Carlos Raventós (Clinical Associated Investigator)
José Placer (Clinical Associated Investigator)
Carlos Salvador (Resident)
Jordi Ropero (Resident)
Marta Alué (Resident)
María Carmen Mir (Resident)
Luis Castro (Resident)
Geisy Delgado (Resident)
Juan M. Bastarós (Resident)
Our overall goal focuses several aspects of translational urology mainly based on the knowledge of the molecular bases of prostate cancer, in particular, but also on the role of infl ammatory mechanisms as critical regulators of tumor progression. Our central hypothesis is that a deeper understanding of these pathways will advance the development of preventive treatment strategies.
Research Lines in Prostate Cancer (PC)
Development of non-invasive methods for the early detection of PC in biological fluids We have determined the specific, differential proteomic profi les to be found in the urine of patients with PC, as compared to age matched controls, with the ultimate goal of settling on a non-invasive diagnostic tool using urine that could help to circumvent the low specifi city of the currentlyused PSA serum measurements. We use liquid chromatography, mass spectrometry and triple quadrupole mass spectrometry (LC/MSMS SRM). The Selected Reaction Monitoring technique (SRM) is an emerging technology that ideally complements the discovery capabilities of shotgun strategies through its unique potential for the reliable quantifi cation of low abundance analytes in complex mixtures, such as urine samples. Using this technique we quantify and detect diff erent selected proteins with high sensitivity and a good chromatographic separation within the complex biological samples. The final goal of this research is the establishment of a reliable diagnostic test, which can be used in hospitals and outpatient routines.
Identification of the molecular markers of bone metastases in prostate cancer
We have developed humanized animal models for metastatic prostate cancer able to mimic the human dissemination of PC cells to bones. We use immunocompromised mice transplanted with human bone. Human prostate cancer cells, which over-express luciferase, are injected, allowing metastasis detection and the continued monitoring of the living animals. This permits the identifi cation of bone metastasis markers, patients with a high risk of recurrence and could defi ne new therapeutic targets that will act to block bone lesions through conventional therapies
Development of improved bone metastasis animal models, very close to the clinics, in order to monitor the process of in vivo metastasis
We use an animal model of immunocompromised mice with a transplantation of human bone fragments. Subsequently, human PCa cell lines, over-expressing luciferase, are injected. This allows the detection and monitoring of metastasis in the living animal, since the implanted bone fragments maintain their human microenvironment.
Identification of the molecules responsible for the formation of human bone metastasis
By analyzing the changes in protein expression levels by proteomics in the metastases obtained from this animal model, we examine whether
the reinjection of bone metastasis cells aff ects the specifi city or phenotype, due to reprogramming. We attempt to identify the factors that attract human prostate cancer cells to human bone and the mechanisms that are involved in the process of metastasis. This is accomplished by proteomics, using a fl uorescence-based differential gel electrophoresis (DIGE) with mass spectrometry (MALDI / TOF), as well as isotope-based techniques (iTRAC etc.) and LC-MS/ MS with SRM.
Efficacy of new adjuvant therapies for PC bone metastasis
We use intra-tibial injection of prostate cancer cells overexpressing luciferase in immunocompromised balb/c nude mice, a straightforward method to induce local growth in bone marrow. This Bioluminescent Imaging (BLI)- based metastasis model allows us a regular monitoring of the development and progression of experimental bone metastases in living animals with high sensitivity. Fewer laboratory animals are needed as due to the noninvasive nature of the methods repetitive measurements can be taken from the same animal, which also increases the reliability of observed eff ects. This approach will enable us to include the micro-environmental growth support system of the bone for the treatment of metastatic disease.
Extracellular matrix and infl ammatory mechanisms regulated by prostate cancer-associated fibroblasts
We are interested in understanding extracellular matrix and inflammatory mechanisms regulated by cancer-associated fi broblasts (CAFs) as
promoting forces for prostate cancer progression. Cancer-associated fibroblasts support tumorigenesis by stimulating angiogenesis, cancer
cell proliferation, invasion and tumor-enhancing infl ammation. Using primary cell cultures, we have learned that prostate CAFs
display signifi cant phenotypic and transcriptional diff erences from their normal associated fi broblast (NAF) counterparts:
i) an invasive and migratory phenotype,
ii) expression of epithelial-mesenchymal transition genes and
iii) enhanced expression of inflammatory molecules.
