Figure S7: Translational study endpoints (IHC) and subcutaneous in-vivo model. (A) Representative photos of primary tumor pEGFR and pAkt immunohistochemistry in each of the 6 subrenal model experiments. (B) Mean and sem of tumor volume of dual blockade (ERL+ BYL) versus single blockade (ERL, BYL) versus control (NT) over 28 days in the subcutaneous model (n=5 in each treatment group).

PDF file - 206K, Table S1 Numbers of test positive cases and controls using CA19-9 37 U/mL and CA125 30 U/mL cut-offs Figure S1 Scatter plots showing distribution of CA19-9, CA125, CEACAM1 and REG3A levels against time to diagnosis for discovery set. Zero represents the point of clinical diagnosis. Figure S2 Examples of CA19-9 and CA125 levels in individual cases with serial/longitudinal samples. Figure S3 Box and whisker plots showing serum levels of CA19-9 and CA125 for case control validation samples grouped into different time to diagnosis groups. Whisker limits represent the 5th and 95th percentiles, the box limits represent interquartile range, the horizontal line the median and the cross the mean. Case and control groups were compared using the Mann-Whitney test; significant P values (<0.05) are shown above the plots.

Figure S1: EGFR and PI3K signaling in 5 PDAC cell lines. (A) Densitometric quantitation of Western blotting of pAkt/ total Akt (left panel) and pERK/ total ERK (right panel), with no treatment (NT), or stimulation by EGF, IGF-1, or both growth factors: average  SEM of 3 experiments. (B) Quantitation of Western blot of pEGFR, pIGF1R, pERK and pAkt in response to increasing dose of erlotinib or NVP-AEW541, average of 3 experiments. Drug doses were selected based on relative potency from proliferation assay and investigator brochure. Note that all signals were adjusted to EGF-stimulated control, so for PANC-1 which had a much more highly EGF-stimulated pAkt, the relative fold-change with erlotinib inhibition appeared smaller than for BxPC-3. * P<0.05, ** P<0.01

Figure S6: Effect of single and dual blockade (DB) on apoptosis in resistant and parental cell lines. (A) Percentage of cells undergoing apoptosis and necrosis measured by flow cytometry: paired t-test statistics calculated for DB versus no treatment. (NT) (B) Representative confocal microscopy of cleaved caspase-3 (red), after 24 hours of treatment. Blue = DAPI (nuclei). (C) Cleaved caspase-3 immunofluorescence, mean intensity averaged from 4 experiments, with t-test statistics *P<0.05; **P<0.001; ***P<0.0001. (D) Relative migration rate, expressed as increasing percentage cell density across a monolayer wound over 44 hours, mean values from 3 experiments {plus minus} SEM. Treatments are control (black solid), erlotinib 10 µM (E10- black dotted), NVP-AEW541 1 µM (A1- black dashed), NVP-BYL719 5 µM (B5- grey solid), E10A1 (E10+ A1- grey dotted), and E10B5 (E10+ B5- grey dashed). Proliferation was inhibited by 10 µg/ml mitomycin.

Supplementary Methods and Legends

Table S1: IC50 values for inhibition of proliferation of (A) 5 PDAC cell lines and (B) 2 derived erlotinib-resistant (ER) cell lines, in response to inhibitors of EGFR (erlotinib, gefitinib), IGF1R (NVP-AEW541), MEK (PD-98059), PI3K (LY-294002), PI3Kï¡ (NVP-BYL719) and PI3K/mTOR (NVP-BEZ235). Average  SEM of 6 experiments. Highly insensitive cell lines are shaded in grey in (A). Paired t-test statistics was calculated for resistance in (B).

Figure S3: Phospho-RTK array on ER versus parental cell lines (A) Phosphorylated protein chemiluminescence intensity in untreated (NT) parental and resistant cells after adjusting to positive and negative controls, with the largest differences in signal intensity shaded in grey (B) Quantitation of Western blotting results of pERK, pAkt and pS6 adjusted to total proteins in ER and parental cell lines, either untreated (NT) or after stimulation by EGF, IGF-1, or both growth factors. * P<0.05

Table S2: Cell cycle dual blockade experiments. (A) Cell cycle: Quantitative analysis of proportion of cells in G1, S and G2/M phases, calculated as mean +/- SEM of 3 experiments (at 24 hours). Average CV is provided: a CV <5% is acceptable for cell cycle analysis. (B) Apoptosis assay, average total apoptosis + necrosis by flow cytometry, expressed as a percentage of total cells; mean of 5 experiments with SEM. NT: untreated; E10: erlotinib 10 µM; A1: AEW541 1 µM; B5: BYL719 5 µM.

