Evaluation of the NOD/SCID xenograft model for glucocorticoid-regulated gene expression in childhood B-cell precursor acute lymphoblastic leukemia

  • Vivek A Bhadri1, 3,

    Affiliated with

    • Mark J Cowley2,

      Affiliated with

      • Warren Kaplan2,

        Affiliated with

        • Toby N Trahair1, 3 and

          Affiliated with

          • Richard B Lock1Email author

            Affiliated with

            BMC Genomics201112:565

            DOI: 10.1186/1471-2164-12-565

            Received: 16 August 2011

            Accepted: 17 November 2011

            Published: 17 November 2011

            Abstract

            Background

            Glucocorticoids such as prednisolone and dexamethasone are critical drugs used in multi-agent chemotherapy protocols used to treat acute lymphoblastic leukemia (ALL), and response to glucocorticoids is highly predictive of outcome. The NOD/SCID xenograft mouse model of ALL is a clinically relevant model in which the mice develop a systemic leukemia which retains the fundamental biological characteristics of the original disease. Here we report a study evaluating the NOD/SCID xenograft mouse model to investigate glucocorticoid-induced gene expression. Cells from a glucocorticoid-sensitive xenograft derived from a child with B-cell precursor ALL were inoculated into NOD/SCID mice. When highly engrafted the mice were randomized into groups of 4 to receive dexamethasone 15 mg/kg by intraperitoneal injection or vehicle control. Leukemia cells were harvested from mice spleens at 0, 8, 24 or 48 hours thereafter, and gene expression analyzed on Illumina WG-6_V3 chips, comparing all groups to time 0 hours.

            Results

            The 8 hour dexamethasone-treated timepoint had the highest number of significantly differentially expressed genes, with fewer observed at the 24 and 48 hour timepoints, and with minimal changes seen across the time-matched controls. When compared to publicly available datasets of glucocorticoid-induced gene expression from an in vitro cell line study and from an in vivo study of patients with ALL, at the level of pathways, expression changes in the 8 hour xenograft samples showed a similar response to patients treated with glucocorticoids. Replicate analysis revealed that at the 8 hour timepoint, a dataset with high signal and differential expression, using data from 3 replicates instead of 4 resulted in excellent recovery scores of > 0.9. However at other timepoints with less signal very poor recovery scores were obtained with 3 replicates.

            Conclusions

            The NOD/SCID xenograft mouse model provides a reproducible experimental system in which to investigate clinically-relevant mechanisms of drug-induced gene regulation in ALL; the 8 hour timepoint provides the highest number of significantly differentially expressed genes; time-matched controls are redundant and excellent recovery scores can be obtained with 3 replicates.

            Background

            Glucocorticoids such as prednisolone and dexamethasone are critical components of multi-agent chemotherapy protocols used in the treatment of acute lymphoblastic leukemia (ALL) [1] due to their ability to induce apoptosis in lymphoid cells. Despite their use for over 50 years their mechanism of action is not completely understood. Glucocorticoids are steroid hormones that act on target cells through interaction with a specific glucocorticoid receptor (GR) [2]. The GR is held in a cytosolic complex by a number of co-chaperone molecules including HSP-90 and HSP-70 [3], and on ligand binding dissociates from the co-chaperone complex, dimerizes and is transported to the nucleus where it binds to palindromic DNA sequences known as glucocorticoid response elements (GREs) found in the promoter regions of target genes [4]. This leads to the activation of transcription of primary target genes, repression of transcription through interaction with negative GREs [5] or of gene activation through transcription factors such as AP-1 and NF-ΚB [6]. In lymphoid cells, this results in repression of cell cycle progression through cyclin-D3 and C-MYC [7], and cell death through the activation of apoptosis. Glucocorticoids also induce other non-apoptotic mechanisms of programmed cell death including autophagy [8] and mediate a number of pathways involved in the metabolism of carbohydrates, lipids and proteins.

            A number of studies have published microarray data of glucocorticoid-induced genes in lymphoid cells, but comparison of the data is complicated by technical differences in platform and chip type. Previous studies of glucocorticoid-induced genes in ALL have been carried out using in vitro cell-line models [915] and patient-derived cells, both in vivo [16] and in vitro [17]. Cell lines are extensively used in the study of ALL but in the process of immortalization acquire multiple genetic defects, particularly in the p53 pathway [18], and mechanisms demonstrated in cell lines are often not replicated in more clinically relevant models. Primary patient cells have a finite supply and rarely survive ex vivo for more than a few days. The non-obese diabetic/severe combined immunodeficient (NOD/SCID) xenograft mouse model is widely used to study ALL. In this model, human leukemia cells obtained from diagnostic bone marrow biopsies are inoculated into NOD/SCID mice, and on engraftment establish a systemic leukemia which retains the fundamental biological characteristics of the original disease [19]. It has also been shown that the in vivo responses to chemotherapeutic agents, including dexamethasone, correlates with patient outcome [20], and thus the NOD/SCID xenograft mouse model provides a stable, reproducible and clinically relevant model with which to study ALL. Here we report the first study investigating glucocorticoid-induced gene expression in ALL using the NOD/SCID xenograft model, the optimal experimental design, and a comparison of our microarray data to publicly available datasets of glucocorticoid-induced genes in other experimental models.

            Methods

            NOD/SCID xenograft mouse model

            All experimental studies were approved by the Human Research Ethics Committee and the Animal Care and Ethics Committee of the University of New South Wales. ALL-3, a glucocorticoid-sensitive xenograft derived from a 12 year old girl with mixed lineage leukemia (MLL)-rearranged BCP-ALL, was chosen for this study. Although MLL-rearranged ALL is associated with a poor prednisolone response and an inferior outcome [21], this patient is currently a long-term survivor. ALL-3 demonstrates in vitro glucocorticoid sensitivity, with an IC50 of 9.4 nM on exposure to dexamethasone. In the in vivo NOD/SCID xenograft mouse model, ALL-3 is highly responsive to 4 weeks of treatment with single agent dexamethasone, with rapid clearance of leukemic blasts from the peripheral blood and recurrence of leukemia delayed by 63.4 days compared to vehicle-treated controls [20].

            Cells from ALL-3 were inoculated by tail-vein injection into 28 NOD/SCID mice. The mice were bled weekly and the samples stained with fluorescein isothiocyanate (FITC)-conjugated anti-murine CD45 and allophycocyanin (APC)-conjugated anti-human CD45 (BioLegend, San Diego, CA). Following lysis of erythrocytes with FACS lysing solution (BD Biosciences, San Jose, CA), samples were analyzed by multiparametric flow cytometry on a FACSCanto cytometer (BD Biosciences, San Jose, CA). Engraftment was calculated as the proportion of human versus total CD45+ cells.

