Abstract
The escape of cancer cells from host immunosurveillance involves a shift in immune responses, including an imbalance in Th1 and Th2 cells. A Th1-dominated immune response predicts positive outcomes in colorectal cancer. The E3 ubiquitin ligase, Asb2α, is expressed in Th2 cells, but its roles in T-cell maturation and cancer are unclear. We provide evidence that the Th2 master regulator, Gata3, induces Asb2. Loss of Asb2 did not affect Th differentiation ex vivo, but reduced IL4 production from Th2 cells. We found that high ASB2 expression was associated with poor outcome in colorectal cancer. Loss of Asb2 from hematopoietic cells promoted a Th1 response and attenuated colitis-associated tumorigenesis in mice. Diminished Th2 function correlated with increased IFNγ production and an enhanced type 1 antitumor immune response in Asb2-deficient mice. Our work suggests that Asb2α promotes a Th2 phenotype in vivo, which in turn is associated with tumor progression in a mouse model of colitis.
Introduction
The key roles played by the tumor microenvironment, which is mainly characterized by the various stromal and immune cell subsets and the cytokines they produce (1), in the development, evolution, and outcome of cancers is increasingly appreciated. The quality of the immune response is a prognostic factor for patients with cancer (2–4). This is particularly true for patients with colorectal cancer (5). Colorectal cancer, which includes hereditary, sporadic, and colitis-associated colorectal cancer is a leading cause of cancer and cancer-related deaths (6). Colitis-associated colorectal cancer represents about 2% of colorectal cancer cases and develop in patients with a history of inflammatory bowel disease, including ulcerative colitis and Crohn's disease (7). The risk of developing colitis-associated colorectal cancer is approaching 20% in patients with prolonged and extensive colitis (8). In addition to somatic and epigenetic abnormalities, the inflammatory microenvironment surrounding intestinal epithelial cells plays a pivotal role in carcinogenesis. Indeed, inflammation-related epithelial cell injury and repair contribute to the progression of colitis-associated colorectal cancer (9–11). Therefore, deciphering the mechanisms linking inflammation and colorectal cancer may facilitate the development of new therapeutic approaches.
Studies have highlighted the role played by CD4+ Th cells in the generation and control of cancer-related inflammation (12–14). CD4+ T cells support cytotoxic CD8+ T-cell (CTL) responses (15), but may also exert tumor suppressive or promoting effects, depending on the cytokines they produce (16). CD4+ T cells include IFNγ-producing Th1 cells, IL4, IL5-, and IL13-producing Th2 cells, IL17-producing Th17 cells, and IL10- and TGFβ-producing T regulatory (Treg) cells. An immune response dominated by Th1 cells is associated with a positive outcome in patients with colorectal cancer, although the role of Th17 and Treg cells in colorectal cancer pathogenesis remains controversial (9, 17–19). The contribution of Th2 cells to colorectal cancer development remains poorly understood.
In adults, the ASB2 gene encodes two isoforms, the hematopoietic-type ASB2α and the muscle-type ASB2β (20, 21). ASB2α is the specificity subunit of a Cullin 5-RING E3 ubiquitin ligase that triggers ubiquitylation and degradation by the proteasome of the actin-binding protein filamins A and B (22–25). Although ASB2α transcript and protein were initially identified as induced in differentiating human myeloid leukemia cells (20), they are also expressed in nonmalignant immune cells including dendritic cells (DC; refs. 23, 24) and CD4+ T lymphocytes (26, 27).
In this study, we asked whether ASB2α is involved in the immune response to colorectal cancer and investigated the impact of ASB2α deficiency in tumor development using a pathologically relevant mouse model of colorectal cancer.
Materials and Methods
Human dataset and relapse-free survival analysis
We used published microarray and clinical outcome data deposited in the Gene Expression Omnibus (GSE39582; ref. 28) to perform correlation and survival analyses. Tumor samples were split into ASB2-high, -intermediate, and -low expressers based on gene expression value using the R software. Relapse-free survival of patients was analyzed according to the Kaplan–Meier method and differences between relapse-free survival distributions were assessed with the log-rank test.
Mice
All mice were specific pathogen free. Mice studies were handled according to the Centre National de la Recherche Scientifique ethical guidelines and were approved by the Comité d’éthique de la Fédération de Recherche en Biologie de Toulouse (C2EA-01). Polyinosinic-polycytidylic acid [poly(I·C); Sigma-Aldrich; 300 μg] was injected intraperitoneally three times at 2-day intervals to 5-week-old Mx1-Cre;Asb2fl/fl (23) and Mx1-Cre control mice. Female mice were injected intraperitoneally 2 weeks after the last injection of poly(I·C) with a single 10 mg/kg dose of azoxymethane (AOM; Sigma-Aldrich). Dextran sodium sulfate (DSS; MP Biochemicals; molecular weight 36,000–50,000 kDa) was dissolved in drinking water, filtered, and administered at 2% to mice for 8 days at 9, 13, and 16 weeks of age. Naïve groups that were not exposed to AOM/DSS were also included as noncancer controls. Mice were euthanized and analyzed 43 or 69 days after AOM injection. Bone marrow chimeras were generated by lethally irradiating CD45.1 C57BL/6 recipient mice (9 Gy) using a γ-irradiation system (Biobeam 8000). Irradiated mice were reconstituted by retro-orbital injection of 5 × 106 bone marrow cells of Mx1-Cre;Asb2fl/fl or Mx1-Cre control mice previously flushed from femurs, subjected to red blood cell lysis, and filtered through a 30-μm filter. Chimeric mice were kept on antibiotic-containing water (enrofloxacin) for 2 weeks. Eight weeks after transplantation, mice were injected with poly(I·C) and subjected to the AOM/DSS protocol as described above. For in vivo Th2 cell transfer, naïve T cells from CD45.1 C57BL/6 mice were differentiated for 6 days in Th2-polarizing conditions and then were transferred into AOM/DSS-treated cKO mice (2 × 106 Th2 cells for the first retro-orbital injection, 1 × 106 Th2 cells for the others). The reconstitution of bone marrow and the presence of Th2 cells in the colon after Th2 cell transfer were confirmed by flow cytometry analysis using anti-CD45.1 and anti-CD45.2. For antibody neutralization, mice were injected intraperitoneally with 75 μg anti-IFNγ neutralizing antibodies or isotype-matched control IgG1κ (Ultra-LEAF purified antibodies, BioLegend) every 4 days after the second DSS cycle. Colons were dissected and measured. Colonic contents were removed and colons were cleaned with PBS. Tumors were counted and their area was measured using the AxioVision Software (Zeiss). A portion of the colon was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned with a Leica RM2245 microtome for alcian blue and nuclear fast red staining to examine tumor morphology. For the analysis of peripheral blood parameters, blood was collected retro-orbitally and analyzed with an ABX Micros 60 (Horiba) hematology analyzer.
