Figure 7.
ActD treatment promotes Trp53+/+;Eμ-Myc lymphomas regression in vivo. (A) C57BL/6J and C57BL/6J-Ly5.1 mice intravenously injected with 200 000 Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells, respectively, were treated with either ActD (0.1 mg/kg) or vehicle, starting 9 to 10 days after injection. Drug was delivered in 3 discontinuous cycles, with the dosing days indicated by the arrows, resuming at day 32 posttransplantation. Tumor burden was assessed by immunostaining, and flow cytometric analyses from peripheral blood and hematopoietic tissues were collected from 5 to 7 mice per group after the third administration (red arrow) as described in “Methods.” (B) Number of lymphoma cells in circulating blood from mice after 3 days of ActD or vehicle administration determined by flow cytometry as described in the legend for Figure 6D and calculated as detailed in “Methods.” ***P < .000 (2-way ANOVA test). Dotted line is set at y = 0. (C) Representative dot plots from peripheral blood of a vehicle-treated (top) and an ActD-treated (bottom) Trp53+/+;Eμ-Myc-bearing mouse from panel B showing the Trp53+/+;Eμ-Myc lymphoma cell population (GFP+B220+B220low) in orange. (D) Percentage of live and dead Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells (left) wt-B cells (center), and non-B cells (right panel), from the peripheral blood of mice analyzed in panel B. The proportion of dead cells from each population was determined as described in the legend for Figure 6D, with the markers identifying each population indicated on the y-axis of the corresponding graph. A, ActD; V, vehicle. ****P < .0001 (2-way ANOVA test). (E-F) Number of Trp53+/+;Eμ-Myc lymphoma cells (E) and white blood cells (WBCs) (F) contained in the spleen (Spl), bone marrow (BM), and thymus (Thy) of Trp53+/+;Eμ-Myc lymphoma-bearing mice after 3 days of ActD or vehicle administration compared with ActD- or vehicle-treated C57BL/6J control mice (n = 5 mice per group), determined by multicolor flow cytometry panel evaluation as detailed in supplemental Figure 7G. Data are presented as individual values with error bars displaying mean ± SEM. *P < .05; **P < .01; ****P < .0001. Statistical analyses for unpaired Student t test (E) and 1-way ANOVA test (F) were performed separately in each tissue. Dotted line (E) is set at y = 0. (G) Number of major immune cell populations contained in the spleen, bone marrow, thymus, and peripheral blood (PB) of Trp53+/+;Eμ-Myc lymphoma-bearing mice (n = 5-7 mice per treatment group) and of C57BL/6J control mice (n = 5 mice per treatment group) treated as in panel E. Box plots represent the median and the 95% CI for each cell population, as indicated on the x-axis. DC, dendritic cell; CD4, CD4+ T cells; CD8, CD8+ T cells; DN, double-negative; DP, double-positive; Eos, eosinophils; i, immature; iSP, immature single-positive thymocytes; m, mature; Mo/MФ, monocytes/macrophages; Neut, neutrophils; NK, natural killer; SP4, single-positive CD4 thymocytes; SP8, single-positive CD8 thymocytes. *P < .05; **P < .01; ***P < .001; ****P < .0001 (1-way ANOVA test performed separately for each population). (H) Photomicrographs (original magnification ×40) of hematoxylin- and eosin-stained paraffin-embedded sections of inguinal lymph nodes, liver, and lungs from representative C57BL/6J control and ActD-treated mice and from lymphoma-burdened sick control and ActD-treated Eμ-Myc mice (n = 3 mice per group) treated as in panel E. Al, alveoli; F, follicle; T, infiltrating tumor mass. (I) Kaplan-Meier survival curves of C57BL/6J and C57BL/6J-Ly5.1 mice harboring Trp53+/+;Eμ-Myc and Trp53–/–;Eμ-Myc lymphomas, respectively, and treated with ActD or vehicle (n = 9 mice per group) as indicated in panel A. Data are presented as mean ± SEM. ****P < .0001 (log-rank Mantel-Cox and Gehan-Breslow-Wilcoxon text).