            When high level (> 70%) engraftment was achieved in the peripheral blood, between 8 and 10 weeks post-transplantation, the mice were randomized and split into groups of 4 to receive either dexamethasone 15 mg/kg (Sigma-Aldrich, St Louis, MO) or vehicle control by intraperitoneal injection. Mice were culled by CO2 asphyxiation at 0 hours (pre-treatment, group 1), 8 hours (groups 2 and 3), 24 hours (groups 4 and 5) or 48 hours (groups 6 and 7) following treatment. The mice in groups 6 and 7 received a second dose of dexamethasone or vehicle control at 24 hours. Two mice succumbed early to thymoma, a well-recognized complication in NOD/SCID mice, resulting in 3 mice in each of groups 6 and 7. Cell suspensions of spleens were prepared and mononuclear cells enriched and purified to > 97% human by density gradient centrifugation using LymphoPrep (Axis-Shield, Norway), and cell viability assessed by trypan blue exclusion. RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany) and the RNA integrity verified (Agilent Bioanalyzer, Santa Clara, CA). The RNA was amplified using the Illumina TotalPrep RNA amplification kit (Ambion, Austin, TX) and hybridized onto Illumina WG-6_V3 chips (Illumina, San Diego, CA). The chips were scanned on the Illumina Bead Array Reader (Illumina, San Diego, CA) and gene expression analyzed. The data have been deposited in NCBI's Gene Expression Omnibus [22] and are accessible through GEO Series accession number GSE30392 http://​www.​ncbi.​nlm.​nih.​gov/​geo/​query/​acc.​cgi?​acc=​GSE30392.

            Gene expression and functional analysis

            The sample probe profiles with no normalization or background correction were exported from BeadStudio (version 3.0.14, Illumina, San Diego, CA). The data were pre-processed using variance stabilizing transformation [23] and robust spline normalization in lumi [24] which takes advantage of each probe being represented by > 25 beads. Differential gene expression was determined using limma [25] by comparing all treated groups to time 0 hours, with the positive False Discovery Rate correction for multiple testing [26]. Complete linkage hierarchical clustering using Euclidian distance was used to compare groups to each other. Functional analysis was performed using gene set enrichment analysis (GSEA) version 2.04 [27], comparing the limma moderated t-statistic for each probe in a pre-ranked file, against the c2_all collection of gene sets from the Molecular Signatures Database [27] version 2.5 with 1000 permutations. The similarity of the top 100 up- and down-regulated genesets was assessed using meta-GSEA (Cowley et al, manuscript in preparation).

            Comparison of models

            The molecular response to glucocorticoids in xenografts was compared to publicly available microarray data [13, 16] using parametric analysis of gene set enrichment [28] implemented in the PGSEA package (version 1.20.1, Furge and Dykema) from the Bioconductor project [29], with some modifications to the algorithm to assess significance of the genes that are in the geneset and represented on the microarray, and to allow more control over control sample specification (available upon request). Expression levels of each gene in each sample were converted to expression ratios relative to patient matched controls before glucocorticoid treatment (Schmidt et al), time-matched controls (Rainer et al), or time 0 hours (xenografts). Within each dataset, these gene-level ratios were summarized into geneset-level Z-scores, using PGSEA with genesets from the c2_all collection [27]. The Z-scores from each sample from the 3 studies were combined and then compared by hierarchical clustering of the top 100 gene sets demonstrating the greatest variance across the combined studies.

            Replicate analysis

            The stability of results when reducing the number of replicates was assessed using the Recovery Score method [30] from the GeneSelector package (version 1.4.0) of the Bioconductor project [29].

            Results and Discussion

            It has been demonstrated that changes in gene expression can be detected as early as 6 hours after treatment of ALL with glucocorticoids in vivo [16] and in vitro [11], although earlier timepoints show few, if any, significantly differentially expressed genes [17]. In this study the 8 hour dexamethasone-treated timepoint demonstrated the highest number of differentially expressed genes compared to baseline control, with far fewer observed at the 24 and 48 hour dexamethasone-treated timepoints (Tables 1 and 2, and Figure 1). Whilst a similar proportion of up- and down-regulated genes were identified at the 8 hour dexamethasone-treated timepoint (1158 vs 1072 respectively, FDR < 0.05), of those with large fold changes (FC > 2 or FC < 0.5, red dots in Figure 1A), 75% were up regulated (199 vs 65 respectively), consistent with the predominant role of glucocorticoids as transcriptional activators. The large numbers of statistically differentially expressed genes (FDR < 0.05) with small fold changes (0.5 < FC < 2) are indicative of both small measurement error across replicates, and thus the high reproducibility of the xenograft model, and good experimental power resulting from using 4 replicates. There was minimal significant differential gene expression across the time-matched controls (Tables 1 and 2). This demonstrates that in the xenograft mouse model, the 8 hour timepoint provides the greatest information, and that these changes are not sustained over later timepoints. The handling of the mice and intraperitoneal vehicle control injections had minimal effect on gene expression, and thus time-matched controls are redundant.
            Table 1

            Number of differentially expressed genes by False Discovery Rate (FDR), compared to time 0 hours.

            Timepoint (hours)

            FDR < 0.25

            FDR < 0.1

            FDR < 0.05

             

            +

            -

            +

            -

            +

            -

            Dex 8

            2313

            2434

            1470

            1423

            1158

            1072

            Dex 24

            970

            1087

            273

            421

            75

            195

            Dex 48

            321

            327

            41

            95

            12

            44

            Con 8

            0

            0

            0

            0

            0

            0

            Con 24

            0

            1

            0

            1

            0

            1

            Con 48

            0

            1

            0

            1

            0

            1

            + upregulated; - downregulated; Dex, dexamethasone-treated; and Con, control

            Table 2

            Number of differentially expressed genes by Fold Change (FC), compared to time 0 hours.

            Timepoint (hours)

            FC > 1.5

            FC > 2

            FC > 4

             

            +

            -

            +

            -

            +

            -

            Dex 8

            501

            429

            201

            68

            38

            0

            Dex 24

            137

            341

            15

            90

            0

            0

            Dex 48

            79

            234

            5

            69

            0

            3

            Con 8

            1

            37

            1

            2

            0

            0

            Con 24

            1

            5

            0

            0

            0

            0

            Con 48

            7

            34

            0

            2

            0

            0

            + upregulated; - downregulated; Dex, dexamethasone-treated; and Con, control

            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-565/MediaObjects/12864_2011_3723_Fig1_HTML.jpg
            Figure 1

            Volcano plots of significantly differentially expressed genes following treatment with dexamethasone at 8 hours (A), 24 hours (B), 48 hours (C). Significance was defined as log2 Fold Change > 1 or < -1 with False Discovery Rate (FDR) < 0.05. Each dot represents a single gene, and significant genes indicated by red dots.

            At the 8 hour timepoint, there were 173 genes upregulated with a t-statistic (the ratio of fold change to standard error) > 10 and 25 genes downregulated with a t-statistic < -10 (corresponding to P < 1.74 × 10-9 and FDR < 2.95 × 10-7, table 3). None of these genes had sustained expression changes at 24 or 48 hours, and although this could potentially reflect the early death of sensitive cells, there was no significant difference in the total number of cells harvested from the spleens at any timepoint compared to the time-matched controls (data not shown), and all harvests had a cell viability of ≥ 96%.
            Table 3

            Genes regulated 8 hours following dexamethasone treatment.

