Deformability Measurement of Single-Cells at High-Throughput With Imaging Flow Cytometry

Disease conditions like malaria, sickle cell anemia, diabetes mellitus, cancer, etc., are known to significantly alter the deformability of certain types of cells (red blood cells, white blood cells, circulating tumor cells, etc.). To determine the cellular deformability, techniques like micropipette aspiration, atomic force microscopy, optical tweezers, quantitative phase imaging have been developed. Many of these techniques have an advantage of determining the single cell deformability with ultrahigh precision. However, the suitability of these techniques for the realization of a deformability based diagnostic tool is questionable as they are expensive and extremely slow to operate on a huge population of cells. In this paper, we propose a technique for high-throughput (800 cells/s) determination of cellular deformability on a single cell basis. This technique involves capturing the image(s) of cells in flow that have undergone deformation under the influence of shear gradient generated by the fluid flowing through the microfluidic channels. Deformability indices of these cells can be computed by performing morphological operations on these images. We demonstrate the applicability of this technique for examining the deformability index on healthy, diabetic, and sphered red blood cells. We believe that this technique has a strong role to play in the realization of a potential tool that uses deformability as one of the important criteria in disease diagnosis.

Differential activity of GSK-3 isoforms regulates NF-kappa B and TRAIL- or TNF alpha induced apoptosis in pancreatic cancer cells

While TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in neoplastic cells, many tumors, including pancreatic ductal adenocarcinoma (PDA), display intrinsic resistance, highlighting the need for TRAIL-sensitizing agents. Here we report that TRAIL-induced apoptosis in PDA cell lines is enhanced by pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) or by shRNA-mediated depletion of either GSK-3 alpha or GSK-3 beta. In contrast, depletion of GSK-3 beta, but not GSK-3 alpha, sensitized PDA cell lines to TNF alpha-induced cell death. Further experiments demonstrated that TNF alpha-stimulated I kappa B alpha phosphorylation and degradation as well as p65 nuclear translocation were normal in GSK-3 beta-deficient MEFs. Nonetheless, inhibition of GSK-3 beta function in MEFs or PDA cell lines impaired the expression of the NF-kappa B target genes Bcl-xL and cIAP2, but not I kappa B alpha. Significantly, the expression of Bcl-xL and cIAP2 could be reestablished by expression of GSK-3 beta targeted to the nucleus but not GSK-3 beta targeted to the cytoplasm, suggesting that GSK-3 beta regulates NF-kappa B function within the nucleus. Consistent with this notion, chromatin immunoprecipitation demonstrated that GSK-3 inhibition resulted in either decreased p65 binding to the promoter of BIR3, which encodes cIAP2, or increased p50 binding as well as recruitment of SIRT1 and HDAC3 to the promoter of BCL2L1, which encodes Bcl-xL. Importantly, depletion of Bcl-xL but not cIAP2, mimicked the sensitizing effect of GSK-3 inhibition on TRAIL-induced apoptosis, whereas Bcl-xL overexpression ameliorated the sensitization by GSK-3 inhibition. These results not only suggest that GSK-3 beta overexpression and nuclear localization contribute to TNF alpha and TRAIL resistance via anti-apoptotic NF-kappa B genes such as Bcl-xL, but also provide a rationale for further exploration of GSK-3 inhibitors combined with TRAIL for the treatment of PDA.

Stem cells, vascular smooth muscle cells and atherosclerosis

Stem cells have the ability to differentiate into a variety of cells to replace dead cells or to repair tissue. Recently, accumulating evidence indicates that mechanical forces, cytokines and other factors can influence stem cell differentiation into vascular smooth muscle cells (SMCs). In developmental process, SMCs originate from several sources, which show a great heterogenicity in different vessel walls. In adult vessels, SMCs display a less proliferative nature, but are altered in response to risk factors for atherosclerosis. Traditional view on SMC origins in atherosclerotic lesions is challenged by the recent findings that stem cells and smooth muscle progenitors contribute to the development of atherosclerotic lesions. Vascular progenitor cells circulating in human blood and the presence of adventitia in animals are recent discoveries, but the source of these cells is still unknown. The present review gives an update on the progress of stem cell and SMC research in atherosclerosis, and discusses possible mechanisms of stem/progenitor cell differentiation that contribute to the disease process.

