Specific Aptamer Functionalization of Dense Ceramic by Click Chemistry Towards Circulating Tumor Cells Apheresis.

BACKGROUND/AIM: Circulating tumor cells (CTC) are tumor cells which can be disseminated at distance of the primary tumor and form metastatic niche. Moreover, their quantity is an important parameter which can induce cluster metastasis. A solution, can be the creation of a system that allow the capture and elimination from the blood of patients by using the medical device developed which is an inert bioceramic functionalized by aptamer target to CTC. MATERIALS AND METHODS: Herein we develop chemical reactions to bind a modified MUC1 specific DNA aptamer on an alumina (Al2O3) dense ceramic surface. In fact, MUC1 biomarker is very present on the surface of tumor cells. RESULTS: The specific developed chemical reactions led to the covalent binding of the aptamer while preserving its biological characteristics. CONCLUSION: This functionalization of dense alumina would allow the potential capture of circulating tumor cells.

Splicing factor ratio as an index of epithelial-mesenchymal transition and tumor aggressiveness in breast cancer.

Epithelial-to-mesenchymal transition (EMT) has been shown to be associated with tumor progression and metastasis. During this process in breast cancer, a crucial role is played by alternative splicing systems. To identify a new early prognostic marker of metastasis, we evaluated EMT-related gene expression in breast cell lines, and in primary tumor tissue from 31 patients with early breast cancer, focusing our attention on EMT-related splicing factors ESRP1, ESRP2 and RBFOX2. Results showed that the expression patterns of these genes were indicative of the onset of EMT in in-vitro models, but not in tissue samples. However, the ratio between ESRP1 or ESRP2 and RBFOX2 significantly decreased during EMT and positively correlated with the EMT-specific phenotype in cell models, representing a promising prognostic markers. Low ESRP1/RBFOX2 ratio value was associated with a higher risk of metastasis (p < 0.005) in early breast cancer patients, regardless other clinical features. A cut-off of ratio of 1.067 was determined by ROC curve analysis (AUC 0.8375; 95% CI 0.6963-0.9787). Our study show evidence that a decrease in this ratio correlates with cancer progression. The results provide a rationale for using ESRP1/RBFOX2 ratio as a new prognostic biomarker for the early prediction of metastatic potential in breast cancer.

Epithelial Mesenchymal Transition: a double-edged sword.

Epithelial mesenchymal transition (EMT) is a physiological process necessary to normal embryologic development. However in genesis of pathological situations, this transition can be perverted and signaling pathways have different regulations from those of normal physiology. In cancer invasion, such a mechanism leads to generation of circulating tumor cells. Epithelial cancer cells become motile mesenchymal cells able to shed from the primary tumor and enter in the blood circulation. This is the major part of the invasive way of cancer. EMT is also implicated in chronic diseases like fibrosis and particularly renal fibrosis. In adult organisms, healing is based on EMT which is beneficial to repair wounds even if it can sometimes exceed its goal and elicit fibrosis. In this review, we delineate the clinical significance of EMT in both physiological and pathological circumstances.

Circulating tumor cells and epithelial, mesenchymal and stemness markers: characterization of cell subpopulations.

Until now detection and numeration of circulating tumor cells (CTCs) were essentially used as a prognostic factor in cancer progression. To extend the role of these kinds of analysis, it seems necessary to improve analytical methods related to isolation and characterization of CTCs. Discrepancies between published results corroborates this requirement. In this review we suggest a combination of markers able to reach the goal. Moreover to improve the clinical utility of CTC analysis, particularly in the therapeutic follow up of the disease, epithelial mesenchymal transition (EMT) level of a global CTC population should be studied.

Metformin: a rising star to fight the epithelial mesenchymal transition in oncology.

Metformin is a biguanide derivative which is widely prescribed as an oral drug for diabetes mellitus type 2. This old molecule has recently received a new attention because of its therapeutic properties in oncology, that seem to be independent of its action on glycemia homeostasis. The reappraisal of its pharmacological effects was supported by delineation of signaling pathways and more recently clinical trials. Numerous epidemiological studies showed that diabetics have an increased risk of several types of cancer and cancer mortality. Complex relationship between cancer and type 2 diabetes is going to be unraveled and recent observations revealed a significant action of metformin, but not other anti-diabetic agents, on cancer cells. As metformin may act as an anticancer drug through inhibition of mTOR, it might have greater benefice than suggested by insulin lowering alone. This review summarizes major publications on the link between cancer and metformin underscoring new implications of this chemical drug in oncology field. New perspectives about utilization of this molecule in clinical oncological routine, are described, particularly for patients without disturbance of glucose homeostasis. As the epithelial mesenchymal transition (EMT) seems implicated into invasive process and metastasis in cancer, and as metformin is able to inhibit EMT pathways, it is important to highlight cellular mechanisms of metformin.

