IFN-γ Mediates the Antitumor Effects of Radiation Therapy in a Murine Colon Tumor

• DOI: 10.1016/j.ajpath.2013.02.041
• Publication Date: 2013-04-17T03:43:47Z
• Authors: Scott A. Gerber; Abigail L. Sedlacek; Kyle R. Cron; Shawn P. Murphy; John G. Frelinger; Edith M. Lord
• Keywords: Cytotoxicity, Immunologic; Mice, Knockout; 0303 health sciences; Adenocarcinoma; CD8-Positive T-Lymphocytes; Pathology and Forensic Medicine; 3. Good health; Mice, Inbred C57BL; Interferon-gamma; Mice; 03 medical and health sciences; Colonic Neoplasms; Animals; Neoplasm Transplantation

Cancer treatments using ionizing radiation (IR) therapy are thought to act primarily through the induction of tumor cell damage at a molecular level. However, new concept has recently emerged, suggesting that immune system is required for effective IR therapy. Our work here identified interferon gamma (IFN-γ) as an essential cytokine efficacy Local (15 Gy) mice bearing Colon38, colon adenocarcinoma, decreases burden in wild-type animals. Interestingly, had no effect on IFNγKO mice. We further determined intratumoral levels IFN-γ increased 2 days following IR, which directly correlated with decrease was not result direct cytotoxic effects cells. T cells from IR-treated tumors exhibited far greater capacity lyse 51Cr release assay, process dependent IFN-γ. CD8+ were predominant producers IFN-γ, demonstrated by intracellular staining and studies reporter Elimination antibody treatment reduced over 90%. 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C57BL/6J B6.129S7-Ifngtm1Ts (IFNγ−/−) purchased Jackson Laboratory (Bar Harbor, ME). Bicistronic [Yeti (yellow-enhanced transcript IFN-γ)] mice19Stetson D.B. Mohrs Reinhardt R.L. Baron Z.E. Gapin Kronenberg Locksley R.M. Constitutive mRNAs mark natural killer (NK) poised rapid function.J Exp 2003; 198: 1069-1076Crossref (509) obtained Dr. Troy Randall (University Alabama Birmingham). All treated accordance guidelines approved University Committee Animal Resources. Colon38,18Corbett Edward Brown maintained MAT/P medium (US patent 4.816.401) supplemented 100 U/mL penicillin, mg/mL streptomycin, 2% fetal calf serum. Colon38/dominant negative (DNR) clones (0.1 0.2) generated stably transfecting parental Colon38 plasmid encoding DNR (pcDNA.mugR) described.10Lugade Mice injected 1 × 105 i.m. left thigh. size measured calipers mean thigh diameter calculated (square root quantity W H, width H height). restrained received 15-Gy irradiation tumor-bearing leg, 7 challenge described.1Lugade sacrificed analysis indicated days. To deplete 200 μg anti-CD8 (53-6.7) diluted balanced salt solution (Sigma-Aldrich, St. Louis, MO) i.p. day before then every 4 until sacrifice. Rat IgG vehicle control. measure i.t. approximately mg tissue removed placed 500 μL Lysis 11 buffer20Gerber Pober J.S. IFN-alpha transcription hypoxia-inducible factor-1alpha inhibit human endothelial cells.J 181: 1052-1062Crossref (47) containing protease inhibitors (BioVision, Milpitas, CA) homogenized ice 30 60 seconds Bio-Gen PRO200 homogenizer (PRO Scientific, Oxford, CT). Samples centrifuged (2300 g) 4°C 8 minutes, supernatant collected tested total protein BCA kit (Pierce Biotechnology, Rockford, IL) enzyme-linked immunosorbent assay (ELISA) (PeproTech, Rocky Hill, NJ) according manufacturer's instructions. concentration normalized protein. Tumors dissociated described.21Blieden T.M. McAdam Mechanism lymphocyte killing low I-expressing 1991; 147: 1433-1438Crossref Briefly, pieces weighed, minced, digested 45 minutes collagenase D (Sigma-Aldrich). Single-cell suspensions counted, blocked Fc Block, stained various antibodies listed Table samples analyzed FACSCanto Flow Cytometer (BD Biosciences, San Jose, FlowJo software version 9.4.11 (Tree Star, Ashland, OR) software. tumor. For staining, single-cell prepared described above (no restimulation) fixed/permeabilized CytoFix/CytoPerm instructions Biosciences). above. Fluorescence minus (FMO) controls. Percentages subsets first gating CD45+ cells.Table 1Antibodies Used CytometryAntibody againstCloneManufacturerCD4L3T4BD BiosciencesCD853-6.7eBioscience (San Diego, CA)CD11cHL3BD BiosciencesCD11bM1/70eBioscienceCD19ID3BD BiosciencesCD25PC61BD BiosciencesCD4530-F11BD BiosciencesCD119GR20BD BiosciencesNK1.1PK136BD BiosciencesF4/80BM8eBioscienceFoxP3 