EX VIVO LYMPH NODE NATIVE MICROENVIRONMENT ASSAY SHOWS NOVEL ANTIPROLIFERATIVE ACTIVITY FOR IDELALISIB AND IBRUTINIB ON CLL CELLS
(Abstract release date: 05/19/16)
EHA Library. Ballesteros J. 06/09/16; 132586; E1037
Mrs. Juan Ballesteros
Contributions
Contributions
Abstract
Abstract: E1037
Type: Eposter Presentation
Background
Survival and proliferation of chronic lymphocytic leukemia (CLL) cells is favored by the essential role of the tumor microenvironment (TME) that is similarly responsible at least in part for disease progression and drug resistance.
Aims
We planned to evaluate and predict the efficacy of therapeutic compounds in vivo, by reproducing in a co-culture system the different microenvironmental components that enable B cells to survive and proliferate, mimicking in particular the lymph node microenvironment where most of the crucial events of the pathogenesis of CLL occur.
Methods
To this purpose, cryopreserved peripheral blood (PB) mononuclear cells from CLL patients in need of treatment were tested with the Exvitech® proprietary automated flow cytometry-based platform. Different components have been evaluated to reproduce the ME and induce proliferation and survival of CLL cells: (i) 3 backbone stimulations: CD40L+CpG, CD40L+IL21, CpG+IL2; (ii) “Native Environment”, defined as the plasma & erythrocyte/granulocyte fraction of a Ficoll gradient: (iii) the stroma cell line H5S, added at different ratios (1:10 or 1:100); (iv) both human and bovine fetal serum (at 10 or 20% total volume); (v) stimulatory B cell factors, including IL-21, soluble CD40L, BAFF, and B cell receptor stimulation (anti-IG).
Results
Of all the described combinations, the addition of CpG+IL2, HS5 (at 1:100 ratio), human serum 10%, and “native environment” from PB of CLL samples (pooled samples to prevent interpatient variability) were the best to promote CLL proliferation (30±3%) and viability (60±5%). We then tested dose responses of the novel BCR inhibitors, idelalisib (PI3kδ inhibitor) and ibrutininb (BTK inhibitor) in 29 and 25 cryopreserved progressive CLL samples, respectively (Figure) and showed that resting CLL cells were virtually unaffected by either drug (EC50 of 12,5µM and 28.3µM), suggesting a limited direct pro-apoptotic activity of the drugs. In contrast, marked inhibition of proliferation was observed (EC50 of 30 nM and 550 nM, respectively) in the presence of either inhibitor, leaving only a median of 5% CLL cells that continued proliferating even at the highest doses.
Conclusion
We here report a novel ex vivo assay that incorporates TME stimuli thus more accurately simulating in vivo interactions and enabling high-throughput pharmacological characterization in more physiological conditions. This assay demonstrated a previously unreported anti-proliferative activity for both idelalisib and ibrutinib that may explain the efficacy of both drugs in patients.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Ex vivo, Microenvironment
Type: Eposter Presentation
Background
Survival and proliferation of chronic lymphocytic leukemia (CLL) cells is favored by the essential role of the tumor microenvironment (TME) that is similarly responsible at least in part for disease progression and drug resistance.
Aims
We planned to evaluate and predict the efficacy of therapeutic compounds in vivo, by reproducing in a co-culture system the different microenvironmental components that enable B cells to survive and proliferate, mimicking in particular the lymph node microenvironment where most of the crucial events of the pathogenesis of CLL occur.
Methods
To this purpose, cryopreserved peripheral blood (PB) mononuclear cells from CLL patients in need of treatment were tested with the Exvitech® proprietary automated flow cytometry-based platform. Different components have been evaluated to reproduce the ME and induce proliferation and survival of CLL cells: (i) 3 backbone stimulations: CD40L+CpG, CD40L+IL21, CpG+IL2; (ii) “Native Environment”, defined as the plasma & erythrocyte/granulocyte fraction of a Ficoll gradient: (iii) the stroma cell line H5S, added at different ratios (1:10 or 1:100); (iv) both human and bovine fetal serum (at 10 or 20% total volume); (v) stimulatory B cell factors, including IL-21, soluble CD40L, BAFF, and B cell receptor stimulation (anti-IG).
Results
Of all the described combinations, the addition of CpG+IL2, HS5 (at 1:100 ratio), human serum 10%, and “native environment” from PB of CLL samples (pooled samples to prevent interpatient variability) were the best to promote CLL proliferation (30±3%) and viability (60±5%). We then tested dose responses of the novel BCR inhibitors, idelalisib (PI3kδ inhibitor) and ibrutininb (BTK inhibitor) in 29 and 25 cryopreserved progressive CLL samples, respectively (Figure) and showed that resting CLL cells were virtually unaffected by either drug (EC50 of 12,5µM and 28.3µM), suggesting a limited direct pro-apoptotic activity of the drugs. In contrast, marked inhibition of proliferation was observed (EC50 of 30 nM and 550 nM, respectively) in the presence of either inhibitor, leaving only a median of 5% CLL cells that continued proliferating even at the highest doses.
