Virginia Tirino

Detection and Characterization of CD133 + Cancer Stem Cells in Human Solid Tumours

Background Osteosarcoma is the most common primary tumour of bone. Solid tumours are made of heterogeneous cell populations, which display different goals and roles in tumour economy. A rather small cell subset can hold or acquire stem potentials, gaining aggressiveness and increasing expectancy of recurrence. The CD133 antigen is a pentaspan membrane glycoprotein, which has been proposed as a cancer stem cell marker, since it has been previously demonstrated to be capable of identifying a cancer initiating subpopulation in brain, colon, melanoma and other solid tumours. Therefore, our aim was to observe the possible presence of cells expressing the CD133 antigen within solid tumour cell lines of osteosarcoma and, then, understand their biological characteristics and performances. Methodology and Principal Findings In this study, using SAOS2, MG63 and U2OS, three human sarcoma cell lines isolated from young Caucasian subjects, we were able to identify and characterize, among them, CD133+ cells showing the following features: high proliferation rate, cell cycle detection in a G2\M phase, positivity for Ki-67, and expression of ABCG2 transporters. In addition, at the FACS, we were able to observe the CD133+ cell fraction showing side population profile and forming sphere-clusters in serum-free medium with a high clonogenic efficiency. Conclusions Taken together, our findings lead to the thought that we can assume that we have identified, for the first time, CD133+ cells within osteosarcoma cell lines, showing many features of cancer stem cells. This can be of rather interest in order to design new therapies against the bone cancer.

Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization

Primary tumors are responsible for 10% of cancer deaths. In most cases, the main cause of mortality is the formation of metastases. Accumulating evidence suggests that a subpopulation of tumor cells with distinct stem‐like properties is responsible for tumor initiation, invasive growth, and metastasis formation. This population is defined as cancer stem cells (CSCs). Existing therapies have enhanced the length of survival after diagnosis of cancer but have completely failed in terms of recovery. CSCs appear to be resistant to chemotherapy, may remain quiescent for extended periods, and have affinity for hypoxic environments. The CSCs can be identified and isolated by different methodologies, including isolation by CSC‐specific cell surface marker expression, detection of side population phenotype by Hoechst 33342 exclusion, assessment of their ability to grow as floating spheres, and aldehyde dehydrogenase (ALDH) activity assay. None of the methods mentioned are exclusively used to isolate the solid tumor CSCs, highlighting the imperative to delineate more specific markers or to use combinatorial markers and methodologies. This review provides an overview of the main characteristics and approaches used to identify, isolate, and characterize CSCs from solid tumors.—Tirino, V., Desiderio, V., Paino, F., De Rosa, A., Papaccio, F., La Noce, M., Laino, L., De Francesco, F., Papaccio, G. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 27, 13–24 (2013). www.fasebj.org

Activating E17K mutation in the gene encoding the protein kinase AKT1 in a subset of squamous cell carcinoma of the lung

Somatic mutation (E17K) that constitutively activates the protein kinase AKT1 has been found in human cancer patients. We determined the role of the E17K mutation of AKT1 in lung cancer, through sequencing of AKT1 exon 4 in 105 resected, clinically annotated non-small cell lung cancer specimens. We detected a missense mutations G->A transition at nucleotide 49 (that results in the E17K substitution) in two squamous cell carcinoma (2/36) but not in adenocarcinoma (0/53). The activity of the endogenous kinase carrying the E17K mutation immunoprecipitated by tumour tissue was significantly higher compared with the wild-type kinase immunoprecipitated by the adjacent normal tissue as determined both by in vitro kinase assay using a consensus peptide as substrate and by in vivo analysis of the phosphorylation status of AKT1 itself (pT308, pS473) or of known downstream substrates such as GSK3 (pS9/S22) and p27 (T198). Immunostaining or immunoblot analysis on membrane-enriched extracts indicated that the enhanced membrane localization exhibited by the endogenous E17K-AKT1 may account for the observed increased activity of mutant E17K kinase in comparison with the wild-type AKT1 from adjacent normal tissue. In conclusion, this is the first report of AKT1 mutation in lung cancer. Our data provide evidence that, although AKT1 mutations are apparently rare in lung cancer (1.9%), the oncogenic properties of E17K-AKT1 may contribute to the development of a fraction of lung carcinoma with squamous histotype (5.5%).

