The colorectal cancer stem-like cell hypothesis: a pathologist's point of view.

Colorectal cancer is the fourth most common cancer in men and the third most common cancer in women worldwide. The partial failure of classic therapeutic options makes scientists to doubt the efficacy of systemic treatments in targeting the essential cell populations and achieving cure as a final goal. Overgrowing data suggest that cancer is a disease closely linked to stem cells (SCs). It is well known that the first identification of cancer stem-like cells in acute myeloid leukaemia was soon followed by similar results in solid malignancies, including colorectal cancer, and the classic model for colon carcinogenesis supports the development of sudden mutations that will lead to the activation or inactivation of certain oncogenes or tumor suppressors. Thus, this process may go on for years before the first symptoms and the only cells able to withstand for many years, avoid apoptosis and have a high regenerative capacity are the progenitor cells found at the lower part of colon crypts. A more profound study of the mechanisms and molecular signalling pathways that control the basic characteristics of SCs, such as asymmetrical division or self-renewal, may help comprehend the basic mechanisms of cancer genesis and progression. This will result in the development of new therapeutic agents that may target chemoresistant cell populations and improve the therapeutic results. In the current review we point out the importance of cancer stem-like cells in colorectal oncology from a pathologist's point of view, stating the obvious correlation between histology, embryology and surgical pathology.

Antidiabetic pharmacology: a link between metabolic syndrome and neuro-oncology?

One of the main topics of the annual meeting of the American Society for Clinical Oncology in 2011 were the results presented on breast cancer chemotherapy and concomitant administration of the oral antidiabetic metformin. The overall agreement was that current evidence is just enough to dramatically change the clinical practice of oncology, and in our case, brain cancer treatment, and that further research is needed to address the relationship between diabetes, metabolism, insulin analogues and neoplasia. Still, it is very interesting to explore the potentially beneficial effects of metformin in glioma chemo/immunotherapy and wait for results in the clinic. In the current paper we present the cell and molecular aspects of the metabolic syndrome, metformin administration and cancer chemotherapy, with a special emphasis in neuro-oncology, since brain tumors are usually devastating diseases with an extremely high mortality within two years of diagnosis even when surgical, radiotherapeutic and chemotherapeutic interventions are applied.

Metformin plus temozolomide-based chemotherapy as adjuvant treatment for WHO grade III and IV malignant gliomas.

PURPOSE: Glioblastoma multiforme (GBM) remains one of the most devastating diseases known to man and affects more than 17,000 patients in the United States alone every year. This malignancy infiltrates the brain early in its course and makes complete neurosurgical resection almost impossible. Recent years have brought significant advances in tumor biology, including the discovery that many cancers, including gliomas, appear to be supported by cells with stem-like properties. In the current study we have investigated the effects of combining metformin with the standard treatment-of-care, as this drug, already used in the treatment of diabetes mellitus, has shown surprising results in the treatment of breast cancer, being also associated with lower mortality in several other malignancies. METHODS: The subjects of the current study were 8 patients with newly diagnosed high-grade gliomas, operated at the Department of Neurosurgery - Clinical University Emergency Hospital, Cluj Napoca. Tumor tissue cultures were established and characterized using immunofluorescence microscopy and PCR analysis and the sensitivity to metformin, epidermal growth factor (EGF) and temozolomide (TMZ) was tested. Microvascular density (MVD) assay was performed on the tumor samples. RESULTS: Seven of the 8 cases had a positive correlation between the number of endothelial cells, the phenotype of isolated tumor cells and the response to adjuvant chemoradiotherapy. The isolated tumor cells had a stem-like behavior, being resistant to conventional drugs. In most cases there was no statistical significant difference between TMZ alone and TMZ plus EGF arms, but there was a important difference between TMZ alone and TMZ plus metformin arms in 6 of the cases. CONCLUSION: New drugs and targeted molecular therapies are important for future therapeutics, but sometimes we must not exclude drugs already used in the clinic that might have remarkable results. Such is the case of metformin, a drug used for decades in the treatment of type 2 diabetes mellitus that has proven to enhance the effect of TMZ in the treatment of breast cancer and, starting with this paper, of brain cancer.

Functional and molecular characterization of glioblastoma multiforme-derived cancer stem cells.

