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gene expression between these subsets and in relation to
mono-cultured cells.
Adhesion to stromal cells affects gene expression in MCL cells
Physical separation of adherent MCL cells from stromal cells in co-cultures was avoided by direct RNA isolation from the mixed cell population in order to minimize sep- aration-associated artifacts in the RNAseq data. The mixed-species short RNAseq reads were separated in silico by species-specific read separation using reference genomes for human (MCL cells) and mouse (stromal cells). Read counts per sample prior to and following species- specific read separation are presented in Online Supplementary Table S1.
The experimental design allowed for global transcript level comparisons between three distinct MCL fractions: mono-cultured MCL cells (SEP), adherent MCL cells in co- culture (ADH), and suspension MCL cells in co-culture (SUSP) (Figure 1B). Pairwise comparisons of transcript lev- els for the three MCL fractions identified a total 3697 genes that are differentially expressed in at least one com- parison after 24 h mono-/co-culture (FDR q-value ≤0.05). The heat map in Figure 1C shows relative transcript levels across the three cell populations. Overall, the largest dif- ference is between the ADH fraction and the suspension cells in mono-culture (SEP) and co-culture (SUSP), which while distinct, are more similar to each other (column den- drogram, Figure 1C).
The Venn diagram sets in Figure 1D show the number of genes that are differentially expressed between each pair of MCL cell populations: 3453, 1471 and 1050 genes were differentially expressed in the respective compar- isons of ADH relative to SEP (ADH/SEP), SUSP relative to SEP (SUSP/SEP) and ADH relative to SUSP (ADH/SUSP). The three comparison groups of differentially regulated genes are highly overlapping with half the genes occurring in two or more groups (Figure 1D). Given the previously reported survival advantage conferred to lymphoma cells adhering to stromal cells in co-culture,6,8 and as this com- parison provides the best opportunity for specifically understanding molecular aspects associated with adhesion to stromal cells, we focused the analysis on the 1050 genes with altered transcript level between the two MCL cell fractions within the co-culture system (ADH/SUSP). Altogether, 137 of these genes were changed more than 2- fold at the transcript level (Figure 1E). Significantly changed genes in the ADH fraction with the highest fold change are presented in Table 1 (Complete lists of signifi- cantly changed genes are available in Online Supplementary Tables S2-S4).
Gene expression and co-culture dependent changes in stromal cells
The study design did not allow for an in-depth com- parison of transcript levels in stromal cells analogous to the analysis of cancer cells above. Thus a detailed analy- sis of differentially expressed stromal cell genes is not reported here. Nonetheless, 100 genes were significantly changed at the transcript level between mono- and co- cultured stromal cells (Figure 2). These include the chemotactic molecules Ccl2 and Ccl7, both with higher transcript levels in the co-cultured MS-5 cells. These can interact with the CCR2 cytokine receptor, which is expressed in Jeko-1 MCL cells. A complete list with sig-
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B
Figure 2. Significant transcript level changes between mono- and co-cultured stromal cells. (A) Heatmap representation of the 100 genes with significant transcript level changes between co-cultured (COCULT) and mono-cultured (SEP) and MS-5 stromal cells (FDR q-value ≤0.05) after hierarchical clustering. (B) Ranked fold changes for the 100 genes with altered transcript levels between COCULT and SEP MS-5 stromal cell fractions.
haematologica | 2018; 103(4)