            ProbeSet ID

            Gene

            t

            P

            FDR

            Definition

            Upregulated

                 

            ILMN_5080450

            ZBTB16

            83.77

            < 2.2E-16

            < 2.2E-16

            zinc finger and BTB domain containing 16

            ILMN_3800088

            MMP7

            53.22

            < 2.2E-16

            < 2.2E-16

            matrix metallopeptidase 7

            ILMN_1770593

            CH25H

            53.14

            < 2.2E-16

            < 2.2E-16

            cholesterol 25-hydroxylase

            ILMN_6560328

            C6orf85

            44.60

            < 2.2E-16

            < 2.2E-16

            chromosome 6 open reading frame 85

            ILMN_7570561

            TSC22D3

            39.16

            < 2.2E-16

            < 2.2E-16

            TSC22 domain family, member 3

            ILMN_580187

            PDE8B

            33.88

            < 2.2E-16

            3.90E-16

            phosphodiesterase 8B

            ILMN_5130066

            C8orf61

            33.82

            < 2.2E-16

            3.90E-16

            chromosome 8 open reading frame 61

            ILMN_4120431

            TMEM100

            31.38

            < 2.2E-16

            1.64E-15

            transmembrane protein 100

            ILMN_650553

            BIN1

            29.76

            < 2.2E-16

            4.43E-15

            bridging integrator 1

            ILMN_1400373

            SLA

            29.57

            < 2.2E-16

            4.63E-15

            Src-like-adaptor

            ILMN_6330593

            PTHR1

            29.28

            < 2.2E-16

            5.22E-15

            parathyroid hormone receptor 1

            ILMN_6110037

            LILRA3

            29.04

            < 2.2E-16

            5.75E-15

            leukocyte immunoglobulin-like receptor subfamily A, member 3

            ILMN_4150477

            LOXL4

            28.67

            < 2.2E-16

            6.66E-15

            lysyl oxidase-like 4

            ILMN_2680079

            OGFRL1

            28.65

            < 2.2E-16

            6.66E-15

            opioid growth factor receptor-like 1

            ILMN_4210411

            NDRG2

            28.20

            < 2.2E-16

            8.62E-15

            NDRG family member 2

            ILMN_3780093

            LILRA1

            27.86

            < 2.2E-16

            1.05E-14

            leukocyte immunoglobulin-like receptor subfamily A, member 1

            ILMN_240441

            IL1R2

            27.46

            < 2.2E-16

            1.33E-14

            interleukin 1 receptor, type II

            ILMN_4730315

            MERTK

            26.14

            < 2.2E-16

            3.31E-14

            c-mer proto-oncogene tyrosine kinase

            ILMN_3800538

            ACPL2

            25.90

            < 2.2E-16

            3.72E-14

            acid phosphatase-like 2

            ILMN_6860392

            UGT2B17

            25.83

            < 2.2E-16

            3.72E-14

            UDP glucuronosyltransferase 2 family, polypeptide B17

            ILMN_4730541

            SLC44A1

            25.82

            < 2.2E-16

            3.72E-14

            solute carrier family 44, member 1

            ILMN_4860546

            CTHRC1

            25.64

            < 2.2E-16

            4.10E-14

            collagen triple helix repeat containing 1

            ILMN_3460270

            ZHX3

            24.56

            < 2.2E-16

            8.79E-14

            zinc fingers and homeoboxes 3

            ILMN_10639

            RASSF4

            23.21

            < 2.2E-16

            2.57E-13

            Ras association (RalGDS/AF-6) domain family 4

            ILMN_1190064

            UGT2B7

            23.13

            < 2.2E-16

            2.67E-13

            UDP glucuronosyltransferase 2 family, polypeptide B7

            ILMN_6400603

            MGC2463

            23.06

            < 2.2E-16

            2.71E-13

            poliovirus receptor related immunoglobulin domain containing

            ILMN_3450187

            IRGM

            23.04

            < 2.2E-16

            2.71E-13

            immunity-related GTPase family, M

            ILMN_6620528

            MT1X

            22.95

            2.40E-16

            2.85E-13

            metallothionein 1X

            ILMN_1260341

            IL13RA1

            22.47

            3.67E-16

            4.13E-13

            interleukin 13 receptor, alpha 1

            ILMN_2650112

            SLC16A2

            22.25

            4.48E-16

            4.91E-13

            solute carrier family 16, member 2

            ILMN_5570170

            PNMT

            22.01

            5.59E-16

            5.95E-13

            phenylethanolamine N-methyltransferase

            ILMN_870376

            C9orf152

            21.93

            6.02E-16

            6.25E-13

            chromosome 9 open reading frame 152

            ILMN_3190379

            TGFBR3

            21.52

            8.78E-16

            8.89E-13

            transforming growth factor, beta receptor III

            ILMN_1780142

            DSCR1

            21.08

            1.33E-15

            1.31E-12

            Down syndrome critical region gene 1

            ILMN_2640341

            FKBP5

            20.63

            2.05E-15

            1.89E-12

            FK506 binding protein 5

            ILMN_7610136

            LOC652626

            20.43

            2.48E-15

            2.23E-12

            Leukocyte immunoglobulin-like receptor subfamily B member 2

            ILMN_1410609

            CORO2A

            20.34

            2.72E-15

            2.35E-12

            coronin, actin binding protein, 2A

            ILMN_1780088

            TBXA2R

            20.29

            2.84E-15

            2.40E-12

            thromboxane A2 receptor

            ILMN_270431

            BAALC

            20.23

            3.02E-15

            2.50E-12

            brain and acute leukemia, cytoplasmic

            ILMN_6280176

            GBE1

            20.02

            3.72E-15

            3.01E-12

            glucan (1,4-alpha-), branching enzyme 1

            ILMN_6060113

            TBX15

            19.81

            4.62E-15

            3.67E-12

            T-box 15

            ILMN_4890743

            IQSEC1

            19.71

            5.09E-15

            3.97E-12

            IQ motif and Sec7 domain 1

            ILMN_150056

            DPEP1

            19.65

            5.41E-15

            4.13E-12

            dipeptidase 1

            ILMN_2060364

            BTNL9

            19.26

            8.04E-15

            5.91E-12

            butyrophilin-like 9

            ILMN_3830735

            UPB1

            19.23

            8.30E-15

            5.91E-12

            ureidopropionase, beta

            ILMN_5670377

            STYK1

            19.15

            9.09E-15

            6.35E-12

            serine/threonine/tyrosine kinase 1

            ILMN_4390630

            STAG3

            18.72

            1.42E-14

            9.39E-12

            stromal antigen 3

            ILMN_4070048

            NPHP4

            18.44

            1.91E-14

            1.25E-11

            nephronophthisis 4

            ILMN_4220474

            C6orf81

            18.31

            2.16E-14

            1.39E-11

            chromosome 6 open reading frame 81

            ILMN_1470746

            PTPN3

            18.30

            2.23E-14

            1.41E-11

            protein tyrosine phosphatase, non-receptor type 3

            ILMN_5860576

            C20orf133

            18.25

            2.36E-14

            1.47E-11

            MACRO domain containing 2

            ILMN_6020468

            PPP1R14A

