A NOVEL TOOL TO MONITOR ANGIOGENESIS IN BONE MARROW SMEARS: DETECTION AND QUANTIFICATION OF ENDOTHELIAL PROGENITOR CELLS BY MULTIPLE-LABELING IMMUNOFLUORESCENCE ANALYSIS
(Abstract release date: 05/19/16)
EHA Library. Tenreiro M. 06/09/16; 134846; PB1946
Ms. Maria Tenreiro
Contributions
Contributions
Abstract
Abstract: PB1946
Type: Publication Only
Background
Angiogenesis is a crucial event in cancer, as it is present in tumor growth, invasion and metastasis. It is considered to have a key role in several haematological malignancies that affect the bone marrow, such as multiple myeloma. Tumor neovascularization and angiogenesis involve the recruitment of endothelial progenitor cells (EPCs), which are bone-marrow derived circulating progenitors with the potential to differentiate into cells of the endothelial lineage. Two types of EPCs have been described: early EPCs, which can also be called angiogenic cells, and mature EPCs or endothelial outgrowth cells. These two types of cells differ in markers expression, morphology, proliferative potential and in vitro function, like vascular tube formation. Identification of EPCs is still a challenging task, since these cells display no specific cell-surface antigen. Even so, it is widely accepted that early EPCs are positive for the following markers: cluster of differentiation (CD)133, which is expressed by haematopoietic and progenitor cells; CD34, characteristic of hematopoietic stem cells and activated endothelium of small vessels; and vascular endothelial growth factor receptor (VEGFR)-2, present in endothelial lineage. Regarding mature EPCs, they are considered CD133-/CD34+/VEGFR-2+.
Aims
To overcome the difficulty in the assessment of EPCs, usually by flow cytometry and cell culture, we decided to develop a new triple-labelling immunofluorescence analysis protocol to assess EPCs in bone marrow smears as a tool to monitor the angiogenesis process and disease progression in pathologies like multiple myeloma.
Methods
Bone marrow smears archived at the Hematology Service of Instituto Português de Oncologia Dr. Francisco Gentil, Lisbon, from multiple myeloma patients were used. Smears were fixed and permeabilized with a 75% methanol/25% acetone/0.01% Triton X-100 solution and blocked with 10% goat serum/1% bovine serum albumin in PBS or 10% rabbit serum serum/1% bovine serum albumin in PBS, according to the host species of the secondary antibodies. Triple labelling was performed using antibodies against CD133, CD34 and VEGFR-2, followed by species-specific secondary antibodies, and nuclei labelling with DAPI. Smears were analysed under a confocal laser microscope and/or a widefield epifluorescence microscope with appropriate excitation and emission filters.
Results
With this protocol, it was possible to identify cells that were triple (CD133+, CD34+ and VEGFR-2+) and double labelled (CD133-, CD34+ and VEGFR-2+), which correspond to early EPCs and mature EPCs, respectively. Furthermore, quantification of the total number of labelled nuclei allowed the establishment of the percentage of early EPCs and mature EPCs relatively to the cellular population of the bone marrow smear. Moreover, analysis of samples of the same patient collected at different time points allowed the establishment of differences in EPCs subsets along the course of the disease.
Conclusion
Our protocol is an easy and accessible method to monitor angiogenesis based on analysis of EPCs, which can be implemented in any laboratory equipped with a suitable microscope. This protocol may be used to study multiple myeloma, as well as other pathologies involving enhanced angiogenesis in the bone marrow, therefore constituting a novel tool for early detection of angiogenesis and efficient monitoring of neovascularization along disease progression and treatment.
Session topic: E-poster
Keyword(s): Bone marrow biopsy, Endothelial progenitor cell, Immunohistochemistry, Multiple myeloma
Type: Publication Only
Background
Angiogenesis is a crucial event in cancer, as it is present in tumor growth, invasion and metastasis. It is considered to have a key role in several haematological malignancies that affect the bone marrow, such as multiple myeloma. Tumor neovascularization and angiogenesis involve the recruitment of endothelial progenitor cells (EPCs), which are bone-marrow derived circulating progenitors with the potential to differentiate into cells of the endothelial lineage. Two types of EPCs have been described: early EPCs, which can also be called angiogenic cells, and mature EPCs or endothelial outgrowth cells. These two types of cells differ in markers expression, morphology, proliferative potential and in vitro function, like vascular tube formation. Identification of EPCs is still a challenging task, since these cells display no specific cell-surface antigen. Even so, it is widely accepted that early EPCs are positive for the following markers: cluster of differentiation (CD)133, which is expressed by haematopoietic and progenitor cells; CD34, characteristic of hematopoietic stem cells and activated endothelium of small vessels; and vascular endothelial growth factor receptor (VEGFR)-2, present in endothelial lineage. Regarding mature EPCs, they are considered CD133-/CD34+/VEGFR-2+.