Micro-array and chromatin immunoprecipitation sequencing analysis
Micro-array ASB2 expression data and chromatin immunoprecipitation sequencing (ChIP-seq) data showing global histone H3 lysine 4 (H3K4me3) and lysine 27 trimethylation (H3K27me3) histone modification status in the Asb2 locus in naïve CD4+ T cells or cells cultured under Th1, Th2, Treg, and Th17 conditions were retrieved from the publicly available GSE14308 dataset (29). The patterns of GATA3 and Fli1 binding, as well as H3K4me1, H3K4me2, H3K4me3, and H3K27me3 modifications at the Asb2 gene locus were retrieved from the GSE20898 (30).
Isolation of hematopoietic cells from organs
Isolation of bone marrow cells and splenocytes was performed as described previously (24). For isolation of immune cells from colonic mucosa, colon tissues were minced in 1 to 3-mm pieces and incubated at 37°C in RPMI medium containing 5 mmol/L EDTA, 1% penicillin/streptomycin, and 3% FCS (Biowest) for 20 minutes and then agitated three times for 30 seconds in RPMI, 2 mmol/L EDTA, and 1% penicillin/streptomycin. Colon pieces were then washed in PBS, cut in smaller pieces, and digested with collagenase D (0.5 mg/mL, Roche) and DNase I (0.1 mg/mL, Roche) in RPMI for 60 minutes at 37°C. Single-cell suspensions were generated from digested tissues after filtration through a 40-μm strainer.
Antibodies
Antibodies to CD34 (RAM34), FoxP3 (FJK16S), RORγt (AFKJS-9), and IL13 (eBio13A) were from eBiosciences. All other antibodies were from BioLegend: CD3ϵ (145-2C11), CD4 (RM4-5), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), CD16/CD32 (93), CD19 (6D5), CD28 (37.51), CD41 (MWReg30), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), CD45R (RA3-6B2), CD103 (2E7), CD117 (2B8), CD135 (A2F10), F4/80 (BM8), GATA-3 (16E10A23), Gr-1 (RB6-8C5), I-A/I-E (M5/114), IFNγ (XMG1.2), IL4 (11B11), IL10 (JES5-16E3), IL17A (TC11-18H10.1), Ly-6C (HK1.4), Ly-6G (1A8), NK1.1 (PK136), Sca-1 (D7), Tbet (4B10), and TCRγδ (H57-597). Isotype-matched antibodies were used as controls.
Flow cytometry
For multicolor flow cytometry analysis and cell sorting, cells were first incubated with the Zombi-yellow viability dye (BioLegend) for 15 minutes at room temperature and then incubated for 30 minutes a 4°C with the appropriate combination of antibodies. For hematopoietic stem and progenitor cell analyses, bone marrow cells and splenocytes were stained with biotinylated lineage antibodies and enriched for Lin− cells using Anti-Biotin Microbeads (Miltenyi Biotec) according to the manufacturer's protocol, followed by staining with c-Kit (CD117), Sca-1, CD34, CD135, and CD16/CD32 antibodies for LSK cells (Lin−Sca-1+c-Kit+) including LT-HSCs (LSK CD34−CD135−), ST-HSCs (LSK CD34+CD135−), MPPs (LSK CD34+CD135+), and for myeloid progenitors (GMPs: Lin−Sca-1−c-Kit+CD16/CD32+CD34+ and MEPs: Lin−Sca-1−c-Kit+CD16/CD32−CD34−). For the analysis of Th1, Th2, Th9, Th17, and Treg, cells were isolated from colonic immunocytes. IFNγ, IL4, IL10, IL17A, IL13, and FoxP3 were measured by intracellular staining on CD4+ cells after stimulation for 5 hours with phorbol 12-myristate 13-acetate (50 ng/mL, Sigma-Aldrich) and ionomycin (500 ng/mL, Sigma-Aldrich). Cells were incubated with Monensin (BioLegend) during the last 4 hours of stimulation. Flow cytometry analysis was performed with a LSRII Cytometer (BD Biosciences) and cell sorting on a FACS ARIA II Cytometer (BD Biosciences). Analyses of flow cytometry data were performed using FlowJo (TreeStar).
In vitro differentiation of Th1, Th2, Treg, and Th17 cells
Mouse naïve CD4+ cells were isolated from spleen and lymph node immunocytes of male mice using a CD4+ T-cell Isolation Kit (BioLegend) and stimulated for 3 days with plate-bound anti-CD3ϵ (5 μg/mL) and were differentiated in the presence of polarizing cytokines and antibody cocktails (Th1: 10 ng/mL IL2, 10 ng/mL IL12, 2 μg/mL anti-CD28, and 10 μg/mL anti-IL4; Th2: 10 ng/mL IL2, 40 ng/mL IL4, 1 μg/mL anti-CD28, and 10 μg/mL anti-IFNγ; Treg: 10 ng/mL IL2, 3 ng/mL TGFβ, and 2 μg/mL anti-CD28; and Th17: 2 ng/mL TGFβ, 20 ng/mL IL6, 10 ng/mL IL23, 10 ng/mL IL1β, 1 μg/mL anti-CD28, 6 μg/mL anti-IFNγ, and 10 μg/mL anti-IL4) in RPMI containing 10% FCS, 1% glutamine, 0.1% β-mercaptoethanol, and 1% penicillin/streptomycin in 24-well plates. Cells were then split across four noncoated plates and cultured for 3 additional days in fresh differentiating medium without antibodies (Th1: 20 ng/mL IL2 and 10 ng/mL IL12; Th2: 20 ng/mL IL2 and 40 ng/mL IL4; Treg: 20 ng/mL IL2 and 3 ng/mL TGFβ1; and Th17: 10 ng/mL IL2, 2 ng/mL TGFβ1, 20 ng/mL IL6, 10 ng/mL IL23, and 10 ng/mL IL1β). Recombinant mouse cytokines were from Miltenyi Biotec (IL2, IL4, IL6, IL1β, and IL23) and PeproTech (IL12). TGFβ was from Active Bioscience.
