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Modeling tumor-host interactions of chronic lymphocytic leukemia in xenografted mice to study tumor biology and evaluate targeted therapy.

Herman SE, Sun X, McAuley EM, Hsieh MM, Pittaluga S, Raffeld M, Liu D, Keyvanfar K, Chapman CM, Chen J, Buggy JJ, Aue G, Tisdale JF, Pérez-Galán P, Wiestner A - Leukemia (2013)

Bottom Line: We found that the murine spleen (SP) microenvironment supported CLL cell proliferation and activation to a similar degree than the human LN, including induction of BCR and NF-κB signaling in the xenografted cells.Next, we used this model to study ibrutinib, a Bruton's tyrosine kinase inhibitor in clinical development.Ibrutinib inhibited BCR and NF-κB signaling induced by the microenvironment, decreased proliferation, induced apoptosis and reduced the tumor burden in vivo.

View Article: PubMed Central - PubMed

Affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

ABSTRACT
Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, the B-cell receptor (BCR) and nuclear factor- κB (NF-κB) pathways are activated in the lymph node (LN) microenvironment. Thus, model systems mimicking tumor-host interactions are important tools to study CLL biology and pathogenesis. We investigated whether the recently established NOD/scid/γc() (NSG) mouse xenograft model can recapitulate the effects of the human microenvironment. We assessed, therefore, tumor characteristics previously defined in LN-resident CLL cells, including proliferation, and activation of the BCR and NF-κB pathways. We found that the murine spleen (SP) microenvironment supported CLL cell proliferation and activation to a similar degree than the human LN, including induction of BCR and NF-κB signaling in the xenografted cells. Next, we used this model to study ibrutinib, a Bruton's tyrosine kinase inhibitor in clinical development. Ibrutinib inhibited BCR and NF-κB signaling induced by the microenvironment, decreased proliferation, induced apoptosis and reduced the tumor burden in vivo. Thus, our data demonstrate that the SP of xenografted NSG mice can, in part, recapitulate the role of the human LN for CLL cells. In addition, we show that ibrutinib effectively disrupts tumor-host interactions essential for CLL cell proliferation and survival in vivo.

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Ibrutinib inhibits CLL but not T-cell proliferation in vivo. (a) NSG mice (n=24) injected with CFSE labeled PBMCs from six patients were treated with vehicle (control) or ibrutinib and sacrificed 3-4 weeks later. Symbols identify patients (Table 1), and each data point represents one mouse. The percentage of CFSE low cells (cells having completed cell division) in PB and spleen (SP) for each treatment group is shown for CLL cells (left panel) and T-cells (right panel). In one patient (coded by open circles) the T-cell count in the PB was too low for analysis. (b) Overlay histogram showing decreased expression of Ki67 in CLL cells from the mouse spleen of treated as compared to untreated mice at time of sacrifice, 3-4 weeks post xenograft. (c) NSG mice (n=22) injected with unlabeled (no CFSE) PBMCs from five patients were treated with vehicle or ibrutinib and sacrificed 3-4 weeks later. Ibrutinib significantly decreased the percentage of Ki67+ CLL cells in PB and spleen (SP). The multivariable analysis used to test for significance in all panels is described in Materials and Methods. (d) The percentage of Ki67+ CLL cells is decreased by ibrutinib in all patients studied. Shown is the mean (± SEM) percentage Ki67+ CLL cells in PB (left panel) and spleen (right panel) of 2-3 mice in each treatment group injected with cells from the five different patients.
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Figure 6: Ibrutinib inhibits CLL but not T-cell proliferation in vivo. (a) NSG mice (n=24) injected with CFSE labeled PBMCs from six patients were treated with vehicle (control) or ibrutinib and sacrificed 3-4 weeks later. Symbols identify patients (Table 1), and each data point represents one mouse. The percentage of CFSE low cells (cells having completed cell division) in PB and spleen (SP) for each treatment group is shown for CLL cells (left panel) and T-cells (right panel). In one patient (coded by open circles) the T-cell count in the PB was too low for analysis. (b) Overlay histogram showing decreased expression of Ki67 in CLL cells from the mouse spleen of treated as compared to untreated mice at time of sacrifice, 3-4 weeks post xenograft. (c) NSG mice (n=22) injected with unlabeled (no CFSE) PBMCs from five patients were treated with vehicle or ibrutinib and sacrificed 3-4 weeks later. Ibrutinib significantly decreased the percentage of Ki67+ CLL cells in PB and spleen (SP). The multivariable analysis used to test for significance in all panels is described in Materials and Methods. (d) The percentage of Ki67+ CLL cells is decreased by ibrutinib in all patients studied. Shown is the mean (± SEM) percentage Ki67+ CLL cells in PB (left panel) and spleen (right panel) of 2-3 mice in each treatment group injected with cells from the five different patients.

Mentions: Finally, we determined the effect of ibrutinib on tumor proliferation. Ibrutinib decreased CLL cell proliferation measured using CFSE dilution by >80% compared to controls (Figure 6a and Supplementary Figure S6a; P<.001). In contrast, ibrutinib had no effect on T-cell proliferation (Figure 6a and Supplementary Figure S6b). In order to assess the proportion of actively cycling cells we measured Ki67 by flow cytometry and determined the percentage of CLL cells that expressed Ki67 (Supplementary Figure S7). Figure 6b shows a representative histogram demonstrating the reduction in Ki67 in CLL cells from ibrutinib treated mice compared to control. Ibrutinib significantly inhibited tumor proliferation as reflected in decreased Ki67 expression in CLL cells in both the PB and spleen of ibrutinib treated mice (mean reduction >50%, Figure 6c-e; P≤.006). Thus ibrutinib potently and selectively inhibits tumor proliferation in vivo.


