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CXCR4-targeted nanocarrier to DLBCL cells
Statistical analysis
In vitro experiments were performed in biological triplicates while in vivo experiments were performed in triplicates/quadrupli- cates. The data for all experiments were reported as mean ±Standard Error of Mean (SEM). All results were analyzed using the Student t-test. P<0.05 was considered statistically significant. Statistical calculations were performed using SPSS software ver- sion.21
Results
CXCR4-dependent internalization of T22-GFP-H6 in human CXCR4+ diffuse large B-cell lymphoma cell lines CXCR4 membrane levels were evaluated in four human DLBCL cell lines by flow cytometry (Figure 1A) and IHC (Online Supplementary Figure S1). CXCR4 expression was highest in Toledo cells, followed by U-2932 and RIVA, whereas CXCR4 expression in the SUDHL-2 cell line was undetectable. CXCR4-transfected SUDHL-2 cells
(CXCR4+ SUDHL-2) showed average CXCR4 levels. T22-GFP-H6 nanocarrier internalization correlated with CXCR4 expression. Thus, T22-GFP-H6 internalized the
most in Toledo cells, followed by U-2932 and RIVA, whereas it did not internalize in SUDHL-2 (Figure 1B). Moreover, T22-GFP-H6 nanocarrier internalization was CXCR4-dependent. So, after preincubation with CXCR4 antagonist AMD3100, T22-GFP-H6 internalization decreased significantly in Toledo, U-2932 and RIVA cells (Figure 1C). As expected, T22-GFP-H6 did not internalize in SUDHL-2 cells (only background FLI was detected), whereas high internalization was registered in CXCR4+ SUDHL-2 cells. Similarly, AMD3100 preincubation had no effect on nanocarrier internalization in SUDHL-2 cells but led to a significant decrease in CXCR4+ SUDHL-2 cells (Figure 1D). Thus, we showed specific in vitro entry of T22-GFP-H6 into CXCR4+ DLBCL cells through the CXCR4 receptor.
Non-cytotoxic effect of T22-GFP-H6 in diffuse large B-cell lymphoma cell lines in vitro
After exposure to T22-GFP-H6 (50-500nM range), cell viability for all four evaluated DLBCL cell lines was approximately or above 100% (Figure 1E). Therefore, T22-GFP-H6 nanocarrier has no in vitro antineoplastic effect against these DLBCL cell lines.
A
B
CDE
Figure 1. In vitro T22-GFP-H6 nanocarrier internalization in CXCR4+ diffuse large B-cell lymphoma (DLBCL) cell lines and its dependence on the CXCR4 receptor. (A) CXCR4 membrane expression of different DLBCL cell lines (Toledo, U-2932, RIVA and SUDHL-2) and the SUDHL-2 cell line transfected with a CXCR4 plasmid (CXCR4+ SUDHL-2) measured by flow cytometry. (B) Levels of intracellular fluorescence quantified by flow cytometry in Toledo, U-2932, RIVA and SUDHL-2 cells after 1 hour (h) exposure to T22-GFP-H6 nanocarrier at different concentrations (range: 0.1nM-250nM). (C) T22-GFP-H6 internalization, measured by flow cytometry, in Toledo, U-2932 and RIVA cells after 1h pretreatment with the antagonist AMD3100 (50nM T22-GFP-H6:500nM AMD3100). (D) Competition assays with AMD3100 (250nM T22-GFP-H6: 2500nM AMD3100) in SUDHL-2 cells and CXCR4+ SUDHL-2 cells. (E) Lack of cytotoxicity (measured as percentage of cell viability) after 48h exposure to high concentrations of T22-GFP-H6 nanocarrier (range: 50nM-500nM) in Toledo, U-2932, RIVA and SUDHL-2 cells. *P<0.05; **P<0.01; ***P<0.001; ns: non-significant; MFI: mean fluorescence intensity.
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