qRT-PCR
RNA isolation, cDNA synthesis, and real-time PCR with the Power SYBR Green mix were carried out as described previously (23). Gene expression was determined using the ΔΔCt method and data are presented as relative amounts of mRNA normalized to Rplp0 (ribosomal protein, large, P0). The following primers were used: for Rplp0: 5′-CGCGTCCTGGCATTGTCTG-3′ (forward) and 5′- GGCCTTGACCTTTTCAGTAAGT-3′ (reverse); for Asb2α: 5′-CACTCTGGCTCTGCACCTTC-3′ (forward) and 5′-GGGCTCTGCAAGATTCTTCC-3′ (reverse); for Gata3: 5′-CTCGGCCATTCGTACATGGAA-3′ (forward) and 5′-GGATACCTCTGCACCGTAGC-3′ (reverse); for Granzyme B: 5′-CTCTCGAATAAGGAAGCCCC-3′ (forward) and 5′-CTGACCTTGTCTCTGGCCTC-3′ (reverse); for Ifn-γ: 5′-CAGCAACAGCAAGGCGAA-3′ (forward) and 5′-GGACCTGTGGGTTGTTGACCT-3′ (reverse); for Perforin 1: 5′-TAGCCAATTTTGCAGCTGAG-3′ (forward) and 5′-TGGAGGTTTTTGTACCAGGC-3′ (reverse); and for Tbet/Tbx21: 5′-CAACAACCCCTTTGCCAAAG-3′ (forward) and 5′-TCCCCCAAGCAGTTGACAGT-3′ (reverse).
Statistical analysis
All experiments were repeated as indicated. n indicates the numbers of independent biological repeats. All P values were calculated using the nonparametric Mann–Whitney t test using the GraphPad Prism software, unless otherwise indicated. A P < 0.05 was considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Results
Asb2α is expressed in Th2 cells
To determine whether Asb2α contributes to T-cell maturation, we evaluated its expression upon differentiation of wild-type naïve CD4+ T cells in vitro. By qRT-qPCR with primers specific to the Asb2α isoform, we showed that Asb2α transcripts were more expressed in Th2 cells than in naïve, Th1, Treg, or Th17 cells (Fig. 1A), in agreement with Affymetrix gene chip data (Fig. 1B). We therefore searched for the molecular mechanisms that may control Asb2 expression in Th2 cells. Global mapping of H3K4me3 and H3K27me3 in naïve and CD4+ effector T cells indicated that H3K4me3-active marks were associated with Asb2 in Th2 cells (Fig. 1C). A genome-wide ChIP-seq analysis of Th2 master regulator Gata3 in naïve and CD4+ T cells (30) revealed Gata3 binding within the Asb2 locus in Th2 cells (Fig. 1D). The expression of Asb2 transcripts in Th2 cells was reduced following Gata3 deletion (ratio of Asb2 mRNA in wild-type to Gata3-deleted Th2 cells = 2.24 ± 0.12; ref. 30). In addition, the genomic regions surrounding Gata3 binding sites in Asb2 were associated with H3K4me1-, H3K4me2-, and H3K4me3-active marks, but the H3K27me3 repressive mark was not evident in Th2 cells (Fig. 1D). Bioinformatics analysis of the Asb2 locus further indicated that the three Gata3 binding sites (Fig. 1E) are surrounded by motifs for the Ets (AGGAAG), Runx (ACCACA), and AP1 (TGACTCA) families of transcription factors known to be involved in T-cell differentiation and function. As shown in Fig. 1D, the Ets family member, Fli1, colocalizes with Gata3 in the Asb2 locus as previously shown for the IL4/IL13 locus (30). Taken together, these results suggest that Gata3 positively modulates Asb2 expression by regulating histone methylation in Th2 cells.
Asb2α expression was positively regulated by Gata3 in Th2 cells. A, Relative expression of Asb2α mRNA in naïve CD4+ T cells, Th1, Th2, Treg, and Th17 cells of wild-type mice assessed by qRT-PCR. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. B, Asb2 expression detected using Affymetrix gene chip in naïve CD4+ T cells, Th1, Th2, Treg, and Th17 cells are plotted from the GSE14308 (29). C, Total island tag counts of lineage-specific H3K4me3 and H3K27me3 in the Asb2 locus in naïve CD4+ T cells, Th1, Th2, Treg, and Th17 cells are plotted from the GSE14308 (29). D, Genome browser image showing Gata3 binding pattern at the genomic region containing Prima1, Fam181a, Asb2, Otub2, Ddx24, and Ifi27 genes in Th2 cells. Genome browser images showing the patterns of Gata3 and Fli1 binding, as well as H3K4me1, H3K4me2, H3K4me3, and H3K27me3 modifications at the Asb2 gene in Th2 cells. Peaks identified with P ≤ 10−13 are plotted from the GSE20898 (30) using the Integrative Genome Viewer. E, Sequences of the Gata3 and Ets binding sites in the Asb2 locus are aligned to their respective top motifs in Th2 cells (30). **, P < 0.01; ***, P < 0.001.
Asb2α expression was positively regulated by Gata3 in Th2 cells. A, Relative expression of Asb2α mRNA in naïve CD4+ T cells, Th1, Th2, Treg, and Th17 cells of wild-type mice assessed by qRT-PCR. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. B, Asb2 expression detected using Affymetrix gene chip in naïve CD4+ T cells, Th1, Th2, Treg, and Th17 cells are plotted from the GSE14308 (29). C, Total island tag counts of lineage-specific H3K4me3 and H3K27me3 in the Asb2 locus in naïve CD4+ T cells, Th1, Th2, Treg, and Th17 cells are plotted from the GSE14308 (29). D, Genome browser image showing Gata3 binding pattern at the genomic region containing Prima1, Fam181a, Asb2, Otub2, Ddx24, and Ifi27 genes in Th2 cells. Genome browser images showing the patterns of Gata3 and Fli1 binding, as well as H3K4me1, H3K4me2, H3K4me3, and H3K27me3 modifications at the Asb2 gene in Th2 cells. Peaks identified with P ≤ 10−13 are plotted from the GSE20898 (30) using the Integrative Genome Viewer. E, Sequences of the Gata3 and Ets binding sites in the Asb2 locus are aligned to their respective top motifs in Th2 cells (30). **, P < 0.01; ***, P < 0.001.
