Page 132 - Haematologica-April 2018

A. Arvidsson et al.
ent in vitro systems reflect different aspects of microenvi-
ronment interactions.
Cell adhesion, migration and homing
Mantle cell lymphoma cell adhesion to stromal cells is associated with induction of genes involved in cellular adhesion and cell motility/migration. The extracellular matrix cluster (c6) consists entirely of matrisome proteins,33 where 9 encode matrisome core proteins (glycoproteins, collagens or proteoglycans) that constitute structural com- ponents of extracellular matrix. The remaining 29 genes encode matrisome-associated proteins, which modulate extracellular matrix function, including cellular adhesion. Other clusters contain structural components important for cell rigidity and motility such as ACTB, ACTG1 and TUBA4A. Additional cell-cell adhesion-related genes with significantly higher transcript levels in adherent MCL cells include ICAM1, ITGB2 and AMIGO2. These may be important for homing of MCL cells to microenvironmental niches and their retention in the niche.
The CXCR4 gene encoding a homing-related cytokine receptor is down-regulated in adherent cells, consistent with previous reports on microenvironment-associated downregulation of its mRNA and protein level in MCL and CLL patients as well as cell culture systems.17,30,34-36
The CXCR4 ligand, CXCL12, is expressed by the stro- mal cells and can promote adhesion of MCL cells to fibronectin and VCAM-1.17 Thus, this receptor-ligand pair could facilitate homing and subsequent adhesion of MCL cells to microenvironments.
Anti-apoptosis and cell survival
Anti-apoptosis clusters are up-regulated in adherent
cells and include genes involved in CD40 signaling (c1),
which is known to have an anti-apoptotic effect on MCL
cells.37 A second example (c5) includes upregulation of
BCL2-family members (eg. BCL2A1, BCL2L1, and MCL1)
as well as BCL2 that is up-regulated in the co-cultured
MCL cells relative to mono-cultured cells. Importantly,
increased level of alternative family members causes
resistance to the BCL2 inhibitor ABT-737 in CLL,38 thus
indicating their significance for designing therapies target-
ing the BCL2-family. Results expand previous knowledge
showing that stromal cell interaction protects MCL and
CLL cells from spontaneous and drug-induced apopto- sis.8,10, 11
Cell proliferation
The observed downregulation of early mitosis genes is consistent with previous observations showing cell cycle arrest in MCL and diffuse large B-cell lymphoma (DLBCL) cells upon interaction with stromal cells in vitro15 as well as lower response rates to cytostatic drugs observed for adherent lymphoma cells compared to cells in suspension.8 Reduced proliferation of MCL cells co-cul- tured with stromal cells lacking CD40L relative to cells expressing CD40L37 may indicate interaction partner-spe- cific differences in proliferative response. While adherent MCL cells up-regulate CD40, the CD40L was not detect- ed, consistent with downregulation of cell cycle genes in this system.
Immune cell recruitment
Adherent MCL cells up-regulate genes involved in B-cell receptor (BCR) downstream signaling, seen in BCR and
NF-κB gene signatures, including genes involved in immune-modulation via recruitment of immune cells, such as CCL3 and CCL4 that are known to be induced in adherent MCL cells.17,20 CCL3 and CCL4 attract activated T cells and monocytes, and CCL4 has been shown to attract regulatory T cells.39 T-cell infiltration has prognostic relevance in MCL40 and elevated serum levels of CCL3 and CCL4 are correlated with an inferior prognosis in DLBCL and CLL.41,42
A further example is the increased level of the immunoregulatory chemokine IL-10 in adherent cells, which permits autocrine survival signaling via the IL-10 receptor (IL10RA, 2.9-fold up-regulated in co-cultured cells) and STAT3 (cluster c17).43 BCR activation can induce such an autocrine survival loop in MCL which is attenuat- ed by the proteasome-inhibitor drug, bortezomib.44 Elevated serum levels of IL-10 are associated with poor disease outcome in DLBCL.45 Regulatory B cells common- ly express IL-10 and phenotypically-related CLL cells have been ascribed similar immunosuppressive attributes.46
The importance of BCR-signaling is supported by obser- vations that blocking BCR signaling by the BTK inhibitor ibrutinib (PCI-32765) both lowered plasma levels of CCL22, CCL4, TNF and IL-10 and caused MCL cells to leave their protective microenvironmental niches and enter the peripheral blood.17 BTK-dependent secretion of the affected cytokines was induced in vitro by co-culture with stromal cells or IgM stimulation. Reduced transcript and secreted level of IL-10 has also been observed in DLBCL cell lines treated with ibrutinib or the mTOR inhibitor, AZD2014.47
Ccl2, which could play a role in monocyte recruitment and polarization similar to that shown in follicular lym- phoma,48 and Ccl7, which promotes and directs the migra- tion of macrophages,49 were up-regulated in adherent stro- mal cells. Thus adherence of MCL cells to stromal cells appears to be sufficient to establish the cytokine produc- tion needed for recruitment of monocytic cells and T-cell subsets even in the absence of such cells.
Utility of the co-culture model and identification of a core set of microenvironment genes
The utility of the co-culture model system is supported by the systematic identification of functional themes that correspond well with current knowledge about how lymphomas in general and MCL in particular develop and survive. Relevance was further supported by overlap of the 1050 adherence-regulated genes with microenvi- ronment (lymph node) regulated genes in MCL and CLL patients.30,31 The overlap is similar in extent to the overlap between the MCL and CLL data, indicating that the in vitro model system reciprocates relevant aspects of microenvironment-mediated gene regulation in lym- phoma cells. The direction of regulation (up or down) was not conserved between the three datasets in 51 of the 116 genes that overlap between all three datasets, and, perhaps unsurprisingly, it is most often the in vitro data (43 of 116 genes) that differ from the in vivo studies. Interestingly, most of these genes are associated with the cell cycle, consistent with previous reports of cell cycle arrest in MCL and DLBCL cells adhering to stromal cells.15 The effect of cell adherence on the cell cycle may depend on specific properties of the interacting stromal cells, as discussed above in relation to expression of CD40L.37
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haematologica | 2018; 103(4)