SOLEIL: single-objective lens inclined light sheet localization microscopy

High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the usability of these approaches. Here, we developed a single-objective lens-inclined light sheet microscope (SOLEIL) that is capable of 2D and 3D SMLM in thick samples. SOLEIL combines oblique illumination with point spread function PSF engineering to enable dSTORM imaging in a wide variety of samples. SOLEIL is compatible with standard sample holders and off-the-shelve optics and standard high NA objectives. To accomplish optimal optical sectioning we show that there is an ideal oblique angle and sheet thickness. Furthermore, to show what optical sectioning delivers for SMLM we benchmark SOLEIL against widefield and HILO microscopy with several biological samples. SOLEIL delivers in 15 μm thick Caco2-BBE cells a 374% higher intensity to background ratio and a 54% improvement in the estimated CRLB compared to widefield illumination, and a 184% higher intensity to background ratio and a 20% improvement in the estimated CRLB compared to HILO illumination.     

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression

Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C–induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C–induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.     

Diagnostic value of immunohistochemical IMP3 expression in core needle biopsies of pancreatic ductal adenocarcinoma

The oncofetal protein, insulin-like growth factor II messenger ribonucleic acid-binding protein 3 (IMP3), has been analyzed in many different tumors. Various studies have found that IMP3 is a marker for malignancy and is correlated with increased tumor aggressiveness and reduced overall survival. The diagnosis of pancreatic ductal adenocarcinoma (PDAC) in core needle biopsies can be challenging, and immunohistochemical markers are needed. We studied IMP3 expression in 177 core needle biopsies of the pancreas, including 112 PDACs, 55 cases with chronic sclerosing pancreatitis, and 10 biopsies with tumor-free pancreatic tissue without inflammation. An additional 18 biopsies of PDAC metastases (16 liver biopsies and 2 lymph node biopsies) were analyzed. To study IMP3 expression in large tissue sections, 45 pancreatic resection specimens (26 with PDAC and 19 with chronic sclerosing pancreatitis) were investigated. In contrast to normal or inflamed pancreatic tissue, which was negative in 47 of 65 (72.3%) cases and weakly positive in 15 of 65 (23.1%) cases, strong IMP3 expression was found in 99 of 112 (88.4%) PDACs. Therefore, sensitivity and specificity of IMP3 expression in the differential diagnosis of PDAC and chronic sclerosing pancreatitis using core needle biopsies were found to be 88.4% and 94.6%, respectively. These results were confirmed in the pancreas resection specimens. Furthermore, strong IMP3 expression was found in 17 of 18 (94.4%) of the PDAC metastases that were analyzed. Our study shows that IMP3 is an easy to use and potentially new immunohistochemical marker for the diagnosis of PDAC in core needle biopsies.     

Short-term preoperative supplementation of an immunoenriched diet does not improve clinical outcome in well-nourished patients undergoing abdominal cancer surgery

ObjectiveA recent study suggested that the anti-inflammatory effect of immunonutrition starts after only two d. We therefore investigated the effect of an immunoenriched oral diet administered for three d preoperatively.MethodsIn this prospective, randomized, double-blind, placebo-controlled study, well-nourished patients (Nutrition Risk Screening 2002 <3) with gastrointestinal cancer who were scheduled for major elective abdominal cancer surgery were randomly assigned to either 750 mL of an immunoenriched formula (IEF group) or 750 mL of an isocaloric, isonitrogenous placebo diet (Con group) for 3 consecutive d preoperatively.ResultsA total of 108 patients (IEF group: n = 55; Con group: n = 53) were randomized. The two groups were comparable for all baseline and surgical characteristics. The overall mortality was 2.8% and not significantly different between the two groups (IEF group: 3.6% vs. Con group: 1.9%, P = 1.00). Intention-to-treat analysis showed no difference for the incidence of postoperative overall (IEF group: 29% vs. Con group: 30%; P = 1.00) and infectious (IEF group: 15% vs. Con group: 17%; P = 0.79) complications. Length of hospital stay was 12 ± 4.9 d in the IEF group and 11.6 ± 5.3 d in the Con group (P = 0.68).ConclusionsPreoperative oral supplementation with an immunoenriched diet for 3 d preoperatively did not improve postoperative outcome compared with the placebo in well-nourished patients with elective gastrointestinal cancer surgery.