            18.18

            2.52E-14

            1.55E-11

            protein phosphatase 1, regulatory (inhibitor) subunit 14A

            ILMN_1400634

            MT1M

            18.10

            2.76E-14

            1.64E-11

            metallothionein 1M

            ILMN_4250315

            ITGA9

            17.90

            3.46E-14

            2.03E-11

            integrin, alpha 9

            ILMN_5080471

            MAP3K6

            17.40

            6.02E-14

            3.44E-11

            mitogen-activated protein kinase 6

            ILMN_5360242

            FLJ42461

            17.36

            6.28E-14

            3.53E-11

            smoothelin-like 2

            ILMN_6620402

            NUDT16

            17.33

            6.50E-14

            3.60E-11

            nudix (nucleoside diphosphate linked moiety X)-type motif 16

            ILMN_3360112

            TMEM2

            17.26

            7.04E-14

            3.85E-11

            transmembrane protein 2

            ILMN_6840743

            PER1

            17.22

            7.41E-14

            3.99E-11

            period homolog 1

            ILMN_4220347

            LRRC1

            17.12

            8.29E-14

            4.33E-11

            leucine rich repeat containing 1

            ILMN_4850592

            P2RY14

            17.11

            8.35E-14

            4.33E-11

            purinergic receptor P2Y, G-protein coupled, 14

            ILMN_6560300

            SLC31A2

            16.91

            1.05E-13

            5.39E-11

            solute carrier family 31 member 2

            ILMN_4060091

            DKFZ

            16.87

            1.11E-13

            5.62E-11

            DKFZp451A211 protein

            ILMN_6770370

            LOC92196

            16.28

            2.23E-13

            1.11E-10

            death associated protein-like 1

            ILMN_580487

            IL9R

            16.21

            2.40E-13

            1.18E-10

            interleukin 9 receptor

            ILMN_1990300

            SOCS1

            16.18

            2.49E-13

            1.21E-10

            suppressor of cytokine signaling 1

            ILMN_5720424

            NRP1

            16.17

            2.54E-13

            1.22E-10

            neuropilin 1

            ILMN_4180427

            CIB4

            16.11

            2.74E-13

            1.30E-10

            calcium and integrin binding family member 4

            ILMN_4180544

            ROPN1L

            16.08

            2.81E-13

            1.32E-10

            ropporin 1-like

            ILMN_4250167

            SOX13

            16.04

            2.95E-13

            1.37E-10

            SRY (sex determining region Y)-box 13

            ILMN_6330170

            CHKA

            15.81

            3.94E-13

            1.81E-10

            choline kinase alpha, 3

            ILMN_4560192

            SFXN5

            15.62

            4.95E-13

            2.25E-10

            sideroflexin 5

            ILMN_2810136

            CAPN11

            15.56

            5.33E-13

            2.40E-10

            calpain 11

            ILMN_2690709

            VIPR1

            15.38

            6.68E-13

            2.91E-10

            vasoactive intestinal peptide receptor 1

            ILMN_630091

            NCOA7

            15.38

            6.69E-13

            2.91E-10

            nuclear receptor coactivator 7

            ILMN_5390730

            MGC17330

            15.21

            8.25E-13

            3.55E-10

            phosphoinositide-3-kinase interacting protein 1

            ILMN_130364

            MST150

            15.19

            8.49E-13

            3.62E-10

            MSTP150

            ILMN_3450241

            KIAA0774

            14.95

            1.16E-12

            4.77E-10

            KIAA0774

            ILMN_2230678

            ACACB

            14.80

            1.41E-12

            5.76E-10

            acetyl-Coenzyme A carboxylase beta

            ILMN_5870307

            LOC440359

            14.78

            1.44E-12

            5.83E-10

            similar to muscle Y-box protein YB2

            ILMN_3840554

            SPOCK2

            14.76

            1.49E-12

            5.95E-10

            sparc/osteonectin, cwcv and kazal-like domains 2

            ILMN_5810600

            MAP3K5

            14.69

            1.63E-12

            6.47E-10

            mitogen-activated protein kinase 5

            ILMN_2360719

            IRAK3

            14.65

            1.71E-12

            6.65E-10

            interleukin-1 receptor-associated kinase 3

            ILMN_1510121

            MTSS1

            14.64

            1.73E-12

            6.66E-10

            metastasis suppressor 1

            ILMN_540671

            LILRB2

            14.54

            1.98E-12

            7.41E-10

            leukocyte immunoglobulin-like receptor subfamily B, member 2

            ILMN_6980377

            MTMR15

            14.44

            2.26E-12

            8.39E-10

            myotubularin related protein 15

            ILMN_6220288

            PRDM1

            14.43

            2.28E-12

            8.39E-10

            PR domain containing 1, with ZNF domain

            ILMN_7330739

            NDRG4

            14.42

            2.30E-12

            8.39E-10

            NDRG family member 4

            ILMN_2600470

            WDR60

            14.20

            3.10E-12

            1.12E-09

            WD repeat domain 60

            ILMN_4050441

            SH3MD4

            14.16

            3.27E-12

            1.17E-09

            SH3 multiple domains 4

            ILMN_6760546

            TIPARP

            13.89

            4.74E-12

            1.64E-09

            TCDD-inducible poly(ADP-ribose) polymerase

            ILMN_2760537

            MTE

            13.89

            4.75E-12

            1.64E-09

            metallothionein E

            ILMN_160019

            SORT1

            13.79

            5.44E-12

            1.83E-09

            sortilin 1

            ILMN_6330132

            ISG20

            13.60

            7.00E-12

            2.32E-09

            interferon stimulated exonuclease gene 20 kDa

            ILMN_1510685

            DOK4

            13.52

            7.86E-12

            2.58E-09

            docking protein 4

            ILMN_1240228

            PAG1

            13.47

            8.50E-12

            2.77E-09

            phosphoprotein associated glycosphingolipid microdomains 1

            ILMN_580592

            CPNE8

            13.32

            1.04E-11

            3.31E-09

            copine VIII

            ILMN_5870301

            KIAA0513

            13.32

            1.05E-11

            3.31E-09

            KIAA0513

            ILMN_20129

            CD52

            13.32

            1.05E-11

            3.31E-09

            CD52 molecule

            ILMN_1820386

            PARVB

            13.31

            1.06E-11

            3.31E-09

            parvin, beta

            ILMN_6200402

            MT1A

            13.24

            1.17E-11

            3.64E-09

            metallothionein 1A

            ILMN_290661

            CLN8

            13.10

            1.43E-11

            4.36E-09

            ceroid-lipofuscinosis, neuronal 8

            ILMN_670082

            GNA12

            13.08

            1.47E-11

            4.43E-09

            guanine nucleotide binding protein (G protein) alpha 12

            ILMN_5570286

            TACC2

            12.99

            1.67E-11

            5.00E-09

            transforming, acidic coiled-coil containing protein 2

            ILMN_3190411

            STARD13

            12.93

            1.81E-11

            5.32E-09

            START domain containing 13

