HETEROTYPIC SIGNALLING BETWEEN BONE MARROW STROMAL CELLS AND BLASTS PROVIDES INSIGHT INTO THE CELLULAR COMMUNICATION IN ACUTE MYELOID LEUKAEMIA
(Abstract release date: 05/19/16)
EHA Library. Dokal A. 06/09/16; 132451; E902
Mr. Arran Dokal
Contributions
Contributions
Abstract
Abstract: E902
Type: Eposter Presentation
Background
The bone marrow stromal microenvironment (BMSM) has a well-established role in the pathophysiology of acute myeloid leukaemia (AML). This is demonstrated by primary AML blasts dependence on stromal conditioned media to survive in culture. Although it is clear that there are components of the stromal secretome that augment AML blast survival, we are currently only aware of a fraction of these factors and the precise dynamics of the stromal-blast interactions are not fully understood. In this study we were interested in dissecting the secretome and identifying novel secreted proteins that may contribute to AML cell survival outside of the current understanding (e.g. SDF-1).
Aims
The ultimate aim of this project is to determine proteins secreted by AML and bone marrow stromal cells that may mediate communication between AML blasts and the stromal environment. Further, we wanted to identify secreted proteins in AML cells harbouring different karyotypes to understand how these cells illicit heterotypic activation or inhibition of signalling pathways in cells across the different AML-subtypes.
Methods
To assess the feasibility of this approach, the secretome of 4 human AML cell lines and 1 mouse bone marrow stromal cell line were characterised by harvesting the conditioned media from each cell line individually and in co-culture (9 conditions) in triplicate (n=3). The secretomes were then purified from the conditioned media before undergoing global quantitative proteomic analysis using Liquid Chromatography Tandem Mass Spectrometry. MASCOT searches against both mouse and human proteomes allowed for discrimination between the stromal and AML proteins during bioinformatic analysis. In parallel the viability of these cell populations was recorded using a Beckman Coulter Vi-Cell cell viability analyser, functionally assessing the supportive capabilities of the BMSM after the AML cells were subjected to 24 hours of serum starvation.
Results
Secretomic analysis of the chemical crosstalk identified 520 bone marrow stromal proteins (including Osteopontin, Fibronectin, ECM proteins) and 293 AML blast proteins that satisfied identification criteria across the samples. 98 of such proteins have known functions as cytokines/growth factors. Analysis of the stromal secretome revealed a set of 20 stromal proteins whose secretion was modulated as a result of AML-stromal co-culture. These putative extracellular signalling proteins have known roles in cell adhesion, migration or microenvironment remodelling. In addition, the modulations in the composition of the AML secretome are suggestive of reactive crosstalk with the BMSM leading to stromal cell complicity. Finally, we display how serum starvation only leads to a drop in viability for AML cells cultured in isolation. It appears the BMSM is able to maintain if not increase basal AML cell viability under the same conditions. These findings are complimented by the reduction in extracellular markers of stress in the co-cultured secretomes.
Conclusion
This proteomic approach has allowed for the identification of a panel of proteins secreted by the stromal cells that can affect cell signalling and therefore behaviour of AML blast cells. Interestingly there are a number of novel signalling proteins secreted by the blasts that are indicative of orchestrating the BMSM. Analysis is now underway to fully map the progression of signalling in AML blasts that is initiated by the binding of these stromal signalling proteins. When complete this study will provide insight as to how the stromal microenvironment helps propagate leukaemic blasts across the different AML-subtypes.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Microenvironment, Proteomics, Signaling
Type: Eposter Presentation
Background
The bone marrow stromal microenvironment (BMSM) has a well-established role in the pathophysiology of acute myeloid leukaemia (AML). This is demonstrated by primary AML blasts dependence on stromal conditioned media to survive in culture. Although it is clear that there are components of the stromal secretome that augment AML blast survival, we are currently only aware of a fraction of these factors and the precise dynamics of the stromal-blast interactions are not fully understood. In this study we were interested in dissecting the secretome and identifying novel secreted proteins that may contribute to AML cell survival outside of the current understanding (e.g. SDF-1).