PURPOSE: Brain tumors are the leading cause of cancer mortality in children and remain incurable despite advances in surgery and adjuvant therapies. The failure of malignant gliomas to respond to conventional treatment reflects the unique biology of these tumors, linked to a small population of stem-like precursors. This study describes the characteristics of stem cells isolated from glioblastoma multiforme (GM) and gives insight into the mechanism of brain tumorigenesis. METHODS: Tumor stem-like precursors were identified from primary human GM-derived cell culture using immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). Cells were cultured in vitro in stem cell medium supplemented with growth factors and then the capacity of the surviving stem-like precursors to form tumor spheres and to continue to proliferate after chemoradiotherapy were tested. RESULTS: The tumor cells expressed the cellular markers CD133, CD105, CD90, Nanog, Oct 3/4, CXCR4, nestin, glial fibrillary acidic protein (GFAP), neurofilament protein (NF) and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cells also displayed a high proliferative potential despite chemotherapy and irradiation and also had the ability to form spheroids in suspension. CONCLUSION: High grade gliomas contain stem-like precursors, which exhibit neural stem cell properties with tumorigenicity, establishing a novel developmental paradigm in the study of brain carcinogenesis and providing a powerful tool to develop patient-tailored therapy for this devastating disease.

Placental stem cell differentiation into islets of Langerhans-like glucagon-secreting cells.

BACKGROUND AND PURPOSE: For the past few years, in an attempt to find new sources of cells that may be used in cell therapy, numerous researchers have highlighted the particular properties of mesenchymal stem cells. Mesenchymal stem cells can be isolated from adult tissues such as the bone marrow or adipose tissue, but also from other organs such as the human placenta. Our study focuses adult stem cells isolated from the chorionic villi in an attempt to differentiate them into islets of Langerhans in order to study their differentiation potential, as a future background for cell therapy. EXPERIMENTAL DESIGN: Full-term placentas were prelevated from volunteer women that have just delivered a normal pregnancy. After a mechanical fragmentation of the placenta, the chorion fragments are transferred in a dish with dispase before the enzyme is inactivated using fetal calf serum. The cell suspension is filtered in order to obtain a single-cell suspension. After the adherence of the first cells, the proliferation rate increased progressively and cell morphology is kept the same for several passages. In order to correctly differentiate placental stem cells into glucagon-secreting cells, we used a culture method on a scaffold with sequential exposure to different growth factors. The underlying substrate used contained type IV collagen, chytosan, Matrigel and laminin. Molecular biology techniques were carried out to investigate the gene expression of the stem cells. RESULTS: Our results show that exendin-4 is able to induce the differentiation of placental stem cells into glucagon-secreting cells. We also notice the absence of the insulin gene, a conclusion that may be explained by the fact that our phenotype is a partial one, incomplete, closer to islet cell progenitors than to insulin-producing progenitors. CONCLUSIONS: The identification of the placenta as a valid source for stem cells has important practical advantages because it is easily accessible, it raises no ethical issues and cells are easily to isolate in a large enough number to use. The future knowledge and manipulation of the signaling pathways that determines the dramatic phenotype shift may provide the basis for efficient cell differentiation, with great impact on regenerative medicine and tissue engineering.

Aspects of placental morphogenesis and angiogenesis.

Placental morphology and vascularization are important stages in the evolution of pregnancies. Placental morphogenesis and angiogenesis processes are studied by two-dimensional, three-dimensional and Doppler ultrasound. Ultrasound methods provide important data on the physiology and pathophysiology of fetal-placental exchange. The macroscopic and microscopic study of the placenta brings valuable information on the possible structural changes and implicitly allows assessing fetal-placental circulation. The ultrasound and microscopic evaluation of the placenta are complementary means of examination for the assessment of fetal-maternal exchange. These methods of investigation can be applied in the context of a strict knowledge of placental morphogenesis and angiogenesis.

Isolation and characterization of mesenchymal stem cells from the amniotic membrane.

The aim of the study was the isolation and the genotypic and phenotypic characterization of mesenchymal stem cells from the amniotic membrane. The placentas included in the study were derived from pregnancies with a normal evolution. Along with the placentas, umbilical cord blood and maternal peripheral blood samples were taken. The isolation and the culture of cells from the amniotic membrane was followed by the determination of the markers of these cells. The cells expressed markers characteristic of stem cells. Immunofluorescence and evaluation of the gene expression evidenced the pluripotential properties of these cells. HLA expression provides information that might help explain the immunological mechanisms of tolerance between the maternal organism and fetal structures.

