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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

Co-cultured CLL cells display a marked chemoresistance to fludarabine and bendamustine treatmentPrimary CLL cells from 7 patients were cultured for 48 hours in suspension or in co-culture with BMSC, CD40L and CpG ODN; subsequently, increasing doses of fludarabine and bendamustine were added for additional 24 hours. LD50 curves for fludarabine (A) and bendamustine (B) are plotted on a logarithmic scale. (C) Quantification of Mcl-1 and Bcl-2 expression analyzed by western blot relative to GAPDH. Each bar represents the mean ± SEM from 7 patients (**P<0.01, ***P<0.001, two-way ANOVA, Bonferroni's post-test). (D) Mcl-1 and Bcl-2 expression relative to primary CLL cells in suspension. Each bar represents the mean ± SEM from 7 patients (**P<0.01, two-way ANOVA, Bonferroni's post-test). (E) One representative immunoblot analysis of Mcl-1 and Bcl-2 expression.
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Figure 5: Co-cultured CLL cells display a marked chemoresistance to fludarabine and bendamustine treatmentPrimary CLL cells from 7 patients were cultured for 48 hours in suspension or in co-culture with BMSC, CD40L and CpG ODN; subsequently, increasing doses of fludarabine and bendamustine were added for additional 24 hours. LD50 curves for fludarabine (A) and bendamustine (B) are plotted on a logarithmic scale. (C) Quantification of Mcl-1 and Bcl-2 expression analyzed by western blot relative to GAPDH. Each bar represents the mean ± SEM from 7 patients (**P<0.01, ***P<0.001, two-way ANOVA, Bonferroni's post-test). (D) Mcl-1 and Bcl-2 expression relative to primary CLL cells in suspension. Each bar represents the mean ± SEM from 7 patients (**P<0.01, two-way ANOVA, Bonferroni's post-test). (E) One representative immunoblot analysis of Mcl-1 and Bcl-2 expression.

Mentions: The microenvironment found in the proliferative centers has been shown to provide direct pro-survival signals and to protect CLL cells from the effect of chemotherapeutical agents[7],[25]. Consequently, this proliferative and chemoresistant compartment of CLL cells has been hypothesized to be potentially responsible for MRD persistence and disease relapse. In this regard, to evaluate the role of co-culture on the chemoresistance of primary CLL cells, we co-cultured primary CLL cells for 48 hours and subsequently treated them with increasing doses of fludarabine or bendamustine for additional 24 hours. Interestingly, as we previously described[15], the co-culture of CLL cells in these conditions inhibited at such extend the capacity of fludarabine to induce apoptosis that it was not possible to calculate its LD50, whereas LD50 of fludarabine in CLL cells in suspension was 416μM (95% CI 125.5-1379) (Figure 5A). For CLL cells treated with bendamustine, LD50 of CLL cells in proliferative conditions was 5.18 times higher than for CLL cells in suspension (Figure 5B).


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)

Co-cultured CLL cells display a marked chemoresistance to fludarabine and bendamustine treatmentPrimary CLL cells from 7 patients were cultured for 48 hours in suspension or in co-culture with BMSC, CD40L and CpG ODN; subsequently, increasing doses of fludarabine and bendamustine were added for additional 24 hours. LD50 curves for fludarabine (A) and bendamustine (B) are plotted on a logarithmic scale. (C) Quantification of Mcl-1 and Bcl-2 expression analyzed by western blot relative to GAPDH. Each bar represents the mean ± SEM from 7 patients (**P<0.01, ***P<0.001, two-way ANOVA, Bonferroni's post-test). (D) Mcl-1 and Bcl-2 expression relative to primary CLL cells in suspension. Each bar represents the mean ± SEM from 7 patients (**P<0.01, two-way ANOVA, Bonferroni's post-test). (E) One representative immunoblot analysis of Mcl-1 and Bcl-2 expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Co-cultured CLL cells display a marked chemoresistance to fludarabine and bendamustine treatmentPrimary CLL cells from 7 patients were cultured for 48 hours in suspension or in co-culture with BMSC, CD40L and CpG ODN; subsequently, increasing doses of fludarabine and bendamustine were added for additional 24 hours. LD50 curves for fludarabine (A) and bendamustine (B) are plotted on a logarithmic scale. (C) Quantification of Mcl-1 and Bcl-2 expression analyzed by western blot relative to GAPDH. Each bar represents the mean ± SEM from 7 patients (**P<0.01, ***P<0.001, two-way ANOVA, Bonferroni's post-test). (D) Mcl-1 and Bcl-2 expression relative to primary CLL cells in suspension. Each bar represents the mean ± SEM from 7 patients (**P<0.01, two-way ANOVA, Bonferroni's post-test). (E) One representative immunoblot analysis of Mcl-1 and Bcl-2 expression.
Mentions: The microenvironment found in the proliferative centers has been shown to provide direct pro-survival signals and to protect CLL cells from the effect of chemotherapeutical agents[7],[25]. Consequently, this proliferative and chemoresistant compartment of CLL cells has been hypothesized to be potentially responsible for MRD persistence and disease relapse. In this regard, to evaluate the role of co-culture on the chemoresistance of primary CLL cells, we co-cultured primary CLL cells for 48 hours and subsequently treated them with increasing doses of fludarabine or bendamustine for additional 24 hours. Interestingly, as we previously described[15], the co-culture of CLL cells in these conditions inhibited at such extend the capacity of fludarabine to induce apoptosis that it was not possible to calculate its LD50, whereas LD50 of fludarabine in CLL cells in suspension was 416μM (95% CI 125.5-1379) (Figure 5A). For CLL cells treated with bendamustine, LD50 of CLL cells in proliferative conditions was 5.18 times higher than for CLL cells in suspension (Figure 5B).

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