Aims
To overcome the difficulty in the assessment of EPCs, usually by flow cytometry and cell culture, we decided to develop a new triple-labelling immunofluorescence analysis protocol to assess EPCs in bone marrow smears as a tool to monitor the angiogenesis process and disease progression in pathologies like multiple myeloma.
Methods
Bone marrow smears archived at the Hematology Service of Instituto Português de Oncologia Dr. Francisco Gentil, Lisbon, from multiple myeloma patients were used. Smears were fixed and permeabilized with a 75% methanol/25% acetone/0.01% Triton X-100 solution and blocked with 10% goat serum/1% bovine serum albumin in PBS or 10% rabbit serum serum/1% bovine serum albumin in PBS, according to the host species of the secondary antibodies. Triple labelling was performed using antibodies against CD133, CD34 and VEGFR-2, followed by species-specific secondary antibodies, and nuclei labelling with DAPI. Smears were analysed under a confocal laser microscope and/or a widefield epifluorescence microscope with appropriate excitation and emission filters.
Results
With this protocol, it was possible to identify cells that were triple (CD133+, CD34+ and VEGFR-2+) and double labelled (CD133-, CD34+ and VEGFR-2+), which correspond to early EPCs and mature EPCs, respectively. Furthermore, quantification of the total number of labelled nuclei allowed the establishment of the percentage of early EPCs and mature EPCs relatively to the cellular population of the bone marrow smear. Moreover, analysis of samples of the same patient collected at different time points allowed the establishment of differences in EPCs subsets along the course of the disease.
Conclusion
Our protocol is an easy and accessible method to monitor angiogenesis based on analysis of EPCs, which can be implemented in any laboratory equipped with a suitable microscope. This protocol may be used to study multiple myeloma, as well as other pathologies involving enhanced angiogenesis in the bone marrow, therefore constituting a novel tool for early detection of angiogenesis and efficient monitoring of neovascularization along disease progression and treatment.
Session topic: E-poster
Keyword(s): Bone marrow biopsy, Endothelial progenitor cell, Immunohistochemistry, Multiple myeloma
Abstract: PB1946
Type: Publication Only
Background
Angiogenesis is a crucial event in cancer, as it is present in tumor growth, invasion and metastasis. It is considered to have a key role in several haematological malignancies that affect the bone marrow, such as multiple myeloma. Tumor neovascularization and angiogenesis involve the recruitment of endothelial progenitor cells (EPCs), which are bone-marrow derived circulating progenitors with the potential to differentiate into cells of the endothelial lineage. Two types of EPCs have been described: early EPCs, which can also be called angiogenic cells, and mature EPCs or endothelial outgrowth cells. These two types of cells differ in markers expression, morphology, proliferative potential and in vitro function, like vascular tube formation. Identification of EPCs is still a challenging task, since these cells display no specific cell-surface antigen. Even so, it is widely accepted that early EPCs are positive for the following markers: cluster of differentiation (CD)133, which is expressed by haematopoietic and progenitor cells; CD34, characteristic of hematopoietic stem cells and activated endothelium of small vessels; and vascular endothelial growth factor receptor (VEGFR)-2, present in endothelial lineage. Regarding mature EPCs, they are considered CD133-/CD34+/VEGFR-2+.
Aims
To overcome the difficulty in the assessment of EPCs, usually by flow cytometry and cell culture, we decided to develop a new triple-labelling immunofluorescence analysis protocol to assess EPCs in bone marrow smears as a tool to monitor the angiogenesis process and disease progression in pathologies like multiple myeloma.
Methods
Bone marrow smears archived at the Hematology Service of Instituto Português de Oncologia Dr. Francisco Gentil, Lisbon, from multiple myeloma patients were used. Smears were fixed and permeabilized with a 75% methanol/25% acetone/0.01% Triton X-100 solution and blocked with 10% goat serum/1% bovine serum albumin in PBS or 10% rabbit serum serum/1% bovine serum albumin in PBS, according to the host species of the secondary antibodies. Triple labelling was performed using antibodies against CD133, CD34 and VEGFR-2, followed by species-specific secondary antibodies, and nuclei labelling with DAPI. Smears were analysed under a confocal laser microscope and/or a widefield epifluorescence microscope with appropriate excitation and emission filters.