Isolation and characterization of stem cells from the placenta and the umbilical cord.

In addition to its essential role in the development, nutrition and immunological tolerance of the product of conception, human placenta is an important source of stem cells. Over the past years, scientific research has been aimed at isolating and characterizing mesenchymal cells and amniocytes, which show a high plasticity and are found in the chorionic villi and the membranes. At the level of the umbilical cord, two types of stem cells can be found: hematopoietic and mesenchymal. The blood of the umbilical cord is already in the focus of attention of researchers, as an important source of hematopoietic stem cells that can be used for transplantation.

In vitro expansion and characterization of corneal stem cells isolated from an eye with malignant melanoma.

PURPOSE: The objective of this study was the identification, characterization and in vitro replication of the human corneal stem cells, taking into consideration the difficulties in obtaining sufficient corneal material from living donors. The study explored a variety of stem cell markers, usually found in embryonic or adult mesenchymal stem cells. Culture medium and replication substrates had to be identified, with no data available on this subject in our country (there are no other reports on corneal stem cells in Romania, to our knowledge). MATERIALS AND METHODS: Corneal epithelial limbus was harvested from an enucleated eye, containing also a choroid malignant melanoma. Stem cells from the limbus were isolated and cultivated in vitro. Expression of specific stem cell markers was evaluated with immunocytochemistry. RESULTS: Corneal stem cell expansion in primary culture was slow, achieving 70-80% confluence after 28 days. Stem cells were easily isolated in standard medium, showed fibroblastoid morphology and were positive for certain stem cell specific markers in immunocytochemical staining: Oct3÷4, SOX2, Nanog, SSEA4, CD44, CD90, CD133, and CD34. They also expressed pan-cytokeratin. Donor age (72 years) and the presence of a malignant tumor close to limbal stem niche could have had an impact on the proliferation rate and the characteristics of the corneal stem cells. CONCLUSIONS: Isolated limbal cells were adult type stem cells with an epithelial orientation. The characterization of these cells with immunocytochemistry allowed us to observe surface markers that other stem cells also express.

Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma.

OBJECTIVES: Metformin, the most used oral antidiabetic drug for the treatment of type 2 diabetus mellitus, has proved encouraging results when used in the treatment of various types of cancer such as triple-negative breast cancer. Despite compelling evidence of a role of metformin as an anticancer drug, the mechanisms by which metformin exerts its oncostatic actions are not fully understood yet. Therefore, we tried to bring new insights by analyzing the anti-neoplastic effect of metformin for hepatocellular carcinoma-derived stem-like cells treated with conventional combination chemotherapy. METHODS: Cancer stem-like cells previusly isolated from a hepatocellular carcinoma biopsy were treated with metformin, PIAF chemotherapy regimen and the combination of these two protocols. Measurements of lipid peroxidation, reduced glutathione, fluorescein diacetate and proliferation rates were determined, apart from the autophagy assay and apoptosis determination by chip flow cytometry. RESULTS: Metformin alone and especially metformin in association with PIAF increases oxidative stress within the cells by increasing the levels of lipid peroxids as well as decreasing the levels of reduced glutathione. The MTT cell proliferation assay showed decreased prolife-ration rates for the arm treated with metformin and with the combination of drugs in comparison with the control arm, proving high correlation with the oxidative stress results. The autophagy assay and determination of apoptosis by chip flow cytometry confirmed the results obtained in the previous assays. CONCLUSION: Metformin could be used in chemotherapy treatments to induce reactive oxygen species and increase the cytostatics effects within the tumor cell. Still, further experiments must be carried out on murine models before we can move on and use this drugs in the adjuvant setting for unresectable primary liver cancer.

Isolation and characterization of chorionic mesenchyal stem cells from the placenta.

The aim of the study was the isolation and characterization of mesenchymal stem cells from the placental chorion from a genotypical and phenotypical point of view. The placentas included in the study were derived from term pregnancies with a normal evolution. Along with the placentas, umbilical cord blood, maternal and newborn peripheral blood samples were taken. The isolation and culture of chorionic and, incidentally, trophoblastic cells was followed by the determination of markers of the former cells. They expressed proteins and genes characteristic of stem cells. Immunofluorescence and evaluation of gene expression evidenced the pluripotential properties of these cells and also their higher position on the differentiation pathway. HLA expression provides information that might help explain the immunological mechanisms of tolerance between the maternal organism and fetal structures.