KITCat#.77-5775-40eBioscienceGR-1RB6-8C5eBioscienceIFNγXMB1.2BD BiosciencesMHC (H-2kb)AF6-88.5BD BiosciencesFc Block2.4G2BD Biosciences Open table tab 2Cell Number Intratumoral Subsets per (×106)Tumor CellsTotal CD45+CD8+ cellsTotal CD4+ cellsTregsNK cellsMacrophagesMonocytesDendritic cellsDay 713 ± 211 10.3 0.040.2 0.030.1 0.010.6 0.084.8 0.72.5 0.60.4 0.06Day 9 UT14 20.7 0.20.4 0.090.3 0.030.4 0.085.7 12.9 0.80.4 0.08Day IR8.7 19 0.050.1 0.02∗P < 0.05 one-way variance Bonferroni post hoc test.0.07 0.004∗P test.0.3 0.064.4 0.42.5 0.70.3 0.05Day UT16 113 22.4 0.060.3 0.040.4 0.076.7 0.92.2 0.40.2 0.04Day IR4.7 0.8∗P test.18 42.7 0.060.1 0.04∗P test.0.6 0.16.8 14.3 0.90.4 0.1Cell populations defined follows: CD45−, forward scatterhi; CD45+, CD8+; CD4+; Tregs: CD4+, FoxP3+; NK1.1+; macrophages: F480hi, monocytes: F480intermediate, scatterlow; F480-, CD11b+, CD11c+. Data least six individual group determine means SEM each subset except Tregs, three used. Comparisons made between UT values time points (but across groups).IR, irradiated; UT, unirradiated.∗ P test. Cell groups). unirradiated. Protein 20 μg) homogenate loaded lane subjected SDS-PAGE immunoblotted described.20Gerber Antibodies immunoblots phosphorylated signal transducer activator (pSTAT1) (sc-7988; Santa Cruz Cruz, CA), STAT1 (sc-345; Biotechnology), (IRF1) (sc-640; β-actin Preparations anti–CD8-PE, anti–CD45-PerCP, anti–CD3-FITC. CD3+, sorted FACSAria fluorescence-activated sorter (FACS) Biosciences) immediately lysed Buffer RLT lysis buffer (Qiagen, Valencia, CA). RNA purified RNeasy mini (Qiagen) quantified spectrophotometer. mRNA Tbet, FasL, CTLA4, Prf1 (perforin) real-time quantitative RT-PCR described20Gerber nested primers designed span intron (CTLA-4: 5′-ATTCTGACTTCCTCCTTTGG-3′; 5′-CCTGTTGTAAGAGGACTTCTT-3′. FasL: 5′-CACCAACCAAAGCCTTAAAG-3′; 5′-ATATGTGTCTTCCCATTCC-3′. GAPDH: 5′-CATTGCTCTCAATGACAACT-3′; 5′-GGGTTTCTTACTCCTTGGAG-3′. Perforin: 5′-AAGACCTATCAGGACCAGTA-3′; 5′-CTGTGGAGCTGTTAAAGTTG-3′. Tbet: 5′-GATCATCACTAAGCAAGGAC-3′; 5′-ACATCCACAAACATCCTGTA-3′). GAPDH expressed fold increase examined whole-mount histology described.22Gerber J.A. Fenton B.M. IL-12 alterations tumour blood vessel morphology: whole-tissue mounts.Br Cancer. 88: 1453-1461Crossref (37) 23Gerber Rybalko Bigelow C.E. Lugade Foster Preferential attachment peritoneal omental aggregates possible vascular survival growth.Am Pathol. 2006; 169: 1739-1752Abstract (150) above, tumor-infiltrating lymphocytes magnetically separated beads conjugated anti–Thy-1 (clone T24/40.7). Tumor-infiltrating assayed ability target standard 6-hour assay. Results presented SE replicates. significance paired Student's t-test, followed test when appropriate. previous necessity model.10Lugade examine effects, C57BL/6 (WT) untreated locally γ-irradiation later, monitored time. single high dose because our showed 15 Gy compared 5 3-Gy fractionated regimen.1Lugade Unirradiated WT grew progressively animals around 14 (Figure 1). Radiation did immediate controlling (day 9). after 9, significant seen. Most eventually grew, albeit slower rate unirradiated tumors. both similarly, ineffective present microenvironment, homogenates ELISA. standardized significantly induced 10, 2A ) Figure 2B represents illustrates minimal seen (24 hours therapy). remain elevated out 11. rise level correlates same point These demonstrate mediate assess mechanism radiation-induced Parental transfected vector, rendering unresponsive their inability phosphorylate induce IRF1 1- 3-hour vitro 3A ). Colon38/DNR unable (MHC) (data shown). proved stable vivo subsequent flow cytometry performed ex (as Materials Methods) 13 retained mutant CD119 (IFN-γ receptor) (Supplemental S1A) lower MHC S1B). Colon38/DNR.1 without Both lines similarly 3B). similar fashion acting next focused modulate function, particular, microscopy abundant CD8+, 4, B Surprisingly, apparent difference number groups images 4A 4B). observation verified (described below (Table 2). FACS-sorted four genes associated status (T-bet, Prf1, CTLA4) PCR. Unexpectedly, differences observed enhance function. versus (without culture stimulation) basis Methods. modest lytic activity (∼25% highest effector/target ratio); therapy, 4C). experiment repeated isolated growing grown little Colon38. enhances type(s) responsible producing 7, i