Induction of signalling in non-erythroid cells by pharmacological levels of erythropoietin

Erythropoiesis is maintained by the hormone erythropoietin (Epo) binding to its cognate receptor (EpoR) on erythroid progenitor cells. The Epo-EpoR interaction initiates a signal transduction process that regulates the survival, growth and differentiation of these cells. Originally perceived as highly lineage-restricted, Epo is now recognised to have pleiotropic effects extending beyond the maintenance of red cell mass. Functional interactions between Epo and EpoR have been demonstrated in numerous cells and tissues. EpoR expression on neoplastic cells leads to concern that recombinant human erythropoietin, used to treat anaemia in cancer patients, may augment tumour growth. Here we demonstrate that EPO, at pharmacological concentrations, can activate three major signalling cascades, viz. the Jak2/STAT5, Ras/ERK and PI3K/Akt pathways in non-small cell lung carcinoma (NSCLC) cell lines. EpoR synthesis is normally under the control of GATA-1, but NSCLC cells exhibit decreased GATA-1 levels compared to GATA-2, -3 and -6, suggesting that GATA-1 is not essential for EpoR production. The increased Epo-induced signalling was not associated with a growth advantage for the NSCLC cells.

The role of immune-related myeloid cells in angiogenesis

Macrophage function is not restricted to the innate and adaptive immune responses, but also includes host defence, wound healing, angiogenesis and homeostatic processes. Within the spectrum of macrophage activation there are two extremes: M1 classically activated macrophages which have a pro-inflammatory phenotype, and M2 alternatively activated macrophages which are pro-angiogenic and anti-inflammatory. An important property of macrophages is their plasticity to switch from one phenotype to the other and they can be defined in their polarisation state at any point between the two extremes. In order to determine what stage of activation macrophages are in, it is essential to profile various phenotypic markers for their identification. This review describes the angiogenic role for myeloid cells: circulating monocytes, Tie-2 expressing monocytes (TEMs), myeloid-derived suppressor cells (MDSCs), tumour associated macrophages (TAMs), and neutrophils. Each cell type is discussed by phenotype, roles within angiogenesis and possible targets as a cell therapy. In addition, we also refer to our own research on myeloid angiogenic cells (MACs), outlining their ability to induce angiogenesis and their similarities to alternatively activated M2 macrophages. MACs significantly contribute to vascular repair through paracrine mechanisms as they lack the capacity to differentiate into endothelial cells. Since MACs also retain plasticity, phenotypic changes can occur according to disease states and the surrounding microenvironment. This pro-angiogenic potential of MACs could be harnessed as a novel cellular therapy for the treatment of ischaemic diseases, such as diabetic retinopathy, hind limb ischaemia and myocardial infarction; however, caution needs to be taken when MACs are delivered into an inflammatory milieu.

Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38±9 and 67±8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

High sensitivity analysis of BRAF mutations in neoplastic and non-neoplastic thyroid lesions

The clonal distribution of BRAFV600E in papillary thyroid carcinoma (PTC) has been recently debated. No information is currently available about precursor lesions of PTCs. My first aim was to establish whether the BRAFV600E mutation occurs as a subclonal event in PTCs. My second aim was to screen BRAF mutations in histologically benign tissue of cases with BRAFV600E or BRAFwt PTCs in order to identify putative precursor lesions of PTCs. Highly sensitive semi-quantitative methods were used: Allele Specific LNA quantitative PCR (ASLNAqPCR) and 454 Next-Generation Sequencing (NGS). For the first aim 155 consecutive formalin-fixed and paraffin-embedded (FFPE) specimens of PTCs were analyzed. The percentage of mutated cells obtained was normalized to the estimated number of neoplastic cells. Three groups of tumors were identified: a first had a percentage of BRAF mutated neoplastic cells > 80%; a second group showed a number of BRAF mutated neoplastic cells < 30%; a third group had a distribution of BRAFV600E between 30-80%. The large presence of BRAFV600E mutated neoplastic cell sub-populations suggests that BRAFV600E may be acquired early during tumorigenesis: therefore, BRAFV600E can be heterogeneously distributed in PTC. For the second aim, two groups were studied: one consisted of 20 cases with BRAFV600E mutated PTC, the other of 9 BRAFwt PTCs. Seventy-five and 23 histologically benign FFPE thyroid specimens were analyzed from the BRAFV600E mutated and BRAFwt PTC groups, respectively. The screening of BRAF mutations identified BRAFV600E in “atypical” cell foci from both groups of patients. “Unusual” BRAF substitutions were observed in histologically benign thyroid associated with BRAFV600E PTCs. These mutations were very uncommon in the group with BRAFwt PTCs and in BRAFV600E PTCs. Therefore, lesions carrying BRAF mutations may represent “abortive” attempts at cancer development: only BRAFV600E boosts neoplastic transformation to PTC. BRAFV600E mutated “atypical foci” may represent precursor lesions of BRAFV600E mutated PTCs.

