A summary of the Petriz lab research interests
Fast-screening flow cytometry method for detecting nanoplastics in
human peripheral blood
Objective:
Plastic pollution is a global problem. Animals and humans can
ingest and inhale plastic particles, with uncertain health
consequences. Potential effects of nanoplastics (NPs) exposure may
be associated with alterations in the xenobiotic metabolism,
nutrients absorption, energy metabolism, cytotoxicity, and
behavior. In humans, no data on nanoplastic absorptions has been
reported previously. Nanoplastics are particles ranging from 1 nm
to 1000 nm that result from the erosion or breakage of larger
plastic debris, and can be highly polydisperse in physical
properties and heterogeneous in composition. Given that their
detection relies significantly on environmental exposure, we have
prospectively studied the presence of NPs in human peripheral blood
(PB).
Methodology:
Specifically, we use fluorescence techniques and nanocytometry,
together with the staining of the lipophilic dye Nile Red (NR), to
demonstrate that NPs can be accurately detected using flow
cytometry.
Expected results:
We have demonstrated for the first time the existence of NPs in
human peripheral blood detected by flow cytometry. While MPs can be
easily seen using microscopy, flow cytometry is a highly sensitive
tool to study plastic nanoparticles, with advantages over other
techniques, such as Raman spectroscopy, which can resolve generally
1-2 μm particle sizes. Exposure to NPs may lead to accumulation by
inhalation and ingestion. Further analyses will beneeded to
determine association of NP pollution with lifestyle, health and
pathology
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.
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.
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.
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.
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.
Methodology:
We use murine xenotransplantation models developed by the research
group of Dr. Gabriel Capellà (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).
