MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF ALK+ AND ALK- ANAPLASTIC LARGE CELL LYMPHOMA (ALCL)-DERIVED EXOSOMES AND THEIR INTERACTIONS WITH TUMOR MICROENVIRONMENT
(Abstract release date: 05/19/16)
EHA Library. Rassidakis G. 06/09/16; 132925; E1376
Assoc. Prof. George Rassidakis
Contributions
Contributions
Abstract
Abstract: E1376
Type: Eposter Presentation
Background
Anaplastic large cell lymphoma (ALCL) is an aggressive type of T-cell non-Hodgkin lymphoma with high frequency in childhood. The current WHO classification recognizes two distinct types of ALCL based on the expression of the anaplastic lymphoma kinase (ALK), the ALK+ and the ALK- ALCL. The ALK+ ALCL is characterized by chromosomal translocations involving the alk gene locus, the most frequent being the t(2;5) leading to aberrant expression and activation of NPM.ALK oncoprotein. The latter is known to activate multiple oncogenic pathways including the Ras/ERK, JAK/STAT3, PI3K/AKT/MTOR, JNK/Jun, Sonic Hedgehog and others, resulting in cell cycle and apoptosis deregulation. However, the potential role of bi-directional crosstalk between the microenvironment (TME) and ALCL cells is not yet known. Exosomes are endosome-derived vesicles that contain DNA, RNA, proteins, lipids and other factors that have been reported to play significant role in intercellular signaling. Their role in the ALCL-TME interactions has not been investigated to date.
Aims
To characterize the ALCL-derived exosomes and investigate their possible functional interactions with microenvironment using an in vitro system as well as an ex vivo mouse model of ALK+ and ALK- ALCL.
Methods
Exosomes derived from ALK+ and ALK- ALCL cell lines were isolated using well established ultracentrifugation protocols. The exosomes were subsequently characterized by nanoparticle tracking analysis (NanoSight) and transmission electron microscopy for their size and shape. The molecular composition of exosomes at the RNA and protein level was assessed by real time RT-PCR and Western blotting, respectively. Stromal cells including bone marrow-derived fibroblasts and mesenchymal stem cells were co-cultured with ALK+ and ALK- ALCL cells or they were educated with exosomes and the biologic effects were investigated. Uptake levels of exosomes by recipient lymphoma or stromal cells were assessed by DIR labeling and flow cytometry. Expression of proteins associated with the Cancer-Associated Fibroblasts (CAF) was evaluated by immunofluorescence and confocal microscopy. The biologic effects of co-cultured lymphoma and stromal cells after ALK (Crizotinib) and STAT3 (Stattic, XIII) inhibition were analyzed with standard cell viability and proliferation assays and flow cytometry in our in vitro ALCL system. In addition, an ex vivo mouse model for ALK+ and ALK- ALCL (xenografts) was used in this study.
Results
We characterized for first time the ALK+ and ALK- ALCL-derived exosomes. Transmission electron microscopy and nanoparticle tracking analysis showed significant differences in exosome size among various ALCL cell lines. Western blot analysis performed on both, whole cellular and exosomal lysates, confirmed the expression of various exosomal markers including Rab5, Alix, and CD82. Lack of AIF in the exosomal lysates served as a quality control. More importantly, Western blot analysis revealed the presence of critical components of the ALK signaling in NPM-ALK positive ALCL-derived exosomes including pALK, pSTAT3, AP-1 transcription factors, pAKT and mTOR pathway kinases. ALCL-derived exosomes were uptaken by ALK+ and ALK- ALCL cells at a high level ranging from 30% to 80% at 6 hours, and similarly, they were uptaken by bone marrow-derived fibroblasts and mesenchymal stem cells at a high level as well. Co-culture of the ALCL cells with stromal cells resulted in activation of stromal fibroblasts that acquired a CAF phenotype (aSMA+) as shown by immunofluorescence and confocal microscopy. Furthermore, co-culturing of ALK+ ALCL and stromal cells altered response (sensitivity) of ALK+ ALCL cells to Crizotinib treatment at a moderate level.