After clouds sun again shines on circulating tumor cells research.

In the Science issue of first February 2013 Yu M et al. characterized epithelial and mesenchymal circulating tumor cells (CTC) by RNA-in situ hybridization. In this editorial we comment their results and emphasize the different CTC subpopulations arising from epithelial mesenchymal transition (EMT).

Mesenchymal characterization: alternative to simple CTC detection in two clinical trials.

BACKGROUND: Breast cancer is one of the most common malignancies in women. Approximately 25% of patients with early-stage disease will develop metastatic recurrence. Two clinical trials were undertaken in order to detect circulating tumor cells (CTCs) in primary breast cancer. PATIENTS AND METHODS: Four-hundred patients with early breast cancer were enrolled in the trial. After enrichment from their peripheral blood, their CTCs were characterized by gene expression of cancer cell markers. RESULTS: CTCs had a predominant epithelial phenotype in 8.75% of patients and de-differentiated characteristics (mesenchymal, stem phenotypes alone or both) in 37.6%. CONCLUSION: Tumor epithelial cells undergoing epithelial-mesenchymal transition give rise to cells with mesenchymal aggressive phenotype. Detection of mesenchymal and cancer stem cells, which are tumor-initiating cells, is more relevant than simple counting of CTCs to assess their presence in the blood of patients with breast cancer. This study will be the basis for future evaluation of the outcome of the disease and the prognostic value of early-detected CTCs.

Epithelial mesenchymal transition: a new insight into the detection of circulating tumor cells.

Many research groups reported on the relation between circulating tumor cells (CTCs) in peripheral blood and worse prognosis for metastatic cancer patients. These results are based on CTCs counting and did not take into account molecular characteristics of cells. To establish CTCs as a reliable and accurate biological marker, new technologies must be focused on CTC subpopulations: dedifferentiated circulating tumor cells (ddCTCs) arising from epithelial mesenchymal transition (EMT). To select and detect them, different methods have been proposed but none has still reached the goal. Technical progress and translational research are expected to establish CTCs as a real marker. Thus CTC evaluation profiling for each patient will lead to personalize followup and therapy.

Mesenchymal and stemness circulating tumor cells in early breast cancer diagnosis.

BACKGROUND: Epithelial mesenchymal transition (EMT) is a crucial event likely involved in dissemination of epithelial cancer cells. This process enables them to acquire migratory/invasive properties, contributing to tumor and metastatic spread. To know if this event is an early one in breast cancer, we developed a clinical trial. The aim of this protocol was to detect circulating tumor cells endowed with mesenchymal and/or stemness characteristics, at the time of initial diagnosis. Breast cancer patients (n = 61), without visceral or bone metastasis were enrolled and analysis of these dedifferentiated circulating tumor cells (ddCTC) was realized. METHODS: AdnaGen method was used for enrichment cell selection. Then, ddCTC were characterized by RT-PCR study of the following genes: PI3Kα, Akt-2, Twist1 (EMT markers) and ALDH1, Bmi1 and CD44 (stemness indicators). RESULTS: Among the studied primary breast cancer cohort, presence of ddCTC was detected in 39% of cases. This positivity is independant from tumor clinicopathological factors apart from the lymph node status. CONCLUSIONS: Our data uniquely demonstrated that in vivo EMT occurs in the primary tumors and is associated with an enhanced ability of tumor cells to intravasate in the early phase of cancer disease. These results suggest that analysis of circulating tumor cells focused on cells showing mesenchymal or stemness characteristics might facilitate assessment of new drugs in clinical trials.

FDG-PET/CT of head and neck squamous cell carcinoma in a rat model.

