ABCG2, a novel antigen to sort luminal progenitors of BRCA1- breast cancer cells.

INTRODUCTION: Tumor-initiating cells (TICs), aka "cancer stem cells", are believed to fuel tumors and to sustain therapy resistance and systemic metastasis. Breast cancer is the first human carcinoma in which a subpopulation of cells displaying a specific CD44+/CD24-/low/ESA+ antigenic phenotype was found to have TIC properties. However, CD44+/CD24-/low/ESA+ is not a universal marker phenotype of TICs in all breast cancer subtypes. The aim of this study was to identify novel antigens with which to isolate the TIC population of the basal-A/basal-like breast cancer cell lines. METHODS: We used polychromatic flow-cytometry to characterize the cell surface of several breast cancer cell lines that may represent different tumor molecular subtypes. We next used fluorescence-activated cell sorting to isolate the cell subpopulations of interest from the cell lines. Finally, we explored the stem-like and tumorigenic properties of the sorted cell subpopulations using complementary in vitro and in vivo approaches: mammosphere formation assays, soft-agar colony assays, and tumorigenic assays in NOD/SCID mice. RESULTS: The CD44+/CD24+ subpopulation of the BRCA1-mutated basal-A/basal-like cell line HCC1937 is enriched in several stemness markers, including the ABCG2 transporter (i.e., the CD338 antigen). Consistently, CD338-expressing cells were also enriched in CD24 expression, suggesting that coexpression of these two antigenic markers may segregate TICs in this cell line. In support of ABCG2 expression in TICs, culturing of HCC1937 cells in ultra-low adherent conditions to enrich them in precursor/stem-cells resulted in an increase in CD338-expressing cells. Furthermore, CD338-expressing cells, unlike their CD338-negative counterparts, displayed stemness and transformation potential, as assessed in mammosphere and colony formation assays. Lastly, CD338-expressing cells cultured in ultra-low adherent conditions maintained the expression of CD326/EpCAM and CD49f/α6-integrin, which is a combination of antigens previously assigned to luminal progenitors. CONCLUSION: Collectively, our data suggest that CD338 expression is specific to the tumor-initiating luminal progenitor subpopulation of BRCA1-mutated cells and is a novel antigen with which to sort this subpopulation.

Surface proteomic analysis of differentiated versus stem-like osteosarcoma human cells.

Cancer stem cell characterization represents a breakthrough in cancer research. Despite evidence showing the existence and the role of cancer stem cells in osteosarcoma (OS) onset and progression, little is known about their specific surface phenotype. To address this issue, we carried out a cytometric analysis with an antibody-array comprising 245 membrane proteins comparing the stem and differentiated OS cells. As experimental model, we chose the stem-like cell line 3aminobenzamide-OS and its parental, differentiated, cell line MG63. We identified 50 differentially expressed, 23 homogeneously expressed, and 172 not expressed proteins in the two cell line models, thus defining a surface protein signature specific for each of them. Furthermore, we selected ERK1/2 (p44/42 mitogen-activated protein kinases) as a potential pathway correlated with processes that characterize tumorigenic potential and stemness of 3aminobenzamide-OS cells.

The secretome signature of colon cancer cell lines.

The definition of the secretome signature of a cancer cell line can be considered a potential tool to investigate tumor aggressiveness and a preclinical exploratory study required to optimize the search of cancer biomarkers. Dealing with a cell-specific secretome limits the contamination by the major components of the human serum and reduces the range of dynamic concentrations among the secreted proteins, thus favouring under-represented tissue-specific species. The aim of the present study is to characterize the secretome of two human colon carcinoma cell lines, CaCo-2 and HCT-GEO, in order to evaluate differences and similarities of two colorectal cancer model systems. In this study, we identified more than 170 protein species, 64 more expressed in the secretome of CaCo-2 cells and 54 more expressed in the secretome of HCT-GEO cells; 58 proteins were shared by the two systems. Among them, more than 50% were deemed to be secretory according to their Gene Ontology annotation and/or to their SignalP or SecretomeP scores. Such a characterization allowed corroborating the potential of a cell culture-based model in order to describe the cell-specific invasive properties and to provide a list of putative cancer biomarkers.

CD66c is a novel marker for colorectal cancer stem cell isolation, and its silencing halts tumor growth in vivo.