High ASB2 expression in human colorectal cancer is associated with shorter relapse-free survival
To further investigate the Th1/Th2 balance in colorectal cancer and uncover a role for ASB2, we analyzed ASB2 expression in fresh frozen primary tumor samples containing tumor, stromal, and infiltrating cells, from a large multicenter cohort with stage I–IV colorectal cancer (28). Transcript amounts for ASB2 and for a marker indicative of total leukocytes (CD45/PTPRC) were positively correlated (Fig. 2A). mRNA expression of master Th2 transcription factors, GATA3, c-MAF, and IRF4, showed positive correlation with those of ASB2 in samples from human colorectal cancer (Fig. 2A). We found that ASB2 expression was higher in the C4 and C6 subtypes, which are associated with shorter relapse-free survival (28), than in the C1, C2, C3, and C5 subtypes (Fig. 2B). When patients were categorized according to ASB2 expression, high ASB2 expression was associated with shorter relapse-free survival (Fig. 2C). Accordingly, patients of the C4 and C6 subtypes mainly clustered in the ASB2high and ASB2int groups (Fig. 2D). These results reveal that low expression of ASB2 in colorectal cancer biopsies is associated with a positive outcome for patients.
ASB2 expression in human colorectal cancer biopsies correlated with shorter relapse-free survival. Microarray expression data (GSE39582) from human colorectal cancer biopsies containing tumor cells, stroma, and infiltrate (28) were analyzed for expression of ASB2, CD45/PTPRC, c-MAF, GATA3, and IRF4. A, Scatter plots showing correlation data for ASB2 and CD45/PTPRC, ASB2 and GATA3, ASB2 and c-MAF, and ASB2 and IRF4 transcripts in human colorectal cancer biopsies (n = 250). Linear regression-fit curves are shown as red lines. Correlations between nonparametric variables were evaluated using Spearman rank correlation test (r). B, ASB2 expression in the C1 (n = 55), C2 (n = 55), C3 (n = 27), C4 (n = 26), C5 (n = 61), and C6 (n = 26) subtypes of colorectal cancers as defined by Marisa and colleagues (28). Boxplots (with lower quartile, median, and upper quartile, Tukey whiskers) are shown. C, Kaplan–Meier curves showing relapse-free survival rates in patients with high (ASB2high; n = 8), intermediate (ASB2int; n = 138), and low (ASB2low; n = 104) ASB2 expression. Relapse-free survival curves were assessed for statistically significant differences using the Log-rank (Mantel–Cox) test. D, Percentages of the C1–C6 subtypes in the ASB2high, ASB2int, and ASB2low groups. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
ASB2 expression in human colorectal cancer biopsies correlated with shorter relapse-free survival. Microarray expression data (GSE39582) from human colorectal cancer biopsies containing tumor cells, stroma, and infiltrate (28) were analyzed for expression of ASB2, CD45/PTPRC, c-MAF, GATA3, and IRF4. A, Scatter plots showing correlation data for ASB2 and CD45/PTPRC, ASB2 and GATA3, ASB2 and c-MAF, and ASB2 and IRF4 transcripts in human colorectal cancer biopsies (n = 250). Linear regression-fit curves are shown as red lines. Correlations between nonparametric variables were evaluated using Spearman rank correlation test (r). B, ASB2 expression in the C1 (n = 55), C2 (n = 55), C3 (n = 27), C4 (n = 26), C5 (n = 61), and C6 (n = 26) subtypes of colorectal cancers as defined by Marisa and colleagues (28). Boxplots (with lower quartile, median, and upper quartile, Tukey whiskers) are shown. C, Kaplan–Meier curves showing relapse-free survival rates in patients with high (ASB2high; n = 8), intermediate (ASB2int; n = 138), and low (ASB2low; n = 104) ASB2 expression. Relapse-free survival curves were assessed for statistically significant differences using the Log-rank (Mantel–Cox) test. D, Percentages of the C1–C6 subtypes in the ASB2high, ASB2int, and ASB2low groups. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Loss of Asb2 in hematopoietic cells inhibits colitis-associated colorectal cancer in mice
To elucidate a potential functional relationship between ASB2 expression and colorectal cancer, we used the Mx1-Cre;Asb2fl/fl–mutant mouse model (23). In this model, Asb2 is conditionally inactivated upon treatment with poly(I·C) (hereafter referred to as cKO). Two weeks after the last injection of poly(I·C), we subjected these mutant mice and their Mx1-Cre control counterparts (hereafter referred to as ctrl) to a model of colitis-associated tumorigenesis by administration of a single dose of the carcinogen AOM followed by repeated cycles of DSS in the drinking water (Fig. 3A). This treatment induces inflammation in the large intestine and tumor development in the distal part of the colon (Fig. 3B and C). As expected, expression of Asb2α was decreased in spleen cells of tumor-bearing cKO mice compared with tumor-bearing ctrl mice (Fig. 3D). Furthermore, Asb2α transcripts were mainly detected in CD45+ cells isolated from colons of AOM/DSS-treated mice (Fig. 3E). Deletion of Asb2 attenuated the shortening of the colon and the tumor load 69 days after AOM injection (Fig. 3B, C, F, and G), and tumors were less numerous and smaller in cKO mice compared with in ctrl mice (Fig. 3B, C, and H). However, colon length, tumor load, number, and size of the tumors were similar in cKO and ctrl mice 43 days after AOM injection (Fig. 3I–K), suggesting an impact of Asb2 deletion on tumor progression rather than tumor initiation.