ActD treatment promotes Trp53+/+;Eμ-Myc lymphomas regression in vivo. (A) C57BL/6J and C57BL/6J-Ly5.1 mice intravenously injected with 200 000 Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells, respectively, were treated with either ActD (0.1 mg/kg) or vehicle, starting 9 to 10 days after injection. Drug was delivered in 3 discontinuous cycles, with the dosing days indicated by the arrows, resuming at day 32 posttransplantation. Tumor burden was assessed by immunostaining, and flow cytometric analyses from peripheral blood and hematopoietic tissues were collected from 5 to 7 mice per group after the third administration (red arrow) as described in “Methods.” (B) Number of lymphoma cells in circulating blood from mice after 3 days of ActD or vehicle administration determined by flow cytometry as described in the legend for Figure 6D and calculated as detailed in “Methods.” ***P < .000 (2-way ANOVA test). Dotted line is set at y = 0. (C) Representative dot plots from peripheral blood of a vehicle-treated (top) and an ActD-treated (bottom) Trp53+/+;Eμ-Myc-bearing mouse from panel B showing the Trp53+/+;Eμ-Myc lymphoma cell population (GFP+B220+B220low) in orange. (D) Percentage of live and dead Trp53+/+;Eμ-Myc or Trp53–/–;Eμ-Myc lymphoma cells (left) wt-B cells (center), and non-B cells (right panel), from the peripheral blood of mice analyzed in panel B. The proportion of dead cells from each population was determined as described in the legend for Figure 6D, with the markers identifying each population indicated on the y-axis of the corresponding graph. A, ActD; V, vehicle. ****P < .0001 (2-way ANOVA test). (E-F) Number of Trp53+/+;Eμ-Myc lymphoma cells (E) and white blood cells (WBCs) (F) contained in the spleen (Spl), bone marrow (BM), and thymus (Thy) of Trp53+/+;Eμ-Myc lymphoma-bearing mice after 3 days of ActD or vehicle administration compared with ActD- or vehicle-treated C57BL/6J control mice (n = 5 mice per group), determined by multicolor flow cytometry panel evaluation as detailed in supplemental Figure 7G. Data are presented as individual values with error bars displaying mean ± SEM. *P < .05; **P < .01; ****P < .0001. Statistical analyses for unpaired Student t test (E) and 1-way ANOVA test (F) were performed separately in each tissue. Dotted line (E) is set at y = 0. (G) Number of major immune cell populations contained in the spleen, bone marrow, thymus, and peripheral blood (PB) of Trp53+/+;Eμ-Myc lymphoma-bearing mice (n = 5-7 mice per treatment group) and of C57BL/6J control mice (n = 5 mice per treatment group) treated as in panel E. Box plots represent the median and the 95% CI for each cell population, as indicated on the x-axis. DC, dendritic cell; CD4, CD4+ T cells; CD8, CD8+ T cells; DN, double-negative; DP, double-positive; Eos, eosinophils; i, immature; iSP, immature single-positive thymocytes; m, mature; Mo/MФ, monocytes/macrophages; Neut, neutrophils; NK, natural killer; SP4, single-positive CD4 thymocytes; SP8, single-positive CD8 thymocytes. *P < .05; **P < .01; ***P < .001; ****P < .0001 (1-way ANOVA test performed separately for each population). (H) Photomicrographs (original magnification ×40) of hematoxylin- and eosin-stained paraffin-embedded sections of inguinal lymph nodes, liver, and lungs from representative C57BL/6J control and ActD-treated mice and from lymphoma-burdened sick control and ActD-treated Eμ-Myc mice (n = 3 mice per group) treated as in panel E. Al, alveoli; F, follicle; T, infiltrating tumor mass. (I) Kaplan-Meier survival curves of C57BL/6J and C57BL/6J-Ly5.1 mice harboring Trp53+/+;Eμ-Myc and Trp53–/–;Eμ-Myc lymphomas, respectively, and treated with ActD or vehicle (n = 9 mice per group) as indicated in panel A. Data are presented as mean ± SEM. ****P < .0001 (log-rank Mantel-Cox and Gehan-Breslow-Wilcoxon text).

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