Figure S4: Western blotting dual blockade isobolograms: Isobolograms constructed for pERK, pAkt and pS6 inhibition by ERL* AEW (A) and ERL* BYL (B) for BxPC-3 and PANC-1. An IC50 additivity line was extrapolated based on the predicted single drug IC50 in three separate experiments. The combined IC50 values of dual inhibitors were plotted on the same graph. If the observed points lied below the additivity line, then the two drugs acted synergistically in inhibiting the respective signal.

Figure S5: Effect of single and dual blockade (DB) on cell proliferation (A, B) 3D contour maps for inhibition by ERL+AEW (A) and ERL+BYL (B). Synergy index (SI) was calculated by non-linear regression (PROC NLIN) using the Bliss synergy/antagonism formula. SI>1 indicates synergy and SI<1 indicates antagonism between two drugs. If the 95% confidence interval does not cross 1, the effect is significant. (C) Proliferation of control and ER cell lines was measured in real-time over 70 h of drug treatment. Treatments are control (black solid), erlotinib 10 µM (E10- black dotted), NVP-AEW541 1 µM (A1- black dashed), NVP-BYL719 5 µM (B5- grey solid), E10A1 (E10+ A1- grey dotted), and E10B5 (E10+ B5- grey dashed). Mean values {plus minus} SEM for 4 experiments.

This chapter studies nutritional support for hospital patients. The concept of ‘enteral nutrition’ has developed as a specialized form of nutrition therapy out of the space programme, where a balanced nutrition that resulted in minimal excretion was a distinct advantage. Enteral nutrition is indicated as a means of nutritional support for patients who are unable to sustain themselves with an oral diet and who have a sufficient normal intestine available for absorption of enteral formula. Meanwhile, parenteral feeding is indicated when patients cannot be nourished with oral nutrition or enteral feeding for more than five days. Parenteral nutrition needs to provide all necessary electrolytes, trace elements, and vitamins in balanced amounts. The chapter then looks at the benefit of nutritional support teams.

Abstract Background and Aims Cancer‐associated stroma (CAS) is emerging as a key determinant of metastasis in colorectal cancer (CRC); however, little is known about CAS in colorectal liver metastases (CRLM). This study aimed to validate the prognostic significance of stromal protein biomarkers in primary CRC and CRLM. Secondly, this study aimed to describe the transcriptome of the CAS of CRLM and identify novel targetable pathways of metastasis. Methods A case–control study design from a prospectively maintained database was adopted. The prognostic value of epithelial and stromal CALD1, IGFBP7, POSTN, FAP, TGF‐β and pSMAD2 expression was assessed by immunohistochemistry (IHC) in multivariate models. Pathway enrichment and sparse partial least square‐discriminant analysis (sPLS‐DA) were performed on a nested cohort after isolating epithelial tumour and CAS by laser capture microdissection. Results 110 CRCs with 124 paired CRLMs, and 110 matched non‐metastatic control CRCs were included. Median follow‐up was 62 and 45 months for primary and CRLM groups, respectively. Stromal FAP and POSTN were independent predictors for the development of CRLM. After CRLM resection, stromal IGFBP7 and POSTN were predictors of poorer survival. sPLS‐DA on the nested cohort identified a number of novel targetable stromal genes and pathways that defined poor prognosis CRC and the CAS of CRLM. Conclusions This study is the first to describe key differences in stromal gene expression between paired primary CRC and CRLM as well as identifying several targetable biomarkers and transcriptomic pathways whose relevance specifically in the CAS of CRC and CRLM have not been previously described.

