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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 induces proliferation of CLL cellsPrimary CLL cells were cultured with BMSC, with CD40L, with CpG ODN or with BMSC, CD40L and CpG ODN. Cellular analyses were performed after 24, 48 and 72 hours of culture. (A) Mean % of Ki-67-positive cells from 8 patients was analyzed by flow cytometry. (B) Cell cycle distribution was assessed in primary CLL cells from 8 patients by PI staining by FC. (C) Viability was assessed in primary CLL cells from 8 patients by Annexin V and PI staining (D) MTS assay of co-cultured CLL cells. The mean optical density (OD) values at 490nm ± SEM from triplicates from 4 patients are depicted in the graph.
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Figure 1: The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN induces proliferation of CLL cellsPrimary CLL cells were cultured with BMSC, with CD40L, with CpG ODN or with BMSC, CD40L and CpG ODN. Cellular analyses were performed after 24, 48 and 72 hours of culture. (A) Mean % of Ki-67-positive cells from 8 patients was analyzed by flow cytometry. (B) Cell cycle distribution was assessed in primary CLL cells from 8 patients by PI staining by FC. (C) Viability was assessed in primary CLL cells from 8 patients by Annexin V and PI staining (D) MTS assay of co-cultured CLL cells. The mean optical density (OD) values at 490nm ± SEM from triplicates from 4 patients are depicted in the graph.

Mentions: With the aim of partially ex vivo mimicking the microenvironment found in the proliferative centers, we cultured primary CLL cells in 5 different culture conditions: in suspension, co-cultured with the BMSC cell line UE6E7T-2, with soluble CD40L, with CpG ODN and with the combination of all elements: BMSC, CD40L and CpG ODN. Next, we analyzed the effects in terms of proliferation and survival after 24, 48 and 72 hours of culture. In this setting, proliferative responses, measured as increase in expression of Ki-67, were significantly observed after 48 hours in CLL cells cultured with BMSC, CD40L and CpG ODN (Figure 1A) (mean % Ki-67 positive cells: 4.41±1.41 cultured with BMSC, CD40L and CpG ODN vs. 0.42±0.10 in suspension, P<0.01) and were even higher after 72 hours when, as we had previously observed [15], the co-culture with BMSC, CD40L and CpG ODN induced a Ki-67 expression of 10.71% ± 2.51% vs. 1.18%±0.34% in suspension (P<0.001). Interestingly, when we analyzed the effect of every stimulus independently we observed that none of them increased Ki-67 expression significantly (mean % Ki-67 positive cells: 1.18±0.34 cultured in suspension vs. 2.52±0.74 cultured with BMSC vs. 1.41±0.44 cultured with CD40L vs. 4.42±1.75 cultured with CpG ODN, P=n.s.).


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 induces proliferation of CLL cellsPrimary CLL cells were cultured with BMSC, with CD40L, with CpG ODN or with BMSC, CD40L and CpG ODN. Cellular analyses were performed after 24, 48 and 72 hours of culture. (A) Mean % of Ki-67-positive cells from 8 patients was analyzed by flow cytometry. (B) Cell cycle distribution was assessed in primary CLL cells from 8 patients by PI staining by FC. (C) Viability was assessed in primary CLL cells from 8 patients by Annexin V and PI staining (D) MTS assay of co-cultured CLL cells. The mean optical density (OD) values at 490nm ± SEM from triplicates from 4 patients are depicted in the graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480705&req=5

Figure 1: The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN induces proliferation of CLL cellsPrimary CLL cells were cultured with BMSC, with CD40L, with CpG ODN or with BMSC, CD40L and CpG ODN. Cellular analyses were performed after 24, 48 and 72 hours of culture. (A) Mean % of Ki-67-positive cells from 8 patients was analyzed by flow cytometry. (B) Cell cycle distribution was assessed in primary CLL cells from 8 patients by PI staining by FC. (C) Viability was assessed in primary CLL cells from 8 patients by Annexin V and PI staining (D) MTS assay of co-cultured CLL cells. The mean optical density (OD) values at 490nm ± SEM from triplicates from 4 patients are depicted in the graph.
Mentions: With the aim of partially ex vivo mimicking the microenvironment found in the proliferative centers, we cultured primary CLL cells in 5 different culture conditions: in suspension, co-cultured with the BMSC cell line UE6E7T-2, with soluble CD40L, with CpG ODN and with the combination of all elements: BMSC, CD40L and CpG ODN. Next, we analyzed the effects in terms of proliferation and survival after 24, 48 and 72 hours of culture. In this setting, proliferative responses, measured as increase in expression of Ki-67, were significantly observed after 48 hours in CLL cells cultured with BMSC, CD40L and CpG ODN (Figure 1A) (mean % Ki-67 positive cells: 4.41±1.41 cultured with BMSC, CD40L and CpG ODN vs. 0.42±0.10 in suspension, P<0.01) and were even higher after 72 hours when, as we had previously observed [15], the co-culture with BMSC, CD40L and CpG ODN induced a Ki-67 expression of 10.71% ± 2.51% vs. 1.18%±0.34% in suspension (P<0.001). Interestingly, when we analyzed the effect of every stimulus independently we observed that none of them increased Ki-67 expression significantly (mean % Ki-67 positive cells: 1.18±0.34 cultured in suspension vs. 2.52±0.74 cultured with BMSC vs. 1.41±0.44 cultured with CD40L vs. 4.42±1.75 cultured with CpG ODN, P=n.s.).

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