            ILMN_4540138

            NGB

            12.92

            1.85E-11

            5.39E-09

            neuroglobin

            ILMN_2000646

            B4GALT4

            12.83

            2.10E-11

            6.07E-09

            UDP-galactosyltransferase, polypeptide 4

            ILMN_7100731

            CYGB

            12.81

            2.17E-11

            6.17E-09

            cytoglobin

            ILMN_7050113

            NTRK1

            12.71

            2.52E-11

            7.09E-09

            neurotrophic tyrosine kinase receptor, type 1

            ILMN_2490670

            GNPTAB

            12.66

            2.71E-11

            7.52E-09

            N-acetylglucosamine-1-phosphate transferase, alpha and beta

            ILMN_20170

            ZNF385

            12.48

            3.55E-11

            9.72E-09

            zinc finger protein 385

            ILMN_2630687

            CHPT1

            12.43

            3.80E-11

            1.02E-08

            choline phosphotransferase 1

            ILMN_4120215

            WASF2

            12.43

            3.81E-11

            1.02E-08

            WAS protein family, member 2

            ILMN_5260494

            TMLHE

            12.39

            4.06E-11

            1.08E-08

            trimethyllysine hydroxylase, epsilon

            ILMN_5220333

            C14orf139

            12.31

            4.54E-11

            1.20E-08

            chromosome 14 open reading frame 139

            ILMN_3850440

            FCER1G

            12.12

            6.07E-11

            1.60E-08

            Fc fragment of IgE, receptor for; gamma polypeptide

            ILMN_1030008

            TGFB3

            12.11

            6.21E-11

            1.63E-08

            transforming growth factor, beta 3

            ILMN_1450468

            MYT1

            12.02

            7.04E-11

            1.81E-08

            myelin transcription factor 1

            ILMN_7560541

            SLC2A5

            12.01

            7.19E-11

            1.83E-08

            solute carrier family 2 member 5

            ILMN_2030438

            GBA2

            12.01

            7.21E-11

            1.83E-08

            glucosidase, beta (bile acid) 2

            ILMN_6840328

            SMAD3

            12.00

            7.35E-11

            1.86E-08

            SMAD family member 3

            ILMN_3930390

            SMAP1L

            11.91

            8.40E-11

            2.11E-08

            stromal membrane-associated protein 1-like

            ILMN_7570196

            TSPAN9

            11.90

            8.54E-11

            2.12E-08

            tetraspanin 9

            ILMN_6980546

            CACNA1I

            11.90

            8.56E-11

            2.12E-08

            calcium channel, voltage-dependent, T type, alpha 1I subunit

            ILMN_1710364

            LCN6

            11.89

            8.72E-11

            2.15E-08

            lipocalin 6

            ILMN_5360424

            RPS6KA2

            11.77

            1.04E-10

            2.54E-08

            ribosomal protein S6 kinase, 90 kDa, polypeptide 2

            ILMN_5890193

            MS4A4A

            11.72

            1.14E-10

            2.75E-08

            membrane-spanning 4-domains, subfamily A, member 4

            ILMN_3390292

            KLF9

            11.66

            1.24E-10

            2.98E-08

            Kruppel-like factor 9

            ILMN_5720059

            GFOD1

            11.65

            1.26E-10

            3.02E-08

            glucose-fructose oxidoreductase domain containing 1

            ILMN_7650523

            TMEM46

            11.57

            1.43E-10

            3.39E-08

            transmembrane protein 46

            ILMN_5700392

            LOC654000

            11.46

            1.70E-10

            3.95E-08

            ribosome biogenesis protein BMS1 homolog 2

            ILMN_4810348

            C1orf188

            11.40

            1.88E-10

            4.33E-08

            chromosome 1 open reading frame 188

            ILMN_4280180

            CHRNA3

            11.39

            1.91E-10

            4.37E-08

            cholinergic receptor, nicotinic, alpha 3

            ILMN_270458

            CRISPLD1

            11.37

            1.96E-10

            4.45E-08

            cysteine-rich secretory protein LCCL domain containing 1

            ILMN_450615

            MT2A

            11.37

            1.97E-10

            4.46E-08

            metallothionein 2A

            ILMN_20470

            GRASP

            11.35

            2.02E-10

            4.51E-08

            GRP1-associated scaffold protein

            ILMN_3370594

            LILRA2

            11.35

            2.03E-10

            4.51E-08

            leukocyte immunoglobulin-like receptor subfamily A, member 2

            ILMN_5220397

            RREB1

            11.34

            2.05E-10

            4.53E-08

            ras responsive element binding protein 1

            ILMN_1410192

            TDRD9

            11.34

            2.07E-10

            4.56E-08

            tudor domain containing 9

            ILMN_4070259

            LOC653133

            11.27

            2.30E-10

            4.99E-08

            guanine nucleotide binding protein (G protein) alpha 12

            ILMN_5960682

            RBPMS2

            11.24

            2.41E-10

            5.21E-08

            RNA binding protein with multiple splicing 2

            ILMN_1440300

            SLC27A3

            11.22

            2.50E-10

            5.37E-08

            solute carrier family 27, member 3

            ILMN_5050768

            LONRF1

            11.20

            2.58E-10

            5.53E-08

            LON peptidase N-terminal domain and ring finger 1

            ILMN_6270273

            KHDRBS3

            11.18

            2.67E-10

            5.68E-08

            KH domain, RNA binding, signal transduction associated 3

            ILMN_7100603

            KCNK3

            11.17

            2.70E-10

            5.72E-08

            potassium channel, subfamily K, member 3

            ILMN_2320129

            CSDA

            11.03

            3.38E-10

            7.08E-08

            cold shock domain protein A

            ILMN_3930022

            LOC644739

            10.99

            3.63E-10

            7.54E-08

            Wiskott-Aldrich syndrome protein family member 4

            ILMN_7400133

            CUGBP2

            10.90

            4.20E-10

            8.63E-08

            CUG triplet repeat, RNA binding protein 2

            ILMN_3290301

            FZD8

            10.88

            4.33E-10

            8.76E-08

            frizzled homolog 8

            ILMN_7320520

            MTUS1

            10.88

            4.33E-10

            8.76E-08

            mitochondrial tumor suppressor 1

            ILMN_3780053

            PALLD

            10.82

            4.79E-10

            9.60E-08

            palladin, cytoskeletal associated protein

            ILMN_6860162

            LOC441019

            10.74

            5.49E-10

            1.09E-07

            hypothetical LOC441019

            ILMN_5810154

            ALOX15B

            10.74

            5.50E-10

            1.09E-07

            arachidonate 15-lipoxygenase, type B

            ILMN_3930736

            CHST3

            10.73

            5.59E-10

            1.09E-07

            carbohydrate (chondroitin 6) sulfotransferase 3

            ILMN_60470

            STX11

            10.72

            5.68E-10

            1.10E-07

            syntaxin 11

            ILMN_3390484

            SERINC2

            10.69

            5.95E-10

            1.15E-07

            serine incorporator 2

            ILMN_1430647

            TAX1BP3

            10.61