PURPOSE: The aim of this study was to demonstrate the ability to use human clinical positron emission tomography/computed tomography (PET/CT) to detect and investigate head and neck cancers chemically induced by 4-nitroquinoline-1-oxide (4-NQO) in a rat model. STUDY DESIGN: The study design was prospective animal research. PROCEDURES: A head and neck squamous cell carcinoma was established in 20 immunocompetent rats, who drank a 4-NQO solution during 16 weeks. 2-Deoxy-2-[F-18]fluoro-D: -glucose (FDG)-PET/CT was performed for five of them, 34 weeks after the start of the experiment to characterize the tumors. A day following the FDG-PET/CT, rats were euthanized and pathological features were evaluated by hematoxylin-eosin staining. RESULTS: All rats had head and neck tumor at various locations at 34 weeks. Among the five rats selected for having FDG-PET/CT, the clinical examination detected exophytic tumors grown in the oral cavity for three of them (one on the inferior lip, one on the hard palate, and one on the internal side of the cheek). FDG-PET/CT confirmed the presence of those tumors and detected ones located on the base of tongue for three of them. Tumor extensions were characterized and tumor metabolic volumes were measured. The smallest lesion detected measured 3 x 3 x 4 mm. Pathologic examination using hematoxylin-eosin staining confirmed squamous cell carcinoma. CONCLUSIONS: This study demonstrated that FDG-PET/CT is a feasible examination to detect occult primary tumors in rat models. It is useful to follow tumor progression and evaluate therapeutics efficacy.

Characterization of a new rat model of head and neck squamous cell carcinoma.

AIM: To develop and characterize by imaging and pathological examination a new immunocompetent rat model of head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: Prospective animal research. MATERIALS AND METHODS: Frozen specimens of HNSCC induced chemically by 4-nitroquinoline 1 oxide (4-NQO) in Sprague Dawley rats were used for the first graft. Serial allografts were then performed with fresh specimens of tumor in twenty-five Sprague Dawley rats. A specimen of tumor (100 mm3) was picked up by head and neck dissection during an autopsy. The graft was performed in a subcutaneous manner, in the ventral part of the neck, using an incision of 4 mm, through the masseter muscle. Tumors were clinically measured once a week and volumes were calculated. 2-[18F]Fluoro-2-deoxy-D-glucose positron emission tomography coupled with computed tomography (FDG-PET/CT) was performed on days 14 and 30 after the graft. Rats were euthanized and pathological features were assessed using hematoxylin-eosin (HE) staining and immunohistochemistry markers to characterize the tumor. RESULTS: An 80% take rate was achieved using fresh tumor specimens. Tumors grew rapidly; the mean tumoral volume was 1.013 cm3 on day 14 and 7.994 cm3 on day 30. FDG-PET/CT imaging targeted regions of metabolically active tumor. It showed a uniform uptake of 18F-FDG on day 14 and a large area of central necrosis on day 30. Pathological examinations showed a typical squamous cell carcinoma, with similar immunohistochemical analyses to the human squamous cell carcinoma. CONCLUSION: We propose a new allograft HNSCC rat model which is easily reproducible and rapidly obtained in comparison to that induced chemically with 4-NQO. This model was developed in immunocompetent rats, with similar conditions to human carcinogenesis and could be used for testing new therapeutics.

Improved agarose gel assay for quantification of growth factor-induced cell motility.

Cell chemotaxis is frequently required in normal or pathological situations such as invasion, metastasis, and tumor angiogenesis and may involve many different cell types. At present, no device can simultaneously (i) make morphological observations, (ii) quantify cell migration, (iii) test multiple chemoattracting gradients, and (iv) analyze cell-cell interactions. We developed an agarose-based assay to address these questions. Two glass molds were designed, around which agarose gel could be poured to form specific well shapes. Using a vital nuclear stain (Hoechst 33258), we characterized the migration profile of adherent or suspension cells. Cells could be observed during the entire migration process. We were able to follow cells moving toward chemoattractants or being repulsed by other molecules, and we could estimate average migration speed. Using this inexpensive assay, we were able to obtain precise, reproducible results concerning the chemotactic behavior of different cell types. The resulting data differentiated between chemokinetic and chemotactic movement. Chemotactic potencies could be compared using different criteria, such as the number of attracted cells, induced speed, and morphological aspect. This improved agarose assay appears to be a reliable and inexpensive alternative to other available chemotaxis study tools.

Molecular mechanisms regulating the angiogenic phenotype in tumors: clinical impact in the future.

