STEM CELLS IN SOLID TUMORS: FROM BENCH TO BEDSIDE
De Maria R.
Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Roma, Italy
The existence of cancer cells with stem cell-like features has been documented for the first time in leukemia and, more recently, in a series of solid tumors. It is now accepted that this rare subpopulation of tumor cells, called cancer stem cells (CSCs), is responsible for the onset, maintenance, and possibly relapse of the tumor in patients previously treated with chemotherapy or radio-therapy. Thus, these cells should be the preferred target of effective therapies aimed to eradicate the tumor. The development of technologies that allow the unlimited in vitro expansion of CSCs represents a powerful tool for the study of pathogenic events that cause the initiation and progression of the tumor both for screening and pre-clinical development of novel therapeutic compounds. In recent years our group has developed a technology that allows the isolation and in vitro expansion of CSC from several solid tumors, including glioblastoma, melanoma, breast, lung, colon, thyroid and ovarian cancer. We are currently characterizing these populations of cancer cells at different levels, including gene expression profiling and proteomic analysis of microRNA. The extensive molecular characterization may allow the identification of markers more specific for the CSCs, providing important information on potential drug targets relevant to develop a more effective strategy of target therapy. In addition, the use of CSC to generate xenografts that faithfully reproduce the patients' tumors, as assessed by morphological and molecular analysis, offers a unique opportunity to test new cancer treatments and, potentially, to optimize individual therapy.
Therefore, although the identification of the CSC is relatively recent, this research looks extremely promising for potential clinical development. It's likely that understanding of the molecular mechanisms underlying tumorigenesis and aberrant survival of CSCs can significantly contribute to the rational design of new targeted cancer therapies.
CLINICAL EFFICACY OF CELLULAR TERAPIES: A CRITICAL APPROACH
Cellular Therapy “G. Lanzani” Laboratory, USC Hematology, Ospedali Riuniti Bergamo, Italy
Somatic advanced cell therapy products (ATMP) are emerging as innovative therapeutic possibilities in several different fields of medicine. In previous years, nonetheless, a lot of experimental data on in vitro or animal models have created expectations as to regenerative capacity of different kinds of stem cells and even contributed to the emergence of complex hypotheses of dedifferentiation, transdifferentiation and yet others. These results and their interpretations have reached general public and the press, creating a climate of expectations and also the basis of an ever growing profit-based market including badly identified clinical structures all over the world which were claimed able to offer every sort of therapy for the most devastating human diseases based on unidentified “stem cells”.
Scientific rigorous approach is badly needed to explain to the public what the really available therapies are present today, with which stem cell type and for which disorders.
Clinical solid data have to be shown and discussed with transparent attitude and possibly no conflict of interest. In particular we will show our data with hematopoietic stem cells from bone marrow, peripheral blood or cord blood and with mesenchymal stromal cells. Moreover data with epithelial cells will also be shown and discussed.
Finally we will remember which are the rules and the standards necessary today in Italy to perform ATMP clinical trials with the approval of all the Competent Authorities involved.
THE NEW FLOW CYTOMETRY IN MYELODYSPLASTIC SINDROMES (MDS): FROM CD34+ PRECURSORS TO PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) CLONES
Del Vecchio L.
Istituto CEINGE Biotecnologie Avanzate, Università Federico II, Napoli, Italy email@example.com
Clonal transformation in MDS occurs in a CD34+ myeloid committed stem cell. Interesting results in the perspective of a diagnostic use of FCM in MDS patients derive from the analysis of immunophenotypic abnormalities of CD34+ cells. The vast majority of CD34+ cells in MDS are committed to myeloid lineage (CD38+HLA-DR+CD13+CD33+) and the decrease of hematogones is one of most consistent findings in MDS patients. The evaluation of percent CD34+ B-cell progenitors has negligible inter-operator variability and seems to be reliable in many centers. Asynchronous co-expression of stemcell and late-stage myeloid antigens (CD117/CD15, or CD117/CD11b) or abnormal expression of lymphoid markers (CD56, CD2, CD5, CD7, CD19) have been reported. It has been also evaluated the expression of CD38 on CD34+ cells as a diagnostic tool in MDS. B-cell progenitors highly express CD38, and low numbers of B-cell progenitors in MDS can reduce the mean fluorescence intensity of CD38. The CD38 display on CD34+ cells can be be further reduced in MDS since in high risk patients there are elevated numbers of immature CD34+CD38-progenitors.
