B-CELL RECEPTOR SIGNALING PROFILES IN MANTLE CELL LYMPHOMA: A BARCODING AND PHOSPHO-SPECIFIC FLOW CYTOMETRY APPROACH

Mantle cell lymphoma (MCL) is an aggressive and incurable disease. B-cell receptor (BCR) signaling, constitutively activated in MCL, pro- motes tumor growth and is target of BTK inhibitors (BTKi). BTKi showed high response rates in relapsed/refractory (R/R) MCL but resis- tance inevitably emerges for reasons largely unknown. Recent evidence supports that resistance mechanisms can involve BCR signaling. With the aim to characterize BCR signaling profiles related to lymphoma drug responses in MCL, we used phospho-specific flow cytometry, which measures the phosphorylation status of intracellular signaling proteins at the single-cell level. To improve throughput capacity of this analysis, we combined phospho-specific flow cytometry with fluorescent cell bar- coding (FCB), a multiplexing technique. In FCB each sample is labeled with different fluorescent-dye concentrations obtaining a different sig- nature, or barcode, and then mixed with other samples before antibody staining and analysis by flow cytometry. We analyzed BCR phosphopro- teins (pERK1/2, pp38, pPLCγ2, and pNF-kB p65) in both MCL cell lines and peripheral blood cells from R/R MCL patients, in the basal condi- tions or following BCR or CXCR4 stimulation with anti-IgM or CXCL12, respectively. H2O2, which inhibits phosphatases, or phorbol myristate acetate (PMA) were used as control stimuli. First, we set the FCB: based on their barcode fluorescence we deconvoluted mixed sam- ples back to each individual condition. Then, we observed that in both basal and stimulated conditions phosphoproteins levels were heteroge- neous among MCL samples, with each sample having distinct respon- siveness profiles. Although further studies are needed to associate BCR signaling profiles to drug responsiveness, this study shows that phos- pho-specific flow cytometry combined with FCB is a robust and repro- ducible approach to characterize signaling profiles in complex cells populations such as those found in MCL. Importantly, FCB reduces an- tibody consumption and cells need, eliminating staining variability be- tween samples. These results form the basis for future studies aimed at generating maps of BCR signaling profiles related to lymphoma drug responses. The long-term goal is to define a predictive model based on BCR signaling features in MCL within MANTLE-FIRST BIO Project. We thank Fondazione Italiana Linfomi (PGR Ed. 2019) and the Platform of Flow Cytometry and Cellular Analysis (Verona University).