Single-cell analysis of tumors: Creating new value for molecular biomarker discovery of cancer stem cells and tumor-infiltrating immune cells

Biomarker-driven individualized treatment in oncology has made tremendous progress through technological developments, new therapeutic modalities and a deeper understanding of the molecular biology for tumors, cancer stem cells and tumor-infiltrating immune cells. Recent technical developments have led to the establishment of a variety of cancer-related diagnostic, prognostic and predictive biomarkers. In this regard, different modern OMICs approaches were assessed in order to categorize and classify prognostically different forms of neoplasia. Despite those technical advancements, the extent of molecular heterogeneity at the individual cell level in human tumors remains largely uncharacterized. Each tumor consists of a mixture of heterogeneous cell types. Therefore, it is important to quantify the dynamic cellular variations in order to predict clinical parameters, such as a response to treatment and or potential for disease recurrence. Recently, single-cell based methods have been developed to characterize the heterogeneity in seemingly homogenous cancer cell populations prior to and during treatment. In this review, we highlight the recent advances for single-cell analysis and discuss the challenges and prospects for molecular characterization of cancer cells, cancer stem cells and tumor-infiltrating immune cells.     

Naive T cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP(+) cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP(+) cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 +/- 10%) and ulcerative colitis (66 +/- 1%) expressed CCL21, and many perivascular CD45RA(+) naive T cells were found in these tissues, but not in psoriasis, where CCL21(+) vessels were rare (17 +/- 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.     

Race analysis of Puccinia striiformis f.sp. tritici in Iran

The wheat stripe (yellow) rust is one of the most important diseases in Iran. In this study, 41 races out of 104 isolates in greenhouse were determined from 2008 to 2010. Races 6E6A+, 6E10A+ and 6E0A+ were more common. Races 0E0A+ was less aggressive than races 166E158A+ and 134E158A+ with virulence on 11 known genes. Virulence on plant/s with gene/s Yr1, Yr2, Yr4, Yr6, Yr7, Yr8, Yr9, Yr10, Yr25, Yr27, YrSU, YrSD, YrND, Yr3, Yr2+, Yr6+, Yr9+, Yr7+, YrCV and YrA was detected. The majority of isolates with high frequency (more than 70%) showed virulence on plant/s with Yr2, Yr7, Yr9 and YrA genes. No virulence was detected on plant/s with Yr3, Yr5 and YrSP. In greenhouse test, frequency of virulence to wheat genotypes with Yr1, Yr4, Yr10, YrCV (32+) and YrSD gene was less than 7%. Frequency of virulence to other wheat genotypes was between 8 and 100%.     

Association of T/A polymorphism in miR-1302 binding site in CGA gene with male infertility in Isfahan population

Infertility occurs in 10–15% of couples worldwide and close to half of it is caused by male factors. One of the genes that can affect male infertility is CGA. Polymorphisms in CGA gene may affect gene expression, therefore affecting male infertility by disrupting the regulation of this gene. One of the polymorphisms is the substitution of T with A in the miR-1302 binding site in the 3′ untranslated region of the CGA gene. In this study, we explored this polymorphism in Isfahan population. In this case-control study, by the use of Tetra primer-ARMS–PCR technique, rs6631 has been investigated in 224 infertile men and 196 controls. Infertile men were recruited from Isfahan Fertility and Infertility Center. Analysis of genotype and allele frequencies indicated that the differences between case and control populations were significant for rs6631 because P?=?0.00 which is above the threshold. We found a significant relationship between this polymorphism and male infertility. This study which performed for the first time in Iran suggests that polymorphism in CGA gene can affect male infertility. Also, this polymorphism has high heterozygosity, so it can be used for further studies in different populations.     

Resveratrol treatment reduces expression of MCP-1, IL-6, IL-8 and RANTES in endometriotic stromal cells

Endometriosis is an inflammatory disease affecting reproductive-aged women. Immunologic disturbance, as well as inflammation, have crucial roles in the pathogenesis of endometriosis. In this study, we evaluated the effects of resveratrol treatment on expression of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), IL-8, and regulated upon activation, normal T cell expressed and secreted (RANTES) in endometrial stromal cells from patients with endometriosis compared with non-endometriotic controls. Thirteen eutopic (EuESCs) and nine ectopic (EESCs) endometrial stromal cells from endometriotic patients as well as eleven endometrial stromal cells from non-endometriotic controls (CESCs) were treated with resveratrol (100 μmol/L) or ethanol, and gene and/or protein expression of MCP-1, IL-6, IL-8 and RANTES was examined at 6, 24 and 48 hours following treatment in the cells from all origins. Resveratrol treatment significantly reduced gene and protein expression of MCP-1, IL-6, and IL-8 in EuESCs and EESCs compared with CESCs (P < .05-.001, P < .05-.001 and P < .05-<.01, respectively), and this reduction was more noticeable in EESCs than EuESCs (P < .05-<.001). Besides, resveratrol treatment significantly reduced RANTES protein expression in EESCs in all time intervals (P < .05). Resveratrol treatment significantly reduced the expression of MCP-1, IL-6, IL-8 and RANTES in EESCs.     