All these findings strongly suggest that CD34-related parameters are good candidates as diagnostic markers in MDS. The evaluation of CD34-related parameters could to be useful in patients without specific markers of marrow dysplasia (ring sideroblasts, chromosomal abnormalities). During last years important progresses in the understanding the meaning of PNH clones in MDS have been obtained. Full understanding of PNH will have larger implications than for PNH itself, as indicated by the therapeutic implications of the PNH clone presence in MDS: it could disclose an immunological pathogenesis for low risk MDS, such suggesting an immune system targeted therapy.
FISH LABELLING IN SUSPENSION (ISFISH) AND FLOW CYTOMETRY: NEW ACHIEVEMENTS AND APPLICATIONS
Giorgi D.,1 Farina A.,1 Grosso V.,1 Gennaro A.2 and Lucretti S.1
1ENEA Centro Ricerche Casaccia, Unità Tecnica AGRI, Laboratorio GEN, Via Anguillarese 301, 00123 Roma, Italia 2Dip. Scienze e Tecnologie per l'Agricoltura, le Foreste, laNatura e l'Energia 113: (DAFNE), Università della Tuscia, Via S. Camillo de Lellis snc, 01100 Viterbo (Italy)
Flow sorting is always been rewarded as a very interesting and useful application from the beginning of flow cytometry. Among others, chromosome sorting is one of its remarkable achievements, but useful flow cytometry specific chromosome stains are very limited and a true chromosome discrimination can be only made on the basis of total DNA amount and/or AT/CG relative content. In cytogenetic, fluorescence in situ hybridization (FISH) is an extremely powerful methodology allowing a fine discrimination and identification of single chromosome types and most of their rearrangements up to single gene localization. Many unsuccessful attempts have been carried on to combine the good of both techniques to develop a whole and consistent system for high definition sorting of chromosomes in suspension, with the only exception of interphase nuclei. Labelling chromosomes in suspension with specific probes could help in isolating normal and mutant chromosomes for a number of molecular manipulations both in humans and plants. We focus our research on developing a rapid, reproducible and affordable strategy for in situ fluorescence labelling of in suspension chromosomes (ISFISH). This new procedure allows flow karyotyping and quantitative sorting of single type chromosomes from standard genotypes where chromosomes cannot be identified by DNA content variations. We developed ISFISH on plant chromosomes with the aim to sort large numbers of chromosomes from top quality bread and pasta wheat (Triticum durum and T. aestivum) varieties where genes for quality production, for biotic and abiotic stresses and also for good agronomic performances are present, thus avoiding the use of mutant lines (such as substitution or addition lines) usually breeded from poor wheat materials. We checked and used several probes capable to generate reproducible highly informative DNA banding patterns. Chromosome karyotyping and flow sorting was performed on the basis of DNA content and probe labelling. Samples can be prepared in 60 min about, using 50 ng probe/106 chromosomes. ISFISH works equally well on squashes and banding patterns showed to be true-to-type in respect to classic FISH method. This technique allowed detecting and sorting a single chromosome type in commercial wheat genotypes and all the chromosome types in wild grain species (D. villosum). In our opinion, ISFISH opens new avenues in chromosome flow sorting and its medical and biotech applications.
THE USE OF FLOW CYTOMETRY FOR THE DETECTION OF ENVIRONMENTAL BACTERIA IN DIFFERENT ECOSYSTEMS
Manti A., Boi P. and Papa S.
Department of Earth, Life and Environmental Sciences, University of Urbino “Carlo Bo” Urbino, Italy firstname.lastname@example.org
Flow Cytometry is a powerful tool for the analyses in environmental microbiology. FCM can provide a broad range of information at the single-cell level, including total counts, size measurements, nucleic acid content, membrane potential, cell viability and activity.
Furthermore, the simultaneous use of FCM with specific antibodies permits the detection of bacterial groups or species, increasing the knowledge of microbial community also in marine and in freshwater ecosystems. Data from the application of a polyclonal antibody for the detection of V. parahaemolyticus in pure culture and seawater are presented.