Human primary bone sarcomas contain CD133+ cancer stem cells displaying high tumorigenicity in vivo

This study aimed to identify, isolate, and characterize cancer stem cells from human primary sarcomas. We performed cytometric analyses for stemness and differentiation antigens, including CD29, CD34, CD44, CD90, CD117, and CD133, on 21 human primary sarcomas on the day of surgery. From sarcoma biopsies, we obtained 2 chondrosarcoma‐stabilized cell lines and 2 osteosarcoma stabilized cell lines, on which sphere formation, side population profile, stemness gene expression, and in vivo and in vitro assays were performed. All samples expressed the CD133, CD44, and CD29 markers. Therefore, we selected a CD133+ subpopulation from stabilized cell lines that displayed the capacity to grow as sarcospheres able to initiate and sustain tumor growth in nonobese diabetic/severe combined (NOD/SCID) mice, to express stemness genes, including OCT3/4, Nanog, Sox2, and Nestin, and to differentiate into mesenchymal lineages, such as osteoblasts and adipocytes. Our findings show the existence of cancer stem cells in human primary bone sarcomas and highlight CD133 as a pivotal marker for identification of these cells. This may be of primary importance in the development of new therapeutic strategies and new prognostic procedures against these highly aggressive and metastatic tumors.—Tirino, V., Desiderio, V., Paino, F., De Rosa, A, Papaccio, F., Fazioli, F., Pirozzi, G., Papaccio, G. Human primary bone sarcomas contain CD133+ cancer stem cells displaying high tumorigenicity in vivo. FASEB J. 25, 2022‐2030 (2011). www.fasebj.org

Epithelial to Mesenchymal Transition by TGFβ-1 Induction Increases Stemness Characteristics in Primary Non Small Cell Lung Cancer Cell Line

Background Cancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line. Materials and Methods A549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed. Results TGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable. Conclusions The induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers.

Human CD34 + /CD90 + ASCs Are Capable of Growing as Sphere Clusters, Producing High Levels of VEGF and Forming Capillaries

Background Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells. Methodology/Principal Findings In this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers – including CD34, CD90, CD29, CD44, CD105, and CD117 – and endothelial-progenitor-cell markers – including CD34, CD90, CD44, and CD54. Interestingly, CD34+/CD90+ cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34+/CD90+ quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31+/VEGF+/Flk-1+) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests. Conclusions/Significance Our results demonstrate, for the first time, that CD34+/CD90+ cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.

The osteoblastic differentiation of dental pulp stem cells and bone formation on different titanium surface textures.

Bone Tissue Engineering (BTE) and Dental Implantology (DI) require the integration of implanted structures, with well characterized surfaces, in bone. In this work we have challenged acid-etched titanium (AET) and Laser Sintered Titanium (LST) surfaces with either human osteoblasts or stem cells from human dental pulps (DPSCs), to understand their osteointegration and clinical use capability of derived implants. DPSCs and human osteoblasts were challenged with the two titanium surfaces, either in plane cultures or in a roller apparatus within a culture chamber, for hours up to a month. During the cultures cells on the titanium surfaces were examined for histology, protein secretion and gene expression. Results show that a complete osteointegration using human DPSCs has been obtained: these cells were capable to quickly differentiate into osteoblasts and endotheliocytes and, then, able to produce bone tissue along the implant surfaces. Osteoblast differentiation of DPSCs and bone morphogenetic protein production was obtained in a better and quicker way, when challenging stem cells with the LST surfaces. This successful BTE in a comparatively short time gives interesting data suggesting that LST is a promising alternative for clinical use in DI.

