Characterization of U2932 cell line subpopulations and evaluation of their sensibility to a chemotherapeutic drug.
Author
Miranda Pinheiro, Alana
Term
4. term
Publication year
2018
Submitted on
2018-05-31
Pages
105
Abstract
Diffust storcellet B-cellelymfom (DLBCL) er en aggressiv blodkræft i B-celler. Sygdommen inddeles ofte i ABC- og GCB-undergrupper afhængigt af, hvor i B-cellers modningsproces kræftcellerne befinder sig. ABC-typen er typisk forbundet med dårligere prognose og større modstand mod standardbehandlingen R-CHOP. Kræftceller er samtidig ofte heterogene og plastiske, hvilket kan fremme sygdomsudvikling og behandlingsresistens. I denne undersøgelse fokuserede vi på U2932, en ABC-lignende DLBCL-cellelinje. Vi formodede, at den rummer flere subpopulationer af celler i forskellige B-celle-stadier med forskellige lægemiddelfølsomheder. For at teste dette kortlagde vi cellernes overflademarkører og genaktivitet (ved flowcytometri og mikroarrays) og målte deres følsomhed over for doxorubicin, et centralt lægemiddel i R-CHOP. Vi fandt, at der sameksisterer flere subpopulationer med særskilte fænotyper og genetiske ændringer. To stabile grupper dominerede i den første måned: P4 (høj/positiv for CD38 og CD20) og P5 (lav/negativ for CD38 og CD20). Genekspressionsmønstre på dag 36 tydede på, at P4 lignede ekstratfollikulære plasmaceller (PRDM1/Blimp-1 lav, XBP1s lav, PAX5 høj, BCL6 høj, IRF4 positiv), mens P5 lignede germinalcenter-plasmablast/plasmacelle (PRDM1/Blimp-1 positiv, XBP1s positiv, PAX5 høj, BCL6 intermediær, IRF4 positiv) og udtrykte EBV. Genekspressionsklassifikatorer (HemaClass BAGS og REGS) understøttede ABC-lignende/plasmablast-relaterede kendetegn og indikerede doxorubicinresistens på tværs af normaliseringskohorter. Funktionelt prolifererede P4 mindre og var mere resistent over for doxorubicin end de øvrige subpopulationer og moderlinjen. Forskelle mellem P4 og P5 omfattede aktivering af HIF-signalvejen (hypoxi-inducerbar faktor) i P5 og stærk CD20-eksponering i P4. Pathway-berigelse pegede desuden på immunprocesser, som ofte ses ved autoimmunitet eller infektion. Over tid ændrede U2932-subpopulationerne sig. På dag 107 viste de samlet en umoden B-celle-lignende fænotype (CD19 positiv; CD38 variabel; CD10 og IgM positive; CD5 og CD27 negative), forenelig med dedifferentiering. Samtidigt udviste P3 og P4 heterogent CD43-udtryk, hvilket tyder på, at cellerne stadig var i overgang. Efter dag 100 havde alle subpopulationer lignende resistens over for doxorubicin. CD19 var overvejende positiv, men en lille andel i hver gruppe havde lavt CD19, hvilket kan afspejle receptor-internalisering eller progression i B-celledifferentieringen. Samlet viser resultaterne, at U2932 består af flere, skiftende B-celle-tilstande med forskellige lægemiddelsvar, hvilket understreger, hvordan cellulær plasticitet i ABC-lignende DLBCL kan bidrage til behandlingsresistens.
Diffuse large B‑cell lymphoma (DLBCL) is an aggressive blood cancer of B cells. It is often divided into ABC and GCB subtypes based on where the cancer cells sit along the B‑cell maturation process. The ABC subtype is typically linked to poorer outcomes and greater resistance to the standard R‑CHOP regimen. Cancer cells are also frequently heterogeneous and plastic, which can promote disease progression and drug resistance. This study examined U2932, an ABC‑like DLBCL cell line. We hypothesized that it contains multiple subpopulations at different B‑cell differentiation states with distinct drug sensitivities. To test this, we profiled cell‑surface markers and gene activity (using flow cytometry and microarrays) and measured sensitivity to doxorubicin, a key R‑CHOP drug. We confirmed coexisting subpopulations with distinct phenotypes and genetic alterations. Two stable groups dominated during the first month: P4 (high/positive for CD38 and CD20) and P5 (low/negative for CD38 and CD20). Gene‑expression patterns at day 36 suggested that P4 resembled extrafollicular plasma cells (PRDM1/Blimp‑1 low, XBP1s low, PAX5 high, BCL6 high, IRF4 positive), while P5 resembled germinal center plasmablast/plasma cells (PRDM1/Blimp‑1 positive, XBP1s positive, PAX5 high, BCL6 intermediate, IRF4 positive) and showed EBV expression. Gene‑expression classifiers (HemaClass BAGS and REGS) supported ABC‑like, plasmablast‑related features and indicated doxorubicin resistance across normalization cohorts. Functionally, P4 proliferated less and was more resistant to doxorubicin than the other subpopulations and the parental line. Differences between P4 and P5 included activation of the hypoxia‑inducible factor (HIF) pathway in P5 and strong CD20 expression in P4. Pathway enrichment also pointed to immune processes often seen in autoimmunity or infection. Over time, U2932 subpopulations shifted. By day 107, they shared an immature B‑cell‑like phenotype (CD19 positive; CD38 variable; CD10 and IgM positive; CD5 and CD27 negative), consistent with dedifferentiation. At the same time, P3 and P4 showed heterogeneous CD43 expression, indicating cells were still in transition. After day 100, all subpopulations showed similar doxorubicin resistance. CD19 remained mostly positive, though a small fraction in each group had low CD19, suggesting receptor internalization or movement along the B‑cell maturation pathway. Together, these findings show that U2932 comprises multiple, shifting B‑cell states with distinct drug responses, underscoring how cellular plasticity in ABC‑like DLBCL can support treatment resistance.
[This abstract was generated with the help of AI]
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