CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries

Table S9. Software used in this study. (XLSX 39 kb

A multicenter open-label phase II trial to evaluate nivolumab and ipilimumab for 2nd line therapy in elderly patients with advanced esophageal squamous cell cancer (RAMONA)

Background: Advanced esophageal squamous cell cancer (ESCC) is frequently diagnosed in elderly patients. The impact of 2nd line chemotherapy is poorly defined. Recent data demonstrated effectiveness of checkpoint inhibitors in different squamous cell carcinomas. Therefore, we assess combined nivolumab/ipilimumab as 2nd line therapy in elderly ESCC patients. Methods: RAMONA is a multicenter open-label phase II trial. The primary objective is to demonstrate a significant survival benefit of nivolumab/ipilimumab in advanced ESCC compared to historical data of standard chemotherapy. Primary endpoint is therefore overall survival (OS). Major secondary objective is the evaluation of tolerability. Time to QoL deterioration will thus be determined as key secondary endpoint. Further secondary endpoints are tumor response, PFS and safety. We aim to recruit a total of n = 75 subjects that have to be > 65 years old. Eligibility is determined by the geriatric status (G8 screening and Deficit Accumulation Frailty Index (DAFI)). A safety assessment will be performed after a 3 cycle run-in phase of nivolumab (240 mg Q2W) to justify escalation for eligible patients to combined nivolumab (240 mg Q2W) and ipilimumab (1 mg/kg Q6W), while the other patients will remain on nivolumab only. RAMONA also includes translational research sub-studies to identify predictive biomarkers, including PD-1 and PD-L1 evaluation at different time points, establishment of organoid cultures and microbiome analyses for response prediction. Discussion: The RAMONA trial aims to implement checkpoint inhibitors for elderly patients with advanced ESCC as second line therapy. Novel biomarkers for checkpoint-inhibitor response are analyzed in extensive translational sub-studies. Trial registration: EudraCT Number 2017–002056-86; NCT03416244, registered: 31.1.2018

Outcome of Colorectal Cancer Patients Treated with Combination Bevacizumab Therapy: A Pooled Retrospective Analysis of Three European Cohorts from the Angiopredict Initiative

Background/Aims: This study is aimed at analyzing the survival rates and prognostic factors of stage IV colorectal cancer patients from 3 European cohorts undergoing combination chemotherapy with bevacizumab. Methods: Progression free-survival (PFS) and overall survival (OS) were analyzed in 172 patients using the Kaplan–Meier method and uni- and multivariable Cox proportional hazards regression models. Results: The median PFS was 9.7 and the median OS 27.4 months. Patients treated at centers in Germany (n = 97), Ireland (n = 32), and The Netherlands (n = 43) showed a median PFS of 9.9, 9.2, and 9.7 months, OS of 34.0, 20.5, and 25.1 months, respectively. Patients >65 years had a significantly shorter PFS (9.5 vs. 9.8 months) but not OS (27.4 vs. 27.5 months) than younger patients. High tumor grade (G3/4) was associated with a shorter PFS, T4 classification with both shorter PFS and OS. Fluoropyrimidine (FP) chemotherapy backbones (doublets and single) had comparable outcomes, while patients not receiving FP backbones had a shorter PFS. In multivariable analysis, age and non-FP backbone were associated with inferior PFS, T4 classification and therapy line >2nd were significantly associated with poor PFS and OS. Conclusion: The observed survival rates confirm previous studies and demonstrate reproducible benefits of combination bevacizumab regimens. Classification T4, non-FP chemotherapy backbone, and age >65 were associated with inferior outcome

Overexpressed FATP1, ACSVL4/FATP4 and ACSL1 increase the cellular fatty acid uptake of 3T3-L1 adipocytes but are localized on intracellular membranes.

Long chain acyl-CoA synthetases are essential enzymes of lipid metabolism, and have also been implicated in the cellular uptake of fatty acids. It is controversial if some or all of these enzymes have an additional function as fatty acid transporters at the plasma membrane. The most abundant acyl-CoA synthetases in adipocytes are FATP1, ACSVL4/FATP4 and ACSL1. Previous studies have suggested that they increase fatty acid uptake by direct transport across the plasma membrane. Here, we used a gain-of-function approach and established FATP1, ACSVL4/FATP4 and ACSL1 stably expressing 3T3-L1 adipocytes by retroviral transduction. All overexpressing cell lines showed increased acyl-CoA synthetase activity and fatty acid uptake. FATP1 and ACSVL4/FATP4 localized to the endoplasmic reticulum by confocal microscopy and subcellular fractionation whereas ACSL1 was found on mitochondria. Insulin increased fatty acid uptake but without changing the localization of FATP1 or ACSVL4/FATP4. We conclude that overexpressed acyl-CoA synthetases are able to facilitate fatty acid uptake in 3T3-L1 adipocytes. The intracellular localization of FATP1, ACSVL4/FATP4 and ACSL1 indicates that this is an indirect effect. We suggest that metabolic trapping is the mechanism behind the influence of acyl-CoA synthetases on cellular fatty acid uptake

ACSL1-FLAG is localized to mitochondria in 3T3-L1 adipocytes.

<p>(A–D) Comparative analysis of the intracellular localization of FLAG -tagged ACSL1. (A) ACSL1-FLAG (green); mouse monoclonal anti FLAG M2) does not co-localize with CD36. (red). (B–C) Congruency of ASCL1-FLAG (red) with the mitochondrial marker OCT-GFP is excellent for lowly differentiated (B) but less complete for highly differentiated adipocytes (C). (D) In fibroblasts, ACSL1-FLAG overlaps completely with the mitochondrial marker.</p

Characterization of FATP1 and ACSVL4/FATP4 expression in 3T3-L1 adipocytes.

<p>(A) Quantification of FATP1 and ACSVL4/FATP4 mRNA levels in overexpressing (3T3-FATP1, dark grey and 3T3-FATP4, light grey) and control adipocytes (3T3-pRJ, white bars) by efficiency corrected quantitative real-time PCR relative to general transcription factor 2b (Gtf2b). The relative increase of FATP1 mRNA level is 3.5-fold in 3T3-FATP1 (n = 2) and the increase of ACSVL4/FATP4 mRNA level is 6.9-fold in 3T3-FATP4 (n = 1) compared to 3T3-pRJ. (B, C, D) Analysis of protein expression by Western blotting of total cell lysates from FATP1, ACSVL4/FATP4 and ACSL1-FLAG overexpressing and control adipocytes. Densitometry indicates 11-fold overexpression of FATP1 (n = 1) and 9.0-fold overexpression of ACSVL4/FATP4 (n = 2).</p

Insulin-mediated glucose uptake is enhanced in FATP1 and ACSVL4/FATP4 overexpressing adipocytes.

<p>3T3-FATP1 (dark grey bars) and 3T3-FATP4 adipocytes (light grey bars) and control adipo-cytes (white bars) were incubated with 1 µg/ml insulin for 20 min followed by 10 min of co-incubation with 0.1 mM 2-deoxy-D-glucose [DOG], 1 mCi/ml. Glucose uptake is significantly higher for insulin treated cells (* p<0,05) and this effect is more pronounced in FATP1 and ACSVL4/FATP4 overexpressing adipocytes (* p<0,05), n = 3.</p