The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging approach in aquatic microbiology. Nevertheless, few technical improvements have been proposed in the last 5 years. Recent attempts and efforts on environmental samples analyses are reported to remark the importance and the potentiality in microbial ecology of combining CARD-FISH and Flow Cytometry.
APPLICATION OF FLOW CYTOMETRY, ANALYTICAL CYTOLOGY AND IMMUNOHISTOCHEMISTRY IN THE CHARACTERIZATION OF IONIZING RADIATION EFFECTS ON MALE GERM CELLS
Cordelli E., Bartoleschi C., Benassi B., Eleuteri P., Grollino M.G., Pardini M.C., Pacchierotti F., Spanò M. and Villani P.
Laboratory of Toxicology, ENEA CR Casaccia,Via Anguillarese 301, 00123 Rome, Italy
Sperm DNA integrity is essential for the accurate transmission of paternal genetic information. Spermatozoa are the final result of a complex and ordered differentiation process taking place in the testis. The testis, beside being one of the most radiosensitive organs, has a variety of cells which differ in their degree of radiosensitivity. This different sensitivity depends on the differentiation stage, chromatin condensation, nucleoprotein composition and repair capability. Due to the complexity of spermatogenesis and the heterogeneity of testicular cell subpopulations, an accurate characterization of radio-induced damage in this tissue is difficult and requires a multidisciplinary approach which allows the identification of damage in the different cellular compartments.
We applied neutral comet assay, immunodetection of phosphorylated histone H2AX (γ-H2AX) and sperm chromatin structure assay (SCSA) to detect the production of DNA strand breaks in testicular cells and spermatozoa at different times after in vivo X-ray irradiation. Results show that irradiation of testicular cells induced DNA strand breaks which were repaired within a short time. We also found that the kinetics of H2AX phosphorylation -dephosphorylation was not linked with the DNA strand breaks removal since γ-H2AX foci persisted when DNA damage was no more detectable by comet assay. Spermatozoa were resistant to the induction of DNA damage, but nontargeted DNA breaks were found in spermatozoa deriving from radiation-targeted proliferating spermatogonia, becoming evident when the spermatids started to elongate in testicular seminiferous tubules. We also show that, at that time, transcription of pro-apoptotic genes was enhanced, suggesting that an apoptotic-like process could be involved in the DNA breaks formation.
FLOW CYTOMETRY, IMMUNOHISTOCHEMISTRY AND QUANTITATIVE mRNA EXPRESSION: COMPARATIVE APPROACHES FOR DETECTION OF CD133+ CANCER STEM CELLS IN TUMOURS
Pirozzi G. Collina F.,1 Di Bonito M.,1 Cantile M.1, Camerlingo R.2
1Department of Pathology 2Department of Experimental Oncology, National Cancer Institute, Naples, Italy email@example.com
Background: CD133 marker is the main marker for isolation of cancer stem cells (CSCs). Unfortunately, its correct determination on cellular surface can be prejudiced by several factors. Here, we analyze CD133 expression in breast cancer by cytometric analysis, immunohistochemistry and Quantitative Real Time PCR and we evaluated if three methodologies are efficacy in detecting of CD133 marker.
Methods: In this study, 12 patients were enrolled at the National Cancer Institute of Naples. For each patient, immunohistochemical staining for CD133, E-cad, CD44 markers was performed on embedded paraffin tissues. Real Time-PCR for CD133 on frozen biopsies was also performed. Moreover, the tissue obtained from surgery was tested by flow cytometry for CD44 FITC, CD326 PE, CD133PE and CD45APC.
Results: By immunohistochemistry, the E-cad was expressed in 11 cases. The percentage of CD133+ cells was very heterogeneous. In 5 samples, the expression was very low or absent, in 5 it was moderate (5–15%), in one case it was >20% and only in Tubulobular variant breast cancer, it was about 35%. CD44 expression was also extremely variable. By flow cytometry, the mean percentage of CD133 positive cells was 13,75% and the mean percentage of CD44 positive cells was 17,90%. All cells analyzed were positive for CD326 (EpCAM). In Tubulobular breast variant, the percentage of CD133 was about 70%. Both immunohistochemistry and cytometric data for CD133 were confirmed by Real Time-PCR.