Deletion of Asb2 attenuated colitis-associated tumor formation and development. Control (ctrl) and cKO mice were injected with AOM, subsequently treated with the indicated cycles of DSS, and analyzed 69 (B–H) or 43 (I–K) days after AOM injection. A, Schematic representation of AOM/DSS-induced colorectal cancer treatment. B, Representative image of colons. Scale bar = 0.5 cm. C, Representative colon sections stained with alcian blue and nuclear fast red. Scale bar = 200 μm. D, Expression of Asb2α transcripts in spleen cells. E, Expression of Asb2α transcripts in sorted CD45+ and CD45− cells isolated from colonic tissues of AOM/DSS-treated mice. F, Colon lengths after treatment. G, Tumor loads in the colon. H, Enumeration of total tumor burden and of tumors following classification according to their size. I, Colon lengths after treatment. J, Tumor loads in the colon. K, Enumeration of total tumor burden and of tumors following classification according to their size. L, Schematic representation of AOM/DSS-induced colorectal cancer treatment after bone marrow reconstitution of C57BL/6 CD45.1–irradiated mice with bone marrow cells from Mx1-Cre;Asb2fl/fl or Mx1-Cre mice. Representative colon sections stained with alcian blue and nuclear fast red and tumor loads are shown. Scale bar = 200 μm. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM (sample size, Ctrl = 9 and cKO = 7 for D; Ctrl = 4 and cKO = 4 for E; Ctrl = 25 and cKO = 20 for F–H; Ctrl = 10 and cKO = 8 for I–K; and Mx1-Cre = 5 and Mx1-Cre;Asb2fl/fl = 4 for L). *, P < 0.05; **, P < 0.01.
Deletion of Asb2 attenuated colitis-associated tumor formation and development. Control (ctrl) and cKO mice were injected with AOM, subsequently treated with the indicated cycles of DSS, and analyzed 69 (B–H) or 43 (I–K) days after AOM injection. A, Schematic representation of AOM/DSS-induced colorectal cancer treatment. B, Representative image of colons. Scale bar = 0.5 cm. C, Representative colon sections stained with alcian blue and nuclear fast red. Scale bar = 200 μm. D, Expression of Asb2α transcripts in spleen cells. E, Expression of Asb2α transcripts in sorted CD45+ and CD45− cells isolated from colonic tissues of AOM/DSS-treated mice. F, Colon lengths after treatment. G, Tumor loads in the colon. H, Enumeration of total tumor burden and of tumors following classification according to their size. I, Colon lengths after treatment. J, Tumor loads in the colon. K, Enumeration of total tumor burden and of tumors following classification according to their size. L, Schematic representation of AOM/DSS-induced colorectal cancer treatment after bone marrow reconstitution of C57BL/6 CD45.1–irradiated mice with bone marrow cells from Mx1-Cre;Asb2fl/fl or Mx1-Cre mice. Representative colon sections stained with alcian blue and nuclear fast red and tumor loads are shown. Scale bar = 200 μm. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM (sample size, Ctrl = 9 and cKO = 7 for D; Ctrl = 4 and cKO = 4 for E; Ctrl = 25 and cKO = 20 for F–H; Ctrl = 10 and cKO = 8 for I–K; and Mx1-Cre = 5 and Mx1-Cre;Asb2fl/fl = 4 for L). *, P < 0.05; **, P < 0.01.
To evaluate whether these phenotypes are due to loss of Asb2 in hematopoietic cells, we generated mouse bone marrow chimeras. We irradiated C57Bl/6 mice, reconstituted their bone marrow with cells from Mx1-Cre;Asb2fl/fl or Mx1-Cre animals, and treated them with poly(I·C) to generate hematopoietic cKO and ctrl mice (Fig. 3L). After AOM/DSS treatment, the hematopoietic-cKO mice displayed reduced colon tumor development compared with ctrl mice (2.6-fold reduction; Fig. 3L). Taken together, our results indicate that loss of Asb2α in hematopoietic cells reduced tumor burden in colitis-associated tumorigenesis in mice. These functional findings are consistent with our observation that low expression of ASB2 in colorectal cancer biopsies correlates with a better relapse-free survival of patients.
Myeloid-biased differentiation of hematopoietic stem cells triggered by tumorigenesis
Similar to the human colorectal cancer disease (31, 32), the numbers of monocytes, granulocytes, and platelets in the blood of AOM/DSS-treated mice were increased, whereas the numbers of red blood cells were decreased, but there were no differences between cKO and ctrl mice (Fig. 4A). In contrast, the lymphocyte blood counts were similar in untreated and AOM/DSS-treated cKO or ctrl mice (Fig. 4A). The increased numbers of monocytes and granulocytes in the blood of tumor-bearing cKO and ctrl mice are likely due to an emergency myelopoiesis in the bone marrow (Fig. 4B) and an extramedullar myelopoiesis in the spleen (Fig. 4C). Increases in the LSK cell compartment [including long-term hematopoietic stem cells (HSC), short-term HSCs, and multipotent progenitors], in the granulocyte/macrophage progenitors, in monocytes, and in immature myeloid cells (CD11b+Ly6G+Ly6C− and CD11b+Ly6GlowLy6Chigh) were observed in the bone marrow of tumor-bearing mice (Fig. 4B). Expansion of myeloid cells also occurred in the spleen of AOM/DSS-treated mice (Fig. 4C). In contrast, the numbers of B and T cells in the spleen of cKO and ctrl mice were not affected by tumor development (Fig. 4C). A skewing of HSC differentiation toward the myeloid lineage has been described in colitis (33). We show evidence for myeloid-biased differentiation of HSCs in colitis-associated tumorigenesis in mice. However, no differences in the numbers of hematopoietic stem progenitor cells between cKO and control mice were observed (Fig. 4), indicating that the activation of the most immature HSCs and their myeloid differentiation were similar and could not explain the reduced tumor development observed in cKO mice.
