Long-term in vitro maintenance of clonal abundance and leukaemia-initiating potential in acute lymphoblastic leukaemia

Lack of suitable in vitro culture conditions for primary acute lymphoblastic leukaemia (ALL) cells severely impairs their experimental accessibility and the testing of new drugs on cell material reflecting clonal heterogeneity in patients. We show that Nestin-positive human mesenchymal stem cells (MSCs) support expansion of a range of biologically and clinically distinct patient-derived ALL samples. Adherent ALL cells showed an increased accumulation in the S phase of the cell cycle and diminished apoptosis when compared with cells in the suspension fraction. Moreover, surface expression of adhesion molecules CD34, CDH2 and CD10 increased several fold. Approximately 20% of the ALL cells were in G0 phase of the cell cycle, suggesting that MSCs may support quiescent ALL cells. Cellular barcoding demonstrated long-term preservation of clonal abundance. Expansion of ALL cells for >3 months compromised neither feeder dependence nor cancer initiating ability as judged by their engraftment potential in immunocompromised mice. Finally, we demonstrate the suitability of this co-culture approach for the investigation of drug combinations with luciferase-expressing primograft ALL cells. Taken together, we have developed a preclinical platform with patient-derived material that will facilitate the development of clinically effective combination therapies for ALL

Profiling of functional intercellular interactions in a model of the leukemia microenvironment

During leukemia development malignant cells occupy hematopoietic stem cell niches suppressing normal hematopoiesis and infiltrate other sanctuary niches such as the central nervous system or the testis. Interactions with the microenvironment are critical for leukemia cell survival, but the mechanisms involved in these processes are largely unknown. A better understanding of the patterns of intercellular dependence between leukemia and its microenvironment will possibly provide new options to improve leukemia treatment. To identify new pathways that contribute to the leukemia niche function, I established a large scale high-content (automated image-based) screening platform using co-cultures of primary acute lymphoblastic leukemia cells (ALL) on human mesenchymal stromal cells (MSC). Patient samples were expanded by xenotransplantation in immunodeficient mice to generate a renewable source of leukemic cells for systematic functional investigation. The methodology and analytic pipeline was developed in our laboratory in collaboration with the Light Microscopy and Screening Centre of ETH Zurich. We established a robust workflow to discriminate viable ALL and stromal cells using a fluorescent dye. Detailed protocols were optimized to use this platform for in vitro drug testing and for functional genomic projects. Based on gene expression and cell surface proteomic data that we had obtained from both cellular compartments, I generated a customized siRNA library for 110 candidate genes with a potential function in stromal support. Primary ALL cells were seeded on reversely transfected MSC cells, and ALL cell viability was assessed after 6 days using the established high-content screening platform. From a first screen with three cases with highly resistant disease, 20 candidate genes were identified that reproducibly reduced ALL survival in this assay. These were validated in 10 different patient samples. Importantly, specific and distinct contributions of stromal genes for the survival of individual ALL samples were detected. The strongest effects were observed after RNA interference of the vascular endothelial growth factor C (VEGFC) or of Basigin (BSG, alias CD147) in a subset of patient samples. Dependence from stromal VEGFC predicted sensitivity to two different VEGF receptor kinase inhibitors, confirming the patient specific support pattern of stromal VEGFC. Furthermore, the Notch and Wnt pathways were identified to play an important role for the support of ALL cells, extending experimental data obtained in other model systems of the tumour microenvironment or HSC niches. The largest subset of ALL samples was most dependent on the expression of BSG on stromal cells. I could show that this multifunctional cell surface protein was required to provide metabolic support to a subset of ALL in association with the solute carrier family 3 protein, SLC3A2. This protein forms heterodimeric amino acid transporters (HAT) on MSCs indicating that amino acid transport of stromal cells is important for survival of ALL cells. Leukemic cells are deficient to import cystine, and a subset of leukemic cases requires continuous supply of cysteine for de novo glutathione synthesis. We could show that the metabolic transport function of amino acids by stromal cells maintains glutathione levels and reduces oxidative stress specifically in ALL cells that were dependent on stromal BSG/SLC3A2. Indeed, addition of cysteine but not cystine rescued the effect of RNA interference with BSG/SLC3A2 in stromal cells. Taken together, I describe the development of a new platform for systematic investigation of interactions between primary leukemia and stromal cells. The identification of relevant and leukemia-specific pro-survival cues from stromal cells validates this approach. Strong interaction patterns such as the one reported here suggest new possibilities for targeted therapy provided appropriate biomarkers are developed to select patient cohorts efficiently. The platform can also be used for the analysis of anti-leukemic activity of small molecules as single agents and in combinations. This work will also constitute the basis for a more comprehensive functional genomic screen and will stimulate the development of in vivo models

Le pentecôtisme à l'île de la Réunion : « Protestantisme émotionnel » ou nouvelle « religion populaire » ?