Currently we are studying the differential response of the monocytic cell line THP1 in front of CAFs/ NAFs (cell-cell adhesion, chemotaxis, gene and protein expression, matrix metalloproteinase activation).
Molecular analysis of “proliferative inflammatory atrophy” (PIA) as a premalignant condition in prostate cancer development
We also focus our research on the potential importance of chronic inflammatory microenvironments as premalignant condition in prostate cancer development. Currently we are studying a common lesion, often associated with infl ammation, termed “proliferative inflammatory atrophy” (PIA), which has been postulated to represent an intermediate step between normal tissue and cancer. It may, therefore, serve as a risk factor lesion for prostate cancer. Using microdissection and microarray technology we have performed paired comparative analysis of gene expression in the following prostatic tissues: benign, PIA, high grade prostatic intraepithelial neoplasia (HGPIN) and cancer lesions. Our objective is to test whether:
i) our data support the notion that PIA may be considered a premalignant lesion, and
ii) we can detect and characterize common transcriptionally altered pathways among these pathologies.
These studies have implications for prevention and chemoprevention of prostate cancer.
Decrease of bone mass during androgen deprivation in prostate cancer
Decrease of plasmatic levels of testosterone produced by androgen deprivation indirectly alters the mineral bone metabolism and produces loss of bone mass and there is increased the risk of fractures and mortality. This research line contains studies of prevalence of osteoporosis and osteopenia, prediction of the pace of bone mass loss, study of the molecular mediators, specifi cs diagnosis methods and prevention.
Dyslipemia and metabolic syndrome during androgen deprivation in prostate cancer
Cardiovascular mortality is the leading cause of death in patients with prostate cancer and it is believed that androgen deprivation is the intermediate reason. This research line includes studies of metabolic syndrome prevalence and dyslipemia as the most frequent cause of cardiovascular mortality, molecular mediators analysis, early diagnosis methods of cardiovascular risk and prevention.
Cognitive alterations during androgen deprivation in prostate cancer
Androgen suppression in prostate cancer patients produces cognitive alterations that are not well studied despite being very important for quality of life. The purpose of this research line is to study the cognitive alteration profi le that produces androgen deprivation, the mediators who generate these alterations at central level, early diagnosis and possible forms of prevention.
High grade intraepithelial neoplasia and prostate cancer
High grade intraepithelial neoplasia is preneoplasics prostate cancer damage. Nevertheless, it is unknown the molecular mechanisms who define his neoplasic transformation or his persistence as high grade intraepithelial neoplasia isolated. High grade intraepithelial neoplasia detection in a prostatic biopsy entails a repeat biopsy strategy that has not yet been clearly established. This research line integrates the analysis of molecular predictors of neoplasic transformation (genomics and proteomics), the analysis of metabolic image (RNM and spectroscopy) and possible prevention mechanisms of prostate cancer chemoprofilaxis.
Research Lines in Kidney Diseases
Pneumoperitoneum impact of laparoscopic surgery on renal function
Analysis of molecular mechanisms of renal oncogeny
We are focused on the understanding of the molecular bases of endometrial and ovarian cancers. In particular, in new molecules involved in progression and dissemination. Our search also includes new molecular biomarkers of precocious diagnosis as well as molecular therapeutic targets.
Research Lines in Endometrial Cancer (EC)
Differential gene expression in endometrial cancer: analysis of the roles of transcription factors Runx1 and ETV5 in the progression and dissemination of tumors
Previous research in our lab has identifi ed ETV5 and RUNX1 proteins as key determinants of myometrial invasion and dissemination. The main objective of the project is the investigation of the molecular mechanisms regulated by the ETV5 and RUNX1 transcription factors that are responsible for endometrial cancer cell invasion and dissemination. To achieve these objectives we have already developed some tools such as endometrial cancer cell lines with ETV5 overexpression or downregulation. The identifi cation of proteins involved in the invasion and dissemination processes will lead to the design of new experimental therapies that will be evaluated (tumor growth, metastasis) in the orthotopic animal models that we have developed for endometrial cancer.