Conclusion
This is the first study to characterize the ALK+ and ALK- ALCL-derived exosomes and demonstrate potential exosome-associated interactions between the ALCL and stromal cells. Ongoing functional studies will enrich our understanding for the involvement of the microenvironment in the mechanisms of resistance to targeted therapy that would lead to more efficient therapeutic approaches for the ALCL patients.
Session topic: E-poster
Keyword(s): ALK/ALCL, Microenvironment, Resistance, Signaling molecules
Type: Eposter Presentation
Background
Anaplastic large cell lymphoma (ALCL) is an aggressive type of T-cell non-Hodgkin lymphoma with high frequency in childhood. The current WHO classification recognizes two distinct types of ALCL based on the expression of the anaplastic lymphoma kinase (ALK), the ALK+ and the ALK- ALCL. The ALK+ ALCL is characterized by chromosomal translocations involving the alk gene locus, the most frequent being the t(2;5) leading to aberrant expression and activation of NPM.ALK oncoprotein. The latter is known to activate multiple oncogenic pathways including the Ras/ERK, JAK/STAT3, PI3K/AKT/MTOR, JNK/Jun, Sonic Hedgehog and others, resulting in cell cycle and apoptosis deregulation. However, the potential role of bi-directional crosstalk between the microenvironment (TME) and ALCL cells is not yet known. Exosomes are endosome-derived vesicles that contain DNA, RNA, proteins, lipids and other factors that have been reported to play significant role in intercellular signaling. Their role in the ALCL-TME interactions has not been investigated to date.
Aims
To characterize the ALCL-derived exosomes and investigate their possible functional interactions with microenvironment using an in vitro system as well as an ex vivo mouse model of ALK+ and ALK- ALCL.
Methods
Exosomes derived from ALK+ and ALK- ALCL cell lines were isolated using well established ultracentrifugation protocols. The exosomes were subsequently characterized by nanoparticle tracking analysis (NanoSight) and transmission electron microscopy for their size and shape. The molecular composition of exosomes at the RNA and protein level was assessed by real time RT-PCR and Western blotting, respectively. Stromal cells including bone marrow-derived fibroblasts and mesenchymal stem cells were co-cultured with ALK+ and ALK- ALCL cells or they were educated with exosomes and the biologic effects were investigated. Uptake levels of exosomes by recipient lymphoma or stromal cells were assessed by DIR labeling and flow cytometry. Expression of proteins associated with the Cancer-Associated Fibroblasts (CAF) was evaluated by immunofluorescence and confocal microscopy. The biologic effects of co-cultured lymphoma and stromal cells after ALK (Crizotinib) and STAT3 (Stattic, XIII) inhibition were analyzed with standard cell viability and proliferation assays and flow cytometry in our in vitro ALCL system. In addition, an ex vivo mouse model for ALK+ and ALK- ALCL (xenografts) was used in this study.
Results
We characterized for first time the ALK+ and ALK- ALCL-derived exosomes. Transmission electron microscopy and nanoparticle tracking analysis showed significant differences in exosome size among various ALCL cell lines. Western blot analysis performed on both, whole cellular and exosomal lysates, confirmed the expression of various exosomal markers including Rab5, Alix, and CD82. Lack of AIF in the exosomal lysates served as a quality control. More importantly, Western blot analysis revealed the presence of critical components of the ALK signaling in NPM-ALK positive ALCL-derived exosomes including pALK, pSTAT3, AP-1 transcription factors, pAKT and mTOR pathway kinases. ALCL-derived exosomes were uptaken by ALK+ and ALK- ALCL cells at a high level ranging from 30% to 80% at 6 hours, and similarly, they were uptaken by bone marrow-derived fibroblasts and mesenchymal stem cells at a high level as well. Co-culture of the ALCL cells with stromal cells resulted in activation of stromal fibroblasts that acquired a CAF phenotype (aSMA+) as shown by immunofluorescence and confocal microscopy. Furthermore, co-culturing of ALK+ ALCL and stromal cells altered response (sensitivity) of ALK+ ALCL cells to Crizotinib treatment at a moderate level.
Conclusion
This is the first study to characterize the ALK+ and ALK- ALCL-derived exosomes and demonstrate potential exosome-associated interactions between the ALCL and stromal cells. Ongoing functional studies will enrich our understanding for the involvement of the microenvironment in the mechanisms of resistance to targeted therapy that would lead to more efficient therapeutic approaches for the ALCL patients.