            6.82E-10

            1.31E-07

            Tax1 (human T-cell leukemia virus type I) binding protein 3

            ILMN_5960440

            VDR

            10.60

            6.99E-10

            1.34E-07

            vitamin D (1,25-dihydroxyvitamin D3) receptor

            ILMN_6290735

            EPHB3

            10.51

            8.10E-10

            1.53E-07

            EPH receptor B3

            ILMN_2680372

            SH2D4A

            10.46

            8.78E-10

            1.64E-07

            SH2 domain containing 4A

            ILMN_2480050

            SOX7

            10.44

            9.13E-10

            1.69E-07

            SRY (sex determining region Y)-box 7

            ILMN_130128

            LOC285016

            10.41

            9.61E-10

            1.76E-07

            hypothetical protein LOC285016

            ILMN_4890451

            GRAMD3

            10.39

            9.87E-10

            1.80E-07

            GRAM domain containing 3

            ILMN_770161

            C10orf73

            10.39

            9.92E-10

            1.81E-07

            chromosome 10 open reading frame 73

            ILMN_2450202

            KIF3C

            10.35

            1.05E-09

            1.88E-07

            kinesin family member 3C

            ILMN_6840468

            HAL

            10.35

            1.06E-09

            1.89E-07

            histidine ammonia-lyase

            ILMN_2470070

            TBL1X

            10.30

            1.15E-09

            2.04E-07

            transducin (beta)-like 1X-linked

            ILMN_2320114

            KLF13

            10.27

            1.22E-09

            2.15E-07

            Kruppel-like factor 13

            ILMN_6380112

            DIP

            10.23

            1.31E-09

            2.27E-07

            death-inducing-protein

            ILMN_2470358

            IFNGR1

            10.22

            1.32E-09

            2.30E-07

            interferon gamma receptor 1

            ILMN_4250735

            IL27RA

            10.07

            1.70E-09

            2.91E-07

            interleukin 27 receptor, alpha

            ILMN_1470215

            MAP3K8

            10.07

            1.72E-09

            2.91E-07

            mitogen-activated protein kinase 8

            ILMN_2940373

            TACC1

            10.06

            1.74E-09

            2.94E-07

            transforming, acidic coiled-coil containing protein 1

            Downregulated

                 

            ILMN_770538

            LYSMD2

            -15.49

            5.81E-13

            2.58E-10

            LysM, putative peptidoglycan-binding, domain containing 2

            ILMN_7150059

            STAMBPL1

            -14.61

            1.79E-12

            6.84E-10

            STAM binding protein-like 1

            ILMN_5340692

            STRBP

            -14.56

            1.93E-12

            7.31E-10

            spermatid perinuclear RNA binding protein

            ILMN_4210397

            GLDC

            -14.05

            3.80E-12

            1.34E-09

            glycine dehydrogenase

            ILMN_6980327

            DKC1

            -13.79

            5.44E-12

            1.83E-09

            dyskeratosis congenita 1, dyskerin

            ILMN_50086

            TCF12

            -13.23

            1.19E-11

            3.69E-09

            transcription factor 12

            ILMN_4860356

            BYSL

            -12.81

            2.17E-11

            6.17E-09

            bystin-like

            ILMN_4280228

            IVNS1ABP

            -12.70

            2.55E-11

            7.12E-09

            influenza virus NS1A binding protein

            ILMN_1990379

            SLFN11

            -11.82

            9.63E-11

            2.36E-08

            schlafen family member 11

            ILMN_5220338

            MPEG1

            -11.64

            1.27E-10

            3.03E-08

            macrophage expressed gene 1

            ILMN_450168

            SFRS7

            -11.50

            1.60E-10

            3.74E-08

            splicing factor, arginine/serine-rich 7, 35 kDa

            ILMN_3460687

            KIAA0690

            -11.42

            1.81E-10

            4.19E-08

            ribosomal RNA processing 12 homolog

            ILMN_3400360

            MAPRE2

            -11.36

            1.99E-10

            4.48E-08

            microtubule-associated protein, RP/EB family, member 2

            ILMN_4010414

            PPFIBP1

            -11.12

            2.92E-10

            6.16E-08

            PTPRF interacting protein, binding protein 1 (liprin beta 1)

            ILMN_1190139

            UGT3A2

            -10.99

            3.61E-10

            7.54E-08

            UDP glycosyltransferase 3 family, polypeptide A2

            ILMN_4150201

            BCL2

            -10.93

            3.99E-10

            8.24E-08

            B-cell CLL/lymphoma 2

            ILMN_780240

            C12orf24

            -10.85

            4.53E-10

            9.13E-08

            chromosome 12 open reading frame 24

            ILMN_6760167

            MARCH3

            -10.73

            5.60E-10

            1.09E-07

            membrane-associated ring finger (C3HC4) 3

            ILMN_3940615

            PUS7

            -10.52

            7.99E-10

            1.52E-07

            pseudouridylate synthase 7 homolog

            ILMN_20544

            GART

            -10.41

            9.53E-10

            1.76E-07

            phosphoribosylglycinamide formyltransferase

            ILMN_2480326

            HSP90B1

            -10.36

            1.05E-09

            1.88E-07

            heat shock protein 90 kDa beta (Grp94), member 1

            ILMN_5270367

            CTSC

            -10.25

            1.26E-09

            2.20E-07

            cathepsin C

            ILMN_5420095

            MYC

            -10.21

            1.36E-09

            2.34E-07

            v-myc myelocytomatosis viral oncogene homolog

            ILMN_4610180

            PIK3C2B

            -10.20

            1.38E-09

            2.37E-07

            phosphoinositide-3-kinase, class 2, beta polypeptide

            ILMN_6450300

            GEMIN4

            -10.00

            1.95E-09

            3.27E-07

            gem (nuclear organelle) associated protein 4

            t, t-statistic; and FDR, false discovery rate

            The most significantly differentially expressed gene at the 8 hour dexamethasone-treated timepoint was ZBTB16, a known transcriptional repressor and glucocorticoid response gene, which has been shown to modulate glucocorticoid sensitivity in CEM T-ALL cells [31]. Other known glucocorticoid response genes upregulated included TSC22D3 [32] and SOCS1 [15], both downstream targets of the glucocorticoid receptor, FKBP5 [33], a co-chaperone of the glucocorticoid receptor, and MAP kinases 5, 6 and 8 [34]. Downregulated genes at 8 hours included BCL-2 [35] and C-MYC [36], both previously described, but also HSP90B1, a glucocorticoid receptor co-chaperone and regulator of apoptosis. The only pro-apoptotic gene consistently upregulated across multiple microarray analyses is the BH3-only BCL-2 family member BIM, and it has been shown that BIM has a critical role in glucocorticoid sensitivity and resistance [37], although in this current study BIM was only induced 1.3 fold. Thus these genes identified are consistent with previous reports of glucocorticoid-induced genes in ALL. Within these experimental systems however there are significant potential differences in glucocorticoid exposure between in vitro and in vivo models - a crucial one is that cells in vitro are continuously exposed to glucocorticoid whereas in in vivo models the glucocorticoid is subject to pharmacokinetic and pharmacodynamic changes which more accurately reflect changes in patients.

