Functional Multiplex Reporter Assay Using Tagged Gaussia Luciferase

We have developed a multiplex reporter system to monitor multiple biological variables in real-time. The secreted Gaussia luciferase was fused to ten different epitope tags (Gluc$_{tag}$), each expressed in different tumor cells. By immunobinding of the tags followed by Gluc$_{tag}$ detection, this system allowed the independent and real-time monitoring of mixed cell cultures in vitro and of mixed subcutaneous and intracranial tumor subpopulations in vivo

A new humanized in vivo model of KIT D816V+ advanced systemic mastocytosis monitored using a secreted luciferase

Systemic mastocytosis are rare neoplasms characterized by accumulation of mast cells in at least one internal organ. The majority of systemic mastocytosis patients carry KIT D816V mutation, which activates constitutively the KIT receptor. Patient with advanced forms of systemic mastocytosis, such as aggressive systemic mastocytosis or mast cell leukemia, are poorly treated to date. Unfortunately, the lack of in vivo models reflecting KIT D816V+ advanced disease hampers pathophysiological studies and preclinical development of new therapies for such patients. Here, we describe a new in vivo model of KIT D816V+ advanced systemic mastocytosis developed by transplantation of the human ROSAKIT D816V-Gluc mast cell line in NOD-SCID IL-2R g−/− mice, using Gaussia princeps luciferase as a reporter. Intravenous injection of ROSAKIT D816V-Gluc cells led, in 4 weeks, to engraftment in all injected primary recipient mice. Engrafted cells were found at high levels in bone marrow, and at lower levels in spleen, liver and peripheral blood. Disease progression was easily monitored by repeated quantification of Gaussia princeps luciferase activity in peripheral blood. This quantification evidenced a linear relationship between the number of cells injected and the neoplastic mast cell burden in mice. Interestingly, the secondary transplantation of ROSAKIT D816V-Gluc cells increased their engraftment capability. To conclude, this new in vivo model mimics at the best the features of human KIT D816V+ advanced systemic mastocytosis. In addition, it is a unique and convenient tool to study the kinetics of the disease and the potential in vivo activity of new drugs targeting neoplastic mast cells

Well-founded practice or personal preference: a comparison of established techniques for measuring ulnar variance in healthy children and adolescents

Objectives: Ulnar variance is a clinical measure used to determine the relative difference in length between the radius and ulna. We aimed to examine consistency in ulnar variance measurements and normative data in children and adolescents using the perpendicular and the Hafner methods. Methods: Two raters measured ulnar variance on hand radiographs of 350 healthy children. Participants’ mean calendar and skeletal ages were 12.3 ± 3.6 and 12.0 ± 3.7 years, 52% were female. Raters used the perpendicular method, an adapted version of the perpendicular method (in which the distal radial articular surface is defined as a sclerotic rim) and the Hafner method, being the distance between the most proximal points of the ulnar and radial metaphyses (PRPR) and the distance between the most distal points of both (DIDI). Intraclass correlation coefficients (ICCs) for intermethod consistency and inter- and intrarater agreement were calculated using a two-way ANOVA model. Variability and limits of agreement were determined using the Bland-Altman method. Results: The interrater ICC was 0.75 (95% CI, 0.61–0.84) for the adapted perpendicular method, 0.88 (95% CI, 0.80–0.93) for PRPR, and 0.94 (95% CI, 0.90–0.97) for DIDI. The intermethod consistency ICC was 0.60 (95% CI, 0.48–0.70) for perpendicular versus PRPR and 0.60 (95% CI, 0.49–0.70) for perpendicular versus DIDI. The intrarater ICC was 0.88 (95% CI, 0.70–0.95) for perpendicular, 0.90 (95% CI, 0.83–0.94) for PRPR, and 0.81 (95% CI, 0.69–0.89) for DIDI. The perpendicular method was not useable in 38 cases (skeletal age ≤ 9 years) and the Hafner method in 79 cases (skeletal age ≥ 12 years). Conclusions: The perpendicular and Hafner methods show moderate intermethod consistency. The Hafner method is preferred for children with skeletal ages < 14 years, with good to excellent inter- and intrarater agreement. The adapted perpendicular method is recommended for patients with skeletal ages ≥ 14 years. Key Points: • The perpendicular method for measuring ulnar variance requires extended instructions to ensure good interrater agreement in pediatric and adolescent patients. • The Hafner method is recommended for ulnar variance measurement in children with unfused growth plates and up to a skeletal age of 13 years, and the perpendicular method is recommended for children with fused growth plates and from skeletal age 14 and older. • The mean ulnar variance measured in this study for each skeletal age group (range, 5–18 years) is provided, to serve as a reference for future ulnar variance measurements using both methods in clinical practice

Bioluminescence-mediated longitudinal monitoring of adipose-derived stem cells in a large mammal ex vivo organ culture

Recently, ex vivo three-dimensional organ culture systems have emerged to study the physiology and pathophysiology of human organs. These systems also have potential as a translational tool in tissue engineering; however, this potential is limited by our ability to longitudinally monitor the fate and action of cells used in regenerative therapies. Therefore, we investigated luciferase-mediated bioluminescence imaging (BLI) as a non-invasive technique to continuously monitor cellular behavior in ex vivo whole organ culture. Goat adipose-derived stem cells (gADSCs) were transduced with either Firefly luciferase (Fluc) or Gaussia luciferase (Gluc) reporter genes and injected in isolated goat intervertebral discs (IVD). Luciferase activity was monitored by BLI for at least seven days of culture. Additionally, possible confounders specific to avascular organ culture were investigated. Gluc imaging proved to be more suitable compared to Fluc in monitoring gADSCs in goat IVDs. We conclude that BLI is a promising tool to monitor spatial and temporal cellular behavior in ex vivo organ culture. Hence, ex vivo organ culture systems allow pre-screening and pre-validation of novel therapeutic concepts prior to in vivo large animal experimentation. Thereby, organ culture systems can reduce animal use, and improve the speed of innovation by overcoming technological, ethical and financial challenges

