Limits...
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.

Show MeSH

Related in: MedlinePlus

The BCR and NF-κB pathways are activated in xenografted CLL cells in the murine spleen. (a) BCR and NF-κB target genes are upregulated in xenografted CLL cells from the murine spleen and in CLL cells isolated from human LN. Shown is the mean (± SEM) of the expression average of representative target genes (described in 3, listed in Supplementary Table S1) in CLL cells in tissue sites normalized to that in the PB of the corresponding patients as described in the Materials and Methods. CLL cells from the spleens of xenografted mice (2-4 spleens per patient) and from the LN and PB of the same patients (n=3) were CD19+ purified. Student's paired t-test was used to test for significance. (b) Overlay histogram showing increased phosphorylation of BTK (pBTK) in CLL cells from both the mouse spleen (SP) and human lymph node (LN) compared to the matched patient's PB. (c) Mean (± SEM) of the MFI ratio of pBTK to isotype control in human PB CLL cells and matched human LN and xenografted CLL cells from mouse spleen (SP) (n=6). (d) Mean (± SEM) of the MFI ratio of pPCLγ2 (left panel) and pERK (right panel) to isotype control as in panel c. The multivariable analysis used to test for significance in panels c and d is described in Materials and Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126654&req=5

Figure 3: The BCR and NF-κB pathways are activated in xenografted CLL cells in the murine spleen. (a) BCR and NF-κB target genes are upregulated in xenografted CLL cells from the murine spleen and in CLL cells isolated from human LN. Shown is the mean (± SEM) of the expression average of representative target genes (described in 3, listed in Supplementary Table S1) in CLL cells in tissue sites normalized to that in the PB of the corresponding patients as described in the Materials and Methods. CLL cells from the spleens of xenografted mice (2-4 spleens per patient) and from the LN and PB of the same patients (n=3) were CD19+ purified. Student's paired t-test was used to test for significance. (b) Overlay histogram showing increased phosphorylation of BTK (pBTK) in CLL cells from both the mouse spleen (SP) and human lymph node (LN) compared to the matched patient's PB. (c) Mean (± SEM) of the MFI ratio of pBTK to isotype control in human PB CLL cells and matched human LN and xenografted CLL cells from mouse spleen (SP) (n=6). (d) Mean (± SEM) of the MFI ratio of pPCLγ2 (left panel) and pERK (right panel) to isotype control as in panel c. The multivariable analysis used to test for significance in panels c and d is described in Materials and Methods.

Mentions: LN-resident CLL cells show activation of the BCR and NF-κB pathways resulting in the upregulation of characteristic gene signatures.3 Whether these pathways are also activated in xenografted CLL cells in NSG mice has not been determined. We selected 13 genes representative of gene signatures regulated by BCR and NF-κB activation (Supplementary Table S1) and measured their expression by quantitative PCR. Eleven of these 13 genes were upregulated in xenografted CLL cells from the murine spleen compared to circulating CLL cells in the patient's PB (Supplementary Figure S4a-b). As a quantitative measure of pathway activation, the mRNA expression levels of the respective target genes were averaged into a BCR and NF-κB gene score. Remarkably, the average increase in gene expression of BCR and NF-κB target genes in mice approximated that in the human LN (Figure 3a).


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)

The BCR and NF-κB pathways are activated in xenografted CLL cells in the murine spleen. (a) BCR and NF-κB target genes are upregulated in xenografted CLL cells from the murine spleen and in CLL cells isolated from human LN. Shown is the mean (± SEM) of the expression average of representative target genes (described in 3, listed in Supplementary Table S1) in CLL cells in tissue sites normalized to that in the PB of the corresponding patients as described in the Materials and Methods. CLL cells from the spleens of xenografted mice (2-4 spleens per patient) and from the LN and PB of the same patients (n=3) were CD19+ purified. Student's paired t-test was used to test for significance. (b) Overlay histogram showing increased phosphorylation of BTK (pBTK) in CLL cells from both the mouse spleen (SP) and human lymph node (LN) compared to the matched patient's PB. (c) Mean (± SEM) of the MFI ratio of pBTK to isotype control in human PB CLL cells and matched human LN and xenografted CLL cells from mouse spleen (SP) (n=6). (d) Mean (± SEM) of the MFI ratio of pPCLγ2 (left panel) and pERK (right panel) to isotype control as in panel c. The multivariable analysis used to test for significance in panels c and d is described in Materials and Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126654&req=5

Figure 3: The BCR and NF-κB pathways are activated in xenografted CLL cells in the murine spleen. (a) BCR and NF-κB target genes are upregulated in xenografted CLL cells from the murine spleen and in CLL cells isolated from human LN. Shown is the mean (± SEM) of the expression average of representative target genes (described in 3, listed in Supplementary Table S1) in CLL cells in tissue sites normalized to that in the PB of the corresponding patients as described in the Materials and Methods. CLL cells from the spleens of xenografted mice (2-4 spleens per patient) and from the LN and PB of the same patients (n=3) were CD19+ purified. Student's paired t-test was used to test for significance. (b) Overlay histogram showing increased phosphorylation of BTK (pBTK) in CLL cells from both the mouse spleen (SP) and human lymph node (LN) compared to the matched patient's PB. (c) Mean (± SEM) of the MFI ratio of pBTK to isotype control in human PB CLL cells and matched human LN and xenografted CLL cells from mouse spleen (SP) (n=6). (d) Mean (± SEM) of the MFI ratio of pPCLγ2 (left panel) and pERK (right panel) to isotype control as in panel c. The multivariable analysis used to test for significance in panels c and d is described in Materials and Methods.
Mentions: LN-resident CLL cells show activation of the BCR and NF-κB pathways resulting in the upregulation of characteristic gene signatures.3 Whether these pathways are also activated in xenografted CLL cells in NSG mice has not been determined. We selected 13 genes representative of gene signatures regulated by BCR and NF-κB activation (Supplementary Table S1) and measured their expression by quantitative PCR. Eleven of these 13 genes were upregulated in xenografted CLL cells from the murine spleen compared to circulating CLL cells in the patient's PB (Supplementary Figure S4a-b). As a quantitative measure of pathway activation, the mRNA expression levels of the respective target genes were averaged into a BCR and NF-κB gene score. Remarkably, the average increase in gene expression of BCR and NF-κB target genes in mice approximated that in the human LN (Figure 3a).

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