Emergency myelopoiesis in the bone marrow and the spleen of tumor-bearing mice. Control (ctrl) and cKO mice were left untreated (−) or treated with AOM and DSS (A/D). Cells were isolated from peripheral blood (A), bone marrow (B), and colon (C) 43 (d43) or 69 days (d69) after AOM injection and analyzed by flow cytometry. A, Peripheral blood parameters including numbers of lymphocytes, monocytes, granulocytes, platelets, and red blood cells. B, Cellularity of the bone marrow and numbers of the indicated bone marrow populations (see Materials and Methods or see below for definitions). C, Cellularity of the spleen and numbers of the indicated spleen populations (see Materials and Methods or see below for definitions). The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. In B and C, monocytes/macrophages are CD45+F4/80+CD11b+, dendritic cells are CD45+CD11c+, B cells are CD45+CD19+CD45R+, and T cells are CD45+CD3+. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Emergency myelopoiesis in the bone marrow and the spleen of tumor-bearing mice. Control (ctrl) and cKO mice were left untreated (−) or treated with AOM and DSS (A/D). Cells were isolated from peripheral blood (A), bone marrow (B), and colon (C) 43 (d43) or 69 days (d69) after AOM injection and analyzed by flow cytometry. A, Peripheral blood parameters including numbers of lymphocytes, monocytes, granulocytes, platelets, and red blood cells. B, Cellularity of the bone marrow and numbers of the indicated bone marrow populations (see Materials and Methods or see below for definitions). C, Cellularity of the spleen and numbers of the indicated spleen populations (see Materials and Methods or see below for definitions). The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. In B and C, monocytes/macrophages are CD45+F4/80+CD11b+, dendritic cells are CD45+CD11c+, B cells are CD45+CD19+CD45R+, and T cells are CD45+CD3+. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Mice lacking Asb2 had reduced Th2 and Treg and enhanced Th1, Th17, and CTL responses
We next examined whether Asb2α controlled immune cell infiltration by analyzing immunocyte profiles in the colon of mice treated with AOM/DSS. More CD4+ T cells accumulated in the colonic mucosa of cKO mice than in their ctrl counterparts 43 days after AOM injection, when both genotypes bear similar tumor numbers (Fig. 5A). In contrast, no difference was found between ctrl and cKO mice in the infiltration by DCs, macrophages, and B cells into the colonic mucosa of tumor-bearing mice, or in the numbers of DCs in the draining lymph nodes (Supplementary Fig. S1). Analysis of the differentiation profile of lymphocytes infiltrating the colonic mucosa of cKO versus control tumor-bearing mice revealed altered differentiation programs. First, we observed a decrease in the number of Th2 (CD4+IL4+ or CD4+IL13+; Fig. 5B) and Treg (CD4+FoxP3+IL10+; Fig. 5C) cells in the colonic mucosa of tumor-bearing cKO mice compared with ctrl mice. In contrast, the numbers of Th1 (CD4+IFNγ+; Fig. 5D) and Th17 cells (CD4+IL17A+; Fig. 5E) in the colonic mucosa of tumor-bearing cKO mice were increased. The numbers of γδ T and IFNγ+ γδ T cells, and natural killer (NK) and IFNγ+ NK cells in the colon of tumor-bearing mice did not significantly increase in cKO mice (Supplementary Fig. S2).
Decreased Th2/Treg and increased Th1/Th17/CTL responses in cKO mice trigger antitumor immunity to inhibit colitis-associated tumorigenesis. Control (ctrl) and cKO mice were treated with AOM and DSS and the cells isolated from colons 43 (d43) or 69 days (d69) after AOM injection were analyzed by flow cytometry and qRT-PCR. A, Data represents the numbers of CD45+CD4+ cells. B, Data represents the numbers of CD45+CD4+IL4+ and CD45+CD4+IL13+ cells. C, Data represents the numbers of IL10+ Treg cells (CD45+CD4+FoxP3+IL10+). D, Representative flow cytometry plots for CD4 versus IFNγ within a CD45+CD4+ gate and numbers of CD45+CD4+IFNγ+ cells. Values inside the plots represent the percentages from the CD45+CD4+ gate. E, Data represents the numbers of Th17 cells (CD45+CD4+IL17A+). F, Data represents the numbers of CD45+CD8+ cells. G, Representative flow cytometry plots for CD8 versus IFNγ within a CD45+CD8+ gate and numbers of CD45+CD8+IFNγ+ cells. Values inside the plots represent the percentages from the CD45+CD8+ gate. H, Relative expression of perforin 1 and granzyme B mRNA. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. *, P < 0.05; **, P < 0.01.
Decreased Th2/Treg and increased Th1/Th17/CTL responses in cKO mice trigger antitumor immunity to inhibit colitis-associated tumorigenesis. Control (ctrl) and cKO mice were treated with AOM and DSS and the cells isolated from colons 43 (d43) or 69 days (d69) after AOM injection were analyzed by flow cytometry and qRT-PCR. A, Data represents the numbers of CD45+CD4+ cells. B, Data represents the numbers of CD45+CD4+IL4+ and CD45+CD4+IL13+ cells. C, Data represents the numbers of IL10+ Treg cells (CD45+CD4+FoxP3+IL10+). D, Representative flow cytometry plots for CD4 versus IFNγ within a CD45+CD4+ gate and numbers of CD45+CD4+IFNγ+ cells. Values inside the plots represent the percentages from the CD45+CD4+ gate. E, Data represents the numbers of Th17 cells (CD45+CD4+IL17A+). F, Data represents the numbers of CD45+CD8+ cells. G, Representative flow cytometry plots for CD8 versus IFNγ within a CD45+CD8+ gate and numbers of CD45+CD8+IFNγ+ cells. Values inside the plots represent the percentages from the CD45+CD8+ gate. H, Relative expression of perforin 1 and granzyme B mRNA. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. *, P < 0.05; **, P < 0.01.
More CD8+ T cells tend to accumulate in the colonic mucosa of cKO mice than in their ctrl counterparts 43 days after AOM injection (Fig. 5F). These CD8+ T cells from tumors of cKO mice produced more IFNγ (Fig. 5G) and had increased transcription of the cytotoxicity-related marker perforin 1 and granzyme B in the colonic mucosa (Fig. 5H), when compared with tumor-associated CD8+ T cells from ctrl mice. From these data, we infer that Asb2α controls the balance between Th1/Th17/CTL and Th2/Treg, promoting the Th2/Treg response. The enhanced Th1/Th17/CTL response in cKO mice likely protects against tumor progression.
Deletion of Asb2 impedes Th2 response of mouse CD4+ T cells
To investigate how Asb2α loss altered CD4+ T-cell polarization, we first investigated the impact of Asb2α loss on the ex vivo generation of Th2 cells from naïve CD4+ cells. As shown in Fig. 6A, deletion of Asb2 had no impact on the expression of the master regulator Gata3 mRNA in Th2 cells and had no impact on the expression of the master regulators Tbet/Tbx21 mRNA in Th1 cells after 6 days of differentiation. This was confirmed for protein content by flow cytometry (Fig. 6B and C). However, the percentage of IL4+ cells and the geometric mean fluorescence intensity (geoMFI) for IL4 in CD4+ cells were lower in Th2 cells generated from cKO mice than from ctrl mice (Fig. 6D). In contrast, Asb2 deletion had no impact on the percentage of IFNγ+ cells in CD4+ cells generated in Th1 conditions (Fig. 6B) and no impact on the ex vivo generation of Th17 and Treg cells from naïve CD4+ cells (Fig. 6E and F). Furthermore, the frequencies of CD4+IL17A+ Th17 cells and CD4+FoxP3+IL10+ Treg cells in the spleen, mesenteric lymph nodes, and colons of untreated control and cKO mice were similar (Supplementary Fig. S3). Altogether, our results indicate that the stronger Th1/Th17/CTL response in AOM/DSS-treated cKO mice is secondary to diminished functions of Th2 cells, consistent with their cross-regulation.