Conclusion
We here report a novel ex vivo assay that incorporates TME stimuli thus more accurately simulating in vivo interactions and enabling high-throughput pharmacological characterization in more physiological conditions. This assay demonstrated a previously unreported anti-proliferative activity for both idelalisib and ibrutinib that may explain the efficacy of both drugs in patients.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Ex vivo, Microenvironment
Abstract: E1037
Type: Eposter Presentation
Background
Survival and proliferation of chronic lymphocytic leukemia (CLL) cells is favored by the essential role of the tumor microenvironment (TME) that is similarly responsible at least in part for disease progression and drug resistance.
Aims
We planned to evaluate and predict the efficacy of therapeutic compounds in vivo, by reproducing in a co-culture system the different microenvironmental components that enable B cells to survive and proliferate, mimicking in particular the lymph node microenvironment where most of the crucial events of the pathogenesis of CLL occur.
Methods
To this purpose, cryopreserved peripheral blood (PB) mononuclear cells from CLL patients in need of treatment were tested with the Exvitech® proprietary automated flow cytometry-based platform. Different components have been evaluated to reproduce the ME and induce proliferation and survival of CLL cells: (i) 3 backbone stimulations: CD40L+CpG, CD40L+IL21, CpG+IL2; (ii) “Native Environment”, defined as the plasma & erythrocyte/granulocyte fraction of a Ficoll gradient: (iii) the stroma cell line H5S, added at different ratios (1:10 or 1:100); (iv) both human and bovine fetal serum (at 10 or 20% total volume); (v) stimulatory B cell factors, including IL-21, soluble CD40L, BAFF, and B cell receptor stimulation (anti-IG).
Results
Of all the described combinations, the addition of CpG+IL2, HS5 (at 1:100 ratio), human serum 10%, and “native environment” from PB of CLL samples (pooled samples to prevent interpatient variability) were the best to promote CLL proliferation (30±3%) and viability (60±5%). We then tested dose responses of the novel BCR inhibitors, idelalisib (PI3kδ inhibitor) and ibrutininb (BTK inhibitor) in 29 and 25 cryopreserved progressive CLL samples, respectively (Figure) and showed that resting CLL cells were virtually unaffected by either drug (EC50 of 12,5µM and 28.3µM), suggesting a limited direct pro-apoptotic activity of the drugs. In contrast, marked inhibition of proliferation was observed (EC50 of 30 nM and 550 nM, respectively) in the presence of either inhibitor, leaving only a median of 5% CLL cells that continued proliferating even at the highest doses.
Conclusion
We here report a novel ex vivo assay that incorporates TME stimuli thus more accurately simulating in vivo interactions and enabling high-throughput pharmacological characterization in more physiological conditions. This assay demonstrated a previously unreported anti-proliferative activity for both idelalisib and ibrutinib that may explain the efficacy of both drugs in patients.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Ex vivo, Microenvironment
Type: Eposter Presentation
Background
Survival and proliferation of chronic lymphocytic leukemia (CLL) cells is favored by the essential role of the tumor microenvironment (TME) that is similarly responsible at least in part for disease progression and drug resistance.
Aims
We planned to evaluate and predict the efficacy of therapeutic compounds in vivo, by reproducing in a co-culture system the different microenvironmental components that enable B cells to survive and proliferate, mimicking in particular the lymph node microenvironment where most of the crucial events of the pathogenesis of CLL occur.
Methods
To this purpose, cryopreserved peripheral blood (PB) mononuclear cells from CLL patients in need of treatment were tested with the Exvitech® proprietary automated flow cytometry-based platform. Different components have been evaluated to reproduce the ME and induce proliferation and survival of CLL cells: (i) 3 backbone stimulations: CD40L+CpG, CD40L+IL21, CpG+IL2; (ii) “Native Environment”, defined as the plasma & erythrocyte/granulocyte fraction of a Ficoll gradient: (iii) the stroma cell line H5S, added at different ratios (1:10 or 1:100); (iv) both human and bovine fetal serum (at 10 or 20% total volume); (v) stimulatory B cell factors, including IL-21, soluble CD40L, BAFF, and B cell receptor stimulation (anti-IG).
Results
Of all the described combinations, the addition of CpG+IL2, HS5 (at 1:100 ratio), human serum 10%, and “native environment” from PB of CLL samples (pooled samples to prevent interpatient variability) were the best to promote CLL proliferation (30±3%) and viability (60±5%). We then tested dose responses of the novel BCR inhibitors, idelalisib (PI3kδ inhibitor) and ibrutininb (BTK inhibitor) in 29 and 25 cryopreserved progressive CLL samples, respectively (Figure) and showed that resting CLL cells were virtually unaffected by either drug (EC50 of 12,5µM and 28.3µM), suggesting a limited direct pro-apoptotic activity of the drugs. In contrast, marked inhibition of proliferation was observed (EC50 of 30 nM and 550 nM, respectively) in the presence of either inhibitor, leaving only a median of 5% CLL cells that continued proliferating even at the highest doses.
Conclusion
We here report a novel ex vivo assay that incorporates TME stimuli thus more accurately simulating in vivo interactions and enabling high-throughput pharmacological characterization in more physiological conditions. This assay demonstrated a previously unreported anti-proliferative activity for both idelalisib and ibrutinib that may explain the efficacy of both drugs in patients.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Ex vivo, Microenvironment
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