BACKGROUND: Despite the well recognized expression of the cell surface markers cluster of differentiation 44 (homing cell adhesion molecule) and CD133 (Prominin 1) on human colorectal cancer stem cells (CCSCs), these molecules do not appear to be effective targets for stem cell-directed therapies. Because the surface marker CD66c (also known as carcinoembryonic antigen-related cell adhesion molecule 6) has demonstrated promise as a therapeutic target in pancreatic malignancy, the authors evaluated its potential as a target for stem cell-directed treatment of colorectal cancer. METHODS: First, the authors characterized CD66c expression by flow cytometry and immunohistochemistry in colon cancer samples and in normal colon tissues. Then, the coexpression of CD66c and CD133 was evaluated on putative CCSCs. CD66c expression also was measured in stem cell-enriched colon spheres. Finally, the effects of small-interfering RNA-mediated CD66c silencing on the in vitro and in vivo growth of Caco2 colon cancer cells were evaluated. RESULTS: CD66c expression was significantly higher in colon cancers than in contiguous normal colon tissues and paralleled cancer stage. CD66c was absent in CD133-positive cells that were isolated from normal colon, whereas its expression was brightest (CD66c(bright) ) in CD133-positive cells from colon cancer samples. In vitro experiments demonstrated that colon spheres were considerably enriched in a CD66c(bright) population in a fashion comparable to the enrichment observed in fresh liver metastases. In vitro proliferation and clonogenic potential were hampered when CD66c was silenced in Caco2 cells. Finally, in vivo xenograft experiments demonstrated that CD66c silencing almost completely abrogated the tumorigenic potential of Caco2 cells. CONCLUSIONS: CD66c(bright) expression was associated with colon cancer stem cells and CD66c silencing blocked tumor growth, thereby opening the way to a potential new treatment for colon cancer.

Chimeric beta-defensin analogs, including the novel 3NI analog, display salt-resistant antimicrobial activity and lack toxicity in human epithelial cell lines.

Human beta-defensins (hBDs) are crucial peptides for the innate immune response and are thus prime candidates as therapeutic agents directed against infective diseases. Based on the properties of wild-type hBD1 and hBD3 and of previously synthesized analogs (1C, 3I, and 3N), we have designed a new analog, 3NI, and investigated its potential as an antimicrobial drug. Specifically, we evaluated the antimicrobial activities of 3NI versus those of hBD1, hBD3, 1C, 3I, and 3N. Our results show that 3NI exerted greater antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis than did hBD1 and hBD3, even with elevated salt concentrations. Moreover, its antiviral activity against herpes simplex virus 1 was greater than that of hBD1 and similar to that of hBD3. Subsequently, we investigated the cytotoxic effects of all peptides in three human epithelial carcinoma cell lines: A549 from lung, CaCo-2 from colon, and Capan-1 from pancreas. None of the analogs significantly reduced cell viability versus wild-type hBD1 and hBD3. They did not induce genotoxicity or cause an increase in the number of apoptotic cells. Using confocal microscopy, we also investigated the localization of the peptides during their incubation with epithelial cells and found that they were distributed on the cell surface, from which they were internalized. Finally, we show that hBD1 and hBD3 are characterized by high resistance to serum degradation. In conclusion, the new analog 3NI seems to be a promising anti-infective agent, particularly given its high salt resistance--a feature that is relevant in diseases such as cystic fibrosis.

ImageStream promyelocytic leukemia protein immunolocalization: in search of promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is a hematological emergency in which a rapid diagnosis is essential for early administration of appropriate therapy, including all-trans retinoic acid before the onset of fatal coagulopathy. Currently, the following methodologies are widely used for rapid initial diagnosis of APL: 1) identification of hypergranular leukemic promyelocytes by using classical morphology; 2) identification of cells with diffuse promyelocytic leukemia (PML) protein distribution by immunofluorescence microscopy; 3) evidence of aberrant promyelocyte surface immunophenotype by conventional flow cytometry (FCM). Here, we show a method for immunofluorescent detection of PML localization using ImageStream FCM. This technique provides objective per-cell quantitative image analysis for statistically large sample sizes, enabling precise and operator-independent PML pattern recognition even in electronic and real dilution experiments up to 10% of APL cellular presence. Therefore, we evidence that this method could be helpful for rapid and objective initial diagnosis and the prompt initiation of APL treatment.

Mollicutes contamination: a new strategy for an effective rescue of cancer cell lines.

Mollicutes contaminations of cellular models can have marked effects on gene expression and cell behaviour in vitro leading to the production of unreliable data, unsafe biopharmaceutical drugs or to the loss of cell culture itself. Fortunately, irreplaceable cell culture can be cured by decontamination with the specific antibiotic regimen. Here, we describe the treatment of 35 mycoplasma-positive cell lines by the use of the novel antibiotic Mycozap(®) as well as evaluate its eradication performance versus the well-known routinely employed BM-Cyclins and fluoroquinolones molecules (175 treatments). Our data evidenced: i) the permanent elimination of mycoplasma infection by MycoZap(®), MRA, Enrofloxacin, Ciprofloxacin and BM-Cyclins in 46%, 29%, 40%, 43%, and 57% of the cultures, respectively; ii) a significant correlation between MRA and Ciprofloxacin eradication profile, as determined by the Spearman correlation coefficient (r = 0.3469, p < 0.05); iii) a mycoplasma eradication in 100% of cell lines by the exclusive adoption of MycoZap(®), Ciprofloxacin, Enrofloxacin, BM-Cyclin 1-2 antibiotic regimen, with the MRA exclusion; iv) the MycoZap(®) effectiveness even in case of a mycoplasmal load higher than 50 CFU/mL, as for SH-SY5Y and Neuro2A cells. In conclusion, we want to suggest an optimized antibiotic panel to get 100% mycoplasma-clearance especially in case of unique or treatment-resistant cellular models.