Asb2α was expressed in Th2 cells and its loss impeded IL4 expression in Th2 cells. Naïve CD4+ T cells from ctrl and cKO mice were polarized ex vivo toward Th1 or Th2 and analyzed by flow cytometry and qRT-PCR. A, Relative expression of Gata3 and Tbet/Tbx21 mRNA. B, Data represent the percentages of Tbet+ and IFNγ+ in CD4+ cells. C, Data represent the percentages of Gata3+ in CD4+ cells. D, Data show representative flow cytometry plots for CD4 versus IL4, the percentages of IL4+ in CD4+ cells, and the IL4 geoMFI in CD4+ cells. Values inside the plots represent the percentages of IL4+ in CD4+cells. Naïve CD4+ T cells from control (ctrl) and cKO mice were polarized ex vivo toward Treg or Th17 and analyzed by flow cytometry. E, Data represents the percentage of FoxP3+ or IL10+ in CD4+ cells. F, Data represents the percentage of RORγt+ or IL17A+ in CD4+ cells. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. *, P < 0.05.
Asb2α was expressed in Th2 cells and its loss impeded IL4 expression in Th2 cells. Naïve CD4+ T cells from ctrl and cKO mice were polarized ex vivo toward Th1 or Th2 and analyzed by flow cytometry and qRT-PCR. A, Relative expression of Gata3 and Tbet/Tbx21 mRNA. B, Data represent the percentages of Tbet+ and IFNγ+ in CD4+ cells. C, Data represent the percentages of Gata3+ in CD4+ cells. D, Data show representative flow cytometry plots for CD4 versus IL4, the percentages of IL4+ in CD4+ cells, and the IL4 geoMFI in CD4+ cells. Values inside the plots represent the percentages of IL4+ in CD4+cells. Naïve CD4+ T cells from control (ctrl) and cKO mice were polarized ex vivo toward Treg or Th17 and analyzed by flow cytometry. E, Data represents the percentage of FoxP3+ or IL10+ in CD4+ cells. F, Data represents the percentage of RORγt+ or IL17A+ in CD4+ cells. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. *, P < 0.05.
Enhanced expression of IFNγ upon loss of Asb2 in Th2 cells suppresses colorectal cancer progression in mice
To determine whether the reduced tumor burden observed in AOM/DSS-treated cKO mice was due to Asb2 deficiency in Th2 cells, we transfer wild-type Th2 cells into cKO mice. The transfer of wild-type Th2 cells into cKO mice increased the tumor growth to amounts similar to those observed in control mice (Figs. 3G and H and 7A–C). We then questioned the mechanisms underlying the Th1/Th17/CTL response and the role of IFNγ in antitumor immunity. Indeed, we observed increased numbers of IFNγ+ cells and increased amount of Ifnγ transcripts in the colonic mucosa of cKO mice treated with AOM/DSS (Fig. 7D and E) that are likely to contribute to the reduced tumor burden following Asb2 deletion. To evaluate the role of IFNγ in the reduced tumor progression in cKO mice, anti-IFNγ neutralizing antibodies were injected into cKO mice. As shown in Fig. 7F and G, the tumor load was increased when cKO mice were administered anti-IFNγ neutralizing antibodies. Tumor numbers were also increased in colons of cKO mice treated with anti-IFNγ compared with colons of cKO mice treated with control antibodies (Fig. 7F and H). Furthermore, large tumors were more numerous in mice treated with anti-IFNγ than in mice treated with control antibodies (Fig. 7F and H-I). Together, these results indicate that IFNγ signaling is essential for protection of cKO mice against colitis-associated colorectal cancer development.
Deletion of Asb2 in Th2 cells attenuated colitis-associated tumorigenesis in mice by reducing the tumor-promoting effects of Th2 cells and enhancing the tumor-inhibiting effects of Th1 cells. Control (Ctrl) and/or cKO mice were injected with AOM, subsequently treated with three cycles of DSS, and analyzed 69 days after AOM injection (A–C, F–I). Transfer of wild-type (WT) CD45.1 Th2 cells to cKO mice promoted tumor growth. Mice were injected intravenously with Th2 cells differentiated from wild-type CD4+ cells 41, 48, and 62 days after AOM treatment. Representative images of colons (A), tumor loads in the colon (B), and enumerations of total tumor burden and of tumors following classification according to their size (C). Control (ctrl) and cKO mice were treated with AOM and DSS and the cells isolated from colons 43 (d43) after AOM injection were analyzed by flow cytometry and qRT-PCR. Data represent the numbers of CD45+IFNγ+ cells (D) and the relative expression of Ifnγ mRNA (E). IFNγ signaling is essential for protection of cKO mice against colitis-associated colorectal cancer progression. Mice were injected intraperitoneally with anti-IFNγ neutralizing antibodies or control IgG every 4 days after the second DSS cycle. Representative image of colons (F), tumor loads in the colon (G), and enumerations of total tumor burden and of tumors following classification according to their size (H) and representative colon sections stained with alcian blue and nuclear fast red (I). In A and F, scale bars = 0.5 cm. In I, scale bar = 200 μm. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. *, P < 0.05.
Deletion of Asb2 in Th2 cells attenuated colitis-associated tumorigenesis in mice by reducing the tumor-promoting effects of Th2 cells and enhancing the tumor-inhibiting effects of Th1 cells. Control (Ctrl) and/or cKO mice were injected with AOM, subsequently treated with three cycles of DSS, and analyzed 69 days after AOM injection (A–C, F–I). Transfer of wild-type (WT) CD45.1 Th2 cells to cKO mice promoted tumor growth. Mice were injected intravenously with Th2 cells differentiated from wild-type CD4+ cells 41, 48, and 62 days after AOM treatment. Representative images of colons (A), tumor loads in the colon (B), and enumerations of total tumor burden and of tumors following classification according to their size (C). Control (ctrl) and cKO mice were treated with AOM and DSS and the cells isolated from colons 43 (d43) after AOM injection were analyzed by flow cytometry and qRT-PCR. Data represent the numbers of CD45+IFNγ+ cells (D) and the relative expression of Ifnγ mRNA (E). IFNγ signaling is essential for protection of cKO mice against colitis-associated colorectal cancer progression. Mice were injected intraperitoneally with anti-IFNγ neutralizing antibodies or control IgG every 4 days after the second DSS cycle. Representative image of colons (F), tumor loads in the colon (G), and enumerations of total tumor burden and of tumors following classification according to their size (H) and representative colon sections stained with alcian blue and nuclear fast red (I). In A and F, scale bars = 0.5 cm. In I, scale bar = 200 μm. The numbers of independent biological repeats are indicated in italics. Data are represented as mean ± SEM. *, P < 0.05.
