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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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Xenografted human CLL cells proliferate in the spleen of NSG mice. (a, b) The fraction of human cells having undergone cell division increases with time from xenografting. PBMCs from seven CLL patients (represented by a unique symbol; Table 1) were labeled with 0.5 μM CFSE before injection into NSG mice. The percentage of human CLL (CD45+, CD19+, CD5+; in a) or T-cells (CD45+, CD19−, CD5+; in b) having undergone cell division (low CFSE staining) is shown in peripheral blood (PB) samples at 2-week intervals (2-5 mice per patient). (c-e) Mice were sacrificed 3-4 weeks post xenografting. (c) A representative histogram demonstrates increased Ki67 staining in CLL cells from the spleen (SP) compared to cells in the PB. (d) The percentage of Ki67 positive CLL cells is higher in the spleen than in the PB. Lines connect PB and spleen samples from the same mouse (n = 13; symbols identify individual patients; Table 1). (e) CLL cells in secondary lymphoid tissues upregulate genes typically expressed in proliferating cells. Shown is the mean (± SEM) expression for each gene in CLL cells from the indicated tissue normalized to its expression in the corresponding PB cells. CLL cells were CD19+ purified from the spleen of xenografted mice (2-4 spleens per patient) and from the corresponding patients' LN and PB (n=3).3 (f) The mean (± SEM) proliferation gene score, computed as the averaged expression of CDT1, PCNA, and RRM2 shown in (e), is comparable between CLL cells from the mouse spleen and the human LN. Student's paired t-test was used to test for significance in all panels.
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Figure 1: Xenografted human CLL cells proliferate in the spleen of NSG mice. (a, b) The fraction of human cells having undergone cell division increases with time from xenografting. PBMCs from seven CLL patients (represented by a unique symbol; Table 1) were labeled with 0.5 μM CFSE before injection into NSG mice. The percentage of human CLL (CD45+, CD19+, CD5+; in a) or T-cells (CD45+, CD19−, CD5+; in b) having undergone cell division (low CFSE staining) is shown in peripheral blood (PB) samples at 2-week intervals (2-5 mice per patient). (c-e) Mice were sacrificed 3-4 weeks post xenografting. (c) A representative histogram demonstrates increased Ki67 staining in CLL cells from the spleen (SP) compared to cells in the PB. (d) The percentage of Ki67 positive CLL cells is higher in the spleen than in the PB. Lines connect PB and spleen samples from the same mouse (n = 13; symbols identify individual patients; Table 1). (e) CLL cells in secondary lymphoid tissues upregulate genes typically expressed in proliferating cells. Shown is the mean (± SEM) expression for each gene in CLL cells from the indicated tissue normalized to its expression in the corresponding PB cells. CLL cells were CD19+ purified from the spleen of xenografted mice (2-4 spleens per patient) and from the corresponding patients' LN and PB (n=3).3 (f) The mean (± SEM) proliferation gene score, computed as the averaged expression of CDT1, PCNA, and RRM2 shown in (e), is comparable between CLL cells from the mouse spleen and the human LN. Student's paired t-test was used to test for significance in all panels.

Mentions: In order to measure CLL and T-cell proliferation we injected CFSE stained PBMCs and determined the fraction of proliferating cells identified by a decrease in CFSE staining on flow cytometry (Supplementary Figure S2a-b). One to two weeks after xenografting <5% of the circulating CLL cells showed decreased CFSE staining. However this proliferating fraction increased significantly by weeks 3-4 (Figure 1a; P=.01), consistent with a delayed onset of tumor proliferation as described previously.39 Proliferation of T-cells was also delayed but once established, was faster than in the CLL cells (Figure 1b). The growth rate of CLL cells ranged from 0.23% to 0.91% of the clonal cells per day (estimated from the fraction of cells with low CFSE staining divided by the number of days post xenografting). This range is in good agreement with the proliferation rate found in patients using deuterium labeling.2 The four tumor samples with the highest growth rates were IGHV unmutated, whereas two of three samples with relatively lower proliferation were IGHV mutated (Figure 1a, Table 1). We did not find a correlation between T-cell and CLL cell proliferation rates; however as previously reported,39 CLL proliferation appeared to depend on the presence of autologous T-cells (data not shown). Thus, despite simplifications in the xenografting protocol, the tissue localization and proliferation kinetics of the xenografted CLL cells are in agreement with findings by Bagnara et al.39


