*2.1. Proliferation Inhibition and Apoptosis Induction of Tumoral B-Cells*

Anti-cancer properties of fucoidan (mainly with extracted fucoidan of high molecular weight) were almost exclusively studied on solid tumors and emphasized anti-proliferative and pro-apoptotic events [32]. In order to confirm this role on lymphoma B-cells, we performed cell cycle analysis by flow cytometry on EBV latency III B-cells (three LCLs) and DLBCL cells (two ABCs: U2932 and OCILy10 and two GCBs: SUDHL4 and SUDHL6 cell lines) treated with native or vLMW-F F1 and F2. Estimation of cell population percentage in the different phases of the cell cycle highlighted significant decrease in S phase after treatment with vLMW-F in contrast to the native form (Figure 1A, B). This was consistent with inhibition of cell proliferation. A significant increase in subG1 peak on DNA content histograms (subsequent to fragmentation of nuclear DNA in late apoptosis) was also

sometimes observed, depending on cell types, for treatment by vLMW-F, especially F2 (Figure 1C).

**Figure 1.** vLMW-F decrease the percentage of LCLs and DLBCLs cells in S-phase. LCLs (J1209, C0401, C1504) and DLBCLs (U2932, OCILy10, SUDHL4, SUDHL6) cells were treated or not (control) with 100μg/mL of native fucoidan or vLMW-F (F1 and F2) for 48 h. Flow cytometry analysis was performed to determine cell-cycle distribution in all tested conditions. Results were obtained from three independent experiments. (**A**) Examples of cell cycle profiles for C0401 (LCL) and SUDHL4 (DLBCL). (**B**) Percentage of LCLs or DLBCLs cells in S-phase: fewer cells are in S-phase after 48 h treatment of 100 μg/mL vLMW-F in contrast with the native form and compared to the control. (**C**) Percentage of LCLs or DLBCLs cells in SubG1 phase: the increase in cell percentage in SubG1 phase occasionally observed suggests apoptosis induction. NS: not significant; \* *p*< 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.

To refine the apoptotic response, we evaluated percentage of apoptotic cells, from early to late stages, for the same cell lines and in the same treatment conditions. Annexin-V binding to externalized phosphatidylserine revealed apoptosis induction only with the two vLMW-F (Figure 2). The importance of the process depended on the cell lines (up to 50% for J1209 and SUDHL6) and was similar for the two fractions. Our results emphasized that, unlike the native form, vLMW-F possess anti-proliferative and pro-apoptotic properties for EBV latency III and DLBCL tumoral cells at the tested dose. This suggests a better

cytotoxic effect for vLMW-F. We verified that toxicity (viability and apoptosis) was due to the formulas and not L-Fucose since it is the smallest subunit of fucoidan skeleton (Figure S2).

**Figure 2.** vLMW-F induce apoptosis in LCLs and DLBCLs. LCLs (J1209, C0401, C1504) and DLBCLs (U2932, OCILy10, SUDHL4, SUDHL6) cells were treated or not (control) with 100μg/mL of native fucoidan or vLMW-F (F1 and F2) for 48 h, followed by apoptosis analysis (Annexin V/PI staining) by flow cytometry. Results were obtained from three independent experiments. (**A**) Examples of cell apoptosis for J1209 (LCL) and SUDHL6 (DLBCL) are shown (intact cells: green events–early apoptotic cells: blue events–late apoptotic cells: purple events). (**B**) Percentage of LCLs or DLBCLs total Annexin V+ cells. vLMW-F fractions induce similar apoptosis. NS: not significant; \* *p*< 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.