Discussion
Our study reveals a role for the hematopoietic E3 ubiquitin ligase, Asb2α, in tumor development. Analysis of ASB2 expression in tumor samples of patients with colorectal cancer indicated that ASB2 expression was higher in the subtypes associated with shorter relapse-free survival (28). In addition, Asb2 deficiency in hematopoietic cells reduced tumor development in a mouse model of colitis-associated tumorigenesis. These results are likely due to altered functions of Th2 cells.
CD4+ T-cell subsets play differential roles during tumor development. Among them, Th1 cells can repress tumor growth by secreting IFNγ and supporting the activity of cytotoxic T lymphocytes (34). Here, we show that cKO mice develop fewer and smaller tumors than control mice with increased amounts of IFNγ indicative of a higher Th1 response, and enhanced expression of the cytotoxic markers perforin and granzyme B in the tumor area. In line with this finding, IFNγ neutralization increases tumor development in cKO mice, further supporting a role for IFNγ-dependent signaling in protection of colitis-associated colorectal cancer. High densities of Th1 cells in the tumor have better prognostic value in colorectal cancer than the tumor–node–metastasis classification (35, 36).
Asb2 mRNAs are expressed in Th2 cells (26, 27, 30). Consistent with these findings, Asb2 is a target gene of the Th2 master transcription factor Gata3, and Gata3 binding to WGATAA motifs in the Asb2 locus in Th2 cells likely facilitates H3K4me1-, H3K4me2- and H3K4me3-mediated activation of Asb2. It has been proposed that Gata3-mediated gene regulation in Th subsets depends on specific cofactors (30). Indeed, in addition to the primary WGATAA motif, GATA binding sites in Th2 cells contain secondary motifs for other transcription factors involved in T-cell differentiation, such as members of the Ets or Runx families (30). This is also the case for Asb2. It is therefore tempting to speculate that Asb2 is one of the genes that is functional in Th2 cells.
The number of IL4+Th2 cells was reduced in the colonic mucosa of tumor-bearing cKO mice and also following ex vivo Th2 polarization of cKO naïve CD4+ T cells, which suggests that the balance in Th1/Th2 responses of tumor-bearing mice was affected by Asb2 deletion and had functional consequences in disease development. Indeed, cross regulation by IL4 and IFNγ suppresses Th1 and Th2 differentiation, respectively (37–41). Although Th2 cells represent a component in the activation and regulation of humoral immunity and allergic inflammatory responses, their roles in tumor immunity are yet to be investigated. Indeed, Th2 cells have pro- or antitumor activity depending on the type of cancer. Th2 cells have antitumor activity in a model of melanoma resistant to cytotoxic T lymphocytes (42). In contrast, Th2 cells are associated with tumor progression in renal cell carcinoma (43), in breast cancer (44), in melanoma (45), and in pancreatic carcinogenesis (46, 47). In addition, depletion of IL4 in a mouse model of metastatic melanoma limits tumor growth suggesting a protumor effect for IL4 (48). In agreement with our data in the AOM/DSS mouse model of colitis-associated colorectal cancer, tumor development is increased in the absence of IFNγ in the inflamed colon in another mouse model of colitis-associated colorectal cancer (49). We here provide evidence that in a mouse model of colitis-associated tumorigenesis, in which Th2 cells promoted tumor growth, Asb2 deficiency in Th2 cells blunted Th2 cytokine production leading to enhanced type 1 antitumor immune response.
Altogether, our results not only revealed that Asb2α is a negative regulator of antitumor immune response in colorectal cancer, but also demonstrated that Asb2α plays functional roles in Th2 cells. We propose that Asb2α should be added to the growing list of E3 ubiquitin ligases involved in the regulation of CD4+ T-cell identity and function. Asb2α induces IκBα degradation in T-ALL (acute lymphoblastic leukemia) cell lines (50). Whether IκBα or other proteins are substrates of Asb2α in Th2 cells, the degradation of which mediates Asb2α effects in these cells remain to be determined. Because E3 ubiquitin ligases are druggable, Asb2α might be a promising pharmacologic target to modulate Th2 response in cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: C.A. Spinner, I. Lamsoul, C. Moog-Lutz, P.G. Lutz
Development of methodology: C.A. Spinner, I. Lamsoul, P.G. Lutz
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.A. Spinner, I. Lamsoul, A. Métais, C. Febrissy, P.G. Lutz
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.A. Spinner, I. Lamsoul, A. Métais, C. Febrissy, C. Moog-Lutz, P.G. Lutz
Writing, review, and/or revision of the manuscript: C.A. Spinner, I. Lamsoul, P.G. Lutz
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.A. Spinner, I. Lamsoul, P.G. Lutz
Study supervision: I. Lamsoul, P.G. Lutz
Acknowledgments
We thank the Anexplo-IPBS and the Toulouse Réseau Imagerie. We acknowledge the Non-Invasive Exploration and the Phenotyping Services, US006/CREFRE Inserm/UPS/ENVT. We thank L. Apetoh, D. Hudrisier, B. Lucas, and O. Neyrolles for reading the article and for helpful comments. This work was supported by the Centre National de la Recherche Scientifique, the University of Toulouse, and the ITMO Cancer Aviesan (Alliance Nationale Pour les Sciences de la Vie et de la Santé, National Alliance for Life Science and Health) within the framework of Cancer Plan. This work was also supported by grants to P.G. Lutz from the Comité Midi-Pyrénées de la Ligue contre le Cancer and from the Fondation ARC pour la recherche sur le cancer. C.A. Spinner was supported by fellowships of the French Ministry of Higher Education and Research and of the Société Française d’Hématologie. C. Febrissy was supported by a fellowship of Fonroga (Fondation Roland Garrigou pour la Culture et la Santé).
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