Assessment of DNA flow cytometry as a surrogate end point biomarker in a bladder cancer chemoprevention trial

Although conventional cytology represents the most widely performed cytometric analysis of bladder cancer cells, DNA flow cytometry has, over the past decade, been increasingly used to evaluate cell proliferation and DNA ploidy in cells from bladder washings. We have investigated whether DNA flow cytometry and conventional cytology of epithelial cells obtained from bladder washings provide reliable surrogate endpoint biomarkers in clinical chemoprevention trials. We used cytometric and clinical data from a chemoprevention trial of the synthetic retinoid Fenretinide on 99 patients with superficial bladder cancer. A total of 642 bladder washing specimens obtained from the patients at 4 month intervals was analyzed. Intra-individual agreement and correlation of flow cytometric DNA ploidy (diploid vs. aneuploid), DNA Index, Hyper-Diploid-Fraction (proportion of cells with DNA content higher than 2C), and conventional cytologic examination, as assessed by kappa statistics and Spearman's correlation test, were poor from baseline through 24 months. Moreover, no correlation was found between DNA ploidy and cytology at each time point. The same results were obtained when the analyses were stratified by treatment group. In addition, the association between the results of bladder washing (by either DNA flow cytometry or cytology) and concomitant tumor recurrence was significant only for abnormal cytology, while neither biomarker was predictive of tumor recurrence at the subsequent visit. During the time of this study only four patients progressed to muscle-invasive bladder cancer, indicating the "low-risk" features of the patient population. We conclude that DNA flow cytometry and conventional cytology on epithelial cells obtained from bladder washings do not appear to provide suitable surrogate endpoint biomarkers during the early stages of bladder carcinogenesis.     

Effects of Low Confluency,Serum Starvation and Hypoxia on the Side Population of Cancer Cell Lines

The cancer stem cell theory describes a small subset of cancer cells that have the ability to initiate and drive the growth of a tumor. The niche refers to the environmental factors and the surrounding cells within which the tumor develops. The exact relationship between cancer stem cells and the tumor niche is not known. However, using side population analysis by flow cytometry, it is possible to analyze the relationship between environmental stresses and putative cancer stem cells. The side population is a subpopulation of cells that efflux Hoechst 33342 and has been previously shown to be enriched for cancer stem cells. Using this technique, we characterized the response of side population cells to low confluency, serum starvation and hypoxia using three different human cancer cell lines. We found that these stresses, characteristic of the tumor niche enrich the side population of DLD1, SW480 and MCF7 cancer cell lines, thus possibly predisposing the tumor to a more malignant phenotype.     

c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

Background

Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells.

Methodology/Principal Findings

Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G₀/G₁ phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice.

Conclusions/Significance

These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.     

MHC-multimers and their application in studies of antiviral immune response

Application of main histocompatibility complex tetrames (MHC-tetramers) for antigen specific T-cells detection and analysis coupled with flow cytometry opened new opportunities for T-cell response analysis. MHC-multimers allow the detection of T-cells against viral, cancer and vaccine antigens with exceptional sensitivity and specificity. This approach has become the "gold standard" for quantative analysis of T-cell immune response. Certain aspects of analysis using MHC-tetramer are examined, and importance of this approach in T-cell response efficacy evaluation in anti-HIV vaccine trials as well as in HIV positive patients are discussed.     

Augmented CD133 expression in distal margin correlates with poor prognosis in colorectal cancer

Pathological assessment of excised tumour and surgical margins in colorectal cancer (CRC) play crucial role in prognosis after surgery. Molecular assessment of margins could be more sensitive and informative than conventional histopathological analysis. Considering this view, we evaluated the distal surgical margins for expression of cancer stem cell (CSC) markers. Cellular and molecular assessment of normal, tumour and distal margin tissues were performed by flow cytometry, real‐time q‐PCR and immuno‐histochemical analysis for CRC patients after tumour excision. CRC patients were evaluated for expression of CSC markers in their normal, tumour and distal tissues. Flow cytometry assay revealed CD133 and CD44 enriched cells in distal margin and tumour compared to normal colorectal tissues, which was further confirmed by immunohistochemistry. Most importantly, immunohistochemistry also revealed the enrichment of CSC markers expression in pathologically negative distal margins. Patients with distal margin enriched for CD133 expression showed an increased recurrence rate and decreased disease‐free survival. This study proposes that although distal margin seems to be tumour free in conventional histopathological analysis, it could harbour cells enriched for CSC markers. Further CD133 could be a promising molecule to be used in molecular pathology for disease prognosis after surgery in CRC patients.     