Session topic: E-poster
Keyword(s): ALK/ALCL, Microenvironment, Resistance, Signaling molecules
Abstract: E1376
Type: Eposter Presentation
Background
Anaplastic large cell lymphoma (ALCL) is an aggressive type of T-cell non-Hodgkin lymphoma with high frequency in childhood. The current WHO classification recognizes two distinct types of ALCL based on the expression of the anaplastic lymphoma kinase (ALK), the ALK+ and the ALK- ALCL. The ALK+ ALCL is characterized by chromosomal translocations involving the alk gene locus, the most frequent being the t(2;5) leading to aberrant expression and activation of NPM.ALK oncoprotein. The latter is known to activate multiple oncogenic pathways including the Ras/ERK, JAK/STAT3, PI3K/AKT/MTOR, JNK/Jun, Sonic Hedgehog and others, resulting in cell cycle and apoptosis deregulation. However, the potential role of bi-directional crosstalk between the microenvironment (TME) and ALCL cells is not yet known. Exosomes are endosome-derived vesicles that contain DNA, RNA, proteins, lipids and other factors that have been reported to play significant role in intercellular signaling. Their role in the ALCL-TME interactions has not been investigated to date.
Aims
To characterize the ALCL-derived exosomes and investigate their possible functional interactions with microenvironment using an in vitro system as well as an ex vivo mouse model of ALK+ and ALK- ALCL.
Methods
Exosomes derived from ALK+ and ALK- ALCL cell lines were isolated using well established ultracentrifugation protocols. The exosomes were subsequently characterized by nanoparticle tracking analysis (NanoSight) and transmission electron microscopy for their size and shape. The molecular composition of exosomes at the RNA and protein level was assessed by real time RT-PCR and Western blotting, respectively. Stromal cells including bone marrow-derived fibroblasts and mesenchymal stem cells were co-cultured with ALK+ and ALK- ALCL cells or they were educated with exosomes and the biologic effects were investigated. Uptake levels of exosomes by recipient lymphoma or stromal cells were assessed by DIR labeling and flow cytometry. Expression of proteins associated with the Cancer-Associated Fibroblasts (CAF) was evaluated by immunofluorescence and confocal microscopy. The biologic effects of co-cultured lymphoma and stromal cells after ALK (Crizotinib) and STAT3 (Stattic, XIII) inhibition were analyzed with standard cell viability and proliferation assays and flow cytometry in our in vitro ALCL system. In addition, an ex vivo mouse model for ALK+ and ALK- ALCL (xenografts) was used in this study.
Results
We characterized for first time the ALK+ and ALK- ALCL-derived exosomes. Transmission electron microscopy and nanoparticle tracking analysis showed significant differences in exosome size among various ALCL cell lines. Western blot analysis performed on both, whole cellular and exosomal lysates, confirmed the expression of various exosomal markers including Rab5, Alix, and CD82. Lack of AIF in the exosomal lysates served as a quality control. More importantly, Western blot analysis revealed the presence of critical components of the ALK signaling in NPM-ALK positive ALCL-derived exosomes including pALK, pSTAT3, AP-1 transcription factors, pAKT and mTOR pathway kinases. ALCL-derived exosomes were uptaken by ALK+ and ALK- ALCL cells at a high level ranging from 30% to 80% at 6 hours, and similarly, they were uptaken by bone marrow-derived fibroblasts and mesenchymal stem cells at a high level as well. Co-culture of the ALCL cells with stromal cells resulted in activation of stromal fibroblasts that acquired a CAF phenotype (aSMA+) as shown by immunofluorescence and confocal microscopy. Furthermore, co-culturing of ALK+ ALCL and stromal cells altered response (sensitivity) of ALK+ ALCL cells to Crizotinib treatment at a moderate level.
Conclusion
This is the first study to characterize the ALK+ and ALK- ALCL-derived exosomes and demonstrate potential exosome-associated interactions between the ALCL and stromal cells. Ongoing functional studies will enrich our understanding for the involvement of the microenvironment in the mechanisms of resistance to targeted therapy that would lead to more efficient therapeutic approaches for the ALCL patients.
