Global Industry Reorganization and Market Concentration : Automobiles, Steel, and Airlines

Glioblastoma multiforme (GBM) is a deadly primary brain malignancy. Glioblastoma stem cells (GSC), which have the ability to self-renew and differentiate into tumor lineages, are believed to cause tumor recurrence due to their resistance to current therapies. A subset of GSCs is marked by cell surface expression of CD133, a glycosylated pentaspan transmembrane protein. The study of CD133-expressing GSCs has been limited by the relative paucity of genetic tools that specifically target them. Here, we present CD133-LV, a lentiviral vector presenting a single chain antibody against CD133 on its envelope, as a vehicle for the selective transduction of CD133-expressing GSCs. We show that CD133-LV selectively transduces CD133+ human GSCs in dose-dependent manner and that transduced cells maintain their stem-like properties. The transduction efficiency of CD133-LV is reduced by an antibody that recognizes the same epitope on CD133 as the viral envelope and by shRNA-mediated knockdown of CD133. Conversely, the rate of transduction by CD133-LV is augmented by overexpression of CD133 in primary human GBM cultures. CD133-LV selectively transduces CD133-expressing cells in intracranial human GBM xenografts in NOD.SCID mice, but spares normal mouse brain tissue, neurons derived from human embryonic stem cells and primary human astrocytes. Our findings indicate that CD133-LV represents a novel tool for the selective genetic manipulation of CD133-expressing GSCs, and can be used to answer important questions about how these cells contribute to tumor biology and therapy resistance

Prognosis after surgery for multiple endocrine neoplasia type 1-related pancreatic neuroendocrine tumors: Functionality matters

Background: Metastasized pancreatic neuroendocrine tumors are the leading cause of death in patients with multiple endocrine neoplasia type 1. Aside from tumor size, prognostic factors of pancreatic neuroendocrine tumors are largely unknown. The present study aimed to assess whether the prognosis of patients with resected multiple endocrine neoplasia type 1-related nonfunctioning pancreatic neuroendocrine tumors differs from those with resected multiple endocrine neoplasia type 1-related insulinomas and assessed factors associated with prognosis. Methods: Patients who underwent resection of a multiple endocrine neoplasia type 1-related pancreatic neuroendocrine tumors between 1990 and 2016 were identified in 2 databases: the DutchMEN Study Group and the International MEN1 Insulinoma Study Group databases. Cox regression was performed to compare liver metastases-free survival of patients with a nonfunctioning pancreatic neuroendocrine tumors versus those with an insulinoma and to identify factors associated with liver metastases-free survival. Results: Out of 153 patients with multiple endocrine neoplasia type 1, 61 underwent resection for a nonfunctioning pancreatic neuroendocrine tumor and 92 for an insulinoma. Of the patients with resected lymph nodes, 56% (18/32) of nonfunctioning pancreatic neuroendocrine tumors had lymph node metastases compared to 10% (4/41) of insulinomas (P = .001). Estimated 10-year liver metastases-free survival was 63% (95% confidence interval 42%–76%) for nonfunctioning pancreatic neuroendocrine tumors and 87% (72%–91%) for insulinomas. After adjustment for size, World Health Organization tumor grade, and age, nonfunctioning pancreatic neuroendocrine tumors had an increased risk for liver metastases or death (hazard ratio 3.04 [1.47–6.30]). In pancreatic neuroendocrine tumors ≥2 cm, nonfunctioning pancreatic neuroendocrine tumors (2.99 [1.22–7.33]) and World Health Organization grade 2 (2.95 [1.02–8.50]) were associated with liver metastases-free survival. Conclusion: Patients with resected multiple endocrine neoplasia type 1-related nonfunctioning pancreatic neuroendocrine tumors had a significantly lower liver metastases-free survival than patients with insulinomas. Postoperative counseling and follow-up regimens should be tumor type specific and at least consider size and World Health Organization grade

CD133-LV does not transduce normal mouse brain cells, hESC-derived neurons and primary human astrocytes <i>in vitro</i>.

