Experimental Characterization of MCF-10A Normal Cells Using AFM: Comparison with MCF-7 Cancer Cells

The mechanical properties of single cells have been recently identified as the basis of an emerging approach in medical applications since they are closely related to the biological processes of cells and human health conditions. The problem in hand is how to measure mechanical properties in order to obtain them more accurately and applicably. Some of the cell’s properties such as elasticity module and adhesion have been measured before using various methods; nevertheless, comprehensive tests for two healthy and cancerous cells have not been performed simultaneously. As a Nanoscale device, AFM has been used for some biological cells, however for breast cells, it has been utilized just to measure elasticity module. To provide a more accurate comparison for the healthy and the malignant cancer cells of breast, mechanical properties of MCF-10A cells such as topography, elasticity module, adhesion force, viscoelastic characteristics, bending and axial rigidity were determined and compared to the MCF-7 cells results obtained in previous works. Results revealed that the healthy breast cells are stiffer and less adhesive in comparison with the cancerous ones. Topography images revealed that cancerous cells have bigger radii. These results can help with the diagnosis of malignant cancer cells and even the level of the disease.     

Endothelium-microenvironment interactions in the developing embryo and in the adult

The vascular endothelium is best known for its role in oxygen and nutrient delivery to the various tissues. Growing evidence supports a far more complex role in tissue homeostasis. In particular, reciprocal interactions between endothelial cells and the local microenvironment may regulate organ development and pattern formation. Such interactions appear to be important also in the adult, in normal and pathological conditions.     

Brain monoamines following castration of aggressive muricidal rats

The effect of castration on the levels of brain monoamines and their metabolites has been investigated in rats which became or did not become muricidal following long-term isolation. Fourteen brain areas were explored: olfactory bulbs (OB), olfactory tubercles (OT), septum (Se), striatum (Sr), amygdala (A), thalamus (Th), hypothalamus (Hy), hippocampus (Hi), superior colliculus (SC), inferior colliculus (IC), raphe (Ra), pons-medulla (PM), frontal cortex (FC), temporal cortex (TC) and parietal cortex (PC). Except in the raphe of non muricidal rats and in the striatum of muricidal animals, all other areas examined demonstrate some changes of monoamines neurotransmitter or their metabolites after castration. The strongest changes, always increases, were found in the thalamus. In several brain areas, the changes occurring after castration, differ quantitatively and qualitatively in muricidal and non-muricidal rats.Special issue dedicated to Dr. Claude Baxter.Prof. P. Mandel passed away on October 6th, 1992.     

Long noncoding RNAs expression in gastric cancer

Long noncoding RNAs (lncRNAs) have been involved in the pathogenesis of several human cancers including gastric cancer. In the current study, we selected five lncRNAs namely NEAT1, TUG1, PANDA, UCA1, and GHET1 to assess their expressions in gastric cancer samples compared with adjacent noncancerous tissues (ANCTs) from the same patients. Some previous reports have shown contribution of these lncRNAs in gastric cancer. However, we aimed to explore their associations with patients’ clinicopathological data and their potential as diagnostic biomarkers. Significant associations were found between site of primary tumor and relative expression of all lncRNAs in cancer samples compared with ANCTs. Besides, GHET1 relative expression was associated with lymph node status. The diagnostic power of GHET1 was higher from other lncRNAs. Combination of GHET1, TUG1, UCA1, and PANDA increased the diagnostic power and significance (AUC = 0.8; P < 0.0001). The current study supports participation of lncRNAs in the pathogenesis of gastric cancer and highlights their potential as diagnostic biomarkers.     

Nucleic Acid Template and the Risk of a PCR-Induced HIV-1 Drug Resistance Mutation

Background

The HIV-1 nucleoside RT inhibitor (NRTI)-resistance mutation, K65R confers intermediate to high-level resistance to the NRTIs abacavir, didanosine, emtricitabine, lamivudine, and tenofovir; and low-level resistance to stavudine. Several lines of evidence suggest that K65R is more common in HIV-1 subtype C than subtype B viruses.

