Nestin expression in osteosarcomas and derivation of nestin/CD133 positive osteosarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>Nestin was originally identified as a class VI intermediate filament protein that is expressed in stem cells and progenitor cells in the mammalian CNS during development. This protein is replaced in the adult organism by other intermediate filament proteins; however, nestin may be re-expressed under certain pathological conditions such as ischemia, inflammation, brain injury, and neoplastic transformation. Nestin has been detected in many kinds of tumors, especially in tumors derived from the CNS. Co-expression of nestin and the CD133 surface molecule is considered to be a marker for cancer stem cells in neurogenic tumors. Our work was aimed at a detailed study of nestin expression in osteosarcomas and osteosarcoma-derived cell lines.</p> <p>Methods</p> <p>Using immunodetection methods, we examined nestin in tumor tissue samples from 18 patients with osteosarcomas. We also successfully established permanent cell lines from the tumor tissue of 4 patients and immunodetection of nestin and CD133 was performed on these cell lines.</p> <p>Results</p> <p>Nestin-positive tumor cells were immunohistochemically detected in all of the examined osteosarcomas, but the proportion of these cells that were positively stained as well as the intensity of staining varied. Nestin-positive cells were rarely observed in 2 tumor samples, and the remaining 16 tumor samples showed various nestin expression patterns ranging from very sporadic occurrence to an overwhelming proportion of cells with strong positive staining. Three of the established osteosarcoma cell lines were demonstrated to be nestin-positive, and only one cell line showed no expression of nestin; this finding corresponds with the rare occurrence of nestin-positive cells in the respective tumor sample. Moreover, three of these osteosarcoma cell lines were undoubtedly proven to be Nes+/CD133+.</p> <p>Conclusion</p> <p>Our results represent the first evidence of nestin expression in osteosarcomas and suggest the possible occurrence of cells with a stem-like phenotype in these tumors.</p

Monitoring DNA–Ligand Interactions in Living Human Cells Using NMR Spectroscopy

International audienceStudies on DNA−ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA−ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA−ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands bindin

Monitoring DNA-Ligand Interactions in Living Human Cells Using High-Resolution NMR Spectroscopy

High-resolution studies of DNA–ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this issue, we developed an in-cell NMR-based approach for monitoring DNA–ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of high-resolution NMR data of cells electroporated with pre-formed DNA-ligand complex. The impact of the intracellular environment on the integrity of the complex is assessed on the basis of in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. By using this technique, we studied complexes of model DNA fragments and four ligands, representative of DNA minor-groove binders (netropsin) or ligands binding to DNA pairing defects (naphthalenophanes). We demonstrate that some of the in vitro validated ligands retain their ability to form stable on-target DNA interactions in situ, while other lose this ability due to off-target interactions with genomic DNA as well as cellular metabolic components. Collectively, our data suggest that direct evaluation of behavior of drug-like molecules in the intracellular environment provides important insights for the design and development of DNA-binding ligands with the desired biological action and minimal side effects resulting from off-target binding.</div

Co-Expression of Cancer Stem Cell Markers Corresponds to a Pro-Tumorigenic Expression Profile in Pancreatic Adenocarcinoma

<div><p>Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. Its dismal prognosis is often attributed to the presence of cancer stem cells (CSCs) that have been identified in PDAC using various markers. However, the co-expression of all of these markers has not yet been evaluated. Furthermore, studies that compare the expression levels of CSC markers in PDAC tumor samples and in cell lines derived directly from those tumors are lacking. Here, we analyzed the expression of putative CSC markers—CD24, CD44, epithelial cell adhesion molecule (EpCAM), CD133, and nestin—by immunofluorescence, flow cytometry and quantitative PCR in 3 PDAC-derived cell lines and by immunohistochemistry in 3 corresponding tumor samples. We showed high expression of the examined CSC markers among all of the cell lines and tumor samples, with the exception of CD24 and CD44, which were enriched under <i>in vitro</i> conditions compared with tumor tissues. The proportions of cells positive for the remaining markers were comparable to those detected in the corresponding tumors. Co-expression analysis using flow cytometry revealed that CD24⁺/CD44⁺/EpCAM⁺/CD133⁺ cells represented a significant population of the cells (range, 43 to 72%) among the cell lines. The highest proportion of CD24⁺/CD44⁺/EpCAM⁺/CD133⁺ cells was detected in the cell line derived from the tumor of a patient with the shortest survival. Using gene expression profiling, we further identified the specific pro-tumorigenic expression profile of this cell line compared with the profiles of the other two cell lines. Together, CD24⁺/CD44⁺/EpCAM⁺/CD133⁺ cells are present in PDAC cell lines derived from primary tumors, and their increased proportion corresponds with a pro-tumorigenic gene expression profile.</p></div

Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia

BackgroundWhile achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy.MethodsFirst, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable (IGHV)-unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 IGHV-mutated samples without mutations in the above-mentioned genes. Then, we mimicked the leukemic microenvironment in the primary cells by ‘2S stimulation’ through interleukin-2 and nuclear factor kappa B. Finally, CRISPR/Cas9-generated ATM-knockout and TP53-knockout clones (four and seven, respectively) from CLL-derived cell lines MEC1 and HG3 were used. All these samples were exposed to CAR T cells. In vivo survival study in NSG mice using HG3 wild-type (WT), ATM-knockout or TP53-knockout cells was also performed.ResultsPrimary unstimulated CLL cells were specifically eliminated after &gt;24 hours of coculture with CAR T cells. ‘2S’ stimulated cells showed increased survival when exposed to CAR T cells compared with unstimulated ones, confirming the positive effect of this stimulation on CLL cells’ in vitro fitness. After 96 hours of coculture, there was no difference in survival among the genetic classes. Finally, CAR T cells were specifically activated in vitro in the presence of target knockout cell lines as shown by the production of interferon-γ when compared with control (CTRL) T cells (p=0.0020), but there was no difference in knockout cells’ survival. In vivo, CAR T cells prolonged the survival of mice injected with WT, TP53-knockout and ATM-knockout HG3 tumor cells as compared with CTRL T cells (p=0.0485, 0.0204 and &lt;0.0001, respectively). When compared with ATM-knockout, TP53-knockout disease was associated with an earlier time of onset (p&lt;0.0001), higher tumor burden (p=0.0002) and inefficient T-cell engraftment (p=0.0012).ConclusionsWhile in vitro no differences in survival of CLL cells of various genetic backgrounds were observed, CAR T cells showed a different effectiveness at eradicating tumor cells in vivo depending on the driver mutation. Early disease onset, high-tumor burden and inefficient T-cell engraftment, associated with TP53-knockout tumors in our experimental setting, ultimately led to inferior performance of CAR T cells

Validation of pro-tumorigenic expression profile of P28B cells by qRT-PCR.

<p>Five anti-tumorigenic and five pro-tumorigenic genes were selected based on the microarray data and their expression was validated by qRT-PCR. The graph shows the expression levels of the respective genes in P6B and P34B cells relative to that in P28B cell line, which served as the arbitrary calibrator. The bars represent the mean expression level (RQ value) of three biological replicates; the data are presented in log2 scale. The calculated maximum (RQMax) and minimum (RQMin) expression levels are indicated by error bars. *<i>P</i> < 0.001, indicates significant differences from P28B cell line.</p

qRT-PCR analysis of CSC marker expression.

<p>P6B cell line served as the arbitrary calibrator of the gene expression. The error bars indicate the calculated maximum (RQMax) and minimum (RQMin) expression levels that represent the standard error of the mean expression level (RQ value).</p

Differentially expressed genes in P28B cells grouped by their role in tumorigenesis.

<p>Differentially expressed genes in P28B cells grouped by their role in tumorigenesis.</p

IHC analysis of CSC marker expression in PDAC tumor samples.

<p>IHC analysis of CSC marker expression in PDAC tumor samples.</p