Figure 6.
Figure 6. Expression of the CXCL12/CXCR4 axis in vivo following PBMC transfer to SCID mice and effects of CXCL12 and CXCR4 neutralization in vivo. (A) The specificity of our primer pairs for human and murine CXCL12 was evaluated on human and murine samples. A band corresponding to human CXCL12 was detected only on human samples: HUVECs (lane 1), MRC-5 (lane 2), human microvascular endothelial cells (lane 3), and LCLs (lane 5) but not on murine tissues such as the peritoneal membrane (lane 4). Conversely, primers specific for murine CXCL12 gave a band only on murine tissues (lane 4). Lane 6 corresponds to the water control. (B) The expression of CXCL12 and CXCR4 in cells recovered from the peritoneal cavity of SCID mice injected with PBMCs from EBV-positive donors was evaluated by RT-PCR. The cells were analyzed before and after various time intervals following intraperitoneal injection for the expression of human CXCL12 (i), murine CXCL12 (ii), human CXCR4 (iii), and β-actin (iv). The first left lane corresponds to the profile obtained with freshly isolated PBMCs; the last lane corresponds to a hu/SCID tumor sample. (C) Expression of murine CXCL12 as evaluated by ELISA in the peritoneal washings obtained at various time intervals following PBMC transfer in SCID mice. (D-E) Effect of CXCL12 and CXCR4 neutralization on tumor growth. Freshly isolated hu/SCID tumor cell suspensions were injected intraperitoneally into naive SCID mice. (D) Mice were treated with intraperitoneal injections of goat anti-CXCL12 Ab, PBS, or heat-inactivated goat preimmune serum every day for 3 weeks starting from the day after cell transfer. The effect of anti-CXCL12 Ab and treatment on lymphoma development was assessed by effects on survival. Five to 8 mice were included in each experimental group, and the experiment was repeated twice. (E) In another group of animals following PBMC transfer, Alzet pumps releasing the CXCR4 antagonist 4F-benzoyl-TN14003 were implanted subcutaneously and changed every 2 weeks for a total of 2 implants. The effect of treatment with 4F-benzoyl-TN14003 on lymphoma development was assessed by effects on survival and tumor dissemination. Six to 8 mice were included in each experimental group.

Expression of the CXCL12/CXCR4 axis in vivo following PBMC transfer to SCID mice and effects of CXCL12 and CXCR4 neutralization in vivo. (A) The specificity of our primer pairs for human and murine CXCL12 was evaluated on human and murine samples. A band corresponding to human CXCL12 was detected only on human samples: HUVECs (lane 1), MRC-5 (lane 2), human microvascular endothelial cells (lane 3), and LCLs (lane 5) but not on murine tissues such as the peritoneal membrane (lane 4). Conversely, primers specific for murine CXCL12 gave a band only on murine tissues (lane 4). Lane 6 corresponds to the water control. (B) The expression of CXCL12 and CXCR4 in cells recovered from the peritoneal cavity of SCID mice injected with PBMCs from EBV-positive donors was evaluated by RT-PCR. The cells were analyzed before and after various time intervals following intraperitoneal injection for the expression of human CXCL12 (i), murine CXCL12 (ii), human CXCR4 (iii), and β-actin (iv). The first left lane corresponds to the profile obtained with freshly isolated PBMCs; the last lane corresponds to a hu/SCID tumor sample. (C) Expression of murine CXCL12 as evaluated by ELISA in the peritoneal washings obtained at various time intervals following PBMC transfer in SCID mice. (D-E) Effect of CXCL12 and CXCR4 neutralization on tumor growth. Freshly isolated hu/SCID tumor cell suspensions were injected intraperitoneally into naive SCID mice. (D) Mice were treated with intraperitoneal injections of goat anti-CXCL12 Ab, PBS, or heat-inactivated goat preimmune serum every day for 3 weeks starting from the day after cell transfer. The effect of anti-CXCL12 Ab and treatment on lymphoma development was assessed by effects on survival. Five to 8 mice were included in each experimental group, and the experiment was repeated twice. (E) In another group of animals following PBMC transfer, Alzet pumps releasing the CXCR4 antagonist 4F-benzoyl-TN14003 were implanted subcutaneously and changed every 2 weeks for a total of 2 implants. The effect of treatment with 4F-benzoyl-TN14003 on lymphoma development was assessed by effects on survival and tumor dissemination. Six to 8 mice were included in each experimental group.

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