Methods and Findings

We performed ultra-deep pyrosequencing (UDPS) and clonal dideoxynucleotide sequencing of plasma virus samples to assess the prevalence of minority K65R variants in subtype B and C viruses from untreated individuals. Although UDPS of plasma samples from 18 subtype C and 27 subtype B viruses showed that a higher proportion of subtype C viruses contain K65R (1.04% vs. 0.25%; p<0.001), limiting dilution clonal sequencing failed to corroborate its presence in two of the samples in which K65R was present in >1.5% of UDPS reads. We therefore performed UDPS on clones and site-directed mutants containing subtype B- and C-specific patterns of silent mutations in the conserved KKK motif encompassing RT codons 64 to 66 and found that subtype-specific nucleotide differences were responsible for increased PCR-induced K65R mutation in subtype C viruses.

Conclusions

This study shows that the RT KKK nucleotide template in subtype C viruses can lead to the spurious detection of K65R by highly sensitive PCR-dependent sequencing techniques. However, the study is also consistent with the subtype C nucleotide template being inherently responsible for increased polymerization-induced K65R mutations in vivo.     

Contrast Agents for Quantitative MicroCT of Lung Tumors in Mice

The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced microCT (μCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially available μCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures, and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. μCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4–ALK mice with crizotinib yielded 65% reduction in tumor size and thus demonstrated the utility of quantitative μCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels and lung tumors. Contrast-enhanced μCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.Abbreviations: μCT, microCT, HU, Hounsfield units, RECIST, response evaluation criteria in solid tumorsLung cancer is the leading cause of cancer death worldwide, and non-small–cell lung cancer is the most common form of lung cancer that is diagnosed.¹⁹ Various animal models have been used to mimic these cancers, understand their biology, and evaluate potential therapeutics.^14,20,27 Traditionally, the method to study the disease in vivo has been to inject human tumor derived cell lines subcutaneously in immunodeficient mice (xenografts) or to orthotopically implant tumors in tissues of interest. Xenografts, despite being of human origin, are not ideal models because tumor cell–stroma interactions cannot be reconstituted in the system. Moreover, the tumor microenvironment has been shown to be essential in predicting cancer cell survival, progression, metastasis, and response to therapy.^14,20,27 To overcome these issues, several investigators have propagated tumors orthotopically by either direct injection of tumor cells into the organ of choice, intravenous injection of tumor cell lines, or implantation of patient-derived tumor biopsy samples into immunodeficient mice. These models simulate the tumor microenvironment and bear a closer resemblance to clinical cancer than do xenografts.^11,27 In the past decade, tremendous progress has been made in development of genetically engineered mouse models that reconstitute facets of human disease in the organ of choice. In these models, the tumors are developed in immunocompetent hosts with intact tumor–stroma interactions, and the tumors can be controlled temporally and spatially.^7,14,25,26Despite the many advantages of genetically engineered and orthotopic models, their use has been limited, primarily due to the heterogeneity and technical difficulties associated with monitoring disease progression.¹⁷ Conventional optical imaging is not widely used with genetically engineered and orthotopic models, because these modalities provide low spatial resolution, limited tissue penetration,^1,17 and rarely accomdate reporter genes like luciferase or fluorescent proteins.³⁰ Nuclear imaging (for example, positron-emission tomography and MRI) has been used in evaluating lung tumor models,^7,13,33 but these modalities are not readily available in all facilities, require specialized laboratories (for example, radionucleotide synthesis), and do not achieve accurate quantification of nodular tumors.³³ X-ray CT has been used in human clinical practice to assess lung tumor nodules to predict likelihood of malignancy and to monitor the response of tumors to treatment.^18,23 Response Evaluation Criteria in Solid Tumors (RECIST) scores based on CT imaging data are used routinely in clinical trials and practice.³² Similarly, high-resolution microCT (μCT) scanners have been used successfully to image lung tumors^{6,15,17,24,25,34} in small animals such as rodents. The investigators in the aforementioned studies took advantage of the natural air–tissue contrast within the thorax to identify tumors. However, this method was unable to distinguish tumor and soft tissue from nearby vascular structures.^25,29 This limitation decreased the accuracy of tumor margin demarcation and tumor volumetric measurements.The goal of our study was to compare 3 commercially available μCT contrast agents (products containing iopamidol, iodinated lipid, and inorganic nanoparticulate) to evaluate and accurately quantify lung tumors in preclinical models. Our results showed that, among those we tested, the nanoparticulate product was the best contrast agent for tumor visualization and quantitation. In addition, we demonstrated the utility of contrast-enhanced μCT in a preclinical evaluation of the efficacy of crizotinib (PF-2341066), a small-molecule multikinase inhibitor,^9,10 in a genetically modified mouse model of lung cancer.