            At the later timepoints, significant differential gene expression was much less marked and predominantly downregulated. At 24 hours 5 genes were upregulated (t-statistic > 6) and 10 genes downregulated (t-statistic < -6, table 4), and at 48 hours 1 gene was upregulated (t-statistic > 6) and 15 genes downregulated (t-statistic < -6, table 5). At 24 hours, upregulated genes included NFKBIA, an inhibitor of NF-ΚB, and TRIM74, which was sustained at 48 hours, the significance of which is uncertain. Downregulated genes were those involved in cell cycle progression, including CCNF at 24 hours, and CCNF, CDC20 and AURKA at 48 hours, consistent with growth arrest.
            Table 4

            Genes regulated 24 hours following dexamethasone treatment.

            ProbeSet ID

            Gene

            t

            P

            FDR

            Definition

            Upregulated

                 

            ILMN_3930687

            FAM112A

            6.67

            1.32E-06

            0.0091

            family with sequence similarity 112, member A

            ILMN_6620255

            TRIM74

            6.29

            3.06E-06

            0.0132

            tripartite motif-containing 74

            ILMN_4280113

            NFKBIA

            6.23

            3.48E-06

            0.0138

            nuclear factor kappa B inhibitor, alpha

            ILMN_2140136

            EMR2

            6.10

            4.65E-06

            0.0149

            egf-like containing, mucin-like, hormone receptor-like 2

            ILMN_7000397

            ANKRD15

            6.08

            4.91E-06

            0.0149

            ankyrin repeat domain 15

            Downregulated

                 

            ILMN_870524

            HOXB8

            -8.60

            2.53E-08

            0.0011

            homeo box B8

            ILMN_4830520

            LOC144501

            -6.72

            1.19E-06

            0.0091

            hypothetical protein LOC144501

            ILMN_6110332

            ARHGAP19

            -6.70

            1.24E-06

            0.0091

            Rho GTPase activating protein 19

            ILMN_2970619

            ESPL1

            -6.65

            1.38E-06

            0.0091

            extra spindle pole bodies homolog 1

            ILMN_3130541

            CCNF

            -6.64

            1.43E-06

            0.0091

            cyclin F

            ILMN_4760577

            CENPA

            -6.62

            1.46E-06

            0.0091

            centromere protein A

            ILMN_4810646

            PIF1

            -6.54

            1.76E-06

            0.0095

            PIF1 5'-to-3' DNA helicase homolog

            ILMN_1070762

            PSRC1

            -6.40

            2.38E-06

            0.0114

            proline/serine-rich coiled-coil 1

            ILMN_4860703

            LOC648695

            -6.19

            3.82E-06

            0.0138

            retinoblastoma binding protein 4

            ILMN_1110538

            INCENP

            -6.05

            5.19E-06

            0.0149

            inner centromere protein antigens 135/155 kDa

            t, t-statistic; and FDR, false discovery rate

            Table 5

            Genes regulated 48 hours following dexamethasone treatment.

            ProbeSet ID

            Gene

            t

            P

            FDR

            Definition

            Upregulated

                 

            ILMN_6620255

            TRIM74

            6.30

            3.01E-06

            0.0089

            tripartite motif-containing 74

            Downregulated

                 

            ILMN_4810646

            PIF1

            -8.85

            1.58E-08

            0.0004

            PIF1 5'-to-3' DNA helicase homolog

            ILMN_870524

            HOXB8

            -8.66

            2.26E-08

            0.0004

            homeo box B8

            ILMN_1450193

            LGALS1

            -8.57

            2.66E-08

            0.0004

            lectin, galactoside-binding, soluble, 1 (galectin 1)

            ILMN_4760577

            CENPA

            -7.64

            1.71E-07

            0.0018

            centromere protein A

            ILMN_4730605

            AURKA

            -7.47

            2.42E-07

            0.0021

            aurora kinase A

            ILMN_1500010

            CDC20

            -6.84

            9.09E-07

            0.0053

            CDC20 cell division cycle 20 homolog

            ILMN_4060064

            HMMR

            -6.82

            9.61E-07

            0.0053

            hyaluronan-mediated motility receptor

            ILMN_2070408

            MID1

            -6.80

            9.97E-07

            0.0053

            midline 1 (Opitz/BBB syndrome)

            ILMN_2070288

            MT1E

            -6.66

            1.36E-06

            0.0065

            metallothionein 1E

            ILMN_1070762

            PSRC1

            -6.60

            1.55E-06

            0.0067

            proline/serine-rich coiled-coil 1

            ILMN_150543

            C20orf129

            -6.46

            2.12E-06

            0.0077

            chromosome 20 open reading frame 129

            ILMN_5870193

            FAM64A

            -6.45

            2.14E-06

            0.0077

            family with sequence similarity 64, member A

            ILMN_2810201

            KIF14

            -6.34

            2.77E-06

            0.0089

            kinesin family member 14

            ILMN_1050195

            KIF20A

            -6.28

            3.11E-06

            0.0089

            kinesin family member 20A

            ILMN_3130541

            CCNF

            -6.05

            5.21E-06

            0.0131

            cyclin F

            t, t-statistic; and FDR, false discovery rate

            Functional analysis using GSEA and meta-GSEA on the expression profiles obtained at 8 hours and 24 hours after dexamethasone treatment (additional files 1 and 2), revealed a significant upregulation of metabolic pathways, particularly adipogenesis at 8 hours, and a marked effect on pathways associated with cell cycling and proliferation, particularly downregulation of C-MYC at 8 hours and NF-ΚB at 24 hours, and upregulation of apoptotic pathways at 24 hours. Glucocorticoids are known to have effects on multiple cellular metabolic pathways, including glucose and carbohydrate metabolism, and have pro-adipogenic effects [38]. Suppression of C-MYC is a critical step prior to the initiation of apoptosis by dexamethasone in ALL [39] and suppression of NF-ΚB has been described [40].

            To determine whether the molecular response to glucocorticoids in this xenograft model of ALL mimicked the effects seen in either glucocorticoid-treated patients with ALL [16] or cell-line models of ALL [13], we applied parametric gene set enrichment analysis (PGSEA) [28]. Comparing gene expression profiles from multiple experiments is notoriously difficult and typically any true similarities are swamped by technical differences in microarray vendor, normalization strategies and analytical approach. By summarizing genes at the gene set level (such as genes in the same pathway), these technical differences are mitigated, allowing comparison of samples from multiple studies.

