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Co-culture of primary CLL cells with bone marrow mesenchymal cells, CD40 ligand and CpG ODN promotes proliferation of chemoresistant CLL cells phenotypically comparable to those proliferating in vivo.

Purroy N, Abrisqueta P, Carabia J, Carpio C, Palacio C, Bosch F, Crespo M - Oncotarget (2015)

Bottom Line: Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression.This indicates aggressiveness and capability to interact with surrounding cells, respectively.In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Hematology, Department of Hematology, Vall d'Hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain.

ABSTRACT
Chronic lymphocytic leukemia (CLL) cells residing in the bone marrow (BM) and in secondary lymphoid tissues receive survival and proliferative signals from the microenvironment, resulting in persistence of residual disease after treatment. In this study, we characterized primary CLL cells cultured with BM stromal cells, CD40 ligand and CpG ODN to partially mimic the microenvironment in the proliferative centers. This co-culture system induced proliferation and chemoresistance in primary CLL cells. Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression. This indicates aggressiveness and capability to interact with surrounding cells, respectively. In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells. We suggest that this co-culture system can be used to test drugs and their combinations that target the proliferative and drug resistant CLL cells.

No MeSH data available.


Related in: MedlinePlus

The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN markedly enhances ZAP-70 expression(A) PBMC from 40 patients diagnosed with CLL were used to analyze ZAP-70 expression in Ki-67 negative vs. positive CLL cells (***P<0.001, paired T-test). (B) ZAP-70 expression in CXCR4 and CD5 compartments of CLL cells from PBMC from 40 patients. (**P<0.01, ***P<0.001, one-way ANOVA. Graph shows mean ± SEM). (C) Primary CLL cells from 12 patients were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the expression level of ZAP-70 was analyzed by FC (**P<0.01, paired T-test). (D) One representative histogram of primary CLL cells from one patient after 48 hours in suspension or in co-culture. (E) One representative immunoblot analysis of ZAP-70 expression.
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Figure 4: The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN markedly enhances ZAP-70 expression(A) PBMC from 40 patients diagnosed with CLL were used to analyze ZAP-70 expression in Ki-67 negative vs. positive CLL cells (***P<0.001, paired T-test). (B) ZAP-70 expression in CXCR4 and CD5 compartments of CLL cells from PBMC from 40 patients. (**P<0.01, ***P<0.001, one-way ANOVA. Graph shows mean ± SEM). (C) Primary CLL cells from 12 patients were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the expression level of ZAP-70 was analyzed by FC (**P<0.01, paired T-test). (D) One representative histogram of primary CLL cells from one patient after 48 hours in suspension or in co-culture. (E) One representative immunoblot analysis of ZAP-70 expression.

Mentions: Among the diverse molecular pathways of crosstalk between CLL cells and their microenvironment, BCR signaling has been recognized as one of the most important [17][18]. The expression of the protein tyrosine kinase ZAP-70 has been associated with increased BCR signaling in CLL [19] which translated into increased proliferation and migrative capacity of ZAP-70 positive subclones, based on in vitro and in vivo data [20],[21],[22],[23]. Clinically, ZAP-70 expression has been correlated with IgVH mutational status, disease progression and survival[24]. Therefore, we hypothesized that ZAP-70 expression could be upregulated in proliferating CLL subclones. In order to test this, we assessed ZAP-70 expression in CLL cells from PB according to Ki-67 expression and subsequently in primary CLL cells co-cultured in proliferative conditions. Firstly, we observed that the Ki-67 positive fraction of CLL cells from the PB was significantly enriched in ZAP-70 positive cells (Figure 4A) (mean % of ZAP-70 expression: 83.93±2.40 in Ki-67 positive cells vs. 29.22±4.20 in Ki-67 negative cells, P<0.001). We also determined ZAP-70 expression according to CXCR4 and CD5 expression and interestingly, we observed that the proliferative CXCR4dimCD5bright fraction was also enriched in ZAP-70 positive cells (Figure 4B) (mean % ZAP-70 positive cells: 67.35±3.66 in CXCR4dimCD5bright fraction vs. 38.71±3.87 in CXCR4intCD5int fraction, P<0.001; vs. 19.29±3.04 in CXCR4brightCD5dim fraction, P<0.001). In order to elucidate if signals from the microenvironment could directly modulate the expression of ZAP-70, we cultured primary CLL cells in suspension or co-cultured with BMSC, CD40L and CpG ODN for 48 hours and observed that the percentage of ZAP-70 positive cells was significantly increased (Figure 4C and 4D) (mean % ZAP-70 positive cells: 61.50±7.33 in co-culture vs. 16.91±4.23 in suspension, P<0.01). This was further confirmed by western blot, where we also observed ZAP-70 up-regulation in primary CLL cells co-cultured with BMSC, CD40L and CpG ODN for 48 hours. To clarify which of these stimuli contributed to ZAP-70 up-regulation, we cultured primary CLL cells with BMSC, CD40L or CpG ODN separately, and assessed ZAP-70 expression by western blot. We observed a marked ZAP-70 up-regulation as a consequence of either CD40 or TLR9 stimulation alone (Figure 4E).