Cerebral Cavernous Malformation (CCM) is a brain vascular disease with various neurological symptoms. In this study, we describe the inflammatory profile in CCM and show for the first time the formation of neutrophil extracellular traps (NETs) in rodents and humans with CCM. Through RNA-seq analysis of cerebellum endothelial cells from wild-type mice and mice with an endothelial cell-specific ablation of the Ccm3 gene (Ccm3iECKO), we show that endothelial cells from Ccm3iECKO mice have an increased expression of inflammation-related genes. These genes encode proinflammatory cytokines and chemokines, as well as adhesion molecules, which promote recruitment of inflammatory and immune cells. Similarly, immunoassays showed elevated levels of these cytokines and chemokines in the cerebellum of the Ccm3iECKO mice. Consistently, both flow cytometry and immunofluorescence analysis showed infiltration of different subsets of leukocytes into the CCM lesions. Neutrophils, which are known to fight against infection through different strategies, including the formation of NETs, represented the leukocyte subset within the most pronounced increase in CCM. Here, we detected elevated levels of NETs in the blood and the deposition of NETs in the cerebral cavernomas of Ccm3iECKO mice. Degradation of NETs by DNase I treatment improved the vascular barrier. The deposition of NETs in the cavernomas of patients with CCM confirms the clinical relevance of NETs in CCM.

Sinonasal sarcoidosis is rare, affecting only about 3% of patients with sarcoidosis, and may be difficult to identify without routine use of rhinoscopy. Typically, the presence of symptoms at extra-nasal sites leads to initial consideration of this diagnosis.

We hypothesised that synthetic HDL nanoparticles carrying a gemcitabine prodrug and apolipoprotein A-II (sHDLGemA2) would target scavenger receptor-B1 (SR-B1) to preferentially and safely deliver gemcitabine into pancreatic ductal adenocarcinoma (PDAC). We designed, manufactured and characterised sHDLGemA2 nanoparticles sized-130 nm, incorporating 20 mol% of a gemcitabine prodrug within the lipid bilayer, which strengthens on adding ApoA-II. We measured their ability to inhibit growth in cell lines and cell-derived and patient-derived murine PDAC xenografts. Fluorescent-labelled sHDLGemA2 delivered gemcitabine inside xenografts. Xenograft levels of active gemci-tabine after sHDLGemA2 were similar to levels after high-dose free gemcitabine. Growth inhibition in mice receiving 4.5 mg gemcitabine/kg/d, carried in sHDLGemA2, was equivalent to inhibition after high-dose (75 mg/kg/d) free gemcitabine, and greater than inhibition after low-dose (4.5 mg/kg/d) free gemcitabine. sHDLGemA2 slowed growth in semi-resistant cells and a resistant human xenograft. sHDLGemA2 targeted xenografts more effectively than sHDLGemA1. SR-B1 was over-expressed in PDAC cells and xenografts. Targeting by ApoA-II was suppressed by anti-SR-B1. Because sHDLGemA2 provided only -6% of the free gemcitabine dose for an equivalent response, patient side effects can be greatly reduced, and the sHDLGemA2 concept should be developed through clinical trials.

Gemcitabine (Gem) is a key drug for pancreatic cancer, yet limited by high systemic toxicity, low bioavailability and poor pharmacokinetic profiles. To overcome these limitations, Gem prodrug amphiphiles were synthesised with oleyl, linoleyl and phytanyl chains. Self-assembly and lyotropic mesophase behaviour of these amphiphiles were examined using polarised optical microscopy and Synchrotron SAXS (SSAXS). Gem-phytanyl was found to form liquid crystalline inverse cubic mesophase. This prodrug was combined with phospholipids and cholesterol to create biomimetic Gem-lipid prodrug nanoparticles (Gem-LPNP), verified by SSAXS and cryo-TEM to form liposomes. In vitro testing of the Gem-LPNP in several pancreatic cancer cell lines showed lower toxicity than Gem. However, in a cell line-derived pancreatic cancer mouse model Gem-LPNP displayed greater tumour growth inhibition than Gem using a fraction (<6 %) of the clinical dose and without any systemic toxicity. The easy production, improved efficacy and low toxicity of Gem-LPNP represents a promising new nanomedicine for pancreatic cancer.