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations.

The main causes of familial hypercholesterolemia (FH) are mutations in LDL receptor (LDLR) gene. Functional studies are necessary to demonstrate the LDLR function impairment caused by mutations and would be useful as a diagnostic tool if they allow discrimination between FH patients and controls. In order to identify the best method to detect LDLR activity, we compared continuous Epstein-Barr virus (EBV)-transformed B-lymphocytes and mitogen stimulated T-lymphocytes. In addition, we characterized both novel and known mutations in the LDLR gene. T-lymphocytes and EBV-transformed B-lymphocytes were obtained from peripheral blood of 24 FH patients and 24 control subjects. Functional assays were performed by incubation with fluorescent LDL followed by flow cytometry analysis. Residual LDLR activity was calculated normalizing fluorescence for the mean fluorescence of controls. With stimulated T-lymphocytes we obtained a better discrimination capacity between controls and FH patients compared with EBV-transformed B-lymphocytes as demonstrated by receiver operating characteristic (ROC) curve analysis (the areas under the curve are 1.000 and 0.984 respectively; P < 0.0001 both). The characterization of LDLR activity through T-lymphocytes is more simple and faster than the use of EBV-transformed B-lymphocytes and allows a complete discrimination between controls and FH patients. Therefore the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes.

Solid-phase synthesis and pharmacological evaluation of novel nucleoside-tethered dinuclear platinum(II) complexes.

Three novel inosine-based dinuclear platinum complexes have been synthesized via a solid-phase strategy. In these compounds, the metal is linked both to the N-7 of the purine nucleus and to the terminal amine group of a hexylamine side chain installed on N-1. Cis- or trans- diamine as well as ethylenediamine ligands are coordinated to platinum along with a chloride. The synthesised complexes were tested against four different human tumor cell lines. One of these complexes proved to be more cytotoxic than cisplatin against the MCF7 cancer cell line in a short-term exposure assay.

Novel synthetic, salt-resistant analogs of human beta-defensins 1 and 3 endowed with enhanced antimicrobial activity.

Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

The percentage of CD133+ cells in human colorectal cancer cell lines is influenced by Mycoplasma hyorhinis infection.

BACKGROUND: Mollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of Mycoplasma hyorhinis contamination on CD133 expression in human colon cancer cell lines. METHODS: MycoAlert and mycoplasma agar culture were used to detect mycoplasma contamination on GEO, SW480 and HT-29 cell lines. Restriction fragment length polymorphism assay was used to determine mycoplasma species. All cellular models were decontaminated by the use of a specific antibiotic panel (Enrofloxacin, Ciprofloxacin, BM Cyclin 1 and 2, Mycoplasma Removal Agent and MycoZap). The percentage of CD133 positive cells was analyzed by flow cytometry on GEO, SW480 and HT-29 cell lines, before and after Mycoplasma hyorhinis eradication. RESULTS: Mycoplasma hyorhinis infected colon cancer cell lines showed an increased percentage of CD133+ cells as compared to the same cell lines rendered mycoplasma-free by effective exposure to antibiotic treatment. The percentage of CD133 positive cells increased again when mycoplasma negative cells were re-infected by Mycoplasma hyorhinis. CONCLUSIONS: Mycoplasma hyorhinis infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression. Possible explanations are (i) the direct involvement of Mycoplasma on CD133 expression or (ii) the selective pressure on a subpopulation of cells characterized by constitutive CD133 expression.In keeping with United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines, the present data indicate the mandatory prerequisite, for investigators involved in human colon cancer research area, of employing mycoplasma-free cell lines in order to avoid the production of non-reproducible or even false data.

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies.

BACKGROUND: Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. RESULTS: In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). CONCLUSION: Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.

Diagnostic strategies to investigate cerebrospinal fluid involvement in haematological malignancies.