Co-culture of primary CLL cells with bone marrow mesenchymal cells, CD40 ligand and CpG ODN promotes proliferation of chemoresistant CLL cells phenotypically comparable to those proliferating in vivo.

Purroy N, Abrisqueta P, Carabia J, Carpio C, Palacio C, Bosch F, Crespo M - Oncotarget (2015)

The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN markedly enhances ZAP-70 expression(A) PBMC from 40 patients diagnosed with CLL were used to analyze ZAP-70 expression in Ki-67 negative vs. positive CLL cells (***P<0.001, paired T-test). (B) ZAP-70 expression in CXCR4 and CD5 compartments of CLL cells from PBMC from 40 patients. (**P<0.01, ***P<0.001, one-way ANOVA. Graph shows mean ± SEM). (C) Primary CLL cells from 12 patients were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the expression level of ZAP-70 was analyzed by FC (**P<0.01, paired T-test). (D) One representative histogram of primary CLL cells from one patient after 48 hours in suspension or in co-culture. (E) One representative immunoblot analysis of ZAP-70 expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN markedly enhances ZAP-70 expression(A) PBMC from 40 patients diagnosed with CLL were used to analyze ZAP-70 expression in Ki-67 negative vs. positive CLL cells (***P<0.001, paired T-test). (B) ZAP-70 expression in CXCR4 and CD5 compartments of CLL cells from PBMC from 40 patients. (**P<0.01, ***P<0.001, one-way ANOVA. Graph shows mean ± SEM). (C) Primary CLL cells from 12 patients were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the expression level of ZAP-70 was analyzed by FC (**P<0.01, paired T-test). (D) One representative histogram of primary CLL cells from one patient after 48 hours in suspension or in co-culture. (E) One representative immunoblot analysis of ZAP-70 expression.
Mentions: Among the diverse molecular pathways of crosstalk between CLL cells and their microenvironment, BCR signaling has been recognized as one of the most important [17][18]. The expression of the protein tyrosine kinase ZAP-70 has been associated with increased BCR signaling in CLL [19] which translated into increased proliferation and migrative capacity of ZAP-70 positive subclones, based on in vitro and in vivo data [20],[21],[22],[23]. Clinically, ZAP-70 expression has been correlated with IgVH mutational status, disease progression and survival[24]. Therefore, we hypothesized that ZAP-70 expression could be upregulated in proliferating CLL subclones. In order to test this, we assessed ZAP-70 expression in CLL cells from PB according to Ki-67 expression and subsequently in primary CLL cells co-cultured in proliferative conditions. Firstly, we observed that the Ki-67 positive fraction of CLL cells from the PB was significantly enriched in ZAP-70 positive cells (Figure 4A) (mean % of ZAP-70 expression: 83.93±2.40 in Ki-67 positive cells vs. 29.22±4.20 in Ki-67 negative cells, P<0.001). We also determined ZAP-70 expression according to CXCR4 and CD5 expression and interestingly, we observed that the proliferative CXCR4dimCD5bright fraction was also enriched in ZAP-70 positive cells (Figure 4B) (mean % ZAP-70 positive cells: 67.35±3.66 in CXCR4dimCD5bright fraction vs. 38.71±3.87 in CXCR4intCD5int fraction, P<0.001; vs. 19.29±3.04 in CXCR4brightCD5dim fraction, P<0.001). In order to elucidate if signals from the microenvironment could directly modulate the expression of ZAP-70, we cultured primary CLL cells in suspension or co-cultured with BMSC, CD40L and CpG ODN for 48 hours and observed that the percentage of ZAP-70 positive cells was significantly increased (Figure 4C and 4D) (mean % ZAP-70 positive cells: 61.50±7.33 in co-culture vs. 16.91±4.23 in suspension, P<0.01). This was further confirmed by western blot, where we also observed ZAP-70 up-regulation in primary CLL cells co-cultured with BMSC, CD40L and CpG ODN for 48 hours. To clarify which of these stimuli contributed to ZAP-70 up-regulation, we cultured primary CLL cells with BMSC, CD40L or CpG ODN separately, and assessed ZAP-70 expression by western blot. We observed a marked ZAP-70 up-regulation as a consequence of either CD40 or TLR9 stimulation alone (Figure 4E).

Bottom Line: Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression.This indicates aggressiveness and capability to interact with surrounding cells, respectively.In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Hematology, Department of Hematology, Vall d'Hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain.

ABSTRACT
Chronic lymphocytic leukemia (CLL) cells residing in the bone marrow (BM) and in secondary lymphoid tissues receive survival and proliferative signals from the microenvironment, resulting in persistence of residual disease after treatment. In this study, we characterized primary CLL cells cultured with BM stromal cells, CD40 ligand and CpG ODN to partially mimic the microenvironment in the proliferative centers. This co-culture system induced proliferation and chemoresistance in primary CLL cells. Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression. This indicates aggressiveness and capability to interact with surrounding cells, respectively. In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells. We suggest that this co-culture system can be used to test drugs and their combinations that target the proliferative and drug resistant CLL cells.

No MeSH data available.


Related in: MedlinePlus