Results
With this protocol, it was possible to identify cells that were triple (CD133+, CD34+ and VEGFR-2+) and double labelled (CD133-, CD34+ and VEGFR-2+), which correspond to early EPCs and mature EPCs, respectively. Furthermore, quantification of the total number of labelled nuclei allowed the establishment of the percentage of early EPCs and mature EPCs relatively to the cellular population of the bone marrow smear. Moreover, analysis of samples of the same patient collected at different time points allowed the establishment of differences in EPCs subsets along the course of the disease.
Conclusion
Our protocol is an easy and accessible method to monitor angiogenesis based on analysis of EPCs, which can be implemented in any laboratory equipped with a suitable microscope. This protocol may be used to study multiple myeloma, as well as other pathologies involving enhanced angiogenesis in the bone marrow, therefore constituting a novel tool for early detection of angiogenesis and efficient monitoring of neovascularization along disease progression and treatment.
Session topic: E-poster
Keyword(s): Bone marrow biopsy, Endothelial progenitor cell, Immunohistochemistry, Multiple myeloma
Type: Publication Only
Background
Angiogenesis is a crucial event in cancer, as it is present in tumor growth, invasion and metastasis. It is considered to have a key role in several haematological malignancies that affect the bone marrow, such as multiple myeloma. Tumor neovascularization and angiogenesis involve the recruitment of endothelial progenitor cells (EPCs), which are bone-marrow derived circulating progenitors with the potential to differentiate into cells of the endothelial lineage. Two types of EPCs have been described: early EPCs, which can also be called angiogenic cells, and mature EPCs or endothelial outgrowth cells. These two types of cells differ in markers expression, morphology, proliferative potential and in vitro function, like vascular tube formation. Identification of EPCs is still a challenging task, since these cells display no specific cell-surface antigen. Even so, it is widely accepted that early EPCs are positive for the following markers: cluster of differentiation (CD)133, which is expressed by haematopoietic and progenitor cells; CD34, characteristic of hematopoietic stem cells and activated endothelium of small vessels; and vascular endothelial growth factor receptor (VEGFR)-2, present in endothelial lineage. Regarding mature EPCs, they are considered CD133-/CD34+/VEGFR-2+.
Aims
To overcome the difficulty in the assessment of EPCs, usually by flow cytometry and cell culture, we decided to develop a new triple-labelling immunofluorescence analysis protocol to assess EPCs in bone marrow smears as a tool to monitor the angiogenesis process and disease progression in pathologies like multiple myeloma.
Methods
Bone marrow smears archived at the Hematology Service of Instituto Português de Oncologia Dr. Francisco Gentil, Lisbon, from multiple myeloma patients were used. Smears were fixed and permeabilized with a 75% methanol/25% acetone/0.01% Triton X-100 solution and blocked with 10% goat serum/1% bovine serum albumin in PBS or 10% rabbit serum serum/1% bovine serum albumin in PBS, according to the host species of the secondary antibodies. Triple labelling was performed using antibodies against CD133, CD34 and VEGFR-2, followed by species-specific secondary antibodies, and nuclei labelling with DAPI. Smears were analysed under a confocal laser microscope and/or a widefield epifluorescence microscope with appropriate excitation and emission filters.
Results
With this protocol, it was possible to identify cells that were triple (CD133+, CD34+ and VEGFR-2+) and double labelled (CD133-, CD34+ and VEGFR-2+), which correspond to early EPCs and mature EPCs, respectively. Furthermore, quantification of the total number of labelled nuclei allowed the establishment of the percentage of early EPCs and mature EPCs relatively to the cellular population of the bone marrow smear. Moreover, analysis of samples of the same patient collected at different time points allowed the establishment of differences in EPCs subsets along the course of the disease.
Conclusion
Our protocol is an easy and accessible method to monitor angiogenesis based on analysis of EPCs, which can be implemented in any laboratory equipped with a suitable microscope. This protocol may be used to study multiple myeloma, as well as other pathologies involving enhanced angiogenesis in the bone marrow, therefore constituting a novel tool for early detection of angiogenesis and efficient monitoring of neovascularization along disease progression and treatment.
Session topic: E-poster
Keyword(s): Bone marrow biopsy, Endothelial progenitor cell, Immunohistochemistry, Multiple myeloma
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