To elucidate the role of endothelial nitric oxide synthase (eNOS) activation in the induction of vascular endothelial growth factor (VEGF)-induced permeability, the underlying mechanism and the contribution to exaggerated vascular leakage in eye disease. Retinal neovascularization and pathological edema in the superficial and deep retinal vascular plexa were investigated using the oxygen-induced retinopathy (OIR) mouse model. eNOS activity was blocked either chemically by using the NO inhibitor or genetically by using mutant mice (Mut) in which serine 1176 in eNOS is replaced by alanine (Nos3 S1176A/S1176A ,therefore is unable to induce eNOS activity). Retina avascular area and proliferative vessel tuft formation were assessed at postnatal (P) day 17. Leakage was determined by injecting 25 nm fluorescent microspheres in the circulation followed by microscopy. Isolectin B4, eNOS, vascular endothelial cadherin (VEC), and phosphoY685-VEC immunostaining was assessed by confocal imaging followed by image analysis using ImageJ. Students t -test was used for statistical evaluation. p < 0.05 was regarded as statistically significant. Administration of the NO inhibitor during P12-P17 reduced overall pathological tuft area (p < 0.05), and decreased the area of individual tufts (p < 0.01) compared to control. Mut displayed reduced neovascular tuft formation (p < 0.01) and decreased tuft size compared to wildtype mice (WT) (p < 0.05). Mice unable to activate eNOS displayed a junctional phenotype. Firstly, phosphoY685-VEC in tufts was decreased in Mut compared to WT (p < 0.05). Secondly, immunostaining for eNOS colocalized with VEC in vessels of WT but not in Mut (p < 0.05). Thirdly, vascular leakage was reduced in OIR-challanged Mut (p < 0.01). Our data establish that eNOS is critical for VEC-regulated endothelial junction stability and vascular leakage. Thus, eNOS is an important target for the development of therapeutic agents to treat eye disease.

Background: Hepatocellular carcinoma (HCC) is a common cause of cancer death worldwide. Resection offers the best chance of long-term survival, but a consistent adverse prognostic factor is the presence of microvascular invasion (MVI). In this study, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), a high throughput method of analyzing complex samples, was used to explore differentially expressed proteins between HCC and adjacent nontumour liver tissue (ANLT). These findings were correlated with clinical outcomes. Materials and methods: From 2002 to 2011, tumor and ANLT were collected from patients who underwent liver resection and these samples were later prepared for SELDI-TOF MS. Output data were then used to identify proteins capable of discriminating HCC from ANLT. Proteins fromthemultivariate analysiswere then analyzed to determine prognostic factors and the m/z ratios of these proteins were entered into the ExPASy database to infer potential candidates. Results: During the study period, 30 patients had SELDI-TOF MS performed on their HCC and ANLT samples. On multivariate analysis, a panel of four proteinsdm/z 5840, m/z 8921, m/z 9961, and m/z 25,872ddiscriminated HCC from ANLT with an area under the ROC curve of 0.954 (P < 0.001). On prognostic factor assessment, decreased m/z 9961 was significantly associated with the presence of MVI (P = 0.025) and shorter disease-free survival (P = 0.045) in our patients. A potential candidate for this protein was coxsackievirus and adenovirus receptor, isoform 3 (CAR 3/7), which helps maintain tight junction integrity. Conclusions: Using SELDI TOF-MS, we identified a panel of four proteins with excellent discriminative capacity between HCC and ANLT. Of these, m/z 9961 was the only protein significantly associated with a known poor prognostic factor (presence of MVI) and survival (shorter disease-free survival). While loss of CAR 3/7 could lead to MVI, further research is warranted to validate the identity of protein m/z 9961. (C) 2019 Elsevier Inc. All rights reserved.

Background: Few in vivo models for colorectal cancer have been demonstrated to show external validity by accurately predicting clinical patient outcomes. Patient-derived xenograft (PDX) models of cancer have characteristics that might provide a form of translational research leading to personalized cancer care. The aim of this pilot study was to assess the feasibility of using PDXs as a platform for predicting patient colorectal liver metastases responses, in this case by correlating PDX and patient tumor responses to either folinic acid, fluorouracil plus oxaliplatin or folinic acid, fluorouracil plus irinotecan-based regimens. Methods: Sixteen patients underwent potentially curative resection of colorectal liver metastases, and tumors were grafted into NOD.CB17-Prkdc(scid)/Arc mice. Mice were divided into groups to determine relative tumor growth in response to treatment. Tumors were analyzed by immunohistochemistry for Ki67 and Excision repair cross-complementation group 1. Results: An engraftment rate of 81% was achieved. Overall, there was a 67% positive match rate between eligible patient and PDX chemosensitivity profiles. There was a significant difference in relative decrease in Ki67 expression between sensitive/stable versus resistant PDXs for both treatment regimens. There was no statistically significant correlation between baseline ERCC1 expression and response to Oxaliplatin thorn 5-Fluorouracil in the PDXs. Conclusions: This pilot study supports the feasibility of using PDX models of advanced colorectal cancer in larger studies to potentially predict patient chemosensitivity profiles. (C) 2018 Elsevier Inc. All rights reserved.