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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 proliferative CXCR4dimCD5br compartment of CLL cells is promoted by the co-culture with BMSC, CD40L and CpG ODNPBMC from 40 patients diagnosed with CLL were used to analyze the expression of CXCR4 and CD5 by FC. (A) Representative contour plot of CXCR4 and CD5 expression by CLL cells from one patient. (B) Mean percentage ± SEM of CLL cells in the three compartments defined by CXCR4 and CD5 densities are depicted in the graph (**P<0.01, ***P<0.001, one-way ANOVA). (C) Mean percentage ± SEM of Ki-67 expression in the three compartments (*P<0.05, **P<0.01, ***P<0.001, one-way ANOVA). (C) Primary CLL cells from 7 patients cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours were used to analyze the expression of CXCR4 and CD5. (*P<0.05, two-way ANOVA, Bonferroni's post-test. Graph shows mean ± SEM).
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Figure 3: The proliferative CXCR4dimCD5br compartment of CLL cells is promoted by the co-culture with BMSC, CD40L and CpG ODNPBMC from 40 patients diagnosed with CLL were used to analyze the expression of CXCR4 and CD5 by FC. (A) Representative contour plot of CXCR4 and CD5 expression by CLL cells from one patient. (B) Mean percentage ± SEM of CLL cells in the three compartments defined by CXCR4 and CD5 densities are depicted in the graph (**P<0.01, ***P<0.001, one-way ANOVA). (C) Mean percentage ± SEM of Ki-67 expression in the three compartments (*P<0.05, **P<0.01, ***P<0.001, one-way ANOVA). (C) Primary CLL cells from 7 patients cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours were used to analyze the expression of CXCR4 and CD5. (*P<0.05, two-way ANOVA, Bonferroni's post-test. Graph shows mean ± SEM).

Mentions: We also characterized the proliferative and resting compartments of CLL cells found in PB using differences in the expression of CD5 and CXCR4 by FC as previously defined by Calissano, C et al[16] (Figure 3A). The percentage of CLL cells falling into each compartment was similar to the previously observed distribution[16] (Figure 3B) (mean % CLL cells: 4.52±0.82 in CXCR4dimCD5bright fraction vs. 75.78±2.05 in CXCR4intCD5int fraction vs. 13.83±1.53 in CXCR4brightCD5dim fraction, P<0.001). As expected, the proliferative compartment defined as the CXCR4dimCD5bright fraction was significantly enriched in Ki-67 positive cells (Figure 3C) (mean % Ki-67 positive cells: 3.28±1.12 in CXCR4dimCD5bright fraction vs. 2.63±0.85 in CXCR4intCD5int fraction, P=n.s.; vs. 0.36±0.21 in CXCR4brightCD5dim fraction, P<0.01). As we previously reported[15], co-culturing CLL cells for 48 hours induced an increase in the percentage of CLL cells within the CXCR4dimCD5brightcompartment (Figure 3D) whereas the proportion of CXCR4intCD5int and CXCR4brightCD5dim cells was not significantly affected (mean % CXCR4dimCD5bright cells: 23.16±7.25 in co-culture vs. 5.67±2.52 in suspension, P<0.05).


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 proliferative CXCR4dimCD5br compartment of CLL cells is promoted by the co-culture with BMSC, CD40L and CpG ODNPBMC from 40 patients diagnosed with CLL were used to analyze the expression of CXCR4 and CD5 by FC. (A) Representative contour plot of CXCR4 and CD5 expression by CLL cells from one patient. (B) Mean percentage ± SEM of CLL cells in the three compartments defined by CXCR4 and CD5 densities are depicted in the graph (**P<0.01, ***P<0.001, one-way ANOVA). (C) Mean percentage ± SEM of Ki-67 expression in the three compartments (*P<0.05, **P<0.01, ***P<0.001, one-way ANOVA). (C) Primary CLL cells from 7 patients cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours were used to analyze the expression of CXCR4 and CD5. (*P<0.05, two-way ANOVA, Bonferroni's post-test. Graph shows mean ± SEM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: The proliferative CXCR4dimCD5br compartment of CLL cells is promoted by the co-culture with BMSC, CD40L and CpG ODNPBMC from 40 patients diagnosed with CLL were used to analyze the expression of CXCR4 and CD5 by FC. (A) Representative contour plot of CXCR4 and CD5 expression by CLL cells from one patient. (B) Mean percentage ± SEM of CLL cells in the three compartments defined by CXCR4 and CD5 densities are depicted in the graph (**P<0.01, ***P<0.001, one-way ANOVA). (C) Mean percentage ± SEM of Ki-67 expression in the three compartments (*P<0.05, **P<0.01, ***P<0.001, one-way ANOVA). (C) Primary CLL cells from 7 patients cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours were used to analyze the expression of CXCR4 and CD5. (*P<0.05, two-way ANOVA, Bonferroni's post-test. Graph shows mean ± SEM).
Mentions: We also characterized the proliferative and resting compartments of CLL cells found in PB using differences in the expression of CD5 and CXCR4 by FC as previously defined by Calissano, C et al[16] (Figure 3A). The percentage of CLL cells falling into each compartment was similar to the previously observed distribution[16] (Figure 3B) (mean % CLL cells: 4.52±0.82 in CXCR4dimCD5bright fraction vs. 75.78±2.05 in CXCR4intCD5int fraction vs. 13.83±1.53 in CXCR4brightCD5dim fraction, P<0.001). As expected, the proliferative compartment defined as the CXCR4dimCD5bright fraction was significantly enriched in Ki-67 positive cells (Figure 3C) (mean % Ki-67 positive cells: 3.28±1.12 in CXCR4dimCD5bright fraction vs. 2.63±0.85 in CXCR4intCD5int fraction, P=n.s.; vs. 0.36±0.21 in CXCR4brightCD5dim fraction, P<0.01). As we previously reported[15], co-culturing CLL cells for 48 hours induced an increase in the percentage of CLL cells within the CXCR4dimCD5brightcompartment (Figure 3D) whereas the proportion of CXCR4intCD5int and CXCR4brightCD5dim cells was not significantly affected (mean % CXCR4dimCD5bright cells: 23.16±7.25 in co-culture vs. 5.67±2.52 in suspension, P<0.05).

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