Three Years After Transplants in Human Mandibles, Histological and In-Line Holotomography Revealed That Stem Cells Regenerated a Compact Rather Than a Spongy Bone: Biological and Clinical Implications

Mesenchymal stem cells deriving from dental pulp differentiate into osteoblasts capable of producing bone. In previous studies, we extensively demonstrated that, when seeded on collagen I scaffolds, these cells can be conveniently used for the repair of human mandible defects. Here, we assess the stability and quality of the regenerated bone and vessel network 3 years after the grafting intervention, with conventional procedures and in‐line holotomography, an advanced phase‐imaging method using synchrotron radiation that offers improved sensitivity toward low‐absorbing structures. We found that the regenerated tissue from the graft sites was composed of a fully compact bone with a higher matrix density than control human alveolar spongy bone from the same patient. Thus, the regenerated bone, being entirely compact, is completely different from normal alveolar bone. Although the bone regenerated at the graft sites is not of the proper type found in the mandible, it does seem to have a positive clinical impact. In fact, it creates steadier mandibles, may well increase implant stability, and, additionally, may improve resistance to mechanical, physical, chemical, and pharmacological agents.

Dental pulp stem cells: State of the art and suggestions for a true translation of research into therapy

OBJECTIVES Stem cells have the ability to rescue and/or repair injured tissue. In humans, it is possible to isolate different types of stem cells from the body. Among these, dental pulp stem cells (DPSCs) are relatively easily obtainable and exhibit high plasticity and multipotential capabilities. In particular they represent a gold standard for neural-crest-derived bone reconstruction in humans and can be used for the repair of body defects in low-risk autologous therapeutic strategies. SOURCES An electronic search was conducted on PubMed databases and supplemented with a manual study of relevant references. RESULTS All research described in this review highlight that DPSCs are mesenchymal stem cells that could be used in clinical applications. Unfortunately, very few clinical trials have been reported. Major obstacles imposed on researchers are hindering the translation of potentially effective therapies to the clinic. Both researchers and regulatory institutions need to develop a new approach to this problem, drawing up a new policy for good manufacturing practice (GMP) procedures. We strongly suggest that only general rules be standardized rather than everything. Importantly, this would not have an effect on the safety of patients, but may very well affect the results, which cannot be identical for all patients, due to physiological diversity in the biology of each patient. Alternatively, it would be important to study the role of specific molecules that recruit endogenous stem cells for tissue regeneration. In this way, the clinical use of stem cells could be successfully developed. CONCLUSIONS DPSCs are mesenchymal stem cells that differentiate into different tissues, maintain their characteristics after cryopreservation, differentiate into bone-like tissues when loaded on scaffolds in animal models, and regenerate bone in human grafts. In summary, all data reported up to now should encourage the development of clinical procedures using DPSCs.

Identification and expansion of human osteosarcoma-cancer-stem cells by long-term 3-aminobenzamide treatment

A novel cancer stem‐like cell line (3AB‐OS), expressing a number of pluripotent stem cell markers, was irreversibly selected from human osteosarcoma MG‐63 cells by long‐term treatment (100 days) with 3‐aminobenzamide (3AB). 3AB‐OS cells are a heterogeneous and stable cell population composed by three types of fibroblastoid cells, spindle‐shaped, polygonal‐shaped, and rounded‐shaped. With respect to MG‐63 cells, 3AB‐OS cells are extremely smaller, possess a much greater capacity to form spheres, a stronger self‐renewal ability and much higher levels of cell cycle markers which account for G1‐S/G2‐M phases progression. Differently from MG‐63 cells, 3AB‐OS cells can be reseeded unlimitedly without losing their proliferative potential. They show an ATP‐binding cassette transporter ABCG2‐dependent phenotype with high drug efflux capacity, and a strong positivity for CD133, marker for pluripotent stem cells, which are almost unmeasurable in MG‐63 cells. 3AB‐OS cells are much less committed to osteogenic and adipogenic differentiation than MG‐63 cells and highly express genes required for maintaining stem cell state (Oct3/4, hTERT, nucleostemin, Nanog) and for inhibiting apoptosis (HIF‐1α, FLIP‐L, Bcl‐2, XIAP, IAPs, and survivin). 3AB‐OS may be a novel tumor cell line useful for investigating the mechanisms by which stem cells enrichment may be induced in a tumor cell line. The identification of a subpopulation of cancer stem cells that drives tumorigenesis and chemoresistance in osteosarcoma may lead to prognosis and optimal therapy determination. Expression patterns of stem cell markers, especially CD133 and ABCG2, may indicate the undifferentiated state of osteosarcoma tumors, and may correlate with unfavorable prognosis in the clinical setting. J. Cell. Physiol. 219: 301–313, 2009.