MiR-17-5p and MKL-1 modulate stem cell characteristics of gastric cancer cells

Effectively targeting cancer stem cells to treat cancer has great therapeutic prospects. However, the effect of microRNA miR-17/MKL-1 on gastric cancer stem cells has not been studied yet. This study preliminarily explored the mechanism of miR-17/MKL-1 in gastric cancer stem cells. Many previous reports have indicated that microRNA and EMT regulated cancer stem cell characteristics, and miR-17 and MKL-1 were involved as a critical gene in migration and invasion in the EMT pathway. Through RT-PCR, Western Blot, flow cytometry, immunofluorescence, sphere formation xenograft tumor assays and drug resistance, the role of miR-17-5p and MKL-1 on promoting stem cell-like properties of gastric cancer were verified in vivo and vitro. Next, MKL-1 targets CD44, EpCAM, and miR -17-5p promoter verified by luciferase assay and ChIP. Besides, the TCGA database analysis found that both miR-17-5p and MKL-1 increased in gastric cancer, and the prognostic survival of the MKL-1 high expression group was reduced. It is found that MKL-1 promotes expression by targeting miR-17, CD44 and EpCAM promoters. Besides, the TCGA database analysis found that both miR-17-5p and MKL-1 increased in gastric cancer, and the prognostic survival of the MKL-1 high expression group was reduced. These findings reveal new regulatory signaling pathways for gastric cancer stem cells, thus it give new insights on potential early diagnosis and/or molecular therapy for gastric cancer.     

溶瘤腺病毒ZD55对类肝癌干细胞的抑制效应

溶瘤腺病毒能够靶向和杀死癌症干细胞,被认为是一种很有前景的抗癌药物.已有研究表明,溶瘤腺病毒ZD55能够靶向肝癌,并且表现出明显的细胞毒性效应.然而,其对肝癌干细胞是否具有同样地杀伤效力仍需进一步探讨.利用悬浮培养富集类肝癌干细胞,并验证其肝癌干细胞的特征.进一步通过MTT、结晶紫染色、Hoechst染色、Western blot和流式细胞术等检测ZD55对类肝癌干细胞的细胞存活率、凋亡诱导和病理效应等.结果发现,悬浮培养的类肝癌干细胞具有自我更新和分化能力、高表达干细胞相关转录因子(如NANOG和OCT4)、处于静息状态和具有耐药性等特性,溶瘤腺病毒处理后表现出明显的细胞毒性效应和杀伤特性,类肝癌干细胞的最低生存率仅为26.7%.ZD55能够非常明显地诱导类肝癌干细胞凋亡,其凋亡率最高达到60%.因此,ZD55可能会成为靶向肝癌干细胞的一种很有前景的治疗药物,对肝癌的临床治疗具有一定的应用价值.     

Nicotine increases cancer stem cell population in MCF-7 cells

Epidemiological studies have suggested that cigarette smoking is related to increased breast cancer risk. Nicotine is most likely related to the risk in cigarette smoking. However, the mechanisms by which nicotine promotes cancer development are not fully understood. It has recently been suggested that development of breast cancer are originated from cancer stem cells, which are a minor population of breast cancer. In the present study, we investigated the effects of nicotine on the population of cancer stem cells in MCF-7 human breast cancer cells, using flow cytometry with a cancer stem cell marker aldehyde dehydrogenase (ALDH). We found that nicotine increased ALDH-positive cell population in a dose-dependent manner. We further demonstrated that a PKC-Notch pathway is involved in the effect of nicotine. In addition, the effect of nicotine was blocked by treatment with the α7 subunit-selective antagonist of nicotinic acetylcholine receptors (nAChR) α-Bungarotoxin. These data suggest that nicotine increases the stem cell population via α7-nAChR and the PKC-Notch dependent pathway in MCF-7 cells. These findings reveal a relationship between nicotine and the cancer stem cells in human breast cancer.     