<p><b>A</b>. Injection of CD133-LV expressing mCherry into the mouse basal ganglia did not lead to transduction of normal brain tissue, as opposed to VSVG-LV (BG: basal ganglia, Cx: cortex, CC: corpus callosum, LV: lateral ventricle). <b>Bi,ii</b>. hESC-derived neurons were transduced with either CD133-LV or VSVG-LV expressing mCherry. VSVG-LV led to transduction of MAP2A+ neurons, as opposed to CD133-LV. <b>Ci,ii</b>. Primary human astrocytes were transduced with either CD133-LV or VSVG-LV expressing mCherry (MOI = 10). VSVG-LV led to transduction of GFAP+ astrocytes, as opposed to CD133-LV. (NeuN: neural nuclei, MAP2A: microtubule associated protein 2A, GFAP: glial fibrillary acidic protein). Nuclei were counterstained with DAPI.</p

Selectivity of transduction by CD133-LV.

<p><b>A</b>. Flow cytometry analysis with GBML20 (CD133 content is 68.4±9.8%) shows that CD133-LV - transduced cells (TagBFP+) are also positive for CD133 (right top). In contrast, CD133+ cells are not enriched in the cohort transduced with VSVG-LV (right bottom). Untransduced cells show no TagBFP expression as expected (left panel) <b>B</b>. <b>i</b>. Percent CD133 positivity of cells transduced with either CD133-LV or VSVG-LV. <b>ii</b>. Population statistics for enrichment of CD133+ cells within populations transduced by either CD133-LV or VSVG-LV (MOI = 1). <b>C</b>. Schematic representation of epitopes on the second extracellular loop of CD133 recognized by antibodies predicted to block (recognizing 293C3 epitope) or not block (recognizing AC133 epitope) the interaction of CD133-LV's envelope with CD133 on the cell surface. Primary GBM cells were treated with varying amounts of blocking or non-blocking antibody prior to transduction with CD133-LV (MOI = 0.5). Transduction efficiency of CD133-LV was significantly reduced with blocking antibody. In contrast, non-blocking antibody did not show any significant effect (*, p<10⁻⁷). <b>D,E</b>. Primary GBM lines were modified with lentiviral constructs to either knockdown (<b>D</b>) or overexpress CD133 (<b>E</b>). GBML20 (CD133 content 68.9±9.8%) was used for shRNA-mediated knockdown of CD133. GBML27 (CD133 content 1.4±0.4%) was used for CD133 overexpression after transduction with lentiviral vector CD133-OE. <b>i-ii</b>. Flow cytometric analysis showing the CD133 content of primary lines expressing shRNA against CD133 or overexpressing CD133. <b>iii</b>. qRT-PCR analysis confirmed knockdown and overexpression of <i>PROM1</i> mRNA. <b>iv</b>. Western Blotting confirmed knockdown and overexpression of CD133 in these lines. β-actin was used as loading control. <b>v</b>. CD133 knockdown in GBML20 led to reduced transduction with CD133-LV (MOI = 5). Conversely, CD133 overexpression in GBML27 increased the rate of transduction by CD133-LV (MOI = 5).</p

CD133-LV transduces CD133+ cells in primary GBM xenografts in the mouse brain.

<p><b>A</b>. Intracranial xenograft tumors were generated using injection of GBML20 cells (5×10⁵ cells/animal) and tumor formation (red circle) was confirmed with small animal MRI 1.5 months after injection. High titer stocks of CD133-LV or VSVG-LV expressing TagBFP were injected into the tumor and animals were sacrificed 7 days later for immunofluorescence analysis. <b>B</b>. CD133-LV - transduced cells expressing TagBFP (red) show cell surface immunoreactivity for CD133 (green). <b>C</b>. CD133+ cells were significantly more enriched among TagBFP+ transduced cells in the case of CD133-LV compared to VSVG-LV.</p

Stem-like properties of GBM cells transduced with CD133-LV.

