P-163: Study of cellular senescence in the bone marrow of Multiple Myeloma patients during disease progression

Introduction: Multiple myeloma (MM) is preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (sMM), which are characterized by significantly heterogeneous biological and clinical features. MM cells accumulate in the bone marrow (BM) during myelomagenesis, where they interact with various cell subsets, which support their growth and proliferation. Nevertheless, the exact mechanisms leading to overt disease remain unknown. We evaluated potential changes in the BM niche during progression from MGUS to sMM and MM, focusing on the levels of cellular senescence, reported to play an active role in tumor development and cancer cell proliferation.

Methods: GLF-16, a fluorescent analogue of the dye Sudan Black B which binds to the characteristic senescence biomarker lipofuscin, was used to detect senescent cells both with flow cytometry and fluorescence microscopy. The NCI-H929 and L-363 MM cell lines were driven to senescence with various concentrations (6.25-100 μM) of hydrogen peroxide (H₂O₂) for 24, 48 and 72 h. Primary BM samples of patients with MGUS, sMM and MM were analysed with the next-generation flow (NGF) cytometry panels together with GLF-16, to evaluate the levels of senescence in normal and clonal plasma cells and in distinct BM cell subsets.

Results: Exposure of both MM cell lines to H₂O₂ increased the levels of GLF-16 positive (+) cells in a dose- and time-dependent manner. The optimal H₂O₂ concentration for inducing maximum cellular senescence and minimum cell apoptosis was 25 μΜ, leading to 8.5%, 15% and 30% GLF-16+ cells at 24, 48 and 72 h after H₂O₂ treatment, respectively. Fluorescence microscopy verified the changes in GLF-16+ cell size and the percentages of senescent cells. Primary BM MM cells stained with GLF-16 showed increased expression of the dye compared with BM T cells, mature B cells, NK cells, monocytes and neutrophils, all of which had similar levels of GLF-16+ cells and were used as internal baseline controls. Of note, B cell precursors in all BM samples analyzed, marginally expressed GLF-16 and thus, were used as an internal negative control. Aberrant plasma cells showed a relevant 1.4-1.6-fold increase in the mean fluorescent intensity (MFI) of GLF-16 compared with their normal plasma cell counterparts for all samples tested. No differences were observed in the levels of senescent aberrant plasma cells between MGUS, sMM and MM, although more samples need to be analyzed to highlight alterations between the three groups.

Conclusions: Senescence has been associated with cancer development, therefore its study and possible implication in MM progression is of great interest. The dye GLF-16 stains senescent clonal plasma cells with high specificity and our preliminary results show that primary BM clonal cells from patients with MM display increased senescence, an observation which can be eventually of clinical significance.