Detection of CD133-marked cancer stem cells by surface plasmon resonance: Its application in leukemia patients

Here, we reported the development of a label-free and real-time surface plasmon resonance (SPR) based biosensor for cancer stem cells (CSCs) detection using cell surface biomarker; CD133. The fabricated biosensor was used for detection of this marker in some acute myeloid leukemia (AML) patients and the results were compared with those obtained from flow cytometry (FC) method. CD133 antibody was immobilized on the gold chip surface via EDC/NHS coupling method and binding of the candidate cells to the modified gold sensor surface was monitored after isolation of mononuclear cells from bone marrow of the patients. The method was validated in terms of various parameters such as CD133- antibody concentration and cell density. The CD133-marked cells were investigated in seven AML patients. All SPR results were compared with those obtained from FC method. A very good correlation (R² = 0.96) was obtained between SPR and FC responses related to CD133-marked cells densities. In conclusion, in this study, a label-free and real-time SPR cytometry method was developed to detect CD133 and it was successfully applied to follow this cancer stem cell biomarker in AML patients.     

Photoacoustic flow cytometry

Conventional flow cytometry using scattering and fluorescent detection methods has been a fundamental tool of biological discoveries for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents the long-term study of cells in their native environment. Here, we summarize recent advances of new generation flow cytometry for in vivo noninvasive label-free or targeted detection of cells in blood, lymph, bone, cerebral and plant vasculatures using photoacoustic (PA) detection techniques, multispectral high-pulse-repetition-rate lasers, tunable ultrasharp (up to 0.8nm) rainbow plasmonic nanoprobes, positive and negative PA contrasts, in vivo magnetic enrichment, time-of-flight cell velocity measurement, PA spectral analysis, and integration of PA, photothermal (PT), fluorescent, and Raman methods. Unique applications of this tool are reviewed with a focus on ultrasensitive detection of normal blood cells at different functional states (e.g., apoptotic and necrotic) and rare abnormal cells including circulating tumor cells (CTCs), cancer stem cells, pathogens, clots, sickle cells as well as pharmokinetics of nanoparticles, dyes, microbubbles and drug nanocarriers. Using this tool we discovered that palpation, biopsy, or surgery can enhance CTC release from primary tumors, increasing the risk of metastasis. The novel fluctuation flow cytometry provided the opportunity for the dynamic study of blood rheology including red blood cell aggregation and clot formation in different medical conditions (e.g., blood disorders, cancer, or surgery). Theranostics, as a combination of PA diagnosis and PT nanobubble-amplified multiplex therapy, was used for eradication of CTCs, purging of infected blood, and thrombolysis of clots using PA guidance to control therapy efficiency. In vivo flow cytometry using a portable fiber-based devices can provide a breakthrough platform for early diagnosis of cancer, infection and cardiovascular disorders with a potential to inhibit, if not prevent, metastasis, sepsis, and strokes or heart attack by well-timed personalized therapy.     

Critical appraisal of the side population assay in stem cell and cancer stem cell research

The "Side Population" (SP) discrimination assay is a flow cytometry method used to detect stem cells based on the dye efflux properties of ABC transporters. We discuss the SP assay and its applications in stem cell?biology, with an emphasis on the technical challenges related to sample preparation, data acquisition, analysis, and interpretation. We highlight the value of multicolor phenotyping, the impact of DNA ploidy, and the importance of distinguishing graft versus host cells for an appropriate SP discrimination. To improve the consistency and reliability of data between laboratories, we propose a set of recommendations for SP assay data reporting.     

The relationship between flow-cytometric and immunohistochemically detected c-erbB-2 expression,grade and DNA ploidy in breast cancer

Quantification of c-erb B-2 and its relationship with other prognostic markers using flow cytometry has been examined. In this study a level for c-erb B-2 expression above which tumours are classified as positive by flow cytometry has been determined by employment of positive cut-off threshold levels. c-erb B-2 expression by both flow cytometry and immunohistochemistry was studied using the monoclonal antibody NCL-CBII. The relationship of c-erb B-2 quantification by flow cytometry was then compared with ploidy, axillary node status, tumour size and grade. Increased c-erb B-2 expression was seen using flow cytometry. Correlation between immunohistochemistry and flow-cytometry methods just failed to reach significance (P=0.06). Immunohistochemistry revealed a significant relationship between c-erb B-2 expression and aneuploidy (P=0.04). Cytokeratinpositive cells from 110 samples obtained from patients with breast cancer were assayed for DNA content and c-erb B-2 expression by flow cytometry. No correlation was seen between these parameters upon application of Mann Whitney analysis. However, examination of fluorescence thresholds showed a positive correlation between grade and c-erb B-2 expression at a level of more than 3200 molecules (P0.03). At the level of 3600 molecules significance was increased (P=0.004). These levels equated with between 15% and 19% of the samples being classified as c-erb B-2 positive. Application of these cut-off points showed no correlation between c-erb B-2 expression and ploidy, tumour size or axillary node status. Comparison of ploidy and grade showed a significant association (P=0.0015), increased grade correlating with aneuploidy.     

