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

<p>Abstract</p> <p>Background</p> <p>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 <it>et al</it>, 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 <it>in vitro </it>induction of erythroid differentiation; iii) detection of ALDH⁺cellular subsets in bone marrow from pure red cell aplasia patients.</p> <p>Results</p> <p>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 CD71^bright, 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).</p> <p>Conclusion</p> <p>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.</p

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

<p>Abstract</p> <p>Background</p> <p><it>Mollicutes </it>contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of <it>Mycoplasma hyorhinis </it>contamination on CD133 expression in human colon cancer cell lines.</p> <p>Methods</p> <p>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 <it>Mycoplasma hyorhinis </it>eradication.</p> <p>Results</p> <p><it>Mycoplasma hyorhinis </it>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 <it>Mycoplasma hyorhinis</it>.</p> <p>Conclusions</p> <p><it>Mycoplasma hyorhinis </it>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.</p> <p>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.</p

Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome

Peer reviewe

TRAP1 regulates stemness through Wnt/β-catenin pathway in human colorectal carcinoma

Colorectal carcinoma (CRC) is a common cause of cancer-related death worldwide. Indeed, treatment failures are triggered by cancer stem cells (CSCs) that give rise to tumor repopulation upon initial remission. Thus, the role of the heat shock protein TRAP1 in stemness was investigated in CRC cell lines and human specimens, based on its involvement in colorectal carcinogenesis, through regulation of apoptosis, protein homeostasis and bioenergetics. Strikingly, co-expression between TRAP1 and stem cell markers was observed in stem cells located at the bottom of intestinal crypts and in CSCs sorted from CRC cell lines. Noteworthy, TRAP1 knockdown reduced the expression of stem cell markers and impaired colony formation, being the CSC phenotype and the anchorage-independent growth conserved in TRAP1-rich cancer cells. Consistently, the gene expression profiling of HCT116 cells showed that TRAP1 silencing results in the loss of the stem-like signature with acquisition of a more-differentiated phenotype and the downregulation of genes encoding for activating ligands and target proteins of Wnt/β-catenin pathway. Mechanistically, TRAP1 maintenance of stemness is mediated by the regulation of Wnt/β-catenin signaling, through the modulation of the expression of frizzled receptor ligands and the control of β-catenin ubiquitination/phosphorylation. Remarkably, TRAP1 is associated with higher expression of β-catenin and several Wnt/β-catenin target genes in human CRCs, thus supporting the relevance of TRAP1 regulation of β-catenin in human pathology. This study is the first demonstration that TRAP1 regulates stemness and Wnt/β-catenin pathway in CRC and provides novel landmarks in cancer biology and therapeutics

Detection of erbB2 copy number variations in plasma of patients with esophageal carcinoma

<p>Abstract</p> <p>Background</p> <p>Mortality is high in patients with esophageal carcinoma as tumors are rarely detected before the disease has progressed to an advanced stage. Here, we sought to isolate cell-free DNA released into the plasma of patients with esophageal carcinoma, to analyze copy number variations of marker genes in the search for early detection of tumor progression.</p> <p>Methods</p> <p>Plasma of 41 patients with esophageal carcinoma was prospectively collected before tumor resection and chemotherapy. Our dataset resulted heterogeneous for clinical data, resembling the characteristics of the tumor. DNA from the plasma was extracted to analyze copy number variations of the <it>erbB2 </it>gene using real-time PCR assays.</p> <p>Results</p> <p>The real-time PCR assays for <it>erbB2 </it>gene showed significant (<it>P </it>= 0.001) copy number variations in the plasma of patients with esophageal carcinoma, as compared to healthy controls with high sensitivity (80%) and specificity (95%). These variations in <it>erbB2 </it>were negatively correlated to the progression free survival of these patients (<it>P </it>= 0.03), and revealed a further risk category stratification of patients with low VEGF expression levels.</p> <p>Conclusion</p> <p>The copy number variation of <it>erbB2 </it>gene from plasma can be used as prognostic marker for early detection of patients at risk of worse clinical outcome in esophageal cancer.</p

Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

Flow cytometry's informative potential has been underestimated for many years because of a lack of adequate instruments, automation, reagents, and know-how to approach, integrate, and also substitute other techniques giving single information per assay. In the last decade, flow cytometers have become capable of performing high-throughput screening and high content analysis, evaluating tens of different samples' features in a single run up to 1536 formats on multiple cell populations. The introduction of imaging flow cytometry has filled the gap between flow cytometry and conventional high content imaging screening, putting flow cytometry at the center of many laboratories, which can now cover with a single instrument the vast majority of needs in research programs. The flow cytometry community is a multidisciplinary and diversified group with many different interests and fields of action. These characteristics have prompted the evolution of the techniques, applications, and instruments that allow the use of complex, sophisticated, and standardized and reliable flow cytometric assays in academic and industrial programs

Isolation and characterization of cancer stem cell subsets by identification of new molecular determinants in colon cancer

