biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicstranscription profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-6749We sought to decipher the molecular landscape of tumor resistance to continuous treatment with VEGF-receptor tyrosine kinase inhibitor (RTKi) sunitinib (SU11248, SU). To address that, we performed serial transplantation of a syngeneic LLC tumor models in C57bl6 mice using a genome-wide lentiviral shRNA library targeting about 39,000 transcripts (150K complexity). The abundance of each single knock down (kd) was longitudinally traced via molecular barcodes towards development of resistant tumors. After three in-vivo panning rounds, distinct shRNA constructs were found to be significantly enriched in tumors passaged under SU selection pressure (p3-SU) compared to those passaged without treatment (p3-ctrl).biostudies-arrayexpressSample Collection - LLC cells (mouse Lewis Lung Carcinoma-ATCC® CRL-1642 ™) were cultured in DMEM media (ATCC) complemented with 10% fetal calf serum (FCS) (GIBCO). For functional analysis of genes, an HIV-based GeneNet™ Mouse 40K siRNA Library (SBI, Cat# SI622B-1) containing a pool of 150,000 siRNA sequences designed to target 39,000 mouse transcripts was applied. Cells were injected in right flank of C57BL/6 mice and tumors were either treated with 50mg/kg/day SID sunitinib or with no treatment. Tumors were recovered afterwards and were snap frozen in liquid nitrogen.Nucleic Acid Extraction - Total RNA, from the transduced LLC cells were isolated in phase lock tubes using the procedure of total RNA isolation with Trizol (Ambion) and the RNeasy Kit (QIAGEN). RNA 6000 Nano Lab on Chip kits and Agilent 2100 Bioanalyzer (Agilent-CA) were utilized to check RNA quality and quantity, respectively.Labeling - Biotin-labeled cRNA samples for hybridization on Illumina Human/Mouse/Rat Sentrix-6/8/12 BeadChip arrays (Illumina, Inc.) were prepared according to Illumina's recommended sample labeling procedure based on the modified Eberwine protocol (Eberwine et al., 1992). In brief, 250 – 500 ng total RNA was used for complementary DNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA according to the MessageAmp II aRNA Amplification kit (Ambion, Inc., Austin, TX). Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was column purified according to TotalPrep RNA Amplification Kit, and eluted in 60-80 µl of water. Quality of cRNA was controlled using the RNA Nano Chip Assay on an Agilent 2100 Bioanalyzer and spectrophotometrically quantified (NanoDrop).Scaning - Scanning and feature extraction werer performed with Illumina BeadChip scanner (IsScan+) and Illumna feature extracton software.Sample Treatment - 50mg/kg/day SID sunitinib with oral gavageHybridization - Hybridization is performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed once in High Temp Wash buffer (Illumina Inc.) at 55°C and then twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes (in between washed with ethanol at room temperature). After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays are dried and scanned.E-MTAB-6745MIAME ScoreAssays and DataorganisationMAGE-TAB FilesArray DesignsChrisitan SchwagerAzadeh Fahim GolestanehfalseDevelopment of Resistant Tumors to Anti-angiogenic Therapy (SU 11248) via in-vivo RNAi ScreenWe sought to decipher the molecular landscape of tumor resistance to continuous treatment with VEGF-receptor tyrosine kinase inhibitor (RTKi) sunitinib (SU11248, SU). To address that, we performed serial transplantation of a syngeneic LLC tumor models in C57bl6 mice using a genome-wide lentiviral shRNA library targeting about 39,000 transcripts (150K complexity). The abundance of each single knock down (kd) was longitudinally traced via molecular barcodes towards development of resistant tumors. After three in-vivo panning rounds, distinct shRNA constructs were found to be significantly enriched in tumors passaged under SU selection pressure (p3-SU) compared to those passaged without treatment (p3-ctrl).2026-09-16T00:00:00Z2026-09-17T10:39:12.92Z2022-01-28T22:04:47.94ZE-MTAB-6749E-MTAB-6745E-MTAB-6747E-MTAB-6746E-MTAB-6748EFO_0002768EFO_0003814EFO_0002944EFO_0003813EFO_0005518EFO_0003815EFO_0003969