An important and challenging problem in leukemia research is the limited ability for many laboratories to perform functional analyses of primary patient cells. In order to increase our understanding of the biology of human leukemic malignancies, we perform advanced experimentation using living-cell systems. Functional information extracted from single-cell analysis, provides crucial data to understand cell-to-cell heterogeneity. By enabling functional cytomics, we are able to evaluate the state of patients with leukemia as well as to examine the changes that occur in the accumulation of drugs into the cells over time. The Functional Cytomics Group is mainly focused in the basic mechanisms that regulate CD34+ and CD34- Side Population stem cells. Stem cells reside in most of tissues in a quiescent state, but rapidly become activated to both repair and regenerate the adjacent tissues. We are studying several genes involved in different aspects of stem cell activation, including some that encode for ABC multidrug resistance transporters, and others that regulate self-renewal and differentiation.
Expression of primitive stem cell markers during origin, progression and maintenance of leukemia. Diagnostic implications.
The goal of the proposed research is to learn more about the pathogenesis, natural history and treatment of human leukemia by analyzing the expression of primitive stem cell markers and their association with malignancy at a phenotypic and functional level, using a long series of leukemic malignancies and leukemic stem cells, with particular emphasis on self-renewal signaling pathways.
Design of consensus protocols for safety, quality and standardization of CD34+ cells after-thawing.
Upon the establishment of a convergence framework for the quality control between the Iberian Society of Cytometry (SIC) and the Spanish Society of Immunology (SEI), we will develop consensus strategies for the counting of CD34+ cells pre- and post-thaw through an external quality program and the CD34 intercalibration working group, now supported by both societies.
a) To analyze the intracenter variability of CD34+ cell counting; b) to standardize the counting of CD34+ after thawing and to analyze pre-infusion product quality by means of polychromatic cytometry in combination with metabolic functional assessment; c) to conduct "in silico" intercomparison studies between participating centers; d) to develop predictive models on pre-infusion product quality considering demographic analysis, pathology and graft-related variables in the study population; e) to agree a single protocol for CD34+ cell counting; f) to integrate different existing protocols for the cryopreservation of CD34+ cell-enriched products; g) to extend the convergence criteria within the EuroFlow framework.
Study of the mechanisms by which ABC transporters differentially activates low- or high-level transduction cell signaling: Potential role to both protect the stem cell compartment.
We study the role of ATP Binding Cassette (ABC) multidrug transporters on signal transduction in stem cells (SC). We analyze their protective potential on cell signaling pathways that are fundamental to many cell types, specially for SC function. We study how ABCB1 and ABCG2, two ABC transporters expressed in normal and leukemic cells, can regulate highly conserved stem cell signaling pathways, with implications for the development of therapeutic targets not only to treat leukemia, but also in other diseases. We analyze the regulation of Sonic Hedgehog, Notch, Wnt/β-catenin, epithelial-mesenchymal transition induced by TGF-β, PTEN/PI3K/Akt, and PPAR α/γ.
The use of anticancer drug libraries to study the reversal of multidrug resistance in Side Population leukemic stem cells.
Very recent data support that 25% of cancers are associated with the existence of cancer stem cells (CSCs), these being responsible for the spread of disease from a minority of cells with properties very similar to the normal stem cells. These CSCs lack a specific phenotype and can be only identified and isolated by functional characteristics, especially by the expression of multidrug transporters belonging to the ABC family (ATP Binding Cassette). The activity of one of these transporters, ABCG2, allows the isolation a very primitive type of stem cells, the so-called Side Population (SP). In turn, ABCG2 enables the SP to be highly refractory to many chemotherapeutic agents.