Modeling tumor-host interactions of chronic lymphocytic leukemia in xenografted mice to study tumor biology and evaluate targeted therapy.

Herman SE, Sun X, McAuley EM, Hsieh MM, Pittaluga S, Raffeld M, Liu D, Keyvanfar K, Chapman CM, Chen J, Buggy JJ, Aue G, Tisdale JF, Pérez-Galán P, Wiestner A - Leukemia (2013)

Ibrutinib inhibits CLL but not T-cell proliferation in vivo. (a) NSG mice (n=24) injected with CFSE labeled PBMCs from six patients were treated with vehicle (control) or ibrutinib and sacrificed 3-4 weeks later. Symbols identify patients (Table 1), and each data point represents one mouse. The percentage of CFSE low cells (cells having completed cell division) in PB and spleen (SP) for each treatment group is shown for CLL cells (left panel) and T-cells (right panel). In one patient (coded by open circles) the T-cell count in the PB was too low for analysis. (b) Overlay histogram showing decreased expression of Ki67 in CLL cells from the mouse spleen of treated as compared to untreated mice at time of sacrifice, 3-4 weeks post xenograft. (c) NSG mice (n=22) injected with unlabeled (no CFSE) PBMCs from five patients were treated with vehicle or ibrutinib and sacrificed 3-4 weeks later. Ibrutinib significantly decreased the percentage of Ki67+ CLL cells in PB and spleen (SP). The multivariable analysis used to test for significance in all panels is described in Materials and Methods. (d) The percentage of Ki67+ CLL cells is decreased by ibrutinib in all patients studied. Shown is the mean (± SEM) percentage Ki67+ CLL cells in PB (left panel) and spleen (right panel) of 2-3 mice in each treatment group injected with cells from the five different patients.
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Figure 6: Ibrutinib inhibits CLL but not T-cell proliferation in vivo. (a) NSG mice (n=24) injected with CFSE labeled PBMCs from six patients were treated with vehicle (control) or ibrutinib and sacrificed 3-4 weeks later. Symbols identify patients (Table 1), and each data point represents one mouse. The percentage of CFSE low cells (cells having completed cell division) in PB and spleen (SP) for each treatment group is shown for CLL cells (left panel) and T-cells (right panel). In one patient (coded by open circles) the T-cell count in the PB was too low for analysis. (b) Overlay histogram showing decreased expression of Ki67 in CLL cells from the mouse spleen of treated as compared to untreated mice at time of sacrifice, 3-4 weeks post xenograft. (c) NSG mice (n=22) injected with unlabeled (no CFSE) PBMCs from five patients were treated with vehicle or ibrutinib and sacrificed 3-4 weeks later. Ibrutinib significantly decreased the percentage of Ki67+ CLL cells in PB and spleen (SP). The multivariable analysis used to test for significance in all panels is described in Materials and Methods. (d) The percentage of Ki67+ CLL cells is decreased by ibrutinib in all patients studied. Shown is the mean (± SEM) percentage Ki67+ CLL cells in PB (left panel) and spleen (right panel) of 2-3 mice in each treatment group injected with cells from the five different patients.
Mentions: Finally, we determined the effect of ibrutinib on tumor proliferation. Ibrutinib decreased CLL cell proliferation measured using CFSE dilution by >80% compared to controls (Figure 6a and Supplementary Figure S6a; P<.001). In contrast, ibrutinib had no effect on T-cell proliferation (Figure 6a and Supplementary Figure S6b). In order to assess the proportion of actively cycling cells we measured Ki67 by flow cytometry and determined the percentage of CLL cells that expressed Ki67 (Supplementary Figure S7). Figure 6b shows a representative histogram demonstrating the reduction in Ki67 in CLL cells from ibrutinib treated mice compared to control. Ibrutinib significantly inhibited tumor proliferation as reflected in decreased Ki67 expression in CLL cells in both the PB and spleen of ibrutinib treated mice (mean reduction >50%, Figure 6c-e; P≤.006). Thus ibrutinib potently and selectively inhibits tumor proliferation in vivo.

Bottom Line: We found that the murine spleen (SP) microenvironment supported CLL cell proliferation and activation to a similar degree than the human LN, including induction of BCR and NF-κB signaling in the xenografted cells.Next, we used this model to study ibrutinib, a Bruton's tyrosine kinase inhibitor in clinical development.Ibrutinib inhibited BCR and NF-κB signaling induced by the microenvironment, decreased proliferation, induced apoptosis and reduced the tumor burden in vivo.

View Article: PubMed Central - PubMed

Affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

ABSTRACT
Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, the B-cell receptor (BCR) and nuclear factor- κB (NF-κB) pathways are activated in the lymph node (LN) microenvironment. Thus, model systems mimicking tumor-host interactions are important tools to study CLL biology and pathogenesis. We investigated whether the recently established NOD/scid/γc() (NSG) mouse xenograft model can recapitulate the effects of the human microenvironment. We assessed, therefore, tumor characteristics previously defined in LN-resident CLL cells, including proliferation, and activation of the BCR and NF-κB pathways. We found that the murine spleen (SP) microenvironment supported CLL cell proliferation and activation to a similar degree than the human LN, including induction of BCR and NF-κB signaling in the xenografted cells. Next, we used this model to study ibrutinib, a Bruton's tyrosine kinase inhibitor in clinical development. Ibrutinib inhibited BCR and NF-κB signaling induced by the microenvironment, decreased proliferation, induced apoptosis and reduced the tumor burden in vivo. Thus, our data demonstrate that the SP of xenografted NSG mice can, in part, recapitulate the role of the human LN for CLL cells. In addition, we show that ibrutinib effectively disrupts tumor-host interactions essential for CLL cell proliferation and survival in vivo.

Show MeSH
Related in: MedlinePlus