Bioluminescence-mediated longitudinal monitoring of adipose-derived stem cells in a large mammal ex vivo organ culture

Recently, ex vivo three-dimensional organ culture systems have emerged to study the physiology and pathophysiology of human organs. These systems also have potential as a translational tool in tissue engineering; however, this potential is limited by our ability to longitudinally monitor the fate and action of cells used in regenerative therapies. Therefore, we investigated luciferase-mediated bioluminescence imaging (BLI) as a non-invasive technique to continuously monitor cellular behavior in ex vivo whole organ culture. Goat adipose-derived stem cells (gADSCs) were transduced with either Firefly luciferase (Fluc) or Gaussia luciferase (Gluc) reporter genes and injected in isolated goat intervertebral discs (IVD). Luciferase activity was monitored by BLI for at least seven days of culture. Additionally, possible confounders specific to avascular organ culture were investigated. Gluc imaging proved to be more suitable compared to Fluc in monitoring gADSCs in goat IVDs. We conclude that BLI is a promising tool to monitor spatial and temporal cellular behavior in ex vivo organ culture. Hence, ex vivo organ culture systems allow pre-screening and pre-validation of novel therapeutic concepts prior to in vivo large animal experimentation. Thereby, organ culture systems can reduce animal use, and improve the speed of innovation by overcoming technological, ethical and financial challenge

EZH2-Regulated DAB2IP Is a Medulloblastoma Tumor Suppressor and a Positive Marker for Survival

Purpose: Medulloblastoma is the most common malignant brain tumor in children. Despite recent improvements, the molecular mechanisms driving medulloblastoma are not fully understood and further elucidation could provide cues to improve outcome prediction and therapeutic approaches. Experimental Design: Here, we conducted a meta-analysis of mouse and human medulloblastoma gene expression data sets, to identify potential medulloblastoma tumor suppressor genes. Results: We identified DAB2IP, a member of the RAS-GTPase-activating protein family (RAS GAP), and showed that DAB2IP expression is repressed in medulloblastoma by EZH2-induced trimethylation. Moreover, we observed that reduced DAB2IP expression correlates significantly with a poor overall survival of patients with medulloblastoma, independent of metastatic stage. Finally, we showed that ectopic DAB2IP expression enhances stress-induced apoptosis in medulloblastoma cells and that reduced expression of DAB2IP in medulloblastoma cells conveys resistance to irradiation-induced cell death. Conclusion: These results suggest that repression of DAB2IP may at least partly protect medulloblastoma cells from apoptotic cell death. Moreover, DAB2IP may represent a molecular marker to distinguish patients with medulloblastoma at high risk from those with a longer survival prognosis. Clin Cancer Res; 18(15); 4048-58. (C)2012 AAC

Supplemental data for Blood platelets contain tumor-derived RNA biomarkers

We report on the growth mechanism of spherical silicon nanocrystals in a remote expanding Ar plasma using a time-modulated SiH4 gas injection in the microsecond time range. Under identical time-modulation parameters, we varied the local density of the SiH4 gas by changing its stagnation pressure on the injection line over the range of 0.1–2.0 bar. We observed that nanocrystals were synthesized in a size range from ∼2 to ∼50 nm with monocrystalline morphology. Smaller nanocrystals (∼2–6 nm) with narrower size distributions and with higher number densities were synthesized with an increase of the SiH4 gas-phase density. We related this observation to the rapid depletion of the number density of the molecules, ions, and radicals in the plasma during nanocrystal growth, which can primarily occur via nucleation with no significant subsequent coagulation. In addition, in our remote plasma environment, rapid cooling of the gas in the particle growth zone from ∼1500 to ∼400 K significantly reduces the coalescence..

A prediction model for response to immune checkpoint inhibition in advanced melanoma

Predicting who will benefit from treatment with immune checkpoint inhibition (ICI) in patients with advanced melanoma is challenging. We developed a multivariable prediction model for response to ICI, using routinely available clinical data including primary melanoma characteristics. We used a population-based cohort of 3525 patients with advanced cutaneous melanoma treated with anti-PD-1-based therapy. Our prediction model for predicting response within 6 months after ICI initiation was internally validated with bootstrap resampling. Performance evaluation included calibration, discrimination and internal-external cross-validation. Included patients received anti-PD-1 monotherapy (n = 2366) or ipilimumab plus nivolumab (n = 1159) in any treatment line. The model included serum lactate dehydrogenase, World Health Organization performance score, type and line of ICI, disease stage and time to first distant recurrence-all at start of ICI-, and location and type of primary melanoma, the presence of satellites and/or in-transit metastases at primary diagnosis and sex. The over-optimism adjusted area under the receiver operating characteristic was 0.66 (95% CI: 0.64-0.66). The range of predicted response probabilities was 7%-81%. Based on these probabilities, patients were categorized into quartiles. Compared to the lowest response quartile, patients in the highest quartile had a significantly longer median progression-free survival (20.0 vs 2.8 months; P &lt; .001) and median overall survival (62.0 vs 8.0 months; P &lt; .001). Our prediction model, based on routinely available clinical variables and primary melanoma characteristics, predicts response to ICI in patients with advanced melanoma and discriminates well between treated patients with a very good and very poor prognosis.</p