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)

Xenografted human CLL cells proliferate in the spleen of NSG mice. (a, b) The fraction of human cells having undergone cell division increases with time from xenografting. PBMCs from seven CLL patients (represented by a unique symbol; Table 1) were labeled with 0.5 μM CFSE before injection into NSG mice. The percentage of human CLL (CD45+, CD19+, CD5+; in a) or T-cells (CD45+, CD19−, CD5+; in b) having undergone cell division (low CFSE staining) is shown in peripheral blood (PB) samples at 2-week intervals (2-5 mice per patient). (c-e) Mice were sacrificed 3-4 weeks post xenografting. (c) A representative histogram demonstrates increased Ki67 staining in CLL cells from the spleen (SP) compared to cells in the PB. (d) The percentage of Ki67 positive CLL cells is higher in the spleen than in the PB. Lines connect PB and spleen samples from the same mouse (n = 13; symbols identify individual patients; Table 1). (e) CLL cells in secondary lymphoid tissues upregulate genes typically expressed in proliferating cells. Shown is the mean (± SEM) expression for each gene in CLL cells from the indicated tissue normalized to its expression in the corresponding PB cells. CLL cells were CD19+ purified from the spleen of xenografted mice (2-4 spleens per patient) and from the corresponding patients' LN and PB (n=3).3 (f) The mean (± SEM) proliferation gene score, computed as the averaged expression of CDT1, PCNA, and RRM2 shown in (e), is comparable between CLL cells from the mouse spleen and the human LN. Student's paired t-test was used to test for significance in all panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Xenografted human CLL cells proliferate in the spleen of NSG mice. (a, b) The fraction of human cells having undergone cell division increases with time from xenografting. PBMCs from seven CLL patients (represented by a unique symbol; Table 1) were labeled with 0.5 μM CFSE before injection into NSG mice. The percentage of human CLL (CD45+, CD19+, CD5+; in a) or T-cells (CD45+, CD19−, CD5+; in b) having undergone cell division (low CFSE staining) is shown in peripheral blood (PB) samples at 2-week intervals (2-5 mice per patient). (c-e) Mice were sacrificed 3-4 weeks post xenografting. (c) A representative histogram demonstrates increased Ki67 staining in CLL cells from the spleen (SP) compared to cells in the PB. (d) The percentage of Ki67 positive CLL cells is higher in the spleen than in the PB. Lines connect PB and spleen samples from the same mouse (n = 13; symbols identify individual patients; Table 1). (e) CLL cells in secondary lymphoid tissues upregulate genes typically expressed in proliferating cells. Shown is the mean (± SEM) expression for each gene in CLL cells from the indicated tissue normalized to its expression in the corresponding PB cells. CLL cells were CD19+ purified from the spleen of xenografted mice (2-4 spleens per patient) and from the corresponding patients' LN and PB (n=3).3 (f) The mean (± SEM) proliferation gene score, computed as the averaged expression of CDT1, PCNA, and RRM2 shown in (e), is comparable between CLL cells from the mouse spleen and the human LN. Student's paired t-test was used to test for significance in all panels.
Mentions: In order to measure CLL and T-cell proliferation we injected CFSE stained PBMCs and determined the fraction of proliferating cells identified by a decrease in CFSE staining on flow cytometry (Supplementary Figure S2a-b). One to two weeks after xenografting <5% of the circulating CLL cells showed decreased CFSE staining. However this proliferating fraction increased significantly by weeks 3-4 (Figure 1a; P=.01), consistent with a delayed onset of tumor proliferation as described previously.39 Proliferation of T-cells was also delayed but once established, was faster than in the CLL cells (Figure 1b). The growth rate of CLL cells ranged from 0.23% to 0.91% of the clonal cells per day (estimated from the fraction of cells with low CFSE staining divided by the number of days post xenografting). This range is in good agreement with the proliferation rate found in patients using deuterium labeling.2 The four tumor samples with the highest growth rates were IGHV unmutated, whereas two of three samples with relatively lower proliferation were IGHV mutated (Figure 1a, Table 1). We did not find a correlation between T-cell and CLL cell proliferation rates; however as previously reported,39 CLL proliferation appeared to depend on the presence of autologous T-cells (data not shown). Thus, despite simplifications in the xenografting protocol, the tissue localization and proliferation kinetics of the xenografted CLL cells are in agreement with findings by Bagnara et al.39

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