Central nervous system (CNS) involvement is a fatal complication of certain haematological malignancies with an incidence as high as 25% in specific leukaemia/lymphoma subtypes. It is often accompanied by 'occult' cerebrospinal fluid (CSF) involvement at diagnosis, which is frequently missed by conventional cytology examination. Unfortunately, a diagnostic gold standard is yet unavailable since CSF morphology may be negative for malignant cells in up to 45% of patients with suspected meningeal involvement. New technologies such as flow cytometry, molecular genetics and newer biomarkers may improve sensitivity and specificity facilitating the diagnosis of CNS involvement as well as effective prophylaxis and successful treatment.

Cytometric profiling of CD133+ cells in human colon carcinoma cell lines identifies a common core phenotype and cell type-specific mosaics.

In colorectal cancer, CD133+ cells from fresh biopsies proved to be more tumorigenic than their CD133- counterparts. Nevertheless, the function of CD133 protein in tumorigenic cells seems only marginal. Moreover, CD133 expression alone is insufficient to isolate true cancer stem cells, since only 1 out of 262 CD133+ cells actually displays stem-cell capacity. Thus, new markers for colorectal cancer stem cells are needed. Here, we show the extensive characterization of CD133+ cells in 5 different colon carcinoma continuous cell lines (HT29, HCT116, Caco2, GEO and LS174T), each representing a different maturation level of colorectal cancer cells. Markers associated with stemness, tumorigenesis and metastatic potential were selected. We identified 6 molecules consistently present on CD133+ cells: CD9, CD29, CD49b, CD59, CD151, and CD326. By contrast, CD24, CD26, CD54, CD66c, CD81, CD90, CD99, CD112, CD164, CD166, and CD200 showed a discontinuous behavior, which led us to identify cell type-specific surface antigen mosaics. Finally, some antigens, e.g. CD227, indicated the possibility of classifying the CD133+ cells into 2 subsets likely exhibiting specific features. This study reports, for the first time, an extended characterization of the CD133+ cells in colon carcinoma cell lines and provides a "dictionary" of antigens to be used in colorectal cancer research.

High aminopeptidase N/CD13 levels characterize human amniotic mesenchymal stem cells and drive their increased adipogenic potential in obese women.

Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P = 0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2 = 0.84, P < 0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P = 0.05 and P = 0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P < 0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.

Combined CD133/CD44 expression as a prognostic indicator of disease-free survival in patients with colorectal cancer.

HYPOTHESIS: Because of some inconsistencies in the traditional model of human colorectal carcinogenesis, the cancer stem cell (CSC) model was recently proposed, in which tumor results from neoplastic transformation of stem cells, which become CSCs. Identification of CSCs by expression of surface antigens remains a critical issue because no biomarker has been shown to be completely reliable. CD133 and CD44 are commonly used as CSC markers, and correlation of their expression with colorectal cancer (CRC) clinicopathological features and outcomes may be useful. DESIGN: Pilot study. SETTING: University hospital. PATIENTS: Thirty-six consecutive patients with CRC. CD133 and CD44 expression (alone or combined) was determined in nontumor cells and in tumor cells by flow cytometry, which identified viable cells only. MAIN OUTCOME MEASURES: Correlation of CD133 and CD44 expression with each other, with other prognostic indicators, and with disease-free survival. RESULTS: CD133 and CD44 expression was significantly higher in tumor cells than in nontumor cells, and expression of one did not necessarily correlate with expression of the other. CD133 or CD44 expression alone was variable, while combined CD133/CD44 expression identified a small subset of cells positive for CRC. CD133 or CD44 overexpression was not associated with CRC recurrence; only high frequencies of CD133(+)/CD44(+) cells were a strong indicator of worse disease-free survival and an independent risk factor for CRC recurrence. CONCLUSION: Evaluation of combined CD133/CD44 expression could be useful to identify putative colorectal CSCs and tumors with a poor prognosis.

Identification and functional characterization of LDLR mutations in familial hypercholesterolemia patients from Southern Italy.

OBJECTIVE: Autosomal dominant hypercholesterolemias are due to defects in the LDL receptor (LDLR) gene, in the apolipoprotein B-100 gene or in the proprotein convertase subtilisin/kexin type 9 gene. The aim of this study was to identify and functionally characterize mutations in the LDLR gene that account for most cases of familial hypercholesterolemia (FH). METHODS: We enrolled 56 unrelated patients from Southern Italy with a clinical diagnosis of FH. The mutation screening was performed by direct sequencing of the promoter and the 18 exons of the LDLR gene and by multiplex ligation-dependent probe amplification (MLPA) analysis to search for large rearrangements. RESULTS AND CONCLUSION: We found 5 new mutations, the causative role of which was demonstrated by functional characterization performed by quantification of fluorescent LDL uptake in EBV-transformed B lymphocytes. These results enlarge the spectrum of FH-causative LDLR mutations. Lastly, screening for large rearrangements is highly recommended for the genetic diagnosis of FH.