Development of highly-sensitive and highly-efficient molecular tools for the diagnosis of endometrial cancer in uterine aspirates
Through a proteomic approach, our lab has identifi ed and validated new robust biomarkers for endometrial carcinoma using human samples obtained from uterine aspirates. Our objective is to develop a reliable tool for screening EC risk using endometrial biopsies, which will enhance sensitivity and specificity, as well as preclude unnecessary hysteroscopy.
Development of endometrial orthotopic murine models to test new therapies
We have developed two different orthotopic endometrial cancer murine models that might be useful tools in endometrial cancer preclinical studies. The generation of these murine models for endometrial cancer has been achieved by inoculation of either a tumor cell line or human tumor tissue intra-uterus.The Hec-1A endometrial cancer cell line derived model represents advanced disease and can be used to test the effi cacy of antimetastastic drugs. In this model, the follow-up of disease progression is performed using bioluminescence in vivo and correlating bioluminescence ex vivo with metastasis generation. The human tissue derived model maintains the histological pattern and represents local and locally-advanced disease, and can be used to test drugs against specific targets of endometrial cancer.
Characterization by proteomics and genomics of markers expressed differentially at the EC invasion front and in metastasis
Using an orthotopic mouse model we have shown the involvement of RUNX1 in distant metastasis in endometrial cancer. Using proteomics, we have also shown a series of promising proteins involved in myometrial invasion that are differently expressed between cancer and age-matched uterine tissue. Our goal is to identify the gene clusters involved in invasion and metastasis and to study their therapeutic potential using preclinical mouse models.
Research Lines in Ovarian Cancer (OC)
New biomarker identifi cation for ovarian cancer diagnosis, prognosis and drug treatment
This project proposes the identification of new molecular biomarkers for the diagnosis and prognosis of ovarian cancer. Microarray technology has been used to identify molecules differentially expressed between tumoral tissue and control samples. The fi nal goal is to identify a panel of biomarkers that can distinguish the presence or absence of ovarian cancer.
Mechanisms of cancer cell dissemination regulated by ETV5 in ovarian cancer
Previous research in our lab has identifi ed ETV5 as a protein overexpressed in ovarian cancer. We have characterized its role in ovarian tumour progression. Currently, we are characterizing ETV5 target genes involved in ovarian cancer dissemination through the peritoneal cavity. The fi nal goal is to design new therapies to stop ovarian cancer cell dissemination.
Molecular pathways involved in ovarian cancer cell dissemination
Ovarian cancer disseminates to secondary sites through the peritoneal cavity. Ovarian cancer cells are shed from the ovarian primary tumor, aggregate as spheroids within the abdominal cavity and subsequently attach to the peritoneal wall. We are interested in the identifi cation of those molecular pathways involved in ovarian cell dissemination through the peritoneal cavity in order to design new therapies that target ovarian cancer dissemination and therefore ovarian cancer spread to secondary sites.
Cohort study comparing surgical vs laparoscopic staging and treatment in primary endometrial cancer (clinical stage I)
Other Clinical Research Projects
• Prospective study of validation of sentinel lymph node detection technique in cervical cancer in initial stages.
• Prospective study of validation of sentinel lymph node detection technique in vulvar cancer in initial stages.
• Prospective comparative study of laparoscopic versus laparotomic radical hysterectomy approach in initial cervical cancer treatment.
• Prospective study of validation of extra-peritoneal aortic laparoscopic or robotic assisted lymphadenectomy in locally advanced or bulky cervical cancer.
• Prospective comparative study of robotic assisted versus laparoscopic versus laparotomic approaches in endometrial cancer (supported by AATRM, Technology and Medical Research Evaluation Agency).
• Pre-neoplasic vaginal and vulvar pathology: VIN and VAIN.
• Study of p-16 as a progression marker in cervical pre-invasive lesions.
• Follow-up in women with HPV 16 infection.
• CIN and pregnancy.