ELUCIDATING THE ROLE OF CD44 EXPRESSION ON MESENCHYMAL STEM CELLS WITHIN THE TUMOR MICROENVIRONMENT

The tumor microenvironment is comprised of a vast array of heterogeneous cells including both normal and neoplastic cells. The tumor stroma recruitment process has been exploited for an effective gene delivery technique using bone marrow derived MSC. Targeted migration of the MSC toward the tumor microenvironment, while successful, is not yet fully understood. This study was designed to assess the role of CD44 in the migration of MSC toward the tumor microenvironment and to determine the implications of CD44-deficient MSC within the tumor stroma. Inhibition of MSC migration was evaluated through a variety of methods in vitro and in vivo including CD44 receptor knockdown, CD44 antagonists, CD44 neutralizing antibodies and small molecule inhibitor of matrix metalloproteinases. Blocking CD44 signaling through MMP inhibition was characterized by lack of intracellular domain cleavage and lead to the decrease in Twist gene expression. A functional relationship between CD44 and Twist expression was confirmed by chromatin immunoprecipitation. Next, a series of murine tumor models were used to examine the role of CD44 deficient stroma within the tumor microenvironment. Labeled transgenic CD44 knockout (KO) MSC or wild type (WT) C57/B6 MSC were used to analyze the stromal incorporation within murine breast carcinomas (EO771 and 4T1). Subsequent tumors were analyzed for vessel formation (CD31), and the presence of tumor associated fibroblast (TAF) markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and fibroblast specific protein (FSP). The tumors with CD44KO MSC cells had less vessel formation than the tumors with WT MSC. The lack of fibroblastic TAF population as defined by FAP/FSP expression by the CD44KO MSC admixed tumors suggest that the bone marrow derived population of MSC were unable to contribute to the fibroblastic stromal population. Subsequently, a bone marrow transplantation experiment confirmed the endogenous migratory deficiencies of the CD44KO bone marrow derived stromal cells toward the tumor microenvironment in vivo. WT mice with CD44KO bone marrow had less CD44KOderived tumor stroma compared to mice with WT bone marrow. These results indicate that CD44 is crucial to stromal cell migration and incorporation to the tumor microenvironment as TAF.

Mechanism of damage sensing and cell killing following the inhibition of nucleotide excision repair by fludarabine in quiescent cells

Inhibition of DNA repair by the nucleoside of fludarabine (F-ara-A) induces toxicity in quiescent human cells. The sensing and signaling mechanisms following DNA repair inhibition by F-ara-A are unknown. The central hypothesis of this project was that the mechanistic interaction of a DNA repair initiating agent and a nucleoside analog initiates an apoptotic signal in quiescent cells. The purpose of this research was to identify the sensing and signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A. Lymphocytes were treated with F-ara-A, to accumulate the active triphosphate metabolite and subsequently DNA repair was activated by UV irradiation. Pre-incubation of lymphocytes with 3 μM F-ara-A inhibited DNA repair initiated by 2 J/m2 UV and induced greater than additive apoptosis after 24 h. Blocking the incorporation of F-ara-A nucleotide into repairing DNA using 30 μM aphidicolin considerably lowered the apoptotic response. ^ Wild-type quiescent cells showed a significant loss in viability than did cells lacking functional sensor kinase DNA-PKcs or p53 as measured by colony formation assays. The functional status of ATM did not appear to affect the apoptotic outcome. Immunoprecipitation studies showed an interaction between the catalytic sub-unit of DNA-PK and p53 following DNA repair inhibition. Confocal fluorescence microscopy studies have indicated the localization pattern of p53, DNA-PK and γ-H2AX in the nucleus following DNA damage. Foci formation by γ-H2AX was seen as an early event that is followed by interaction with DNA-PKcs. p53 serine-15 phosphorylation and accumulation were detected 2 h after treatment. Fas/Fas ligand expression increased significantly after repair inhibition and was dependent on the functional status of p53. Blocking the interaction between Fas and Fas ligand by neutralizing antibodies significantly rescued the apoptotic fraction of cells. ^ Collectively, these results suggest that incorporation of the nucleoside analog into repair patches is critical for cytotoxicity and that the DNA damage, while being sensed by DNA-PK, may induce apoptosis by a p53-mediated signaling mechanism. Based on the results, a model is proposed for the sensing of F-ara-A-induced DNA damage that includes γ-H2AX, DNA-PKcs, and p53. Targeting the cellular DNA repair mechanism can be a potential means of producing cytotoxicity in a quiescent population of neoplastic cells. These results also provide mechanistic support for the success of nucleoside analogs with cyclophosphamide or other agents that initiate excision repair processes, in the clinic. ^

