BM-MSCs mediate malignant B-cell migration and adhesion. (A) Migration assay. Primary LN samples from patients with FL were stained with anti-CD19 mAb and subjected to the chemotaxis assay for migration in response to supernatants from BM-MSCs treated or not with TNF/LT for 3 days. Migration index is calculated as the number of viable CD19⁺TOPRO-3⁻ FL B cells migrating in response to the BM-MSC supernatant divided by the number of viable CD19⁺TOPRO-3⁻ FL B cells migrating in response to culture medium. Results are the mean ± SD from 3 FL patients. (B) Role of CXCL12 in malignant B-cell migration. BL2 cell line was subjected to the chemotaxis assay in response to CXCL12 or to supernatants from BM-MSCs treated or not with TNF/LT for 3 days. When indicated, BL2 was preincubated with the CXCR4 antagonist AMD3100. Results are expressed as the migration index corresponding to the number of cells migrating in response to the chemokine or to the BM-MSC supernatant divided by the number of cells migrating in response to appropriate control medium. Results represent the mean values ± SD from 3 independent experiments. *Mean value is statistically significantly different from that obtained without AMD3100 (P<.05). (C) Adhesion assay. Primary unpurified FL samples were incubated for 2 hours with BM-MSCs pretreated or not with TNF/LT for 7 days. Unbound cells were then removed and adherent cells, including CD19⁺CD45⁺ malignant B cells, CD19⁻CD45⁺ nonmalignant hematopoietic cells, and CD45⁻ stromal cells were collected using trypsin and quantified by flow cytometry. Results are the mean values of the ratio of CD19⁺ to CD45⁻ cells (n = 4). The error bars indicate the SD of the mean. *Mean value is statistically different from that of coculture with unstimulated stromal cells (P<.05).