Page 123 - Haematologica-April 2018

which leads to cyclin D1 overexpression and cell cycle deregulation. Other frequent genetic aberrations are muta- tions in crucial DNA damage response genes, such as ATM and TP53. Relevant for the present work, however, recent studies show an important role for extrinsic, soluble and adhesion-mediated signals from the tumor microenviron- ment in supporting MCL trafficking, homing and suscep- tibility to therapy.5,6
Consistent with the important role of microenviron- ments, primary MCL and chronic lymphocytic leukemia (CLL) cells can only be cultured in vitro for a few days before they undergo spontaneous apoptosis.7,8 If co-cul- tured with mesenchymal stromal cells on the other hand, the in vitro cultures can be sustained for weeks.8,9 Furthermore, stromal cells of both human and murine ori- gin can protect MCL and CLL cells from spontaneous and drug-induced apoptosis.8,10-12 While soluble molecules secreted by stromal cells such as BAFF8,13 and CXCL1214 have been shown to increase survival in malignant B cells, the protective effect is more prominent for lymphoma cells that physically adhere to stromal cells,6,8 and direct interactions between lymphoma cells and stromal cells can induce cell cycle arrest in MCL and diffuse large B-cell lymphoma (DLBCL).15 These mechanisms, involving solu- ble and adhesion-mediated signaling, may specifically confer survival advantages to lymphoma cells that home to protective microenvironmental niches through the acti- vation of anti-apoptotic programs and downregulation of genes involved in proliferation.16
Targeted cell-culture studies have elucidated effects of microenvironment interactions in MCL and CLL. Increased levels of immunomodulatory cytokines, such as CCL3, CCL4, CCL22, IL-10 and TNF, with the capacity to alter microenvironment cellular composition have been report- ed in co-cultures of MCL or CLL cells with stromal cells or under other conditions that mimic microenvironment interactions.17-20 The adhesive properties of non-Hodgkin lymphoma (NHL) cells have been shown to increase upon treatment with anti-IgM, CXCL12 or CXCL13.17 The CXCR4 cytokine receptor protein, central to normal B-cell migration and homing, is down-regulated in adherent CLL cells.14,21 In co-culture and analogous studies, increased expression of anti-apoptotic proteins, such as BCL-XL and MCL-1, have been reported.11,22,23 Co-cultivation of MCL cells with stromal cells has also been reported to increase protein levels of the cell cycle inhibitors p21Cip1 and p27Kip1, along with an increased ratio of G0/G1 cells relative to S- phase cells.15 Many of these effects may be associated with an adhesion-related induction of both the canonical and non-canonical NF-κB pathways.8
While important signaling mechanisms relevant for cell adhesion-mediated survival of lymphoma cells have been revealed by targeted studies, the present work is the first systematic study of global changes in gene expression in a defined model system that allows discrimination of gene expression changes in the different cell types in the co-cul- ture as well as their relationship to the same cells grown in isolation.
Methods
Cell culture
Cells were cultivated in a humidified incubator at 37°C and 5% CO2 in media supplemented with 100 U/mL penicillin and 100
Global mRNA changes in stroma-bound lymphoma cells
mg/mL streptomycin. The mouse stromal cell line MS-5 and the MCL cell line Jeko-1 were purchased from DSMZ and maintained in αMEM-glutamax (Gibco) supplemented with 10% heat-inacti- vated fetal bovine serum (H.I. FBS; Gibco) and 2 mM sodium pyruvate or RPMI-glutamax (Gibco) supplemented with 10% HI FBS, respectively. Co-cultures of Jeko-1 with MS-5 at a 10:1 ratio were maintained under the same conditions as for MS-5 cells alone.
Cell-cell binding assay
Unlabeled Jeko-1 suspension cells were added to established MS-5 monolayers. After 24 h, unlabeled Jeko-1 cells in suspension were removed and replaced with an equivalent number of CFDA- SE labeled Jeko-1 cells. Adhered unlabeled/labeled Jeko-1 cells were counted at 24 h and 48 h. The order of addition of labeled/unlabeled Jeko-1 cells was subsequently reversed.
RNA extraction, library preparation and sequencing
Total RNA was extracted using RNeasy with QIAshredders (Qiagen). Libraries were prepared using TruSeq sample prep kit v.2.0 and included a poly-A enrichment step. Samples were 16-plexed on an Illumina HighSeq 2500 instrument generating 230,700,000 2x101bp short reads (Online Supplementary Table S1).
Species-based read separation and mapping to reference genomes
Reference genomes hg19 and mm10 were obtained from UCSC. Raw reads were separated based on species origin using Xenome (v.1.0.1).24 Separated reads of human and murine ori- gin were aligned to reference genomes hg19 and mm10, respectively, using Tophat2 (2.0.11)25 using default options. Fragments per feature were counted using summarizeOverlaps from the Bioconductor package GenomicAlignments (1.0.6) with counting mode set to “Union”. Gene annotations and co- ordinates were from the Bioconductor packages TxDb.Hsapiens.UCSC.hg19.knownGene (v.2.14.0) and TxDb.Mmusculus.UCSC.mm10.knownGene (v.2.14.0).
Differential gene expression analysis and GSEA
Differential expression was determined by DESeq (1.16.0).26 Normalized count tables were used for Gene Set Enrichment Analysis (GSEA) (Broad Institute, 2.2.0)27 using canonical path- ways (c2.cp.v5.0.symbols.gmt) and GO processes (c5.bp.v5.0.symbols.gmt), from the MsigDB.27 Functional clusters from the leading edge analysis were identified as n ≥10.
genes
Protein interaction network analysis
A unified list with unique gene identifiers based on differential- ly expressed genes (FDR q-value≤0.05) and leading edge genes from the GSEA (n=1458) was uploaded to the STRING interaction database (v.10.0) with the confidence level set to 0.4.28 Node genes were defined as those having 8 or more interactions.
Microarray analysis
Microarray datasets were downloaded from GEO:29 GSE2102930 and GSE70910.31 Overlaps between these datasets and adhesion- related genes from the present study were interrogated by two- sided Fisher’s exact tests.
Data availability
Raw RNA-seq data are available via the gene expression omnibus (GEO) repository29 by accession number: GSE99501. A detailed account of materials and methods used is available in the Online Supplementary Methods.
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