• Follow-up of women treated for H-SIL cervical cancer lesions.
• Endocervical sample as a marker of relapse in cervical intraepithelial neoplasia.
• Results of neo-adjuvant concomitant chemo-radiotherapy in the treatment of locally advanced cervical cancer.
• Validation of robotic assisted and laparoscopic aortic extraperitoneal lymphadenectomy in recurrences of gynaecologic malignancies.
• Evaluation of robotic surgery in the treatment of gynaecologic malignancies.
• Borderline ovarian tumours.
• Endoscopic treatment of ovarian cancer in initial stages.
• Ressecability predictive value of laparoscopic approach in advanced ovarian cancer.
• Results of neo-adjuvant chemotherapy in the treatment of advanced ovarian cancer.
Laboratory of Cell Signalling and Cancer Progression
Marta Sesé (postdoct, PhD)
Neus Marqués (PhD student)
Lide Alaña (PhD student)
Verónica Cánovas (PhD student)
Camilla Faoro (Master student)
Yolanda Puñal (Master student)
Raul Morales (Technician)
The primary interests of the group are the identifi cation of new and selective targets for anti-cancer therapy and markers potentially useful to identify aggressive tumors from non-aggressive. Our studies aim to broaden our knowledge on the biology of aggressive cancer cells and shed light on the molecular circuits that are established in aggressive tumors. We have been approaching our aims studying carcinomas of the exocrine pancreas and, more recently, prostate cancer by:
i) the identifi cation of mis-expressed genes in tumors versus normal tissues using different methodologies to analyze gene expression;
ii) the selected genes are being studied for their contribution to tumor aggressiveness through the analysis of phenotypes obtained with the gain-of-function/knockdown in vitro and in vivo;
iii) the relevant targets are further studied to understand their function and mechanisms of action on major cellular signaling
pathways implicated in cancer progression.
Specific Lines of Research
In pancreas cancer, we are studying tissue plasminogen activator (tPA) specifi cally overexpressed in cancer cells (Paciucci et al., 1998). Our fi ndings indicate that tPA activates cell proliferation in vitro and in vivo, in immuno-defi cient mice, and contributes to pancreas cancer growth and progression (Díaz et al., 2002). tPA is a serine protease that specifi cally activates plasminogen to plasmin. In pancreas cancer cells we identifi ed specifi c binding sites for tPA on the membrane of tumor cells (Díaz et al., 2004.). tPA bound to these receptors induces a proteolytic cascade with the consecutive activation of plasmin and the pro-MMP9. The latter allows heparin-bound EGF to engage the epidermal growth factor receptor (EGFR) producing its activation (Hurtado et al., 2007). This activation event is required for the mitogenic action of tPA on pancreas cancer cells. Using specifi c substrates to detect the activity of tPA in cancer cells, we are screening for specific inhibitors.
In prostate cancer, we are studying the newly identifi ed protein Prostate Tumor OVerxpressed-1 (PTOV1), a protein well conserved in mammals, fl ies and simpler eukaryotes, the protein defi nes a new family of proteins containing a structurally unknown new domain (PTOV), also encountered in other mammalian proteins (i.e. PTOV2/MED25) (Benedit et al., 2001). PTOV1 is overexpressed in pre-malignant lesions of HGPIN and in prostate cancer, where it promotes cell proliferation and invasion (Santamaría et al., 2003).
Its detection in prostate biopsy is useful to predict the presence of cancer (Morote et al., 2008). PTOV1 interacts with Flotillin-1 and both proteins are required for cell proliferation (Santamaría et al., 2005). Our results show that Flotillin-1, a major component of lipid-rafts compartments of cellular membranes, is required for Aurora kinase B function in mitosis (Gómez et al., 2010). PTOV1 protein is overexpressed in numerous other human cancers, including bladder and renal cell carcinoma, colon carcinoma, endometrial and ovary carcinomas.
We are studying the mechanisms implicated in the PTOV1-promoted cell proliferation and invasion. With this aim in mind, are following three major strategies:
• We are studying the interactions of PTOV1 with known cellular proteins implicated in tumor progression obtained by yeast-two-hybrid assays. One such interaction, the PTOV1-RACK1, stimulates the specific translation of several proteins required for the PTOV1-mediated cell invasion eff ect. We are now defi ning the mechanisms implicated in this action.
