Direct In Vivo Evidence for Tumor Propagation by Glioblastoma Cancer Stem Cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC

Novel role of cPLA2α in membrane and actin dynamics

Actin-directed processes such as membrane ruffling and cell migration are regulated by specific signal transduction pathways that become activated by growth factor receptors. The same signaling pathways that lead to modifications in actin dynamics also activate cPLA2α. Moreover, arachidonic acid, the product of cPLA2α activity, is involved in regulation of actin dynamics. Therefore, it was investigated whether cPLA2α plays a role in actin dynamics, more specifically during growth factor-induced membrane ruffling and cell migration. Upon stimulation of ruffling and cell migration by growth factors, endogenous cPLA2α and its active phosphorylated form were shown to relocate at protrusions of the cell membrane involved in actin and membrane dynamics. Inhibition of cPLA2α activity with specific inhibitors blocked growth factor-induced membrane and actin dynamics, suggesting an important role for cPLA2α in these processes

Comparative Effectiveness of Tumor Response Assessment Methods: Standard of Care Versus Computer-Assisted Response Evaluation

Purpose To compare the effectiveness of metastatic tumor response evaluation with computed tomography using computer-assisted versus manual methods. Materials and Methods In this institutional review board–approved, Health Insurance Portability and Accountability Act–compliant retrospective study, 11 readers from 10 different institutions independently categorized tumor response according to three different therapeutic response criteria by using paired baseline and initial post-therapy computed tomography studies from 20 randomly selected patients with metastatic renal cell carcinoma who were treated with sunitinib as part of a completed phase III multi-institutional study. Images were evaluated with a manual tumor response evaluation method (standard of care) and with computer-assisted response evaluation (CARE) that included stepwise guidance, interactive error identification and correction methods, automated tumor metric extraction, calculations, response categorization, and data and image archiving. A crossover design, patient randomization, and 2-week washout period were used to reduce recall bias. Comparative effectiveness metrics included error rate and mean patient evaluation time. Results The standard-of-care method, on average, was associated with one or more errors in 30.5% (6.1 of 20) of patients, whereas CARE had a 0.0% (0.0 of 20) error rate (P < .001). The most common errors were related to data transfer and arithmetic calculation. In patients with errors, the median number of error types was 1 (range, 1 to 3). Mean patient evaluation time with CARE was twice as fast as the standard-of-care method (6.4 minutes v 13.1 minutes; P < .001). Conclusion CARE reduced errors and time of evaluation, which indicated better overall effectiveness than manual tumor response evaluation methods that are the current standard of care

Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification

Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy. We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet (HFD).Rats were exposed to an HFD or a low-fat diet (LFD). In vivo cardiac function was monitored by echocardiography. Substrate uptake and utilisation were determined in isolated cardiomyocytes.Feeding an HFD for 8 weeks induced left ventricular dilation in the systolic phase and decreased fractional shortening and the ejection fraction. Insulin-stimulated glucose uptake and proline-rich Akt substrate 40 phosphorylation were 41% (p <0.001) and 45% (p <0.05) lower, respectively, in cardiomyocytes from rats on the HFD. However, long-chain fatty acid (LCFA) uptake was 1.4-fold increased (p <0.001) and LCFA esterification into triacylglycerols and phospholipids was increased 1.4- and 1.5-fold, respectively (both p <0.05), in cardiomyocytes from HFD compared with LFD hearts. In the presence of the CD36 inhibitor sulfo-N-succinimidyloleate, LCFA uptake and esterification were similar in LFD and HFD cardiomyocytes. In HFD hearts CD36 was relocated to the sarcolemma, and basal phosphorylation of a mediator of CD36-trafficking, i.e. protein kinase B (PKB/Akt), was increased.Feeding rats an HFD induced cardiac contractile dysfunction, which was accompanied by the relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB/Akt. The permanent presence of CD36 at the sarcolemma resulted in enhanced rates of LCFA uptake and myocardial triacylglycerol accumulation, and may contribute to the development of insulin resistance and diabetic cardiomyopathy

Injection of Pseudomonas aeruginosa Exo Toxins into Host Cells Can Be Modulated by Host Factors at the Level of Translocon Assembly and/or Activity

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation

Augmented Osteolysis in SPARC-Deficient Mice with Bone-Residing Prostate Cancer1 2 3

Prostate cancer preferentially metastasizes to bone, which is rich in structural and matricellular proteins capable of altering prostate cancer progression. This study explores the role of the bone stromal matricellular protein SPARC (osteonectin/BM-40) in the progression of bone metastatic prostate cancer. Quantification of bone destruction analyzed by micro-computed tomography showed augmented osteoclastic resorption, characterized by decreases in several morphometric bone parameters in SPARC knock out (KO) tibiae harboring RM1 murine prostate cancer cells compared with wild type (WT) animals. Tumor progression stimulated osteoclast formation, which was augmented in SPARC KO mice. In vitro differentiation of SPARC KO osteoclasts indicated accelerated progenitor expansion and formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells with increased resorptive capacity, a mechanism resulting in enhanced tumor-induced bone loss in vivo. Whereas altered bone structure due to SPARC KO played a role in increased osteolysis, the enhanced osteolysis was primarily the result of increased resorption by SPARC KO osteoclasts. Our findings indicate that bone stromal SPARC suppresses tumor-induced bone lesion expansion by limiting osteoclast maturation and function