Session topic: E-poster
Keyword(s): ALK/ALCL, Microenvironment, Resistance, Signaling molecules
Type: Eposter Presentation
Background
Anaplastic large cell lymphoma (ALCL) is an aggressive type of T-cell non-Hodgkin lymphoma with high frequency in childhood. The current WHO classification recognizes two distinct types of ALCL based on the expression of the anaplastic lymphoma kinase (ALK), the ALK+ and the ALK- ALCL. The ALK+ ALCL is characterized by chromosomal translocations involving the alk gene locus, the most frequent being the t(2;5) leading to aberrant expression and activation of NPM.ALK oncoprotein. The latter is known to activate multiple oncogenic pathways including the Ras/ERK, JAK/STAT3, PI3K/AKT/MTOR, JNK/Jun, Sonic Hedgehog and others, resulting in cell cycle and apoptosis deregulation. However, the potential role of bi-directional crosstalk between the microenvironment (TME) and ALCL cells is not yet known. Exosomes are endosome-derived vesicles that contain DNA, RNA, proteins, lipids and other factors that have been reported to play significant role in intercellular signaling. Their role in the ALCL-TME interactions has not been investigated to date.
Aims
To characterize the ALCL-derived exosomes and investigate their possible functional interactions with microenvironment using an in vitro system as well as an ex vivo mouse model of ALK+ and ALK- ALCL.
Methods
Exosomes derived from ALK+ and ALK- ALCL cell lines were isolated using well established ultracentrifugation protocols. The exosomes were subsequently characterized by nanoparticle tracking analysis (NanoSight) and transmission electron microscopy for their size and shape. The molecular composition of exosomes at the RNA and protein level was assessed by real time RT-PCR and Western blotting, respectively. Stromal cells including bone marrow-derived fibroblasts and mesenchymal stem cells were co-cultured with ALK+ and ALK- ALCL cells or they were educated with exosomes and the biologic effects were investigated. Uptake levels of exosomes by recipient lymphoma or stromal cells were assessed by DIR labeling and flow cytometry. Expression of proteins associated with the Cancer-Associated Fibroblasts (CAF) was evaluated by immunofluorescence and confocal microscopy. The biologic effects of co-cultured lymphoma and stromal cells after ALK (Crizotinib) and STAT3 (Stattic, XIII) inhibition were analyzed with standard cell viability and proliferation assays and flow cytometry in our in vitro ALCL system. In addition, an ex vivo mouse model for ALK+ and ALK- ALCL (xenografts) was used in this study.
Results
We characterized for first time the ALK+ and ALK- ALCL-derived exosomes. Transmission electron microscopy and nanoparticle tracking analysis showed significant differences in exosome size among various ALCL cell lines. Western blot analysis performed on both, whole cellular and exosomal lysates, confirmed the expression of various exosomal markers including Rab5, Alix, and CD82. Lack of AIF in the exosomal lysates served as a quality control. More importantly, Western blot analysis revealed the presence of critical components of the ALK signaling in NPM-ALK positive ALCL-derived exosomes including pALK, pSTAT3, AP-1 transcription factors, pAKT and mTOR pathway kinases. ALCL-derived exosomes were uptaken by ALK+ and ALK- ALCL cells at a high level ranging from 30% to 80% at 6 hours, and similarly, they were uptaken by bone marrow-derived fibroblasts and mesenchymal stem cells at a high level as well. Co-culture of the ALCL cells with stromal cells resulted in activation of stromal fibroblasts that acquired a CAF phenotype (aSMA+) as shown by immunofluorescence and confocal microscopy. Furthermore, co-culturing of ALK+ ALCL and stromal cells altered response (sensitivity) of ALK+ ALCL cells to Crizotinib treatment at a moderate level.
Conclusion
This is the first study to characterize the ALK+ and ALK- ALCL-derived exosomes and demonstrate potential exosome-associated interactions between the ALCL and stromal cells. Ongoing functional studies will enrich our understanding for the involvement of the microenvironment in the mechanisms of resistance to targeted therapy that would lead to more efficient therapeutic approaches for the ALCL patients.
Session topic: E-poster
Keyword(s): ALK/ALCL, Microenvironment, Resistance, Signaling molecules
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