The axis of evil in the fight against cancer.

Over the past years medicine has undergone intensive changes, evolving from classical semiology and internal medicine to individualized treatments, based on recent breakthroughs in immunology and genetics. This concept has had a profound impact in all medical specialties and as a consequence pharmacology and various treatment plans will be based on monoclonal antibodies and targeted cell therapies. One such target is the SDF-1-CXCR4 axis bacause it plays a critical role in many physiological processes that involve cell migration and cell fate decisions, ranging from stem cell homing, angiogenesis and neuronal development to immune cell trafficking. The chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1alpha are also implicated in various pathological conditions, including metastatic spread and HIV infection. In this review, we present the concept that the SDF-1-CXCR4 axis is a master regulator of trafficking of both normal and cancer stem cells, based on the growing evidence that it plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells and their homing to the bone marrow. Because most malignancies originate in the progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and migrate to organs that highly express SDF-1. Hence, we postulate that the metastasis of cancer stem cells and trafficking of normal stem cells involve similar mechanisms, which may be regulated by several small molecules related to inflammation. Consequently, strategies aimed at modulating the SDF-1-CXCR4 axis could have important clinical applications in both tissue engineering and in clinical hematology and oncology to inhibit metastasis of cancer stem cells.

Stem-like cells in colorectal oncology.

Although the treatment for colorectal cancer has seen considerable progress during the past few years, the mortality associated with this type of tumor remains high. This article presents the existing methods of treatment, focusing on the new treatments made possible by the advances in the field of normal and tumor stem cells. Starting from the normal architecture of the colon and the properties of the cells identified in it, we sought to present a few notions concerning these cells which have a direct relevance for both pathology and treatment. The manner in which they divide (symmetrically or asymmetrically) as well as the molecules which control their circulation through the body are just a few examples which are likely to influence the treatment of colorectal cancer in the future.

Pancreatic exocrine adult cells and placental stem cells co-culture. Working together is always the best way to go.

BACKGROUND AND AIM: The progress made in the last few years have managed to come up withy the possibility of using different stem cell types in an endeavor to correct the alterations that appear in different degenerative diseases. The pancreas, an organ with extremely low regenerative capacity, both for the endocrine and for the exocrine component, is an organ perfect for cell therapy in the hope of restoring its function and cure diabetes mellitus or chronic pancreatitis. One main issue in the stem cell transplantation problem is represented by the influence of the cellular niche, formed by completely differentiated cells, on the phenotype and function of the transplanted cells. In this study, we challenge current knowledge in the field by evaluating the influence of exocrine pancreatic cells on placental stem-like cells using the co-culture technique. METHODS: In our experiments, we used two different protocols in which adult pancreatic cells were cultured together with mesenchymal stem cells isolated from human placenta. In the case of the first protocol, we seeded pancreatic cells on a pre-adhered single-cell layer of mesenchymal stem cells and in the second one, the seeding of two cell populations in suspension was done at the same time, after passage. During the experiment, we evaluated the alteration of the morphology of the placental cells using and inverted phase microscope and reverse transcriptase-PCR. RESULTS AND CONCLUSIONS: Based on morphology, in both cases the interaction between epithelial pancreatic cells and placental ones have determined a change in phenotype from mesenchymal to epithelial-like. Taking into consideration the gene expression, placental stem cells have maintained pluripotency gene expression throughout the study. They also expressed pancreatic amylase. These experiments bring out the plasticity of placental stem cells, the cell microenvironment with a decisive part in phenotype and the level of gene expression. The results obtained in vitro can bring a new picture on the effects of the pancreatic stem cell niche.

Cellular interactions in prostate cancer genesis and dissemination. Looking beyond the obvious.

Similar to normal organs arising from normal stem cells, cancers can be viewed as organs composed of heterogeneous cellular populations arising from cancer cells with indefinite proliferation abilities. The continuous malignant progression is maintained by the proliferation of cancer stem cells and not the progeny that undergo limited proliferation before terminally differentiating. Effective therapy must eradicate malignant cells with unlimited clonogenic expansion within the primary tumor bulk. Thus, resolving both the specific cell of origin for prostate cancer and the interactions between the cells and the surrounding microenvironment within the cancer stem cell niche are crucial to appropriately define rational targets for therapeutic intervention and cure prostate cancer.