The expansion of Pentecostalism on the Island of Reunion proceeds mainly from one Evangelical Church, the Mission Salut et Guérison, founded by the Assemblies of God in France. Although, in several respects, this Pentecostalism is close to the local popular religiosity, conversion implies a clean break with the past for the faithful, through the abandonment of earlier religious traditions. Converts take this step without fear of the punishment sent by the guardians of these traditions -the ancestors, from now on regarded as demons -for they are assured of the protection provided by the supreme power of the Holy Spirit. The present author views the Pentecostalism in Reunion less as a type of " emotional protestantism " than as a "new popular religion " aiding the faithful in their adaptation to the new context of their Island on its way to modernity.L'expansion pentecôtiste à l'île de la Réunion repose principalement sur une Église évangélique : la Mission Salut et Guérison, implantée par les Assemblées de Dieu de France. Bien que, par bien des aspects, le pentecôtisme soit proche de la religiosité populaire locale, la conversion implique néanmoins pour les fidèles des ruptures fondamentales avec le passé, par l'abandon des traditions religieuses antérieures. Les adeptes opèrent ces ruptures sans craindre le châtiment envoyé par les gardiens des traditions : les ancêtres -désormais considérés comme des démons -, car ils ont la conviction d'être protégés par la puissance suprême de l' Esprit-Saint. L'auteur considère moins le pentecôtisme à la Réunion comme un «protestantisme émotionnel » que comme une «nouvelle religion populaire », facilitant l'adaptation de ses fidèles à un contexte insulaire en voie de modernisation.Boutter Bernard. Le pentecôtisme à l'île de la Réunion : « Protestantisme émotionnel » ou nouvelle « religion populaire » ?. In: Revue d'histoire et de philosophie religieuses, 81e année n°1, Janvier-mars 2001. pp. 45-61

Characterisation of cholinesterase expression during murine embryonic stem cell differentiation.

It is already established that cholinesterases (ChEs) appear in every embryonic blastema at a very early stage of development, independently from innervation. Embryonic butyrylcholinesterase (BChE) is typically found in cells engaged in proliferation processes, while acetylcholinesterase (AChE) is expressed by cells undergoing morphogenetic processes. In order to better define the regulation of cholinesterases during development, we examined their expressions during in vitro differentiation of two murine embryonic stem cell lines by reverse transcription polymerase chain reaction, histochemistry and enzyme activity measurements. AChE and BChE activity and mRNA were present in the undifferentiated stem cells. To test whether the ChEs expression is regulated during differentiation, we employed the embryoid bodies (EBs) culture method, allowing the cells to differentiate, to then collect them at various stages in culture. Interestingly, phases of differentiation were accompanied by increased AChE transcripts; BChE expression was constant, decreasing at later differentiation stages. Cholinesterase activities showed corresponding patterns, with AChE activity increasing at later stages in culture and BChE slightly decreasing. Histochemistry revealed that AChE and BChE activities were mutually exclusive, being expressed by different cell subpopulations. Thus, we have demonstrated that mouse embryonic stem cells express cholinesterases, the enzymes are functional and their expression is regulated during differentiation. Therefore, it appears that their functions under these conditions are not related to synaptic transmission, but for the developmental processes

Image-based RNA interference screening reveals an individual dependence of acute lymphoblastic leukemia on stromal cysteine support

Interactions with the bone marrow microenvironment are essential for leukemia survival and disease progression. We developed an imaging-based RNAi platform to identify protective cues from bone marrow derived mesenchymal stromal cells (MSC) that promote survival of primary acute lymphoblastic leukemia (ALL) cells. Using a candidate gene approach, we detected distinct responses of individual ALL cases to RNA interference with stromal targets. The strongest effects were observed when interfering with solute carrier family 3 member 2 (SLC3A2) expression, which forms the cystine transporter xc − when associated with SLC7A11. Import of cystine and metabolism to cysteine by stromal cells provides the limiting substrate to generate and maintain glutathione in ALL. This metabolic interaction reduces oxidative stress in ALL cells that depend on stromal xc −. Indeed, cysteine depletion using cysteine dioxygenase resulted in leukemia cell death. Thus, functional evaluation of intercellular interactions between leukemia cells and their microenvironment identifies a selective dependency of ALL cells on stromal metabolism for a relevant subgroup of cases, providing new opportunities to develop more personalized approaches to leukemia treatment.ISSN:1949-255