Vectorcardiography analysis of the repolarization response to pharmacologically induced autonomic nervous system modulation in healthy subjects

Autonomic nervous system activity is essential for regulation of ventricular repolarization (VR) and plays an important role in several arrhythmogenic conditions. This study in 31 healthy adult subjects (16 men, 15 women) evaluated the VR response to pharmacologically modulated autonomic nervous system activity applying vectorcardiography (VCG) analysis. During continuous VCG recording, 0.01-0.1 μg·kg(-1)·min(-1) isoprenaline (Iso) was infused at an increasing flow rate until three targeted heart rates (HR) were reached. After Iso washout, one intravenous bolus of 0.04 mg/kg atropine was given followed by an intravenous bolus of 0.2 mg/kg propranolol. A 5-min steady-state VCG recording was analyzed for each of the seven phases (including baseline 1 and 2). Furthermore, during the first 4 min following atropine, six periods of 10-s VCG were selected for subanalysis to evaluate the time course of change. The analysis included QRS, QT, and T-peak to T-end intervals, measures of the QRS and T vectors and their relation, as well as T-loop morphology parameters. By increasing HR, Iso infusion decreased HR dependent parameters reflecting total heterogeneity of VR (T area) and action potential morphology (ventricular gradient). In contrast, Iso prolonged QT HR corrected according to Bazett and increased the T-peak to T-end-to-QT ratio to levels observed in arrhythmogenic conditions. HR acceleration after atropine was accompanied by a transient paradoxical QT prolongation and delayed HR adaptation of T area and ventricular gradient. In addition to the expected HR adaptation, the VR response to β-adrenoceptor stimulation with Iso and to muscarinic receptor blockade with atropine thus included alterations previously observed in congenital and acquired long QT syndromes, demonstrating substantial overlap between physiological and pathophysiological electrophysiology.     

Changing the convexity and concavity of nasal cartilages and cartilage grafts with horizontal mattress sutures: part II. Clinical results

Horizontal mattress sutures have previously been shown to remove unwanted bulbosity and convexity of nasal tip cartilages. The purpose of this study was to extend that concept by investigating the universal applicability of the horizontal mattress suture to change and control the curvature (e.g., convexity or concavity) of a wide variety of nasal cartilages and warped cartilage grafts. The horizontal mattress suture was applied to a variety of clinical situations, including nasal tip bulbosity caused by convex lateral crura, collapsed external nasal valves, warped grafts and struts, crooked L-shaped septal struts, and collapsed internal nasal valves. Twenty-nine cases were studied over a period of 10 to 23 months. The horizontal mattress suture proved to be a simple, effective means of achieving satisfactory control of the curvature of various cartilages of the nose (including external valves, internal valves, and septum) and warped cartilage grafts. Curvature control was obtained in all cases where the cartilage was supple. Moreover, the resultant strength was increased above normal. Partial recurrence of the curvature was seen in only two cases. Clinical results indicated that the horizontal mattress suture is universally applicable to a variety of situations in which the curvature of nasal cartilage and cartilage grafts needs to be removed or modified. The mattress suture drastically reduces the need for scoring (with its inherent problems of weakness) and the need for cartilage grafting.     

Investigation of Decitabine Effects on HDAC3 and HDAC7 mRNA Expression in NALM-6 and HL-60 Cancer Cell Lines

Background:Decitabine is a potent anticancer hypomethylating agent and changes the gene expression through the gene''s promoter demethylation and also independently from DNA demethylation. So, the present study was designed to distinguish whether Decitabine, in addition to inhibitory effects on DNA methyltransferase, can change HDAC3 and HDAC7 mRNA expression in NALM-6 and HL-60 cancer cell lines.Methods:HL-60, NALM-6, and normal cells were cultured, and the Decitabine treatment dose was obtained (1 µM) through the MTT assay. Finally, HDAC3 and HDAC7 mRNA expression were measured by Real-Time PCR in HL-60 and NALM-6 cancerous cells before and after treatment. Furthermore, HDAC3 and HDAC7 mRNA expression in untreated HL-60 and NALM-6 cancerous cells were compared to normal cells.Results:Our results revealed that the expression of HDAC3 and HDAC7 in HL-60 and NALM-6 cells increases as compared to normal cells. After treatment of HL-60 and NALM-6 cells with Decitabine, HDAC3, and HDAC7 mRNA expression were decreased significantly.Conclusion:Our data confirmed that the effects of Decitabine are not limited to direct hypomethylation of DNMTs, but it can indirectly affect other epigenetic factors, such as HDACs activity, through converging pathways.Key Words: Decitabine, HDAC3, HDAC7, HL-60, NALM-6