Tumor progression depends on the angiogenic switch. In this review, we recapitulate the molecular mechanisms involved in this angiogenic switch. The rat osteosarcoma model employed would permit further studies in the sequential events leading to initial recruitment of blood vessels and could lead to development of an angiogenesis-based panel of circulating blood cells (endothelial cells, endothelial progenitor cells and accessory cells) that can be quantified and used to detect microscopic tumors or to follow the efficacy of antiangiogenic therapy. Such a result would lead to the possibility of early therapy in cancer progression.

Expression of 5-lipoxygenase (5-LOX) in T lymphocytes.

5-Lipoxygenase (5-LOX) is the key enzyme responsible for the synthesis of the biologically active leukotrienes. Its presence has been reported in cells of the myeloid lineage and B lymphocytes but has not been formally defined in T lymphocytes. In this study, we provide evidence for 5-LOX expression on both transcriptional and translational levels in highly purified peripheral blood T cells as well as in human T lymphoblastoid cell lines (MOLT4 and Jurkat). Messenger RNA (mRNA) of 5-LOX was amplified by conventional reverse transcription-polymerase chain reaction (RT-PCR; MOLT4 and Jurkat cells) and by in situ RT-PCR (T lymphocytes). 5-LOX protein expression was confirmed by Western blot and immunofluorescence studies. 5-LOX was present primarily in the cytoplasm with some nuclear localization and was translocated to the nuclear periphery after culture in a mitosis-supporting medium. Fluorescence-activated cell sorter analysis of different T-lymphocyte populations, including CD4, CD8, CD45RO, CD45RA, T helper type 2, and T-cell receptor-alphabeta and -gammadelta expressing cells, did not identify a differential distribution of the enzyme. Purified peripheral blood T lymphocytes were incapable of synthesizing leukotrienes in the absence of exogenous arachidonic acid. Jurkat cells produced leukotriene C(4) and a small amount of leukotriene B(4) in response to CD3-CD28 cross-linking. This synthesis was abolished by two inhibitors of leukotriene synthesis, MK-886 and AA-861. The presence of 5-LOX in T lymphocytes but the absence of endogenous lipoxygenase metabolite production compared to Jurkat cells may constitute a fundamental difference between resting peripheral lymphocytes and leukaemic cells.

Gene expression of HIF-1alpha and XRCC4 measured in human samples by real-time RT-PCR using the sigmoidal curve-fitting method.

Quantitative reverse transcription PCR (RT-PCR) has become an important tool for studying functional gene expression. However the most often used cycle threshold (CT)-based method, primarily related to the required amplification efficiency determination via serial dilution, can call into question the level of quantitative reliability and accuracy that can be achieved, in addition to the impracticalities inherent to CT-based methodologies. In this study, an alternative method, named the sigmoidal curve-fitting (SCF) method, was compared with the classic CT method for two target genes (XRCC4 and HIF-1alpha) and a reference gene (HPRT). The PCR conditions were optimized for each gene on a LightCycler apparatus. Fluorescence data were fitted to a four-parametric sigmoidal function, and the initial messenger RNA (mRNA) copy number was determined by a theoretical fluorescence (F0) value calculated from each fitting curve. The relative expression of the target gene versus that of the reference gene was calculated using an equation based upon these F0 values. The results show that the F0 value had a good linearity with the initial number of target genes between 10(7) and 10(1) copies. The reproducibility tests showed that the variations of initial target quantity were well reflected by F0 values. Relative expression of target gene calculated by the SCF method and by the CT method showed similar results. In our hands, the SCF method gave reliable results and a more precise error description of quantitative RT-PCR.

VEGFR-3, VEGF-C and VEGF-D mRNA quantification by RT-PCR in different human cell types.

The molecular events favoring lymphangiogenic pathways for tumor growth and dissemination are not perfectly understood, nor are the expression patterns of lymphangiogenic biomarkers such as the VEGFR-3 receptor and its ligands, VEGF-C and VEGF-D. In particular, VEGFR-3 expression is not restricted to the lymphatic endothelium, but is found on some cancer cells and other cell types. A quantitative RT-PCR method was set up to measure the mRNA levels of VEGFR-3, VEGF-C and VEGF-D. With this method, a very low detection threshold was obtained when tested on 17 different human cell types. It was found that, in contrast to VEGF-D mRNA, the VEGFR-3 and VEGF-C mRNAs were not expressed in all the cell types studied, and that blood cells expressed high VEGFR-3 mRNA levels compared to solid tumor cells. As a result, quantitative RT-PCR is considered to be a highly reliable and reproducible technique that could help elucidate lymphangiogenic marker patterns of expression and function in cancer.

