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Mixed-species RNAseq analysis of human lymphoma cell adhesion to mouse stromal cells identifies a core gene set that is also differentially expressed in the lymph node microenvironment of MCL and CLL patients.

ABSTRACT: Background:A subset of hematological cancer patients is refractory to treatment or suffer relapse, due in part to minimal residual disease, whereby some cancer cells survive treatment via microenvironment interaction. Cell-adhesion mediated drug resistance is an important mechanism, whereby cancer cells receive survival signals via interaction with e.g. stromal cells. No genome-wide studies of in vitro systems have yet been performed to compare gene expression in different cell subsets within a co-culture and cells grown separately. Results: Using RNAseq and species-specific read mapping, we compared transcript levels in human Jeko-1 mantle cell lymphoma (MCL) cells stably adhered to stromal cells or in suspension within a co-culture and in separate culture as well as mouse MS-5 stromal cells in co-culture or in separate culture. 1050 differentially expressed transcripts in adherent MCL cells identified 24 functional categories that together represent four main functional themes, anti-apoptosis, B-cell signaling, cell adhesion/migration and early mitosis. Comparison with previous MCL and chronic lymphocytic leukemia (CLL) patient data identified 116 genes that are differentially regulated in all three studies. From these genes we suggest a gene signature (CCL3, CCL4, DUSP4, ETV5, ICAM1, IL15RA, IL21R, IL4I1, MFSD2A, NFKB1, NFKBIE, SEMA7A, TMEM2) characteristic of cells undergoing cell-adhesion mediated microenvironment signaling in MCL/ CLL cells. Conclusions: The model system developed and characterized here together with suggested signature genes can be used in future studies of pathways that mediate increased cancer cell survival and drug resistance mechanisms.

OTHER RELATED OMICS DATASETS IN: PRJNA388663

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE99501 | GEO | 2018/06/12

REPOSITORIES: GEO

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Project description:Lymphoma cells interact with specific microenvironment cell subsets, such as stromal cells, from which they receive survival and proliferation stimuli. These interactions negatively influence therapeutic response and increase the risk for drug resistance and relapse. It is still not clear why clinically similar tumors in different patients respond differently to established treatment regimens. Objective: The main aim of this study was to identify and compare key signaling pathways related to stromal cell adhesion in different mantle cell lymphoma (MCL) cell lines.Methods: Human MCL cells (JeKo1 and REC1) were cultured alone or in co-culture with mouse MS-5 stromal cells. Differentially expressed genes in adherent cells were identified by RNA sequencing and species-specific read sorting. The cell surface levels of proteins encoded by selected differentially expressed genes were quantified using flow cytometry in adherent and suspension cells. Selective inhibitors or siRNA were used to assess their significance for adherence to stromal cells. Results: Jeko1 and REC1 cells exhibited different time-dependent patterns of adhesion to stromal cells. 590 genes that were differently regulated upon adhesion in each cell line individually, showed significant differences in the extent or direction of regulation between the two cell lines. From this set, there was a significant increase in transcript levels for B-cell Receptor (BCR) pathway signature genes in adherent JeKo1 cell but not in adherent REC1 cells. Inhibition of BCR signaling using siRNA or the clinically approved inhibitor, Ibrutinib, decreased the number of adherent JeKo1 cells but had no such effect on REC1 cells. The cell surface levels of CXCR4 were higher in JeKo1 cells compared to REC1 cells and CXCR4 inhibition disrupted the adhesion of JeKo1 but not REC1 cells to the stromal cells. ICAM1 cell surface levels were induced in JeKo1 cells upon adhesion while REC1 showed a high level of ICAM1 in non-adherent cells. Depletion of ICAM1 levels by siRNA reduced the stromal cell adhesion of both JeKo1 and REC1 cells. Conclusions: The results suggest that different MCL cell lines exhibit different signaling profiles and pathway dependencies during microenvironment interaction. In this study BCR signaling pathways were shown to be important for stromal cell adhesion of JeKo1 cells but not REC1 cells. This provides a possible mechanism to account for the differential responsiveness of MCL patients to treatment with the Bruton’s Tyrosine Kinase inhibitor, Ibrutinib, an inhibitor of BCR signaling that is clinically approved for MCL.

Project description:Chronic lymphocytic leukemic B cells (CLL) reside in close contact with activated endothelial cells (EC) in infiltrated tissues. Here, we investigated the interactions between EC and CLL cells, highlighting molecular networks involved in this cellular crosstalk. We co-cultured purified CLL cells on HUVEC monolayer (HC) or in medium alone. We found that EC protected CLL from spontaneous apoptosis. A 2.2-fold increase in relative viability in IGHV mutated CLL and a 6.1-fold increase in IGHV unmutated CLL was detected in co-culture. Moreover, the endothelial cell layer decreased the in vitro sensitivity of CLL cells to Fludarabine-induced apoptosis. Physical contact with EC is essential for protection to apoptosis. The insertion of a microporous membrane or blocking adhesion with anti-CD106 and anti-CD18 antibodies determined the complete abrogation of apoptosis protection. On the other hand, a reduction of apoptosis was measured in CLL cells cultured with conditioned medium collected from HC, implying that survival is partially mediated by soluble factors. Overall 1944 genes were modulated in CLL by co-culture. The EC contact seem to determine on CLL a kind of microenvironmental-driven angiogenic switch, improve the secretion of cytokines regulating tissue elements such as stromal cells and macrophages and also increase anti-apoptotic molecules. Our study support the line of evidence indicating endothelial cells as a major player in the CLL-infiltrated microenvironments are able to create a vicious cycle of cooperation that strongly sustains leukemic cell survival, protects CLL from drug-induced apoptosis and widely modifies CLL phenotype. Large-scale gene expression profiling (GEP) was performed on total RNA extracted from purified CD19+ cells isolated from 9 individual CLL patients which were separated in 3 experimental subsets: (i) freshly isolated cells (CLL baseline), (ii) CLL cells cultured in medium alone for 48 hours (CLL only) and (iii) CLL cells co-cultured 48h on HUVEC layer (CLL HC).

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Mixed-species RNAseq analysis of human lymphoma cell adhesion to mouse stromal cells identifies a core gene set that is also differentially expressed in the lymph node microenvironment of MCL and CLL patients.

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