IL6ko in C26 cancer cell clone 4 - effect on Cancer Associated Cachexia

For CRISPR–Cas9-mediated Il6 gene deletion in C26 cells, the following guide (g)RNAs were used: Fw: 5′CACCGAATGATGGATGCTACCAAAC3′; Rev: 5′AAACGTTTGGTAGCATCCATCATTC3′. After annealing, gRNAs were ligated with the vector pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene #62988) using T4 ligase (NEB #M0202L). The construct was amplified in Escherichia coli XL10 competent cells (NEB #C2987), and plasmid isolation was performed using the NucleoBond Xtra plasmid purification kit (Marchery Nagel #740410.50) according to the manufacturer’s protocol. C26 cells were transfected with recombinant vectors and a control vector comprising scrambled RNA using Lipofectamine 3000 (Invitrogen #L3000008). Transfected cells were selected using 6 µg ml−1 puromycin (Sigma-Aldrich #P8833), 100 cells per 10-cm dish were seeded and single cell colonies were picked for further analysis. For verification of gene editing, cellular DNA was obtained by incubating the cells in lysis buffer (Qiagen #102-T) containing 1.5 µl Proteinase K (Roth #7528.2) at 56 °C and 1,500 rpm for 1 h, Proteinase K was subsequently denatured at 85 °C for 45 min. The suspension was centrifuged at 16,000g for 10 min, and the supernatant was collected for PCR. PCR was performed using Q5 high-fidelity DNA polymerase (NEB #M0491) and the following primer pairs: Fw: 5′TGGTAATCTTTTGCAGTTGTGC3′; Rev: 5′AGCTACTGCAGGCCAGTTACA3′ The amplified DNA was sent to Microsynth AG for Sanger sequencing. IL6 deletion from cancer cells was verified using an enzyme-linked immunosorbent assay from C26 cell culture supernatants upon treatment with 100 ng ml−1 lipopolysaccharide for 24 h. To assess IL6 protein levels in tumour tissues, powdered tissues were lysed in 150 µl PBS containing protease inhibitor (pepstatin, Roth, #2936.2; antipain, Roth #2933.2; leupeptin, Roth, #CN33.4), followed by homogenization using an Ultra-Turrax Homogenizer (IKA) and centrifugation for 10 min at 1,000g, and the protein concentration was determined using a bicinchoninic acid assay (Thermo Fisher Scientific Reagent A #23228, Reagent B #23224). Cell culture supernatant, tumour lysates and plasma of tumour-bearing mice were analysed using the mouse IL6 Uncoated ELISA Kit (Invitrogen #88706422). Balb/c mice, bred at the Institute of Molecular Biosciences, Graz, Austria, were maintained in a 14-h light/10-h dark cycle and fed a standard chow diet (ssniff #V1534-000) with ad libitum access to food and water. Then, 12–16-week-old male Balb/c mice were injected with 1 million C26 cells or PBS (100 µl) into the musculus GC of the right hind leg. At study endpoints, blood was drawn via the retro-orbital plexus, mice were euthanised by cervical dislocation, and tissues were excised and snap-frozen in liquid nitrogen. Onset of cachexia was defined by a body weight loss of ≥10% within 2–3 consecutive days. For animal welfare, experiments were discontinued before mice experienced clinically evident functional disorders. If mice underwent rapid weight loss of ≥20 % within a short period, the experiment was terminated and mice were euthanised. Animal handling and experimentation were performed in accordance with the institutional animal welfare officer, and the necessary licences were obtained from by the Austrian Federal Ministry for Science, Research, and Economy (BMBWF-66.007/0005-V/3b/2019).