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140503 IPF Signatures Supplement Withfigs Thorax Supplementary material for Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis Daryle J. DePianto1*, Sanjay Chandriani1⌘*, Alexander R. Abbas1, Guiquan Jia1, Elsa N. N’Diaye1, Patrick Caplazi1, Steven E. Kauder1, Sabyasachi Biswas1, Satyajit K. Karnik1#, Connie Ha1, Zora Modrusan1, Michael A. Matthay2, Jasleen Kukreja3, Harold R. Collard2, Jackson G. Egen1, Paul J. Wolters2§, and Joseph R. Arron1§ 1Genentech Research and Early Development, South San Francisco, CA 2Department of Medicine, University of California, San Francisco, CA 3Department of Surgery, University of California, San Francisco, CA ⌘Current address: Novartis Institutes for Biomedical Research, Emeryville, CA. #Current address: Gilead Sciences, Foster City, CA. *DJD and SC contributed equally to this manuscript §PJW and JRA co-directed this project Address correspondence to Paul J. Wolters, MD University of California, San Francisco Department of Medicine Box 0111 San Francisco, CA 94143-0111 [email protected] or Joseph R. Arron, MD, PhD Genentech, Inc. MS 231C 1 DNA Way South San Francisco, CA 94080 [email protected] 1 METHODS Human lung tissue samples Tissues were obtained at UCSF from clinical samples from IPF patients at the time of biopsy or lung transplantation. All patients were seen at UCSF and the diagnosis of IPF was established through multidisciplinary review of clinical, radiological, and pathological data according to criteria established by the consensus classification of the American Thoracic Society (ATS) and European Respiratory Society (ERS), Japanese Respiratory Society (JRS), and the Latin American Thoracic Association (ALAT) (ref. 5 in main text). Non-diseased normal lung tissues were procured from lungs not used by the Northern California Transplant Donor Network. Clinical and demographic information for the IPF patients is in Supplementary Table 1. Sample and data collection were approved by the UCSF Committee on Human Research and all patients provided written informed consent. RNA preparation Snap frozen lung biopsy samples were pulverized in a pre-cooled (in liquid nitrogen) Bessman tissue pulverizer (Spectrum Laboratories, Rancho Dominguez, CA). Trizol (Life Technologies, Carlsbad, CA) was added to pulverized material and pipetted several times. Lysates were incubated on ice for 10-15 minutes. Lysates were stored at -80 C until further processing. RNA was isolated from Trizol lysates according to the manufacturer’s protocol. Trizol-isolated RNA was then subjected to another purification step using the Qiagen RNeasy columns, as per the manufacturer’s protocol. RNA 2 concentration was determined with a Nanodrop 8000 spectrometer (Thermo Scientific, Waltham, MA) and all microarray samples were analyzed by Bioanalyzer (Agilent, Santa Clara, CA). Series of adjacent 10µm thick cryosections were generated from OCT-embedded fresh frozen lung tissue isolated from healthy controls or IPF patients. Adjacent sections were analyzed for gene expression (qPCR) and histology (H&E and Masson's trichrome staining) as depicted in Figure 4A. For gene expression analysis, RNA was isolated using the RNEasy Mini kit (Qiagen). Frozen lung tissue sections were dissolved in RLT buffer containing 1% beta-mercaptoethanol. RNA column purification was performed as per manufacturer's instructions, including an on-column DNAse digestion. Qualitative analysis of the eluted RNA was assessed using a Bioanalyzer (Agilent 2100) and quantification was performed with a Nanodrop 8000 spectrometer (Thermo Scientific, Waltham, MA). Microarray analysis RNA was amplified and labeled using the Quick Amp labeling kit (Agilent, Santa Clara, CA) to generate labeled cRNA from 1µg of total RNA. Experimental samples were labeled with Cy5; Universal Human Reference RNA (Stratagene, La Jolla, CA) was used for the reference channel and was labeled with Cy3. Cy5 and Cy3 labeled cRNA was competitively hybridized to the two-color Whole Human Genome 4 × 44K gene expression microarray platform. Hybridized microarrays were washed according to the 3 manufacturer's protocol (Agilent, Santa Clara, CA) and all feature intensities were collected using the Agilent Microarray Scanner. TIFF images of scanned slides were analyzed using Feature Extraction Software version 10.7.3.1, protocol GE2-v5_95 (Agilent). Flagged outliers were not included in any subsequent analyses. All data are reported as log2 values of the dye-normalized Cy5/Cy3 ratios. The log2 ratios of all samples were normalized to the average log2 ratios of the corresponding non-IPF controls. Differentially expressed genes were identified by building a linear model that incorporated three factors: diagnosis, source of tissue and