Aims
The ultimate aim of this project is to determine proteins secreted by AML and bone marrow stromal cells that may mediate communication between AML blasts and the stromal environment. Further, we wanted to identify secreted proteins in AML cells harbouring different karyotypes to understand how these cells illicit heterotypic activation or inhibition of signalling pathways in cells across the different AML-subtypes.
Methods
To assess the feasibility of this approach, the secretome of 4 human AML cell lines and 1 mouse bone marrow stromal cell line were characterised by harvesting the conditioned media from each cell line individually and in co-culture (9 conditions) in triplicate (n=3). The secretomes were then purified from the conditioned media before undergoing global quantitative proteomic analysis using Liquid Chromatography Tandem Mass Spectrometry. MASCOT searches against both mouse and human proteomes allowed for discrimination between the stromal and AML proteins during bioinformatic analysis. In parallel the viability of these cell populations was recorded using a Beckman Coulter Vi-Cell cell viability analyser, functionally assessing the supportive capabilities of the BMSM after the AML cells were subjected to 24 hours of serum starvation.
Results
Secretomic analysis of the chemical crosstalk identified 520 bone marrow stromal proteins (including Osteopontin, Fibronectin, ECM proteins) and 293 AML blast proteins that satisfied identification criteria across the samples. 98 of such proteins have known functions as cytokines/growth factors. Analysis of the stromal secretome revealed a set of 20 stromal proteins whose secretion was modulated as a result of AML-stromal co-culture. These putative extracellular signalling proteins have known roles in cell adhesion, migration or microenvironment remodelling. In addition, the modulations in the composition of the AML secretome are suggestive of reactive crosstalk with the BMSM leading to stromal cell complicity. Finally, we display how serum starvation only leads to a drop in viability for AML cells cultured in isolation. It appears the BMSM is able to maintain if not increase basal AML cell viability under the same conditions. These findings are complimented by the reduction in extracellular markers of stress in the co-cultured secretomes.
Conclusion
This proteomic approach has allowed for the identification of a panel of proteins secreted by the stromal cells that can affect cell signalling and therefore behaviour of AML blast cells. Interestingly there are a number of novel signalling proteins secreted by the blasts that are indicative of orchestrating the BMSM. Analysis is now underway to fully map the progression of signalling in AML blasts that is initiated by the binding of these stromal signalling proteins. When complete this study will provide insight as to how the stromal microenvironment helps propagate leukaemic blasts across the different AML-subtypes.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Microenvironment, Proteomics, Signaling
Abstract: E902
Type: Eposter Presentation
Background
The bone marrow stromal microenvironment (BMSM) has a well-established role in the pathophysiology of acute myeloid leukaemia (AML). This is demonstrated by primary AML blasts dependence on stromal conditioned media to survive in culture. Although it is clear that there are components of the stromal secretome that augment AML blast survival, we are currently only aware of a fraction of these factors and the precise dynamics of the stromal-blast interactions are not fully understood. In this study we were interested in dissecting the secretome and identifying novel secreted proteins that may contribute to AML cell survival outside of the current understanding (e.g. SDF-1).
Aims
The ultimate aim of this project is to determine proteins secreted by AML and bone marrow stromal cells that may mediate communication between AML blasts and the stromal environment. Further, we wanted to identify secreted proteins in AML cells harbouring different karyotypes to understand how these cells illicit heterotypic activation or inhibition of signalling pathways in cells across the different AML-subtypes.
Methods
To assess the feasibility of this approach, the secretome of 4 human AML cell lines and 1 mouse bone marrow stromal cell line were characterised by harvesting the conditioned media from each cell line individually and in co-culture (9 conditions) in triplicate (n=3). The secretomes were then purified from the conditioned media before undergoing global quantitative proteomic analysis using Liquid Chromatography Tandem Mass Spectrometry. MASCOT searches against both mouse and human proteomes allowed for discrimination between the stromal and AML proteins during bioinformatic analysis. In parallel the viability of these cell populations was recorded using a Beckman Coulter Vi-Cell cell viability analyser, functionally assessing the supportive capabilities of the BMSM after the AML cells were subjected to 24 hours of serum starvation.
