Figure 2

Effect of RTX and GA101 on MALC. (A) Pictures of treated or UT MALC were taken with an inverted Nikon Eclipse TE200 microscope at magnification × 40 at different times of culture with or without Hoeschst 33342 staining. These pictures are representative of several experiments (d=day). (B) MALC were treated or not (▪) with 10 μg/ml RTX (▴) or GA101 (●). MALC volume was measured at different times of culture. Results are expressed in mm³ and represent the mean±s.d. of nine independent experiments. Asterisk represents significant differences between UT and anti-CD20 MAb treatment (P<0.05); double asterisks represents significant differences between RTX and GA101 treatment (P<0.05); (d=day). (C) Number of viable cells per treated or UT MALC over time. Histograms represent mean±s.d. of nine independent experiments. Asterisk represents significant differences between UT and anti-CD20 MAb treatment (P<0.05); double asterisks represents significant differences between RTX and GA101 treatment (P<0.05); (d=day). (D) Left panel: necrosis was determined by flow cytometry analyzing Annexin V⁺/7AAD⁺ cells in MALC at 5, 10 and 20 days. Right panel: Ki67 immunohistochemistry labelling in MALC at day 10. (Ea) Fibronectin, laminin and vitronectin expression analyzed by western blotting in MALC compared with 2D RL cells. β-Actin expression was used as a control of protein expression. (Eb) Fibronectin and laminin labelling on paraffin sections of RL xenografts or lymph nodes isolated from FL patients. (Ec) Collagen I (yellow arrows) was visualized by second-harmonic generation in a lymph node from FL patients (left) and MALC (right). Results are representative of three independent experiments. (F) TV and tumor weight were measured after RL engraftment onto SCID-Beige mice. Histograms represent the mean±s.d. of 9 animals for PBS, 9 animals for RTX and 10 animals for the GA101-treated group. Double asterisks represents significant differences between RTX and GA101 treatment (P<0.05).