<p><b>A</b>. Cells transduced with CD133-LV expressing TagBFP are clonogenic and produce spheres comprised of TagBFP+ cells. <b>B,C</b>. <i>In vitro</i> tumorsphere formation assays for 3 serial passages did not show any difference in the clonogenic potential of FACS-isolated CD133-LV transduced cells and untransduced CD133+ cells in terms of the number (<b>B</b>) and size (<b>C</b>) of spheres formed.</p

CD133-LV transduces CD133+ cells in primary human GBM cultures <i>in vitro</i>.

<p><b>A</b>. Plasma membrane topology of CD133. CD133-LV recognizes an epitope on the second extracellular loop of the glycoprotein. <b>B</b>. Envelope protein of CD133-LV (Hmut: mutant hemagglutinin, 141.7Fv: single chain antibody against 293C3 epitope on second extracellular loop of CD133). Hmut and 141.7Fv are separated by a linker peptide. <b>C</b>. Observations collected from 3 primary GBM cultures. <b>i</b>. Primary GBM cultures are maintained as tumorspheres in suspension using media supplemented with EGF and bFGF. <b>ii</b>. Cell surface CD133 expression varies among different GBM lines, as assayed by flow cytometry. <b>iii,iv</b>. Flow cytometric analysis of transduction efficiency with CD133-LV (<b>iii</b>) and VSVG-LV (<b>iv</b>) expressing TagBFP. <b>D</b>. CD133-LV transduction efficiency correlates with the CD133 content of three primary GBM cultures (MOI = 5). <b>E</b>. Fluorescent microscopy shows a human GBM tumorsphere with scattered cells transduced with CD133-LV and thereby expressing TagBFP. This image was taken three days after incubation of cells with CD133-LV. <b>F</b>. Transduction of GBML3 cultures (CD133 content is 1.7±0.1%) with CD133-LV is dose-dependent and transduction efficiency is significantly lower than with pantropic VSVG-LV.</p

Prognosis after surgery for multiple endocrine neoplasia type 1-related pancreatic neuroendocrine tumors: Functionality matters

Background: Metastasized pancreatic neuroendocrine tumors are the leading cause of death in patients with multiple endocrine neoplasia type 1. Aside from tumor size, prognostic factors of pancreatic neuroendocrine tumors are largely unknown. The present study aimed to assess whether the prognosis of patients with resected multiple endocrine neoplasia type 1-related nonfunctioning pancreatic neuroendocrine tumors differs from those with resected multiple endocrine neoplasia type 1-related insulinomas and assessed factors associated with prognosis. Methods: Patients who underwent resection of a multiple endocrine neoplasia type 1-related pancreatic neuroendocrine tumors between 1990 and 2016 were identified in 2 databases: the DutchMEN Study Group and the International MEN1 Insulinoma Study Group databases. Cox regression was performed to compare liver metastases-free survival of patients with a nonfunctioning pancreatic neuroendocrine tumors versus those with an insulinoma and to identify factors associated with liver metastases-free survival. Results: Out of 153 patients with multiple endocrine neoplasia type 1, 61 underwent resection for a nonfunctioning pancreatic neuroendocrine tumor and 92 for an insulinoma. Of the patients with resected lymph nodes, 56% (18/32) of nonfunctioning pancreatic neuroendocrine tumors had lymph node metastases compared to 10% (4/41) of insulinomas (P = .001). Estimated 10-year liver metastases-free survival was 63% (95% confidence interval 42%–76%) for nonfunctioning pancreatic neuroendocrine tumors and 87% (72%–91%) for insulinomas. After adjustment for size, World Health Organization tumor grade, and age, nonfunctioning pancreatic neuroendocrine tumors had an increased risk for liver metastases or death (hazard ratio 3.04 [1.47–6.30]). In pancreatic neuroendocrine tumors ≥2 cm, nonfunctioning pancreatic neuroendocrine tumors (2.99 [1.22–7.33]) and World Health Organization grade 2 (2.95 [1.02–8.50]) were associated with liver metastases-free survival. Conclusion: Patients with resected multiple endocrine neoplasia type 1-related nonfunctioning pancreatic neuroendocrine tumors had a significantly lower liver metastases-free survival than patients with insulinomas. Postoperative counseling and follow-up regimens should be tumor type specific and at least consider size and World Health Organization grade