Results
Secretomic analysis of the chemical crosstalk identified 520 bone marrow stromal proteins (including Osteopontin, Fibronectin, ECM proteins) and 293 AML blast proteins that satisfied identification criteria across the samples. 98 of such proteins have known functions as cytokines/growth factors. Analysis of the stromal secretome revealed a set of 20 stromal proteins whose secretion was modulated as a result of AML-stromal co-culture. These putative extracellular signalling proteins have known roles in cell adhesion, migration or microenvironment remodelling. In addition, the modulations in the composition of the AML secretome are suggestive of reactive crosstalk with the BMSM leading to stromal cell complicity. Finally, we display how serum starvation only leads to a drop in viability for AML cells cultured in isolation. It appears the BMSM is able to maintain if not increase basal AML cell viability under the same conditions. These findings are complimented by the reduction in extracellular markers of stress in the co-cultured secretomes.
Conclusion
This proteomic approach has allowed for the identification of a panel of proteins secreted by the stromal cells that can affect cell signalling and therefore behaviour of AML blast cells. Interestingly there are a number of novel signalling proteins secreted by the blasts that are indicative of orchestrating the BMSM. Analysis is now underway to fully map the progression of signalling in AML blasts that is initiated by the binding of these stromal signalling proteins. When complete this study will provide insight as to how the stromal microenvironment helps propagate leukaemic blasts across the different AML-subtypes.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Microenvironment, Proteomics, Signaling
Type: Eposter Presentation
Background
The bone marrow stromal microenvironment (BMSM) has a well-established role in the pathophysiology of acute myeloid leukaemia (AML). This is demonstrated by primary AML blasts dependence on stromal conditioned media to survive in culture. Although it is clear that there are components of the stromal secretome that augment AML blast survival, we are currently only aware of a fraction of these factors and the precise dynamics of the stromal-blast interactions are not fully understood. In this study we were interested in dissecting the secretome and identifying novel secreted proteins that may contribute to AML cell survival outside of the current understanding (e.g. SDF-1).
Aims
The ultimate aim of this project is to determine proteins secreted by AML and bone marrow stromal cells that may mediate communication between AML blasts and the stromal environment. Further, we wanted to identify secreted proteins in AML cells harbouring different karyotypes to understand how these cells illicit heterotypic activation or inhibition of signalling pathways in cells across the different AML-subtypes.
Methods
To assess the feasibility of this approach, the secretome of 4 human AML cell lines and 1 mouse bone marrow stromal cell line were characterised by harvesting the conditioned media from each cell line individually and in co-culture (9 conditions) in triplicate (n=3). The secretomes were then purified from the conditioned media before undergoing global quantitative proteomic analysis using Liquid Chromatography Tandem Mass Spectrometry. MASCOT searches against both mouse and human proteomes allowed for discrimination between the stromal and AML proteins during bioinformatic analysis. In parallel the viability of these cell populations was recorded using a Beckman Coulter Vi-Cell cell viability analyser, functionally assessing the supportive capabilities of the BMSM after the AML cells were subjected to 24 hours of serum starvation.
Results
Secretomic analysis of the chemical crosstalk identified 520 bone marrow stromal proteins (including Osteopontin, Fibronectin, ECM proteins) and 293 AML blast proteins that satisfied identification criteria across the samples. 98 of such proteins have known functions as cytokines/growth factors. Analysis of the stromal secretome revealed a set of 20 stromal proteins whose secretion was modulated as a result of AML-stromal co-culture. These putative extracellular signalling proteins have known roles in cell adhesion, migration or microenvironment remodelling. In addition, the modulations in the composition of the AML secretome are suggestive of reactive crosstalk with the BMSM leading to stromal cell complicity. Finally, we display how serum starvation only leads to a drop in viability for AML cells cultured in isolation. It appears the BMSM is able to maintain if not increase basal AML cell viability under the same conditions. These findings are complimented by the reduction in extracellular markers of stress in the co-cultured secretomes.
Conclusion
This proteomic approach has allowed for the identification of a panel of proteins secreted by the stromal cells that can affect cell signalling and therefore behaviour of AML blast cells. Interestingly there are a number of novel signalling proteins secreted by the blasts that are indicative of orchestrating the BMSM. Analysis is now underway to fully map the progression of signalling in AML blasts that is initiated by the binding of these stromal signalling proteins. When complete this study will provide insight as to how the stromal microenvironment helps propagate leukaemic blasts across the different AML-subtypes.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Microenvironment, Proteomics, Signaling
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