Conclusion: We showed that CD133 antigen can be used as a suitable marker for CSCs detection in breast carcinomas. The analysis was carried out by different methods: flow cytometry, immunohistochemistry and Real Time PCR quantification. Our data were confirmed by all three methods, and the results are overlapping.
CIRCULATING TUMOUR CELLS: TECHNICAL AND CLINICAL CHALLENGES
Danova M.,1 Torchio M.1 and Mazzini G.2
1S.C. Medicina Interna e Oncologia Medica, A.O. di Pavia, Ospedale di Vigevano 2Istituto di Genetica Molecolare IGMCNR, Pavia, Italy firstname.lastname@example.org
Circulating tumour cells (CTCs) may be detected in the blood of patients with epithelial tumours using different analytical approaches. The relative number of CTCs is low and they include a heterogeneous population of cells with diverse biological and molecular characteristics, often different from those of the respective primary tumour. Until recently, they have been difficult to detect and, even though discordant results have been reported when different methods of detection were used, they may provide prognostic and predictive information.
Several antibody-or molecular-based CTC detection methods have been developed, offering hope for individualized risk assessment by utilizing CTCs as biomarkers of disease progression and drug response. Pilot studies have also shown that utilizing methods that permit, besides enumeration, a molecular characterization of CTCs, one could better identify high-risk patients, predict response to targeted therapies, analyze gene expression profiles (in order to identify new potential drug targets), and increase our knowledge of the metastatic process.
In order to achieving these goals, there is a great need for development of more sensitive technologies able to avoid stress on the cells during manipulation, in order to preserve cell viability and proliferation capability and to facilitate subsequent in vitro culture for further molecular analysis of CTCs. Of course, the cost of such methods is another important issue that should be addressed in view of larger clinical applications.
TRABECTEDIN: A SOMETHING NEW IN ONCOLOGY
Erba E., Uboldi S., Romano M., Bernasconi S., Panini N., Marchini S., Fuso Nerini I., Frapolli R. and D'Incalci M.
Mario Negri Institute, Milan, Italy email@example.com
Trabectedin is a tetrahydroisoquinoline alkaloid that was initially isolated from the marine ascidian Ecteinascidia turbinata and is currently prepared synthetically. Trabectedin's mechanism of action seems to be different from that of the available DNA-damaging agents used in cancer chemotherapy to date. In contrast to traditional alkylating agents that bind guanine at the N7 or O6 position in the DNA major groove, trabectedin binds to the exocyclic N2 amino group of guanines in the DNA minor groove. Several studies have been conducted on cell lines with well-defined defects of DNA repair mechanisms suggesting a role for transcription-coupled nucleotide excision repair (TC-NER) and homologous recombination in the cytotoxic activity of trabectedin. In contrast to traditional alkylating agents trabectedin in NER+ cells induced a G2M block of the cell cycle, while in NER−cells did not. Trabectedin inhibits the in vitro production of the proinflammatory mediators CCL2 and interleukin (IL)-6 by monocytes, macrophages, and tumor-associated macrophages isolated from ovarian cancer biopsies. The modulation of cytokines and chemokines occurs at the transcriptional level, thus indicating that the mechanism of trabectedin on transcription regulation can be effective both in cancer cells and in some normal cells, which produce factors that are relevant for the tumor growth and progression. Trabectedin has been shown to be a clinically valid option for the treatment of advanced soft tissue sarcomas or relapsed platinum-sensitive ovarian cancer.
