Database(41). All microarray data are available at the web site http://microarray-pubs.stanford.edu/Gremlin1_BCC.
Data Analysis. We considered only genes for which the cognate array elementhad a fluorescent signal at least 1.5-fold greater than thelocal background signal in both channels. Significance Analysisof Microarrays (29) was then used to identify a set of geneswhose expression levels were significantly different betweenfive tumor- and five nontumor-derived stromal cell culturesat a false discovery rate of 15% or 5%. Resulting expressionpatterns were organized by hierarchical clustering (42).
ISH. Digoxigenin-labeled sense and antisense riboprobes for GREMLIN1 were synthesized by using T7 polymerase-directed in vitrotranscription of linearized plasmid DNA (IMAGE clone 7262108)by using the DIG RNA Labeling Kit (Roche Diagnostics). ISH onparaffin sections was performed by using a biotinyl tyramideamplification procedure, essentially as described (43). Resultswere considered specific when a strong pattern of distinct punctatestaining was seen for the antisense probe, and little or nostaining was observed for the corresponding sense probe. Tissuemicroarrays of tumor samples were made as described (44).
IHC. IHC staining for Gremlin 1 was performed with Dako EnvisionPlus (Glostrup, Denmark). Anti-gremlin 1 antibody (Imgenex,San Diego, CA) was used at 1:10 dilution. IHC for BMPs was performedby using Vectastain ELITE ABC Rabbit IgG (Vector Laboratories,Burlingame, CA). Anti-BMP 2 and 4 antibodies (Santa Cruz Biotechnology,Santa Cruz, CA) were used at 1:50 dilution. IHC for cell lineagespecific markers was performed by using the Vectastain ELITEABC Mouse IgG kit with antibodies against Vimentin (1:200),CD31 (1:30), CD45 (1:100), GFAP (1:100), Desmin (1:100), andpancytokeratin (1:100; Dako). In all cases, antigen retrievalconsisted of a microwave step in 10 mM citrate buffer. Nucleiwere stained with hematoxylin.
As positive and negative controls, each antibody was also testedon a tissue microarray containing a large variety of normaland tumor human tissue samples to confirm the nominal specificity.ISH and IHC images were acquired with the BLISS Microscope System(Bacus Laboratories, Lombard, IL).
In Vitro Expansion and Differentiation Assays. To assess the effects of gremlin 1 and BMP proteins on expansionof cells in vitro, BCC-derived cells were maintained in keratinocytegrowth media containing bovine pituitary extract, human EGF,bovine insulin, hydrocortisone, gentamicin, and amphotericinB (Clonetics, San Diego, CA). Cells were incubated with recombinantmouse gremlin 1 and/or recombinant human BMP 2 or 4 (R&DSystems, Minneapolis, MN) at the concentrations indicated. Cellnumber was assessed by using triplicate counts with a hemacytometer,or RNA was collected for RT-PCR analysis.
Quantitative RT-PCR. Total RNA was isolated from whole tissue, either tumor or adjacentnontumor tissue from the same patient, by using the RNeasy FibrousTissue Mini kit (Qiagen, Chatsworth, CA) and a rotor homogenizer.Total RNA was isolated from cultured cells by using RNeasy Mini(Qiagen). First-strand DNA was generated from mRNA by usingthe SuperScript III First-Strand Synthesis System (Invitrogen).RT-PCR (TaqMan) was performed by using ABI 7300 (Applied Biosystems,Foster City, CA) with duplicate experimental samples for eachsample and each probe/primer set. GAPDH was used for normalizingPCR results.
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