• In the second approach, we are analyzing the functional interaction of PTOV1 with major cellular pathways implicated in cancer progression. We are presently studying the interference of PTOV1 with the signaling of Notch and its importance in prostate cancer establishment and progression.
• Finally, we are using the D. melanogaster model to study and confirm the function of the protein PTOV1 in the signaling circuits identifi ed in cancer cells that might be active during development. In colon cancer, we are studying the contribution of the alteration of protein translation in the expression of genes associated with oncogenesis and progression of carcinomas.
Laboratory of Translational Research in Pediatric Cancer
Josep Sánchez de Toledo
Josep Roma (PhD)
Miguel Segura (PhD)
Anna Almazán (Pre-doctoral investigator)
Isaac Vidal (Technician)
Aroa Soriano (Technician)
Pablo Velasco (Pre-doctoral investigator)
Josep Sánchez de Toledo (Clinical Associated Investigator)
Malignant neoplasms in children and adolescents are rare diseases with different prognoses and biologic behaviour. The prognosis of childhood cancer has improved considerably in recent decades and survival is approximately 70% in western countries. However, even with the current multimodal therapies, a considerable number of these patients still relapse and eventually die due to progressive or refractory neoplasms. Consequently, paediatric oncologists need new approaches to improve the efficacy of anticancer therapies. Molecular diagnosis, detection of microdisseminated disease and the search for new therapeutic strategies would help to improve the results of the current treatments of paediatric cancer.
Our research group is focused on:
• Molecular diagnosis of malignant tumours in children: neuroblastoma, Ewing’s sarcoma, soft tissue sarcomas, nephroblastoma, brain tumours.
• Analysis of the prognostic impact of minimal disseminated disease (MDD).
• Search for new molecular therapeutic targets in children with cancer.
We systematically perform molecular characterisation using PCR of the most common types of cancer in children i.e. neuroblastoma, soft tissue sarcomas, bone sarcomas, non-Hodgkin lymphomas, nephroblastoma and brain tumours. Our laboratory is the National Reference Centre for Biological Studies in soft tissue sarcomas, receiving tumour material from most of the cases included in the current therapeutic protocols in Spain.
Minimal disseminated disease (MDD)
The presence of occult rhabdomyosarcoma cells in peripheral blood and bone marrow is systematically analysed by testing the expression of multiple genes using real-time RT-PCR. In collaboration with Dr. A Rosolen (University of Padua) and Dr. J Stutterheim (AMC, Amsterdam) and under the auspices of the EpSSG (European Pediatric Soft Tissue Sarcoma Group) we have developed the European consensus protocol for the study of MRD in RMS. In neuroblastoma, MDD study is performed by analysing tyrosine hydroxylase gene expression in peripheral blood, and bone marrow using real-time RT-PCR.
• NOTCH and Hedgehog pathways in rhabdomyosarcoma: The main objective of this line is to ascertain the effects of NOTCH and Hedgehog pathways inhibition using in vitro models as well as a murine xenograft model of RMS in an attempt to establish new molecular targets for treating patients with RMS. Our studies in vitro suggest that inhibition of the NOTCH pathway by gamma-secretase inhibitors produces a significant decrease in the invasiveness of rhabdomyosarcoma cells. Moreover, NOTCH and Hedgehog pathways activation seems to play a crucial role in sustaining the rare population of tumour initiating cells in some neoplasms. The main objective of this line is to identify and separate tumour-initiating cells in rhabdomyosarcoma tumours and characterise NOTCH and Hedgehog pathways in this subpopulation as a possible candidates for the development of targeted therapies.
• Cancer stem cells in paediatric cancers: We attempt to isolate progenitor cancer cells (stem cells) in soft tissue sarcomas, bone sarcomas, neuroblastoma, high-grade non-Hodgkin lymphomas and brain tumours. The analysis of the expression profiles of this putative stem cell population could permit us to identify new therapeutic targets that will overcome resistance to chemotherapy.