ADAMTS-Like 2 (ADAMTSL2) Is a Secreted Glycoprotein That Is Widely Expressed During Mouse Embryogenesis and Is Regulated During Skeletal Myogenesis

ADAMTS-like 2 (ADAMTSL2), is a secreted protein resembling the ancillary domains of the ADAMTS proteases, but with distinct structural features. It has 7 thrombospondin type-1 repeats (TSRs), but an unusually long spacer module, which in both humans and mice, contains a novel insertion bearing six N-glycosylation sites. The ADAMTSL2 protein expressed in HEK293F and COS-1 cells, is a cell-surface and extracellular matrix binding glycoprotein, with N-linked carbohydrate constituting ∼ 20% by mass. The 4.0 kb Adamtsl2 mRNA is found most abundantly in adult mouse liver, lung and spleen by northern blotting. During mouse embryogenesis, Adamtsl2 was expressed most strongly in the third week of gestation. Adamtsl2 mRNA was detected by in situ hybridization in developing skeletal muscle, liver, bronchial and arterial smooth muscle, skin, intervertebral disc, perichondrium, pancreas and spinal cord. Immunohistochemical localization of ADAMTSL2 protein was similar to mRNA expression. Detection of Adamtsl2 mRNA and protein in developing skeletal myotubes, but not undifferentiated myogenic precursors led us to investigate its regulation during in vitro myogenic differentiation. In C2C12 and 23A2 myogenic cells, but not in 23A2 cells rendered non-myogenic by expression of G12V:H-Ras (9A2 cells), differentiation induced by serum starvation triggered expression of Adamtsl2 mRNA, coordinately with Myog, a marker of muscle differentiation. Furthermore, activation of the key myogenic determinant MyoD in 10T1/2 fibroblasts also triggered expression of Adamtsl2 mRNA. Collectively, the data suggest that induction of Adamtsl2 mRNA is an integral feature of myogenesis. © 2007 Elsevier B.V. All rights reserved

Micro-CT imaging as a method for comparing perfusion in graduated-height and single-height surgical staple lines

Matthew Eschbach,1 Gregory M Sindberg,2 Marisha L Godek,1 Matthew Nagelschmidt,1 Nicholas Paquette,3 Michael Wegener,1 James Alberino,1 Jane Mayotte,1 Amit Vasanji,4 Andrew M Miesse1 1Minimally Invasive Therapies Group, Medtronic, North Haven,&nbsp;CT, USA; 2Minimally Invasive Therapies Group, Medtronic, Plymouth,&nbsp;MN, USA; 3Minimally Invasive Therapies Group, Medtronic, Mansfield,&nbsp;MA, USA; 4Image IQ, Cleveland, OH, USA Background: Wound healing is a goal for advanced technology in the surgical space to benefit clinical outcomes. Surgical staplers are commonly used in a variety of open and minimally invasive abdominal and thoracic procedures. Assessment of wound healing traits, such as perfusion, has been challenging due to technical limitations. A novel technique that utilizes micro-computed tomography methodology to measure perfusion was designed to compare the micro-perfusion of staple lines between commercial stapler reloads that employ different staple height strategies. Materials and methods: Following an Institutional Animal Care and Use Committee-approved protocol, rats were euthanized and immediately heparinized prior to a subtotal gastrectomy with either graduated-height or single-height staples. Rats were then perfused with barium, following which stomachs were removed and immediately fixed in formalin to prevent degradation. Stomachs were then imaged using micro-computed tomography and subsequent analysis was utilized to quantify fluid volume and patent vasculature proximity to staples within the staple line region for each group. Results: Average perfusion volume was significantly higher with graduated-height staples (0.33% &plusmn; 0.18%) compared to single-height staples (0.16% &plusmn; 0.09%, P=0.011). Average vessel-to-staple line distance was not significant but trended lower with graduated-height staples (0.35&plusmn;0.02&nbsp;mm) compared to single-height staples (0.36&plusmn;0.03&nbsp;mm, P=0.18). Discussion: Graduated-height staples had significantly higher perfusion volume than single-height staples, which likely has a downstream benefit on wound healing and clinical outcomes. Conclusion: This study shows a higher perfusion volume around the staple lines using graduated-height staples as compared to single-height staples and this may contribute to better wound healing in patients. Keywords: micro-computed tomography, wound healing, Tri-Staple, image processing, laparoscopic surgery, microvasculatur