DNA flow cytometry in sperm cells from unilaterally orchiectomized patients with testicular cancer before further treatment

Light microscopic sperm cell analysis and DNA flow cytometry in the seminal fluid were done in 85 testicular cancer patients after orchiectomy before further treatment. The results were compared with those from 26 healthy age-matched males (control group). A computer-based method for analysis of the DNA histograms was developed for evaluation of the percentage of sperm cells within the sub-haploid, haploid (1c), and diploid (2c) and greater than 2c levels. Compared with the control group, testicular cancer patients had a reduced sperm cell density and sperm cell motility. The mobility grade was also significantly reduced as compared with healthy males. In addition, the number of condensed haploid sperm cells (within the subhaploid level) was decreased in testicular cancer patients, whereas the percentages of noncondensed haploid (1c), diploid, and greater than 2c cells were increased. Most of the DNA flow cytometric parameters were significantly correlated with sperm cell density. DNA flow cytometry in human seminal fluid offers a possible means of assessing spermatogenesis, thus providing an objective method for studying fertility disturbances, for example, in cancer patients before and after treatment.     

Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions

Background

The tumor-initiating capacity of many cancers is considered to reside in a small subpopulation of cells (cancer stem cells). We have previously shown that rare prostate epithelial cells with a CD133⁺/α₂β₁ ^hi phenotype have the properties of prostate cancer stem cells. We have compared gene expression in these cells relative to their normal and differentiated (CD133^-/α₂β₁ ^low) counterparts, resulting in an informative cancer stem cell gene-expression signature.

Results

Cell cultures were generated from specimens of human prostate cancers (n = 12) and non-malignant control tissues (n = 7). Affymetrix gene-expression arrays were used to analyze total cell RNA from sorted cell populations, and expression changes were selectively validated by quantitative RT-PCR, flow cytometry and immunocytochemistry. Differential expression of multiple genes associated with inflammation, cellular adhesion, and metastasis was observed. Functional studies, using an inhibitor of nuclear factor κB (NF-κB), revealed preferential targeting of the cancer stem cell and progenitor population for apoptosis whilst sparing normal stem cells. NF-κB is a major factor controlling the ability of tumor cells to resist apoptosis and provides an attractive target for new chemopreventative and chemotherapeutic approaches.

Conclusion

We describe an expression signature of 581 genes whose levels are significantly different in prostate cancer stem cells. Functional annotation of this signature identified the JAK-STAT pathway and focal adhesion signaling as key processes in the biology of cancer stem cells.     

Down-regulation of zeta chain and zeta-associated protein 70 (Zap 70) expression in circulating T lymphocytes in laryngeal squamous cell carcinoma

OBJECTIVE: To evaluate zeta chain and Zap 70 expression in T lymphocytes of patients with laryngeal cancer in relation to surgical treatment. STUDY DESIGN: This study investigated, by dual-color flow cytometry, zeta chain and Zap 70 expression in the circulating T lymphocytes of 13 patients with laryngeal cancer patients before and after surgical treatment. RESULTS: Patients exhibited a significant lower expression of both zeta chain and Zap 70 compared to healthy normal controls; no statistical differences were observed after surgical treatment. CONCLUSION: The results of this investigation seem to indicate that both the zeta chain and the Zap 70 expression in circulating T lymphocytes are down-regulated in patients with laryngeal cancer and that these changes do not immediately return to normal after surgery. Flow cytometry analysis may represent an easy-to-use procedure for monitoring the immune status of patients with laryngeal cancer.     

Cytokinetic effects of cisplatin on cisplatin-resistant ovarian cancer cell lines]

Cytokinetic effects of cisplatin on human ovarian cancer cell lines with natural cisplatin-resistance was examined by means of flow cytometry. These ovarian cancer cell lines derived from patients with clear cell carcinoma and serous cystadenocarcinoma were established and designated "KK" and "MH", respectively. Both KK and MH cells have shown resistance to cisplatin and IC50 of them were 0.95 microM and 3.28 microM, respectively. Cisplatin inhibited cell cycle progression at G2 +M phase up to IC50 of each cell from the analysis of cell cycle. Similar results had been obtained in the case of "KF" cell which was sensitive to cisplatin. Further studies of these cells should be performed to elucidate the mechanism of cisplatin resistance.