Research
A summary of the Petriz lab research interests


Expression of primitive stem cell markers during origin, progression and maintenance of cancer. Diagnostic implications.

Objective:
The goal of the proposed research is to learn more about the pathogenesis, natural history and treatment of human neoplasms 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 myeloid malignancies and cancer stem cells isolated from pancreatic cancer, prostate cancer, glioma and astrocytoma, with particular emphasis on self-renewal signaling pathways.

Methodology:
Immunophenotyping studies: FACS, Mass Cytometry, Flow-FISH, immunomagnetic selection. In agreement with the EuroFlow Consortium (myeloid antigens) we will study the expression of embryonic markers (OCT-4, SOX2, NANOG, TRA-1-60, TRA-1-81), as well as CD133, CD44, EpCAM, ALDH and alkaline phosphatase. 2) Functional studies: Clonogenic assays (progenitors/CFU-L), LTC-ICs, xenotransplantation (immunodeficient). Functional activity of multidrug transporters (ABCB1, ABCG2). 3) Stem cell signaling pathways (Sonic-Hedhehog, Wnt / b-catenin, TGF-b and Notch) real-time PCR, Western- blot, shRNAs. 4) Cell differentiation inhibition under hypoxic conditions (5-10% O2).

Expected results:
It is hoped that the results of the proposed work will contribute to the understanding of human neoplasms and to attainment of the goal of preventing the malignancies or providing a basis for targeted therapy for cancer stem cells. We expect to enlighten the heterogeneity of human neoplasm in differentiative expression of the involved stem cells in the pattern of remissions and in marker expression characteristics of cancer progenitors. Moreover, the gradual implementation of a network that enables cooperative work and knowledge transfer technologies, must address the clinical efficacy and impact of the new technologies that we will develop.

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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.

Objectives:
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.

Methodology:
In general, quality control criovials are representative of the CD34+ cell-infused product. We will recruit 500 criovials from the participating centers in the CD34 SIC/SEI working group. We will use fluorescence microscopy and flow cytometry techniques. We will study functional metabolic parameters, such as mitochondrial activity, cell viability and apoptosis on CD34+ subpopulations after thawing.

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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.

Objectives:
We will study the role of ATP Binding Cassette (ABC) multidrug transporters on signal transduction in stem cells (SC). We will analyze their protective potential on cell signaling pathways that are fundamental to many cell types, specially for SC function. We will study how ABCB1 and ABCG2, two ABC transporters expressed in normal and cancer cells, can regulate highly conserved stem cell signaling pathways, with implications for the development of therapeutic targets not only to treat cancer, but also in other diseases. We will analyze the regulation of Sonic Hedgehog, Notch, Wnt/β-catenin, epithelial-mesenchymal transition induced by TGF-β, PTEN/PI3K/Akt, and PPAR α/γ.

Methodology:
In combination with molecular biology techniques, we will use functional flow cytometry to study the interaction of multiple drugs with the above mentioned cell signaling pathways and ABC transporters. We will analyze the interaction of ABC transporters using competition assays and ABC-specific fluorescent substrates (eg, Rhodamine 123, Hoechst 33342) in highly purified preparations of human hematopoietic CD34+/ABCB1+ CD34-/ABCG2+ stem cells and ABCB1 in Side Population (SP) cancer stem cells derived from gliomas and astrocytomas, as well as in cellular models of transfectants with stable expression of ABCB1, ABCG2 and ABCB1-EGFP.

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The use of anticancer drug libraries to study the reversal of multidrug resistance in Side Population cancer 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.

Objectives:
Based on the efflux of the fluorescent probe Hoechst 33342 (Ho342) by ABCG2, we will isolate SP cells from human neoplasm. Specifically, we will analyze the effects of two large libraries of antineoplastic drugs. We will study primary tumor cells and if exist, we will compare with SP-isolated cells obtained form the same tumor. All cells will be maintained under hypoxic conditions. We will use HTS technology (High-Throughput Screening) study the effectiveness of certain drug combinations on the disruption of specific signaling pathways associated with drug resistance, self-renewal and proliferation of the Side Population. We hope that this information will be helpful to assess the risk of cancer SP cells and their possible metastatic potential.

Methodology:
We will use Ho342 staining for the enrichment of SP and non-SP cells by FACS. HTS will be used in combination with flow cytometry to assess the cytotoxicity of the drug combinations of our libraries. The following parameters will be assessed: plasma membrane potential and cell membrane integrity, number and distribution of mitochondria, efflux of fluorescent substrates for ATP-dependent pumps, cell proliferation markers, ion homeostasis, oxidative stress markers, lipid metabolism, biosynthetic activity and biotransformation. In order to understand the role of multidrug transporters in protecting critic signaling pathways for the stem cell compartment, we will compare the gene expression profiles of SP and non-SP cells.

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Murine xenograft models for human ovary, testis, prostate, pancreas and colon cancers: Detection of bone marrow infiltration by Side Population cells.

1. We isolate Side Population (SP) stem cells from normal tissue and from tumor cells, mainly for cell culture experiments as well as for transplantation in murine xenograft models and for the independent analysis and comparison of gene expression. We also develop non immortalized and non transformed cell models from stem cells with SP phenotype.

2. We study the gene expression profiles to test the hypothesis that the expression of certain genes are associated with an immature cell phenotype as well as with a phenotype of tumor stem cell. We map the signaling pathways, self-renewal, and differentiation of cells SP, as well as stem cell miRNAs and ABC transporters and the mechanisms by which regulate gene expression and resistance to chemotherapy.

3. We study the presence of SP cells in human solid tumors, orthotopically implanted in athymic mice as well as the dissemination and infiltration in different tissues (i.e. bone marrow), with and without the expression of the green fluorescent protein as a marker gene.

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Methodology:
We use murine xenotransplantation models developed by the research group of Dr. Gabriel Capellą (ICO). Specifically, orthotopically implanted human tumors from ovary, testis, prostate and pancreas are selected for their interest and pathological parameters of morbidity and mortality. We analyse the gene expression profiles of infiltrating SP cells in the murine bone marrow using up to 72 xenotransplantation models of human ovarian tumors, testicular, prostate, pancreas and colon. The isolation of cells SP is conducted by FACS (Fluorescence Activated Cell Sorting) under a MoFlo.




Flow cytometry counting of CD34+ cells.

Blood formation is sustained by a population of undifferentiated and metabolically quiescent hematopoietic stem cells (HSC) mainly found in the bone marrow. HSC remain in the G0 compartment of the cell cycle, are able to self-renew, and differentiate into progenitors of all hematopoietic lineages. Their self-renewal and differentiation are regulated by a number of cytokines. A subset of hematopoietic cells presumably containing HSC express the cell surface antigen CD34; CD34+ purified fractions are enriched in colony-forming units and long-term culture initiating cells, whereas CD34 negative fractions are depleted. CD34+ cells obtained from either bone marrow or peripheral blood are commonly used in hemopoietic stem cell transplantation. They can be mobilized from bone marrow into peripheral blood by means of chemotherapy and/or cytokine stimulatory treatments, then collected for use in malignant disease therapy, HSC expansion studies, and gene therapy. The accurate enumeration of CD34+ cells has shown to be important for predicting the success of engraftment after transplantation, as it can assure the presence of sufficient numbers of progenitor cells remaining in the graft. We have developed a new flow cytometry protocols for CD34+ progenitor counting in collaboration with the Quality Assessment of Haematopoietic Stem Cell Grafts Committee from The European Group for Blood and Marrow Transplantation (EBMT).
CD34