The initial aim of our study was to dissect the mosaic of cell populations within the CD133+ putative stem cell compartment in human colon cancer. While addressing this question we built up a panel of 20 monoclonal antibodies chosen on the basis of information provided by selecting in a literature search molecules somewhat involved in the overall malignant behavior of tumor cells. We used an innovative seven-color flow cytometric approach to characterize cells obtained from the mechanic and enzymatic digestion of fresh normal colon and colon cancer tissues. We subdivided our panel into different tubes analyzing two selected markers for each tube and using other fluorescence channels available to keep fixed two putative stem cell markers (CD133 and CD44), a hematopoietic specificity (CD45), an epithelial specific marker (CD326) and a vital dye (Sytox blue). Tumoral population to be analyzed was selected as CD45-/CD326+/Sytox Blue-. This gating was fundamental for the subsequent evaluation of the other markers because of the amount of contaminants composing the cell suspension derived from the digestion of a fresh tissue. These contaminants could easily confuse results leading to ambiguous, misleading and incorrect data. A first analysis of our data revealed a distribution of analyzed markers into three main categories: 1) highly expressed antigens, 2) bimodally expressed antigens and 3) low expressed antigens. A first interesting finding was the observation of a small subset of double positive CD133+/CD44+ cells. The existence of this subset could explain similar results obtained by the three groups which first isolated colorectal cancer stem cells using apparently different approaches (CD133+ or CD44+/CD326+). At the same time we observed this population rapidly appeared in literature studies enforcing the idea that the CD133+/CD44+ population could represent a more purified stem cell subset in human colon cancer. We analyzed the presence of CD133+, CD44+ and CD133+/CD44+ cells in 36 patients correlating these markers to tumor progression and disease free survival. From our analysis CD133 alone seems to be correlated to tumor progression while only the CD133+/CD44+ subset’s presence affected disease free survival in the analyzed patients, suggesting that this cell population was not the main responsible for tumor growth but it was involved in cancer recurrence. From a first analysis of the expression of the analyzed markers another molecule got our attention: CD66c. CD66c is a GPI-linked molecule belonging to the family of CEA antigen known to be involved in carcinogenesis and in colon cancer pathogenesis, however no complete and exhaustive informations are available. From our data, CD66c showed an extremely reproducible behavior and interesting correlations with other cancer related molecules. Its expression correlated with that of all proposed colon cancer stem cell markers (CD133, CD44, CD166, CD24). A correlation was also observed with CD151 (PETA3, involved in the extravasion of cancerous cells), CD227 (MUC1, known as prognostic factor in colon cancer), CD182 (CXCR2, involved in cell migration), CD221 (insulin-like growth factor 1, linked to cell proliferation) and CD55 (Decay accelerating factor, a complement inhibiting molecule). We carefully analyzed the expression of CD66c in normal colon and in colon cancer tissues finding this antigen to be a cancer associate molecule. In fact it was significantly expressed at higher levels in cancer than in normal tissue. We also found a gradient of expression from normal tissues to adenomas and to carcinomas with an increase related to the severity of the lesion. As expected a significantly higher expression was also observed in advanced stages (T3-4 and M1) of colon cancer and in in vitro sphere cultures, which promote the proliferation of stem-like cells. We further analyzed CD66c expression in CD133+ putative stem cell compartment and found a surprisingly sharp distinction between normal and cancer tissues. In normal tissues CD133+ cells are completely CD66c negative while a correlation between the two antigens was observed in cancerous samples. When CD66c was silenced in Caco2 cells, which express high levels of this molecule, their proliferation resulted slowed down while their apoptosis and necrosis appeared increased. Moreover, the in vitro clonogenic and tumorigenic potential of Caco2 cells resulted impaired. Initial hints about the possible use of CD66c as a therapeutic target are suggested by the cCD66c mRNA silencing experiments in Caco2 cells, further studies will be needed to unveil this possibility

Hematopoietic stem/progenitor cells express myoglobin and neuroglobin: adaptation to hypoxia or prevention from oxidative stress?

Oxidative metabolism and redox signaling prove to play a decisional role in controlling adult hematopoietic stem/progenitor cells (HSPCs) biology. However, HSPCs reside in a hypoxic bone marrow microenvironment raising the question of how oxygen metabolism might be ensued. In this study, we provide for the first time novel functional and molecular evidences that human HSPCs express myoglobin (Mb) at level comparable with that of a muscle-derived cell line. Optical spectroscopy and oxymetry enabled to estimate an O2-sensitive heme-containing protein content of approximately 180 ng globin per 10(6) HSPC and a P50 of approximately 3 µM O2. Noticeably, expression of Mb mainly occurs through a HIF-1-induced alternative transcript (Mb-V/Mb-N = 35 ± 15, p < .01). A search for other Mb-related globins unveiled significant expression of neuroglobin (Ngb) but not of cytoglobin. Confocal microscopy immune detection of Mb in HSPCs strikingly revealed nuclear localization in cell subsets expressing high level of CD34 (nuclear/cytoplasmic Mb ratios 1.40 ± 0.02 vs. 0.85 ± 0.05, p < .01) whereas Ngb was homogeneously distributed in all the HSPC population. Dual-color fluorescence flow cytometry indicated that while the Mb content was homogeneously distributed in all the HSPC subsets that of Ngb was twofold higher in more immature HSPC. Moreover, we show that HSPCs exhibit a hypoxic nitrite reductase activity releasing NO consistent with described noncanonical functions of globins. Our finding extends the notion that Mb and Ngb can be expressed in nonmuscle and non-neural contexts, respectively, and is suggestive of a differential role of Mb in HSPC in controlling oxidative metabolism at different stages of commitment

Binding Mode Exploration of B1 Receptor Antagonists’ by the Use of Molecular Dynamics and Docking Simulation—How Different Target Engagement Can Determine Different Biological Effects

The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules&rsquo; mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement