Different Sources of Stromal Cells Diversely Affect Survival and Trafficking of Mantle Cell Lymphoma Cells

Introduction: Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell malignancy characterized by dissemination of lymphoma cells in different compartments, mainly lymph nodes (LN) and bone marrow (BM). The interaction between lymphoma cells and their tumor microenvironment (TME) is known to have an impact on malignant cell behavior. The lymphoma-stroma cross-talk is found to increase cell survival, dissemination and resistance to anti-cancer therapy, but these effects may vary between different TME niches. Several novel drugs are reported to be highly efficient in MCL therapy, inhibiting lymphoma cell proliferation, adhesion and supporting anti-cancer immune attacks. However, the pro-tumor effect of specific TME niches is unknown. The current study aimed to explore in vitro the interaction between MCL cells and stroma cells derived from different sources, focusing on their effects on lymphoma cell survival, trafficking and response to ibrutinib.

Methods: Stroma cells (HS-5; BM cell line, HK; LN-derived cells) were cultured to obtain full confluence. MCL (Mino; ATCC) cells were added and the co-culture was treated with ibrutinib (50nM) for 72 hrs. The expression levels of the SDF1-chemokine receptor, CXCR4, on MCL cells was analyzed by FACS. MCL cell apoptosis was assessed using annexin V/propidium iodide staining after co-culture with stroma cells and exposure to toxic doses of ibrutinib for 48 hrs. Cytokine secretion was quantified using the Human Cytokine Array G5 (RayBio). For migration capacity evaluation, MCL cells, pretreated with ibrutinib, were loaded on the upper chamber of a transwell insert and allowed to migrate through stroma towards SDF-1. The chemotactic potential of stromal conditioned medium was analyzed by evaluating migration of MCL cells pretreated with CXCR4 antagonist AMD3100 towards this medium.

Results: Co-culture of MCL cells with stroma cells increased CXCR4 expression on lymphoma cells in a stroma source-dependent manner. This increase was higher in the presence of BM stroma (9 fold) compared to LN stroma (6 fold). Ibrutinib induced mild CXCR4 up-regulation (1.3 fold) on MCL cells co-cultured with BM-derived cells, while this treatment down-regulated (0.8 fold) the CXCR4 expression on MCL cells co-cultured with LN stroma. MCL cell migration through the stromal barrier towards SDF-1 increased by 20 fold in the BM stroma and only by 5 fold in the LN stroma compared to non-adherent conditions. MCL cells pretreated with ibrutinib demonstrated decreased migration through the BM stromal barrier compared to untreated cells (0.65 fold), while in the LN stromal barrier, the MCL cell migration was increased by 2 fold relative to control. In comparison to serum free medium, conditioned medium obtained from LN stroma facilitated extensive MCL cell migration that was impaired after AMD3100 treatment. In contrast, BM-derived conditioned medium barely promoted lymphoma cell migration, with no effect of AMD3100. The protecting effect of TME on ibrutinib-induced cell apoptosis depended on the TME source. Co-culture of MCL cells with BM and LN stroma resulted in a significant increase in lymphoma cell survival, which was superior in the BM stroma (50% vs. 30%). Resistance to ibrutinib was also observed under non-contact conditions, suggesting the involvement of stroma secreted factors. Consistent with these findings, following co-culture of MCL cells with BM stroma, the secretion of pro-survival cytokines IL-6, IL-8, VEGF and the chemokine RANTES/CCL5 was significantly augmented compared to co-culture with LN stroma.

Conclusions: Our results suggest that the critical role of TME in supporting MCL cell survival and dissemination is context-dependent. LN stroma could secrete higher SDF1 levels and therefore enhance migration that may

be mediated in part by CXCR4-CXCL12 axis. In contrast, in contact conditions, BM stroma induces higher expression of CXCR4 on MCL cell surface, which enables better migration through this stroma. The anti-apoptotic effect of TME is enhanced in the BM-derived stroma compared to the LN cells, which could be related to different secretion of pro-survival cytokines. MCL response to ibrutinib varies between surrounding milieus. Therefore, the therapy should not only target tumor cell-specific mechanisms, but should also be adjusted to particular TME niches. These findings need to be further corroborated in in vivo experiments.