• MicroRNAs as prognostic and therapeutic tools in neuroblastoma: MicroRNAs (miRNAs) are endogenous non-coding small RNAs that interfere with the translation and stability of coding messenger RNAs (mRNAs) in a sequence-specific manner. In recent years it has become apparent that miRNAs are deregulated in a number of different human cancers, including neuroblastoma (NBL), and the over- or under- expression of specific miRNAs correlates with stage, progression and patient outcome. The working hypothesis of this line is that the miRNAs deregulation is contributing to the metastatic and chemoresistant behaviour of high-risk NBL and miRNA(s) levels restoration represents an attractive novel therapeutic approach.
Laboratory of Bioengineering and Cellular Interactions
Maria Antònia Arbós
María Teresa Quiles
Maria Antònia Arbós (MD, PhD, Researcher)
María Teresa Quiles (PhD, Researcher)
Manuel Armengol (MD, PhD, Clinical Associated Investigator)
Manuel López-Cano (MD, PhD, Clinical Associated Investigator)
Marta Rebull (Technician)
Our laboratory is interested in the role of cell-extracellular matrix (ECM) interactions in the fi elds of tissue repair and infl ammation, with a special focus on abdominal wall defects. Our studies are mostly based on patient-derived tissue samples and primary cells, as well as on surgically-induced models. Moreover, soft-tissue repair devices are being investigated by means of “in vitro” and “in vivo” experimental models.
Extracellular matrix, inflammation and abdominal wall The reconstruction of abdominal wall defects is the problem with which surgeons are confronted most often. These defects may have an acute (trauma, cancer, infections) or chronic (hernia pathology) origin. Despite technical advances, both physiopathology and treatment of the disease remains controversial, and further knowledge is needed.
We are trying to unravel the cellular and molecular mechanisms triggering incisional hernia (IH) formation. IH often occurs following laparotomy and can be a source of serious problems. There is evidence that a biological cause may underlie its development, but the mechanistic link between the local tissue microenvironment and tissue rupture is lacking. We have found de-regulated proteolytic and molecular inflammatory signaling in the abdominal wall tissues (fascia and skeletal muscle) of IH patients.
Also, we have identifi ed an ongoing complex interplay of cell death induction, aberrant fi broblast function and tissue loss in IH tissues, which eventually may give rise to tissue rupture in vivo. Currently, we are investigating changes in subsets of genes from IH-derived primary fi broblasts. Overall, these studies may provide a molecular mechanistic framework for better understanding IH formation, and reveal new molecular biomarkers and potential therapeutic targets. Current surgical practice supports the use of permanent prosthetic meshes as the best method for hernia repair. Still, no material has gained a preference for universal use and numerous complications are still reported. The understanding of cell-substrate interactions is fundamental for the improvement of tissue repair and regenerative medicine. We analyze diff erent soft-tissue repair devices. Our approach includes surface and biomechanical characterization, as well as the analyses of host-implant interactions, using both “in-vitro” (primary fi broblasts derived from control and IH patients) and “in-vivo” (rats) experimental models. Our ultimate goal is to impact on the development of new tailored implants based on fi broblasts and biomimetic materials, which are clinically useful to repair damaged organs.
Simulation and virtual reality
In collaboration with Politechnic University of Catalonia and Rovira-Virgili University:
• Virtual reality model of inguinal hernia: Educational purposes: simulator of inguinal area; Clinical Research purposes: protective mechanisms against inguinal hernia formation.
• Virtual reality model of the whole abdominal wall: Educational purposes: simulator of abdominal wall; Clinical Research purposes: abdominal wall mechanical behavior.
• Virtual reality model of synthetic mesh contraction: Clinical/Translational research of physical contraction of synthetic mesh and its influence on hernia recurrence.
Hernia occurrence prevention
• Parastomal hernia prevention with synthetic mesh by laparoscopic approach: clinical study.
• Incisional hernia prevention with synthetic mesh: experimental study.
Surgical devices applied in abdominal wall surgery
In collaboration with Politechnic University of Catalonia and Rovira-Virgili University:
• 3D surgical vision.
