Defining a Characteristic Gene Expression Set Responsible for Cancer Stem Cell-Like Features in a Sub-Population of Ewing Sarcoma Cells CADO-ES1

One of the still open questions in Ewing sarcoma, a rare bone tumor with weak therapeutic options, is to identify the tumor-driving cell (sub) population and to understand the specifics in the biological network of these cells. This basic scientific insight might foster the development of more specific therapeutic target patterns. The experimental approach is based on a side population (SP) of Ewing cells, based on the model cell line CADO-ES1. The SP is established by flow cytometry and defined by the idea that tumor stem-like cells can be identified by the time-course in clearing a given artificial dye. The SP was characterized by a higher colony forming activity, by a higher differentiation potential, by higher resistance to cytotoxic drugs, and by morphology. Several SP and non-SP cell fractions and bone marrow-derived mesenchymal stem cell reference were analyzed by short read sequencing of the full transcriptome. The double-differential analysis leads to an altered expression structure of SP cells centered around the AP-1 and APC/c complex. The SP cells share only a limited proportion of the full mesenchymal stem cell stemness set of genes. This is in line with the expectation that tumor stem-like cells share only a limited subset of stemness features which are relevant for tumor survival

Defining a Characteristic Gene Expression Set Responsible for Cancer Stem Cell-Like Features in a Sub-Population of Ewing Sarcoma Cells CADO-ES1

One of the still open questions in Ewing sarcoma, a rare bone tumor with weak therapeutic options, is to identify the tumor-driving cell (sub) population and to understand the specifics in the biological network of these cells. This basic scientific insight might foster the development of more specific therapeutic target patterns. The experimental approach is based on a side population (SP) of Ewing cells, based on the model cell line CADO-ES1. The SP is established by flow cytometry and defined by the idea that tumor stem-like cells can be identified by the time-course in clearing a given artificial dye. The SP was characterized by a higher colony forming activity, by a higher differentiation potential, by higher resistance to cytotoxic drugs, and by morphology. Several SP and non-SP cell fractions and bone marrow-derived mesenchymal stem cell reference were analyzed by short read sequencing of the full transcriptome. The double-differential analysis leads to an altered expression structure of SP cells centered around the AP-1 and APC/c complex. The SP cells share only a limited proportion of the full mesenchymal stem cell stemness set of genes. This is in line with the expectation that tumor stem-like cells share only a limited subset of stemness features which are relevant for tumor survival

Smarcb1 Loss Results in a Deregulation of esBAF Binding and Impacts the Expression of Neurodevelopmental Genes

The murine esBAF complex plays a major role in the regulation of gene expression during stem cell development and differentiation. As one of its core subunits, Smarcb1 is indispensable for its function and its loss is connected to neurodevelopmental disorders and participates in the carcinogenesis of entities such as rhabdoid tumours. We explored how Smarcb1 regulates gene programs in murine embryonic stem cells (mESC) and in this way orchestrates differentiation. Our data underline the importance of Smarcb1 expression and function for the development of the nervous system along with basic cellular functions, such as cell adhesion and cell organisation. Using ChIP-seq, we were able to portray the consequences of Smarcb1 knockdown (kd) for the binding of esBAF and PRC2 as well as its influence on histone marks H3K27me3, H3K4me3 and H3K27ac. Their signals are changed in gene and enhancer regions of genes connected to nervous system development and offers a plausible explanation for changes in gene expression. Further, we describe a group of genes that are, despite increased BAF binding, suppressed after Smarcb1 kd by mechanisms independent of PRC2 function

Lack of expression of the chondroitin sulphate proteoglycan neuron-glial antigen 2 on candidate stem cell populations in paediatric acute myeloid leukaemia/abn(11q23) and acute lymphoblastic leukaemia/t(4;11)

textabstractIt has increasingly been acknowledged that only a few leukaemic cells possess the capability to renew themselves and that only these self-renewing leukaemic stem cells are able to initiate relapses. Therefore, these leukaemic stem cells should be the target cells for therapy and for minimal residual disease (MRD) detection. Because of its presence on blasts of 11q23-rearranged high-risk leukaemic patients, neuron-glial antigen 2 (NG2) is thought to be a valuable marker for detecting leukaemic stem cells. Six acute myeloid leukaemia (AML)/abn(11q23) and three acute lymphoblastic leukaemia (ALL)/t(4;11) samples were analysed by four-colour flow cytometry for NG2 expression on primitive cell populations. Candidate leukaemic cell populations were defined by the antigen profiles CD34+CD38- in AML and CD34+CD19 -CD117+ in ALL. Surprisingly, in all patients these candidate stem cell populations were shown to lack expression of NG2. Instead, a correlation between the expression of the myeloid differentiation marker CD33 and increasing levels of NG2 on maturing cells could be demonstrated. Similarly, in ALL patients CD34+CD19+ cells showed a higher expression of NG2 mRNA compared with CD34+CD19-. Thus, NG2 appears to be upregulated with differentiation and not to be expressed on primitive disease-maintaining cells. This hampers the clinical use of NG2 as a therapeutic target and as a sensitive marker for MRD detection

Smarcb1 Loss Results in a Deregulation of esBAF Binding and Impacts the Expression of Neurodevelopmental Genes

The murine esBAF complex plays a major role in the regulation of gene expression during stem cell development and differentiation. As one of its core subunits, Smarcb1 is indispensable for its function and its loss is connected to neurodevelopmental disorders and participates in the carcinogenesis of entities such as rhabdoid tumours. We explored how Smarcb1 regulates gene programs in murine embryonic stem cells (mESC) and in this way orchestrates differentiation. Our data underline the importance of Smarcb1 expression and function for the development of the nervous system along with basic cellular functions, such as cell adhesion and cell organisation. Using ChIP-seq, we were able to portray the consequences of Smarcb1 knockdown (kd) for the binding of esBAF and PRC2 as well as its influence on histone marks H3K27me3, H3K4me3 and H3K27ac. Their signals are changed in gene and enhancer regions of genes connected to nervous system development and offers a plausible explanation for changes in gene expression. Further, we describe a group of genes that are, despite increased BAF binding, suppressed after Smarcb1 kd by mechanisms independent of PRC2 function

In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic maturation have stem cell properties

We examined the leukemic stem cell potential of blasts at different stages of maturation in childhood acute lymphoblastic leukemia (ALL). Human leukemic bone marrow was transplanted intrafemorally into NOD/scid mice. Cells sorted using the B precursor differentiation markers CD19, CD20, and CD34 were isolated from patient samples and engrafted mice before serial transplantation into primary or subsequent (up to quaternary) recipients. Surprisingly, blasts representative of all of the different maturational stages were able to reconstitute and reestablish the complete leukemic phenotype in vivo. Sorted blast populations mirrored normal B precursor cells with transcription of a number of stage-appropriate genes. These observations inform a model for leukemia-propagating stem cells in childhood ALL