Flow cytometric quantification of tumour endothelial cells; an objective alternative for microvessel density assessment

Assessment of microvessel density by immunohistochemical staining is subject to a considerable inter-observer variation, and this has led to variability in correlation between microvessel density and clinical outcome in different studies. In order to improve the method of microvessel density measurement in tumour biopsies, we have developed a rapid, objective and quantitative method using flow cytometry on frozen tissues. Frozen tissue sections of archival tumour material were enzymatically digested. The single-cell suspension was stained for CD31 and CD34 for flow cytometry. The number of endothelial cells was quantified using light scatter- and fluorescence-characteristics. Tumour endothelial cells were detectable in a single cell suspension, and the percentage of endothelial cells detected in 32 colon carcinomas correlated highly (r=0.84, P<0.001) with the immunohistochemical assessment of microvessel density. Flow cytometric endothelial cells quantification was found to be more sensitive especially at lower levels of immunohistochemical microvessel density measurement. The current method was found to be applicable for various tumour types and has the major advantage that it provides a retrospective and quantitative approach to the angiogenic potential of tumours

Molecular biology of breast cancer metastasis Molecular expression of vascular markers by aggressive breast cancer cells

During embryogenesis, the formation of primary vascular networks occurs via the processes of vasculogenesis and angiogenesis. In uveal melanoma, vasculogenic mimicry describes the 'embryonic-like' ability of aggressive, but not nonaggressive, tumor cells to form networks surrounding spheroids of tumor cells in three-dimensional culture; these recapitulate the patterned networks seen in patients' aggressive tumors and correlates with poor prognosis. The molecular profile of these aggressive tumor cells suggests that they have a deregulated genotype, capable of expressing vascular phenotypes. Similarly, the embryonic-like phenotype expressed by the aggressive human breast cancer cells is associated with their ability to express a variety of vascular markers. These studies may offer new insights for consideration in breast cancer diagnosis and therapeutic intervention strategies

Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer

High microvessel density, an indirect measure of angiogenesis, has been shown to correlate with increased tumour size, lymph node involvement and poor prognosis in non-small-cell lung cancer (NSCLC). Tumour cell vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) expression correlate with angiogenesis and a poor outcome in this disease. In a retrospective study VEGF and PD-ECGF expression and microvessel density were evaluated immunohistochemically in surgically resected specimens (T1–3, N0–2) from 223 patients with operable NSCLC using the VG1, P-GF.44C and JC70 monoclonal antibodies respectively. High VEGF immunoreactivity was seen in 104 (46.6%) and PD-ECGF in 72 (32.3%) cases and both were associated with high vascular grade tumours (P = 0.009 and P = 0.05 respectively). Linear regression analysis revealed a weak positive correlation between VEGF and PD-ECGF expression in cancer cells (r = 0.21;P = 0.002). Co-expression of VEGF and PD-ECGF was not associated with a higher microvessel density than VEGF or PD-ECGF only expressing tumours. Furthermore a proportion of high vascular grade tumours expressed neither growth factor. Univariate analysis revealed tumour size, nodal status, microvessel density and VEGF and PD-ECGF expression as significant prognostic factors. Tumour size (P< 0.02) and microvessel density (P< 0.04) remained significant on multivariate analysis. In conclusion, VEGF and PD-ECGF are important angiogenic growth factors and have prognostic significance in NSCLC. Furthermore the study underlines the prognostic significance of microvessel density in operable NSCLC. © 2000 Cancer Research Campaig

The role of microvessel density on the survival of patients with lung cancer: a systematic review of the literature with meta-analysis