• Surgical sutures – European Project.
Laboratory of Stem Cells and Cancer
Jordi Pétriz (PhD)
Noelia Purroy (MD)
Verónica Pons (MD)
Jana Balbuena (Graduate Student)
Gisela Pachón (PhD)
Anna Esquerra (Technician)
The focus of the laboratory are the basic mechanisms that regulate Side Population stem cells. Stem cells reside in most tissues in a quiescent state, but rapidly become activated to both repair and regenerate the adjacent tissues. We are studying several genes involved in diff erent aspects of stem cell activation, including some that encode for ABC transporters, and others that regulate self-renewal and diff erentiation. We are also interested in multidrug resistance and we use functional fl ow cytometry to examine the changes that occur in the accumulation of drugs into the cells over time.
Murine xenograft models for human ovary, testis, prostate, pancreas and colon cancers: Detection of bone marrow infi ltration by Side Population cells
We are using xenograft models to examine the genes that regulate bone marrow infi ltration in cancer. 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. 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 diff erentiation of cell SP, as well as stem cell miRNAs and ABC transporters and the mechanisms by which gene expression and resistance to chemotherapy are regulated. We study the presence of SP cells in human solid tumors, orthotopically implanted in athymic mice as well as their dissemination and infiltration in diff erent tissues (i.e. bone marrow), with and without the expression of the green fl uorescent protein as a marker gene.
Development of a new Cytomics Platform for the study of stem cell systems
Cancer is increasingly being viewed as a stem cell disease, both in its propagation by a minority of cells with stem cell-like properties and in its possible derivation from normal tissue stem cells. Recent fi ndings suggest that stem cell biology may be more complex than originally anticipated and a subset of stem cells may alter their function in a manner that is more plastic and dynamic than previously thought. Considerable progress has been made by studying stem cell function based on the high efflux of fluorescent dyes. ABCG2, a half-transporter that belongs to the ATP binding cassette superfamily, is expressed in primitive stem cells, and is responsible for the formation of a Side Population (SP) with a Hoechst 33342 (Ho324) fl uorescent profile blocked in the presence of multidrug reversal agents. SP cells are present in a wide variety of tissues and ABCG2 expression is believed to represent a common molecular mechanism for stem cells possessing multi-organ plasticity. The majority of SC enrichment protocols rely on fl uorescence activated cell sorting (FACS), which allows cells to be selected based on the expression of a set of cell surface proteins. ABCG2 gene is an important determinant of the SP, and it might serve as a marker for stem cells from various sources. Cell sorting for the expression of ABC transporters provides a new strategy for stem-cell purifi cation that could be used for cells from different organ sources. We also study single-cell gene expression profiling to relate the expression of specific genes to a particular cellular phenotype. FACS-based isolation of SP cells, in association with the mRNA expression analysis of gene expression in highly purified preparations of SC subsets on the basis of ABCG2 expression, provides important insights in stem cell biology. We apply the Cytomics Platform for the detection and isolation of SP cells from:
• Adipose tissue (in collaboration with Dr. Simó, MD, PhD, FIR-HUVH).
• Brain tumors (in collaboration with Dr. Sáez Castresana, MD, PhD, CIFA, University of Navarra).
• Xenograft models (in collaboration with Dr. Capellà, MD, PhD, IDIBELL).
• Peripheral blood and bone marrow from leukemic patients (in collaboration with Dr. Prósper, MD, PhD, CIMA and with Dr. Bosch, MD, PhD, HUVH).
Flow cytometry counting of CD34+ cells
Blood formation is sustained by a population of undiff erentiated 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 diff erentiate into progenitors of all hematopoietic lineages. Their self-renewal and diff erentiation are regulated by a number of cytokines. A subset of hematopoietic cells presumably containing HSC express the cell surface antigen CD34; CD34+ purifi ed fractions are enriched in colony-forming units and long-term culture initiating cells, whereas CD34- 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 been shown to be important for predicting the success of engraftment after transplantation, as it can assure the presence of suffi cient numbers of progenitor cells remaining in the graft. We have developed a new fl ow cytometry protocol 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).