            We performed PGSEA on the 6-8 hour samples from the 3 studies, and then two-dimensional hierarchical clustering to identify the relationships between the different ALL models (Figure 2 and annotated in additional file 3). This revealed considerable heterogeneity in the molecular response to glucocorticoids in patients into at least 2, and possibly 4 different groups (green bars, Figure 2), which may represent different modes of response to glucocorticoids in patients. Relative to this inter-patient heterogeneity, both cell lines (purple bars, Figure 2) and xenografts (black bars, Figure 2) are remarkably reproducible; we anticipate that adding additional xenograft models of ALL from distinct patients will mirror the heterogeneity of the patient from whom they were derived. It is also evident that overall, glucocorticoid-treated xenografts co-cluster with a group of 3 patients (B-ALL-37, -38, and -40), all of whom had BCP-ALL and a good early prednisolone response, with varied cytogenetics (hyperploidy, t(12;21), and normal respectively). At more relaxed clustering thresholds, the glucocorticoid-treated xenografts cluster with 4 other patients with BCP-ALL (B-ALL-24, -31, -33, and 43) and the cell lines.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-565/MediaObjects/12864_2011_3723_Fig2_HTML.jpg
            Figure 2

            Parametric GSEA of combined top 100 glucocorticoid-induced gene sets with greatest variance from xenograft, patient and cell line models. Hierarchical clustering with gene sets in rows, samples in columns (xenografts - black, patient - green, cell line - purple). Each colour of each cell represents the Z-score (see legend). Boxes 1-5 represent defined clusters.

            We identified 5 clusters of gene sets with distinct expression profiles, each behaving differently in the 3 models of ALL. Cluster 1 demonstrated the markedly heterogeneous patterns seen in patient samples, with the xenograft samples showing a pattern similar to 8 of the patients; cluster 2 showed genesets that showed strong enrichment in the cell line study, and included a number of genesets associated with cell proliferation; cluster 3 did not show any striking differences across the three ALL models; cluster 4 showed genesets downregulated in both xenografts and cell lines compared to the patient samples, and included a number genesets associated with cell cycle progression, DNA/RNA replication and MYC; cluster 5 showed genesets strongly downregulated in the xenograft and cell line models, and included genesets associated with MYC and metabolic processes, particularly catabolism and energy production. In this limited comparison, it is clear that glucocorticoid-induced gene expression patterns seen in ALL are dependent on the experimental model, and that the patterns derived from the xenograft model show a greater similarity to patient-derived data than to cell lines.

            A search of the TRANSFAC database v8.3 [41] of CoMoDis [42] identified GRE motifs (within 100 kb either side of the gene) in only 25 (14.5%) of the top 173 upregulated genes at the 8 hour timepoint in this study, and no GRE motifs were identified in the upregulated genes at 24 or 48 hours. This supports accumulating evidence that glucocorticoids exert long-range effects through very distal steroid receptor binding sites [43]. Analysis of significantly differentially expressed glucocorticoid-induced genes in an in vitro cell line study [13] revealed a similar number of early response genes after 6 hours of exposure (60 upregulated (t-stat > 10) and 27 downregulated (t-stat < -10)) but a significantly greater number of genes after 24 hours (593 upregulated (t-stat > 10) and 782 downregulated (t-stat < -10)). Interestingly, all but 2 of the genes upregulated at 6 hours remained significantly upregulated at 24 hours, and 17 of the downregulated genes at 6 hours remained downregulated at 24 hours. GRE motifs were identified in 15 (25.0%) of the top 60 upregulated genes at 6 hours, and 87 (14.6%) of the top 593 genes at 24 hours. The observed difference between the studies in gene expression at later timepoints is consistent with continuous rather than physiological glucocorticoid exposure. In addition, in the cell line study, the GR (NR3C1) undergoes highly significant early and sustained autoupregulation, which in the continuous presence of ligand drives downstream gene expression. In contrast, in the xenograft model minimal GR upregulation is seen at the early timepoint but no significant change in GR expression is seen at either of the later timepoints.

            Given the good statistical power observed in Figure 1A, we proceeded to determine whether we could use fewer replicates and still identify a majority of the differentially expressed genes. Replicate analysis (Figure 3) revealed that at the 8 hour dexamethasone-treated timepoint, a dataset with high signal and differential expression, using data from any 3 randomly chosen biological replicates instead of 4 resulted in excellent recovery scores of > 0.9. That is, on average, 90% of the differentially expressed genes identified from all 4 samples were also identified in any combination of 3 arrays. At 24 hours, a timepoint with less signal, the average recovery score was 0.85 with 3 replicates, but was more variable than at 8 hours. Using just 2 biological replicates recovered 88% of the list of differentially expressed genes at 8 hours, which dropped to 14% at 24 hours. This confirms that the 8 hour time point has the strongest signal, which is reproducible across different subsets of biological replicates. We recommend using a minimum of 3 biological replicates, since fewer replicates destabilized our ability to identify differentially expressed genes. This has important considerations for experimental design, and has significant implications on cost and animal numbers.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-565/MediaObjects/12864_2011_3723_Fig3_HTML.jpg
            Figure 3

            Recovery scores at 8 hours and 24 hours when randomly selecting all combinations of 3 replicates (3rep) or 2 replicates (2rep) from the set of 4 biological replicates. The Recovery Score represents the proportion of differentially expressed genes from all 4 replicates recovered when using fewer replicates.

            Conclusions

            We conclude that the NOD/SCID ALL xenograft mouse model provides biologically relevant insights into glucocorticoid-induced gene expression, in a consistent, reproducible and clinically relevant model system. We have demonstrated that the 8 hour timepoint provides the highest number of significantly differentially expressed genes, that time-matched controls are redundant and excellent recovery scores can be obtained with 3 replicates. We have thus established the optimal experimental design, with subsequent important implications for costs and animal numbers.

            Declarations

            Acknowledgements of Research Support

            This research was supported by Children's Cancer Institute Australia for Medical Research (CCIA) and by a grant from the National Health and Medical Research Council (NHMRC). VAB was supported by fellowships from the Leukaemia Foundation and the Steven Walter Foundation. TNT was supported by fellowships from the Cancer Institute NSW and the NHMRC. RBL was supported by a fellowship from the NHMRC. MJC and WK were supported by the Cancer Institute NSW. CCIA is affiliated to Sydney Children's Hospital and the University of New South Wales.

            Authors’ Affiliations

            (1)
            Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of New South Wales
            (2)
            Peter Wills Bioinformatics Centre, Garvan Institute of Medical Research
            (3)
            Centre for Children’s Cancer and Blood Disorders, Sydney Children’s Hospital

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            © Bhadri et al; licensee BioMed Central Ltd. 2011

            This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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