• Alana Miranda Pinheiro
4. semester, Medicin med Industriel Specialisering, Kandidat (Kandidatuddannelse)
DLBCL presents a variety of mutations throughout the B cell differentiation process. Genetic alteration at specific B cells state classify this tumor as ABC or GCB subtype. DLBCL subtypes demonstrate straight relation with patients’ prognostic and overall survival where ABC is considered the most aggressive and with elevated resistance level to R- CHOP regimen. Recently, two subpopulations demonstrating different and steady immunophenotype for 35 days combined with altered expression of genes regulating the B cell differentiation were identified on U2932 cell line, an ABC- like DLBCL. Elevated heterogeneity and plasticity is a marked characteristic of malignant cell in a carcinogenic environment favoring the disease progression, drug resistance and culminating into metastasis. Thus, here we hypothesis that U2932 is a highly heterogenous cell line composed of different subpopulations at distinct B cell states of differentiation marked by a different phenotype and drug resistance. Aiming to confirm or deny this assumption, we characterized U2932 cell line and its subpopulations immunophenotype, transcriptional profiling and sensibility to doxorubicin. Flow cytometry analyses demonstrate the coexistence of different subpopulations on U2932 cell line, each of them marked by a specific phenotype and genetic alterations. Due to their consistent expression during the first month of analyses, P4, expressing CD38/ CD20- high, and P5, expressing CD38/ CD20- low, are the main subpopulations analyses in this study. To precisely determine the state of differentiation and molecularly comprehend the proliferative and drug sensibility features of each subpopulation, gene expression and pathways enrichment was determined by microarrays analyses at day 36. For P4, the phenotype resembled extrafollicular plasma cells demonstrating PRDM1/Blimp-1-low, XBP1s-low, CD38/CD20 -positive, PAX5-high, BCL6-high and IRF4-positive expression. On the other hand, P5 phenotype resembled germinal center plasmablast/plasma cell, demonstrating PRDM1/Blimp-1- positive, XBP1s- positive, CD38/CD20 -negative, PAX5-high, BCL6- intermediate, IRF4- positive and EBV expression. This data is supported by hemaclass BAGS classification of P4 and P5 as plasmablast when only normalized with 17DLBCL + 6U2932, and the ABC classification for all cohorts utilized for normalization. In addition, P4 showed lower proliferation and higher resistance to doxorubicin compared to the other subpopulations and original cell line. This data is associated with the up regulation of HIF- pathway for P5 resulting from P4 vs P5 pathway enrichment and the highly positive expression of CD20 for P4. Accordingly, the hemaclass REGS classification of U2932, P4 and P5 as doxorubicin resistant are common for distinct normalization cohorts. Autoimmune disturbances and/or infectious conditions are common pathways demonstrated between U2932 vs P4 and P4 vs P5 enrichment. Finally, all U2932 subpopulations demonstrate immature B cell like phenotype marked by CD19+, CD38+/-, CD10+, IgM+, CD5- and CD27- at day 107, indicating a process of dedifferentiation. At the same date, P3 an P4 demonstrate an heterogeneous expression for CD43 suggesting that the malignant cells are still in a transiting state. After day 100, all subpopulations demonstrated similar resistance to doxorubicin in accordance with the common phenotype. In all cases, CD19 expression was overall positive even though a minor fraction of all subpopulations demonstrated low expression of this marker, indicating a process of internalization or the progression of the cells in the B cell differentiation.
Udgivelsesdato31 maj 2018
Antal sider105
ID: 280136719