In order to determine whether angiogenesis is a prognostic marker in lung cancer, we performed a systematic review of the literature to assess the prognostic value on survival of microvessel count in patients with lung cancer. Published studies were identified by an electronic search in order to aggregate survival results, after a methodological assessment using a quality scale designed by the European Lung Cancer Working Party. To be eligible, a study had to deal with microvessel count assessment in lung cancer patients on the primary site and to provide survival analysis according to microvessel count expression. Microvessel count has been assessed on surgical samples by immunohistochemistry using factor VIII in 14 studies, CD34 in 10 and CD31 in eight. Respectively 1866, 1440 and 1093 non-small cell lung cancer patients were considered. The overall median quality scores were respectively 52, 59 and 59% for studies assessing microvessel count via factor VIII, CD34 and CD31, without significant difference between studies evaluable or not for meta-analysis nor between studies with significant or non significant results. Seven ‘factor VIII’ studies, nine ‘CD34’ and seven ‘CD31’ provided sufficient data allowing a meta-analysis on survival and were evaluable for results aggregation. This showed that a high microvessel count in the primitive lung tumour was a statistically significant poor prognostic factor for survival in non small cell lung cancer whatever it was assessed by factor VIII (HR: 1.81; 95% CI: 1.16–2.84), CD34 (HR: 1.99; 95% CI: 1.53–2.58) or CD31 (HR: 1.80; 95% CI: 1.10–2.96). Variations in survival among the individual studies can be explained in addition to patients selection criteria by the heterogeneous methodologies used to stain and count microvessels: different antibody clones, identification of ‘hotspots’, Weidner or Chalkey counting method, cut-off selection. Microvessel count, reflecting the angiogenesis, appears to be a poor prognostic factor for survival in surgically treated non small cell lung cancer but standardisation of angiogenesis assessment by the microvessel count is necessary

Decarboxylative Amination: Diazirines as Single and Double Electrophilic Nitrogen Transfer Reagents

The ubiquity of nitrogen-containing small molecules in medicine necessitates the continued search for improved methods for C–N bond formation. Electrophilic amination often requires a disparate toolkit of reagents whose selection depends on the specific structure and functionality of the substrate to be aminated. Further, many of these reagents are challenging to handle, engage in undesired side reactions, and function only within a narrow scope. Here we report the use of diazirines as practical reagents for the decarboxylative amination of simple and complex redox-active esters. The diaziridines thus produced are readily diversifiable to amines, hydrazines, and nitrogen-containing heterocycles in one step. The reaction has also been applied in fluorous phase synthesis with a perfluorinated diazirine.</p

Synthesis of Fully Aliphatic Aziridines with a Macrocyclic Tetracarbene Iron Catalyst

A second-generation aziridination catalyst supported by a borate-based dianionic macrocyclic tetracarbene ligand has been synthesized. The new macrocyclic tetracarbene iron­(II) complex catalyzed the aziridination of alkyl azides and aliphatic alkenes showcasing the first fully aliphatic version of this C₂ + N₁ reaction. High isolated yields were obtained when no functional groups were present on the organic azides and alkenes, while modest yields were achieved when nonprotic functional groups were included. Even multiple functional groups can be added to the azide and alkene fragments to produce the most complex aziridines yet synthesized by this C₂ + N₁ catalytic reaction. The catalyst generated higher yields for aziridination with aryl azides and alkenes than the previously reported catalyst, [(^Me,EtTC^<i>Ph</i>)­Fe­(NCCH₃)₂]­(PF₆)₂. The contrast is particularly apparent with functionalized aryl azides where the second-generation catalyst now provides practical yields for synthetic chemistry. Finally, catalytic intramolecular aziridination was investigated since many natural products with aziridines feature bicyclic tertiary aziridines. For five- and six-membered rings, the bicyclic aziridines were formed catalytically, in contrast to previously studied catalyzed and uncatalyzed reactions

Climate change education: following the information

This chapter explores, through fictionalised story, the nexus of information, climate change, digital geographies, children&#x2019;s geographies, and the future of work. We repurpose a follow-the-thing methodological approach (Cook et al. 2017), normally used for commodities, by applying it to information in order to tell a story about the journeys of information &#x2013; or digital non-objects (Rose, 2016) - about climate change. In one direction the story moves &#x2018;backwards&#x2019; from the classroom, tracing the interfaces, networks and frictions through which this information moves and transforms. In another direction the story moves &#x2018;forwards&#x2019; out of the classroom into the lives of the children to whom it was taught. Children&#x2019;s educational experiences are increasingly digitally mediated, and the unprecedented amounts of information available to teachers presents significant challenges for curriculum making. Children and young people are calling for greater attention to be given to climate change education, and this story highlights opportunities for rethinking the ways in which information about climate change should be navigated to help work towards more equitable, just and sustainable futures