HUMAN TH1 AND TH17 LYMPHOCYTES IN CHRONIC INFLAMMATORY DISEASES
Department of Internal Medicine and DENOTHE Center, University of Florence, Italy firstname.lastname@example.org
Classically, naive CD4+ T cells have been divided into two main lineages, Th1 and Th2 cells. Th1 cells produce IFN-, the cytokine IL-12 being critical for their differentiation. By contrast, Th2 cells produce IL-4, IL-5 and IL-13 and their development is started by IL-4 signalling. More recently, a third subset of CD4+ T cells which produce IL-17 has been described, which was named as Th17. We showed that, in addition to cells producing IL-17 alone (Th17), there is a number of T cells that co-produce IL-17 and IFN- (Th17/Th1). We also found that these two cell types express both RORC and T-bet and that Th17 cells could be shifted to Th1 by the addition of IL-12 suggesting their flexibility. Moreover, we found that CD161 is a marker of human Th17, in comparison with Th1 or Th2, clones. Accordingly, all IL-17-producing cells were found to be included within the CD161+ fraction of adult circulating CD4+ T cells. When CD161+ or CD161-cells were sorted from UCB naïve CD4+ T cells and activated in presence of IL-1beta plus IL-23, IL-17-producing cells could be developed only from the CD161+ fraction. In addition, we found that not only CD4+TCRβ+, but also CD8+TCRβ+, CD4-CD8-TCRβ+, and CD4-CD8-TCR+ circulating cells that produce IL-17 express the distinctive marker CD161 on their surface. Accordingly, we also demonstrated that CD161 expression identifies also CD8+ and CD4-CD8-UCB T cells that already express RORC and IL-23R and that can be differentiated into Th17 cells in the presence of IL-1 and IL-23. Taken together these data allow to conclude that T-cell subsets able to produce IL-17, as well as precursors of IL-17-producing T cells, exhibit surface expression of CD161.
IMAGING CYTOMETRY REQUIRES THE THIRD DIMENSION AND HIGH SPATIAL AND TEMPORAL RESOLUTION
Diaspro A.,1,2,3 Bianchini P.,1 Cella Zanacchi F.,1 Harke B.,1 Vicidomini G.,1 Galiani S.,1,2 Lavagnino Z.,1,2 Chacko J.,1,2 Ronzitti E.,1 Faretta M.,3 Furia L.3 and Mondal PP.4
1Nanophysics, Istituto Italiano di Tecnologia, Genoa 2DIFI, LAMBSMicroScoBio, University of Genoa, Italy 3Dpt. Exp. Oncology, IFOMIEO Campus, Milan Italy 4Dept Instr. Appl. Phys., Indian Institute of Science, Bangalore, India email@example.com
Two-photon excitation (2PE) fluorescence imaging cytometry is a powerful far-field optical approach for the study of the three-dimensional (3D) and dynamic properties of biological systems. The main advantages are given by better penetration in scattering samples and low overall phototoxicity/bleaching coupled to intrinsic 3D optical sectioning properties, while the main drawback lies in the loss of resolution and signal efficiency with respect to the 1PE case. For such reasons we decided to couple 2PE with STED microscopy (STimulated Emission Depletion) and SPIM (Selective Plane Illumination Microscopy). In the former case we aim to augment resolution and at the same time to improve the sample penetration capability of the STED approach. In the latter we aim to boost SPIM penetration depth in thick scattering samples. Results and related characterizations have been obtained by means of 2PE STED-CW and 2PE SPIM adapted architectures available at the Italian Institute of Technology. Other extensions of 2PE are in progress within the farfiled optical super resolution framework. This work was supported by grants from IIT, IFOM-IEO, MIUR PRIN 2008JZ4MLB, European Project SMD FP7-NMP 2800-SMALL-2 proposal no. CP-FP 229375-2.
MICRORNAs AS NEW THERAPEUTIC TARGETS AND TOOLS IN CANCER
Department of Experimental Oncology and Molecular Medicine, Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy firstname.lastname@example.org
MicroRNAs (miRNAs) are a class of endogenous, noncoding small RNAs that negatively regulate gene expression at the post-transcriptional level. Several studies have provided evidence that abnormal expression of selected miRNAs is associated with the pathogenesis of cancer. As they can act as either oncogenes or tumor suppressors, miRNAs have been proposed as potential new therapeutic targets or tools for cancer therapy. A significant body of the experimental data collected to date indicate that specific miRNA inhibition or replacement can successfully modify the proliferative and invasive properties of tumor cells. In addition, a direct involvement of miRNAs in drug resistance has been reported, underlying an entirely new mechanism by which tumor cells may be refractory to the treatment with cytotoxic agents. Based on these findings, in the therapeutic setting, interference with cancer-specific miRNAs could be exploited not only to produce a direct anticancer effect but also to improve the response of tumor cells to conventional treatments. Overall, manipulation of miRNA functions, either by mimicking or inhibiting them, is emerging as a highly
promising therapeutic strategy. However, before miRNA-based therapeutics enters the clinical armamentarium, important issues concerning specific delivery to cells/tissues of interest, safety as well as pharmacokinetic profiles needs to be addressed.