A quantitative RT-PCR method to determine topoisomerase I mRNA levels in human tissue samples.

DNA topoisomerase I (Topo I) is involved in DNA replication, transcription, recombination and repair. Clinical interest has focused on Topo I as it is the molecular target of camptothecin (CPT), used in first and second lines of treatment for different cancer types. Furthermore, it is well demonstrated that the patients who best responded to CPT-based chemotherapy were generally those with the greatest tumoral Topo I expression and/or activity. We developed a sensitive, simple and reproducible method to measure Topo I mRNA expression in human cancer samples. Experiments were performed in two steps. First, we checked the accuracy of the reverse transcription-polymerase chain reaction (RT-PCR) method by testing intra- and interassay reproducibility of Topo I and G6PDH gene amplification in different cell types. We observed that crossing-points (Cps) were different, depending on the cell type, dilution or cDNA concentration, but that the intra- and interassay Cp standard deviation (SD) never exceeded 0.77% and 1.39% for Topo I amplification, or 1.63% and 2.9% for G6PDH amplification, respectively. Secondly, we used our method to measure Topo I mRNA levels in primary tumor samples obtained from 27 patients with advanced colorectal cancer and 10 patients with pharyngeal/laryngeal cancer. The accuracy of G6PDH as a housekeeping gene was tested by analyzing its correlation with the mRNA level of a second housekeeping gene, porphobilinogen deaminase (PBG-D) in the tumoral samples. We found that the normalized Topo I/G6PDH mRNA ratios were significantly correlated with that of Topo I/PBGD in colorectal tumors (r(2)=0.47, p=0.02) but not in pharyngeal/laryngeal tumors (r(2)=0.35, p=0.3). Neither ratio showed any significant association with clinicopathological parameters, such as gender, age, tumor size, or grade and lymph node status. We believe that RT-PCR is a reliable and highly reproducible technique. However, the choice of the reference gene is an important point and must be defined based on the samples studied.

[Lymphatic vessels and cancer]. / Vaisseaux lymphatiques et cancer.

Survival and development of tumors depends on nutritional and respiratory biological events and exchanges ensured by blood and lymph. Tumor proliferation is associated with an increase in the vascular networks either near the tumor or intra-tumorally. Tumor tissues are able to increase their provisionment according to their needs while directing and optimizing the development of peri-tumoral vessels. The production of growth factors stimulating neo-formation of lymphatic vessels by cancer cells constitutes one of the adaptations responsible for metastatic propagation. During tumor development the lymphatic system is considered in many cases of cancer as the primary means of metastasis dissemination. The study of the lymphatic system setting and ways to block it are important points to consider in oncology.

Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells.

As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.

In vivo follow-up of rat tumor models with 2-deoxy-2-[F-18]fluoro-D-glucose/dual-head coincidence gamma camera imaging.

UNLABELLED: Before studying the impact of 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) imaging with a dual-head coincidence gamma camera (DHC) for the follow-up of animal tumor models, we wanted to optimize this technique. METHODS: Three different animal tumor models (osteosarcoma, melanoma, and breast cancer) were studied after FDG injection. Dynamic and dual time point FDG/DHC imaging were studied from one hour to five hours postinjection. In vitro tumor cell FDG uptake was assessed in eight different tumor cell lines. In one model (osteosarcoma), tumor growth, lung metastasis emergence, and survival were assessed by classical clinical follow-up and compared to FDG imaging in a control group (n = 6) and in a group treated by endostatin liposome complexes (n = 6). RESULTS: Images obtained five hours after injection were more reliable for tumor growth follow-up than standard images (one hour). In vitro tumor cell FDG uptake confirmed in vivo imaging studies. In eight different tumor cell lines the FDG uptake was higher after five hours incubation than after one hour (p < 0.002). With FDG follow-up, we found that FDG uptake was strongly correlated with survival and that lung metastasis larger than 5 mm could be detected. CONCLUSION: Using the optimization proposed above, DHC/FDG functional imaging seems to be a powerful tool to study rat tumor models and to help develop novel cancer therapies.