Figure 6.
Activation of IGF1R signaling is associated with myeloid-mediated T-ALL survival. (A) Quantification of viable T-ALL cells and CD8+ T cells, isolated from the spleens of leukemic LN3 mice or tumor-free mice, respectively, 6 or 7 days after culture in the presence or absence of enriched splenic leukemia-associated myeloid cells. Bars represent means + standard error of the mean (SEM) of data compiled from 5 independent experiments with a distinct color-coded primary T-ALL; circles represent the average of 2 or 3 technical replicate wells per experiment. (B) Principal component analysis (PCA) of gene-expression profiles of T-ALL cells from the thymus or spleen of LN3 mice, as well as thymocytes or splenic CD8+ T cells from tumor-free mice. Symbols represent individual biologic replicates. (C) The top 10 pathways that were significantly enriched or depleted in splenic T-ALL cells relative to healthy CD8+ T cells were identified by gene set enrichment analysis, using BioCarta gene sets. (D) Variance-stabilized expression values of Igf1r from control T-lineage vs T-ALL cells from the thymus and spleen. Bars represent means + SEM; circles represent individual biologic replicates. (E) pIGF1R levels in LN3-transplanted T-ALL cells (CD45.2+CD5+) relative to host T cells (CD45.1+CD5+) from the same spleens were quantified by flow cytometry and are displayed as geometric mean fluorescence intensities (GMFI). Data are compiled from 3 independent experiments, each with a distinct color-coded primary T-ALL. Circles represent individual mice. (F) Schematic diagram for acute myeloid depletion in mice with established LN3 T-ALL to assess changes in activation of IGF1R and downstream signals. (G) Levels of pIGF1R and the downstream signaling molecules phosphorylated (p)AKT and pERK were quantified in T-ALL cells (upper panels) and host T cells (lower panels) following acute myeloid depletions, as in (F). GMFIs from clodlip-treated mice were normalized to the mean of control mice in each experiment. Bars represent means + SEM from 9 independent experiments, each with a distinct color-coded primary T-ALL. Circles represent individual mice. The red line indicates the mean of relative GMFIs in control mice. (H) Quantification of viable splenic T-ALL cells, isolated from the spleens of primary LN3 T-ALL–transplanted mice, 6 or 7 days after culture in the presence or absence of leukemia-associated splenic myeloid cells and in the presence or absence of exogenous IGF1 (100 ng/mL). Bars represent means + SEM of data compiled from 7 independent experiments with a distinct color-coded primary T-ALL; circles represent the average of 2 or 3 technical replicate wells per experiment. *P < .05, **P < .01, ***P < .001, repeated measures 2-way ANOVA with the Bonferroni correction (A,H), unpaired Student t test (D,G), paired Student t test (E). ns, not significant.

Activation of IGF1R signaling is associated with myeloid-mediated T-ALL survival. (A) Quantification of viable T-ALL cells and CD8+ T cells, isolated from the spleens of leukemic LN3 mice or tumor-free mice, respectively, 6 or 7 days after culture in the presence or absence of enriched splenic leukemia-associated myeloid cells. Bars represent means + standard error of the mean (SEM) of data compiled from 5 independent experiments with a distinct color-coded primary T-ALL; circles represent the average of 2 or 3 technical replicate wells per experiment. (B) Principal component analysis (PCA) of gene-expression profiles of T-ALL cells from the thymus or spleen of LN3 mice, as well as thymocytes or splenic CD8+ T cells from tumor-free mice. Symbols represent individual biologic replicates. (C) The top 10 pathways that were significantly enriched or depleted in splenic T-ALL cells relative to healthy CD8+ T cells were identified by gene set enrichment analysis, using BioCarta gene sets. (D) Variance-stabilized expression values of Igf1r from control T-lineage vs T-ALL cells from the thymus and spleen. Bars represent means + SEM; circles represent individual biologic replicates. (E) pIGF1R levels in LN3-transplanted T-ALL cells (CD45.2+CD5+) relative to host T cells (CD45.1+CD5+) from the same spleens were quantified by flow cytometry and are displayed as geometric mean fluorescence intensities (GMFI). Data are compiled from 3 independent experiments, each with a distinct color-coded primary T-ALL. Circles represent individual mice. (F) Schematic diagram for acute myeloid depletion in mice with established LN3 T-ALL to assess changes in activation of IGF1R and downstream signals. (G) Levels of pIGF1R and the downstream signaling molecules phosphorylated (p)AKT and pERK were quantified in T-ALL cells (upper panels) and host T cells (lower panels) following acute myeloid depletions, as in (F). GMFIs from clodlip-treated mice were normalized to the mean of control mice in each experiment. Bars represent means + SEM from 9 independent experiments, each with a distinct color-coded primary T-ALL. Circles represent individual mice. The red line indicates the mean of relative GMFIs in control mice. (H) Quantification of viable splenic T-ALL cells, isolated from the spleens of primary LN3 T-ALL–transplanted mice, 6 or 7 days after culture in the presence or absence of leukemia-associated splenic myeloid cells and in the presence or absence of exogenous IGF1 (100 ng/mL). Bars represent means + SEM of data compiled from 7 independent experiments with a distinct color-coded primary T-ALL; circles represent the average of 2 or 3 technical replicate wells per experiment. *P < .05, **P < .01, ***P < .001, repeated measures 2-way ANOVA with the Bonferroni correction (A,H), unpaired Student t test (D,G), paired Student t test (E). ns, not significant.

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