Comment[ArrayExpressAccession] E-GEOD-45609 MAGE-TAB Version 1.1 Public Release Date 2014-01-01 Investigation Title Proton irradiation augments the reduction in tumor progression observed with advanced age Comment[Submitted Name] Proton irradiation augments the reduction in tumor progression observed with advanced age Experiment Description Proton irradiation is touted for its improved tumor targeting due to the physical advantages of ion beams for radiotherapy. Recent studies from our laboratory have shown that, in addition to targeting advantages, proton irradiation can inhibit angiogenic and immune factors and thereby modulate tumor progression. High-energy protons also constitute a principal component of the galactic cosmic rays to which astronauts are exposed. Increased understanding of the biological effects of proton exposure would thus contribute to both improved cancer therapy and carcinogenesis risk assessment for space travel. In addition, age plays a major role in tumor incidence and is a critical consideration for estimating cancer risk. We investigated the effects of host age and proton exposure on tumor progression. Tumor lag time and growth dynamics were tracked following injection of murine Lewis lung carcinoma (LLC) cells into young (68 day) versus old (736 day) mice with or without coincident irradiation. Tumor progression was suppressed in old compared to young mice. Differences in progression were further modulated by proton irradiation (1GeV), with increased inhibition evident in old mice. Through global transcriptome analysis, TGFβ1 and TGFβ2 were determined to be key players that contributed to the tumor dynamics observed. These findings point to older hosts providing decreased systemic tumor support, which can be further inhibited by proton irradiation. For genome-wide expression profiling of tumor tissue, Mouse WG-6 BeadArray chips (Illumina, San Diego, CA) were used. Total RNA was amplified with the Ambion Illumina TotalPrep Amplification Kit (Ambion, Austin, TX) and labeled from all replicate biological samples for each condition. For tumor replicates, thirty tumor samples from adolescent and thirty tumor samples from old mice, for a total of 60 tumor samples, were used. All replicate samples were run individually. For each age group, ten tumor samples had received proton irradiation while twenty tumor samples were from unirradiated mice (as described above). Total RNA was isolated and purified using TRIzol (Invitrogen) and quantified using an Agilent Bioanalyzer. Samples were deemed suitable for amplification and hybridization if they had 28s/18s = 2:1, RIN >7. Total RNA of 500ng per sample was amplified using AmbionTotalPrep, and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C, the chips were washed and then scanned using the Illumina iScan System. The data was checked with GenomeStudio (Illumina) for quality control. In GenomeStudio, data was background subtracted and rank invariant normalization was applied. Data was imported into MultiExperiment Viewer, MeV, for statistical analysis. The statistically significant genes were determined using MeV by applying a one-way ANOVA analysis with standard Bonferroni correction with a FDR <0.05 that resulted in a list of significant genes. Average gene expression signals <10 were filtered out due to signal being Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name briggs Beheshti Peluso Hahnfeldt Hlatky Person First Name christine Afshin Michael Philip Lynn Person Mid Initials elaine Person Email cebriggs7135@gmail.com Person Affiliation christine briggs Person Phone 617 905-9301 Person Address Center for Cancer Systems Biology, christine briggs, 736 Cambridge Street, Boston, MA, USA Person Roles submitter Protocol Name P-GSE45609-1 P-GSE45609-3 P-GSE45609-4 P-GSE45609-2 P-GSE45609-5 Protocol Description The data was checked with GenomeStudio (Illumina) for quality control. In GenomeStudio, data was background subtracted and rank invariant normalization was applied. ID_REF = VALUE = Background-subtracted, rank invariant normalized signal Detection Pval = BEAD_STDERR = Total RNA of 500ng per sample was amplified using Ambion TotalPrep (Ambion), and 1.5µg of the product was loaded onto the chips. Following hybridization at 55C, the chips were washed. RNA was isolated from mouse tumor tissue. Tumor chunks of 1 to 2mm in diameter were placed in TRIzol (Invitrogen, Carlsbad, CA) and homogenized using a Tissue Lyser II (Qiagen, Valencia, CA). Tissue with TRIzol was centrifuged at 1200g and 0.2ml of chloroform was added for 5min. Samples were centrifuged at 10,000g for 15min at 4°C. The aqueous phase with RNA was transferred to a 1.5ml eppendorf tube. Isopropyl alcohol (0.5ml) was added to each sample (room temperature) for 10min and samples were centrifuged at 10,000g for 10min at 4°C. RNA pellets were washed once with 75% ethanol and resuspended in 100μl RNase-free water. Quality and quantity of RNA were measured by an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). Standard Illumina scanning protocol using Illumina iScan. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TREATMENT AGE Experimental Factor Type treatment age Comment[SecondaryAccession] GSE45609 Comment[GEOReleaseDate] 2014-01-01 Comment[ArrayExpressSubmissionDate] 2013-03-28 Comment[GEOLastUpdateDate] 2014-01-01 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE45609_non-normalized.txt SDRF File E-GEOD-45609.sdrf.txt