sex (inferred from expression analysis of Y-linked genes) in R using LIMMA with thresholds of Benjamini-Hochberg adjusted p-value less than 0.05 and absolute value of log fold change greater than 1.5. Clustering was performed on data for the 2940 differentially expressed genes using the cluster (http://genetics.stanford.edu/~sherlock/cluster.html) program with Pearson correlation similarity metric and average linkage node summarization. R-code available as supplementary file ipfDiffExp.R Microarray data are available from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/; GEO accession ID GSE53845). qRT-PCR RNA isolated from lung biopsies utilized for microarray was also used in the qPCR studies. First strand cDNA was generated from 100ng of total RNA using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA). 6.25ng of cDNA was pre-amplified using the Taqman PreAmp Master Mix (Life Technologies) 4 along with diluted Taqman assays (Life Technologies), according to the manufacturer’s instructions. For the analysis of gene expression from tissue sections, 20ng of total RNA was used as input into the reverse transcription reaction and 1.25ng of cDNA was then pre-amplified. qPCR was performed using the Biomark HD system (Fluidigm 96.96 format) and a panel of 24 Taqman assays comprised of genes within the bronchiolar and lymphoid signatures, and HPRT1 as a housekeeping gene (genes and probe IDs listed in Supplementary Table 2). Resultant data was normalized to HPRT1 to yield ΔCt values, while a commercially available human cDNA pool generated from multiple tissue types (Clontech, Mountain View, CA) was used as a reference to calculate ΔΔCt values. Signature score calculation Clusters of genes representing hypothetically relevant biological programs were identified by manually examining the clustering results (using JavaTreeview software) for genes that appeared to be both substantially correlated and of related biological function. Two such clusters were recorded for subsequent investigation, termed "Lymphoid" and "Bronchiolar" signatures. Summary scores for each of these signatures were computed by first centering the data for each gene across all subjects. Each signature was then calculated as the first principal component of the centered expression values for all genes in that signature, yielding a single numerical signature score for each patient. Histology/Immunohistochemistry 5 Ten-µm sections of fresh frozen or formalin-fixed paraffin embedded (FFPE) lung tissue were prepared from lung tissue samples of IPF patients who underwent lung transplantation. Sections were acetone fixed for immunostaining with CD3, CD20 (Dako, Carpinteria, CA), keratin 5 and keratin 14 (Covance, Princeton, NJ), MMP3 (Abcam, Cambridge, MA), and CXCL13 (R&D Systems, Minneapolis, MN) primary antibodies. Immunoreactivity was detected through the use of biotinylated secondary antibody and avidin/biotinylated HRP enzyme complex (Vector Labs, Burlingame, CA). Sections were counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO). Periodic acid-Schiff (PAS) and Masson’s trichrome staining were performed using commercially available kits (Sigma-Aldrich, St. Louis, MO) according to manufacturer’s protocols. Standard hematoxylin and eosin staining was performed on tissue sections post-fixed with formalin. Peripheral biomarkers Serum biomarkers and lung function were assessed in a separate cohort of 80 IPF patients collected at time of presentation to the interstitial lung disease clinic at UCSF. All patients fulfilled the international guideline criteria for diagnosis of IPF (1). Vital status was followed for 2-8 years after sample collection. Serum protein levels of CXCL13 and MMP3 were measured by ELISA and ECLA, respectively. In addition, serum samples from 28 healthy controls were measured concomitantly with the samples from IPF patients. The controls for the soluble biomarkers were obtained from the Genentech employee blood donor donation program. Proper provisions were taken for the recruitment and procurement of these samples under institutional review board approval. 6 Supplementary table 1. IPF lung tissue subject characteristics Variable (N=40) Age, years 60.6 (8.8) Male gender, N (%) 33 (83) VATS biopsy, Explant, N 11, 29 Ever smoKed % 68 FVC, % predicted 55.8 (17.2) TLC, % predicted 55.7 (16.2) DLCO, % predicted 34.1 (13.8) Age, FVC, TLC, and DLCO are reported as mean (standard deviation). Abbreviations: VATS, video assisted thoracoscopic biopsy; FVC, forced vital capacity; TLC, total lung capacity; DLCO, diffusion capacity of carbon monoxide. There were no cUrrent smoKers in the cohort. 7 Supplementary Table 2. Genes in bronchiolar and lymphoid signatures used for qPCR validation
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