br For conditional ablation of IL R in colonic epithelial
For conditional ablation of IL-1R1 in colonic epithelial and tumor cells of CPC-APC mice, Il1rf/fApcf/f mice were crossed to CDX2Cre-Il1D/WT mice, where the null (D, deleted) Il1r allele is derived from the whole-body knockout strain. All mice were maintained
in filter-topped cages on autoclaved food and water at the Fox Chase Cancer Center Animal Facility (FCCC), and all experiments were approved by FCCC IACUC. All experiments used co-housed littermates, unless indicated otherwise, so that consistency of common microflora and genetic background/alterations would be ensured. For tamoxifen-inducible tumorigenesis, CDX2ERT-Apcf/f mice were given 2 injections of tamoxifen 2 mg and 1.5 mg per mouse (Sigma; dissolved in 5% ethanol, 95% corn oil (Sigma)) intraperitoneally (i.p.) every other day. Mice were sacrificed 4-6 weeks after the last dose of tamoxifen for histological analysis and tumor statistics.
Bone Marrow Transplantation
Six- to eight-week-old recipient mice were irradiated twice (600 rad and 500 rad) using cesium irradiator at FCCC during one day to achieve a lethal dose and intravenously injected with a single-cell suspension of donor bone-marrow cells. Recipients were co-housed littermates, which were transplanted with both gene-deficient and wild-type bone marrow. After transplantation, the recipients were placed on neomycin and polymixin B in drinking water for two weeks, followed by transfer to dirty cages that were from the same room and rack to reconstitute potentially depleted microbiota. Mice were sacrificed and analyzed for tumor development 4 months after transplantation; for inducible CDX2ERT-Apcf/f model mice were allowed to reconstitute BM, injected with tamoxifen (as above) and then allowed to develop CRC for 4-6 weeks.
To deplete gut microbiota mice were given mix of U-0126 in drinking water for 4 weeks. Water was changed every 3-4 days. Antibiotics cocktail included: Ciprofloxacin (0.2g/L), Neomycin (1g/L), Vancomycin (0.5g/L), Ampicilin (1g/L), Metronidazol (0.5g/L), Primaxin (Imipenem) (0.5g/L). Antibiotics treatments started after last injection of Tamoxifen and after 4 weeks of treatment mice were sacrificed for tumor analysis.
Immune cells isolation, ex vivo cell stimulation, Flow Cytometry and Cell Sorting
Lamina propria lymphocytes (LPL) and Intraepithelial lymphocytes (IEL) cells from colonic tumors and normal tissues were isolated. Briefly, colonic tumors and normal tissues were cut into small pieces and briefly washed in 1mM DTT-HBSS, followed by mechanical shaking with 10mM EDTA-HBSS in a shaker (200 rpm) at 37 C for 20 min for 2 rounds total. Then IEL/IEC fraction was passed through a metal mesh and cells collected, while the remaining tissue was digested in 20 mL of Collagenese Type VIII (Sigma) solution in 1xHBSS in the shaker (180rpm) at 37 C for 30 min. Digested tissue was passed through a 70 mm Cell Strainer and cell suspension was collected (unpurified LPL). IEL/IEC and LPL fractions were further enriched by Percoll gradient centrifugation (20min, 2200rpm, no brake; Percoll gradient 40%/ 80% in HBSS). Lymphoid cell ring on a border of fraction was collected, washed and re-suspended in FACS buffer (1xPBS, 1mM EDTA, 2%FBS). Single cells suspension was subjected then to flow cytometry analysis, cytokine stimulation or cell sorting.
To detect intracellular cytokine expression in tumor infiltrating immune cells, isolated LPL and IEL cells were stimulated in RPMI 1640 High glucose activation media (10% FBS, 1x Glutamax, HEPES 1mM, 1x penicillin/streptavidin, 1x cell culture b-mercaptoe-thanol) for 5.5 hours with PMA 500 ng/mL and ionomycin 100 ng/mL at 37 C. To inhibit protein transport, Monensin and Brefeldin A at concentration 5 mg/mL were added in activation media after first 40 min of stimulation. Cells were collected, stained for surface markers, washed, fixed and permeabilized (BD Cytofix/Cytoperm Kit), stained for intracellular cytokines and analyzed by flow cytom-etry. All flow samples were acquired on a LSRII (BD Biosciences) and analyzed using FlowJo v. 10 (TreeStar). For cell sorting, samples were stained and sorted on BD FACSAria II sorter (BD Biosciences).
Intestinal epithelial cell isolation for protein analysis
Colonic tumors and normal tissues were cut into small pieces followed by mechanical shaking (200 rpm) with 10mM EDTA in Ca2+ Mg2+ free 1x HBSS at 4 C for 15 min; 3 rounds total. After each round of shaking cells were vortexed for 20 s and put through metal strainer; cell suspension was collected and centrifuged at 1600 rpm for 10 min at 4C. Pellets were resuspended in ice cold PBS con-taining inhibitors of proteases and phosphatases and then subjected to protein nuclear extraction.