Telomere dysfunction alters the chemotherapeutic profile of transformed cells

Telomerase inhibition has been touted as a novel cancer-selective therapeutic goal based on the observation of high telomerase levels in most cancers and the importance of telomere maintenance in long-term cellular growth and survival. Here, the impact of telomere dysfunction on chemotherapeutic responses was assessed in normal and neoplastic cells derived from telomerase RNA null (mTERC−/−) mice. Telomere dysfunction, rather than telomerase per se, was found to be the principal determinant governing chemosensitivity specifically to agents that induced double-stranded DNA breaks (DSB). Enhanced chemosensitivity in telomere dysfunctional cells was linked to therapy-induced fragmentation and multichromosomal fusions, whereas telomerase reconstitution restored genomic integrity and chemoresistance. Loss of p53 function muted the cytotoxic effects of DSB-inducing agents in cells with telomere dysfunction. Together, these results point to the combined use of DSB-inducing agents and telomere maintenance inhibition as an effective anticancer therapeutic approach particularly in cells with intact p53-dependent checkpoint responses.

RhoB is required to mediate apoptosis in neoplastically transformed cells after DNA damage

The effect of neoplastic transformation on the response to genotoxic stress is of significant clinical interest. In this study, we offer genetic evidence that the apoptotic response of neoplastically transformed cells to DNA damage requires RhoB, a member of the Rho family of actin cytoskeletal regulators. Targeted deletion of the rhoB gene did not affect cell cycle arrest in either normal or transformed cells after exposure to doxorubicin or gamma irradiation, but rendered transformed cells resistant to apoptosis. This effect was specific insofar as rhoB deletion did not affect apoptotic susceptibility to agents that do not damage DNA. However, rhoB deletion also affected apoptotic susceptibility to Taxol, an agent that disrupts microtubule dynamics. We have demonstrated that RhoB alteration mediates the proapoptotic and antineoplastic effects of farnesyltransferase inhibitors, and we show here that RhoB alteration is also crucial for farnesyltransferase inhibitors to sensitize neoplastic cells to DNA damage-induced cell death. We found RhoB to be an important determinant of long-term survival in vitro and tumor response in vivo after gamma irradiation. Our findings identify a pivotal role for RhoB in the apoptotic response of neoplastic cells to DNA damage at a novel regulatory point that may involve the actin cytoskeleton.

Differentiation of immortal cells inhibits telomerase activity.

Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

Whole-body protochordate regeneration from totipotent blood cells.

Cell differentiation, tissue formation, and organogenesis are fundamental patterns during the development of multicellular animals from the dividing cells of fertilized eggs. Hence, the complete morphogenesis of any developing organism of the animal kingdom is based on a complex series of interactions that is always associated with the development of a blastula, a one-layered hollow sphere. Here we document an alternative pathway of differentiation, organogenesis, and morphogenesis occurring in an adult protochordate colonial organism. In this system, any minute fragment of peripheral blood vessel containing a limited number of blood cells isolated from Botrylloides, a colonial sea squirt, has the potential to give rise to a fully functional organism possessing all three embryonic layers. Regeneration probably results from a small number of totipotent stem cells circulating in the blood system. The developmental process starts from disorganized, chaotic masses of blood cells. At first an opaque cell mass is formed. Through intensive cell divisions, a hollow, blastula-like structure results, which may produce a whole organism within a short period of a week. This regenerative power of the protochordates may be compared with some of the characteristics associated with the formation of mammalian embryonal carcinomous bodies. It may also serve as an in vivo model system for studying morphogenesis and differentiation by shedding more light on the controversy of the "stem cell" vs. the "dedifferentiation" theories of regeneration and pattern formation.

Immunohistochemical detection of survivin in canine lymphoma

Survivin is a member of the family of proteins known as 'inhibitors of apoptosis proteins'. Survivin has a role in cellular decisions concerning division and survival and is frequently expressed in neoplastic cells. The aim of the present study was to investigate immunohistochemically the expression of survivin in normal canine tissues and in canine lymphoma. A representative range of fetal and adult normal tissues as well as biopsy samples from dogs with lymphoma were assembled in tissue arrays. The lymphomas were classified according to the revised Kiel and to the Revised European American Lymphoma - World Health Organization (REAL-WHO) schemes. Polyclonal and monoclonal antisera cross-reactive with canine survivin identified cytoplasmic expression of the molecule in a broad range of normal canine cells. The same reagents demonstrated cytoplasmic labelling of more than 5% of cells in all 83 lymphoma samples tested with polyclonal antiserum and in 67 of 82 (82%) of samples tested with monoclonal antiserum. Survivin was expressed by a wide range of canine lymphoma subtypes, but the expression of this molecule in normal canine tissues must be considered if novel therapies targeting survivin are applied to the management of canine lymphoma. © 2010 Elsevier Ltd.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.