NextSeq 500 paired end sequencing; Expression analysis of human ex vivo cultivated urothelial carcinoma samples transduced with a trivalent adenoviral vector (Im02) for activation of the immune tumor microenvironment.
Expression analysis of human ex vivo cultivated urothelial carcinoma samples transduced with a trivalent adenoviral vector (Im02) for activation of the immune tumor microenvironment.
All tissue samples were collected in collaboration with the Clinic of Urology, Asklepios Klinik Hamburg-Barmbek, Hamburg (Germany). Urothelial carcinoma and corresponding normal bladder urothelium were collected from a total of 73 patients undergoing radical cystectomy (n=48) or transurethral resection (TUR) (n=25) between May 2013 and October 2016. After resection, entire urinary bladders were assessed by the pathologist and dissected to yield cancerous and non-cancerous tissue specimens. These were immediately transferred to pre-cooled keratinocyte serum-free medium with supplements (K-SFM Kit, Gibco®, Thermo Fisher Scientific) and kept refrigerated during transport to the laboratory. Tissue culture was performed following the protocol described by Bolenz et al., tissue samples cautiously cut into pieces of about 50-100 mg and incubation on a gelatin foam matrix (Gelfoam®, Pfizer, New York City, NY, USA) presoaked with K-SFM media, supplemented with 5 ng/ml recombinant epidermal growth factor, 50 µg/ml bovine pituitary extract, and 160 µg/ml Mezlocillin Carino (Carinopharm, Elze, Germany). After adaptation to culture conditions for 24 h, explants were transferred to a separate 24-well plate and were transduced with adenoviral vectors. The explants were perfused in 500 µl of 10^8 infectious viral particle counts in formulation buffer including 10 µg/ml protamine sulfate for 60 min in the culture atmosphere. Transduced samples were returned to the gelatin foam matrix and incubated for 6 days with complete medium exchange in 3 to 4-day intervals. Specifications of the culture conditions were set on the basis of prior evaluation studies (data not shown). Viability was frequently monitored in culture media using a sensitive enzymatic lactate dehydrogenase (LDH)-release assay (CytoTox-ONE, Promega, Madison, WI, USA), following suppliers' instructions or on cryo-sections by an enzyme activity assay by visualizing mitochondrial nicotinamide adenine dinucleotide (NADH) diaphorase activity. Tissue specimens for RNA preparation were sampled in RNALater® (Ambion™, Thermo Fisher Scientific) and incubated over night at 4°C before freezing at -20°C. Total RNA was purified by homogenization of the samples in Trizol™ (Ambion™, Thermo Fisher Scientific) with the gentleMACS™ Dissociater and corresponding gentleMACS M™ Tubes (Miltenyi Biotec, Bergisch Gladbach, Germany). A phase separation step with chloroform and a spin-column purification (peqGOLD Total RNA, VWR) including DNAse digestion was performed. RNAs were quantified with the Qubit™ RNA HS Assay Kit (Molecular Probes®, Thermo Fisher Scientific) and quality was assessed on the Agilent 2100 bioanalyzer™ (Agilent Technologies, Santa Clara, CA, USA) using the RNA 6000 nano kit, according to the suppliers' instructions. Libraries were prepared automatically from 250 ng total RNA in 50 µL RNAse-free water with the TruSeq Stranded mRNA Library Prep Kit (Illumina Inc.) on the epMotion 5075 (Eppendorf, Hamburg, Germany) using the `Illumina Qualified' methods. Quality and quantity of the libraries were assessed using the DNA 1000 Kit on the Agilent 2100 bioanalyzer™ (Agilent) and the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific).
Experimental Factor: compound
Ad0
Experimental Factor: time
5
Experimental Factor: dose
10^8