Tool For Translational Research Research Articles

OMIXCARE: OMICS technologies solved about 33% of the patients with heterogeneous rare neuro-developmental disorders and negative exome sequencing results and identified 13% additional candidate variants.

Purpose: Patients with rare or ultra-rare genetic diseases, which affect 350 million people worldwide, may experience a diagnostic odyssey. High-throughput sequencing leads to an etiological diagnosis in up to 50% of individuals with heterogeneous neurodevelopmental or malformation disorders. There is a growing interest in additional omics technologies in translational research settings to examine the remaining unsolved cases. Methods: We gathered 30 individuals with malformation syndromes and/or severe neurodevelopmental disorders with negative trio exome sequencing and array comparative genomic hybridization results through a multicenter project. We applied short-read genome sequencing, total RNA sequencing, and DNA methylation analysis, in that order, as complementary translational research tools for a molecular diagnosis. Results: The cohort was mainly composed of pediatric individuals with a median age of 13.7years (4years and 6months to 35years and 1month). Genome sequencing alone identified at least one variant with a high level of evidence of pathogenicity in 8/30 individuals (26.7%) and at least a candidate disease-causing variant in 7/30 other individuals (23.3%). RNA-seq data in 23 individuals allowed two additional individuals (8.7%) to be diagnosed, confirming the implication of two pathogenic variants (8.7%), and excluding one candidate variant (4.3%). Finally, DNA methylation analysis confirmed one diagnosis identified by genome sequencing (Kabuki syndrome) and identified an episignature compatible with a BAFopathy in a patient with a clinical diagnosis of Coffin-Siris with negative genome and RNA-seq results in blood. Conclusion: Overall, our integrated genome, transcriptome, and DNA methylation analysis solved 10/30 (33.3%) cases and identified a strong candidate gene in 4/30 (13.3%) of the patients with rare neurodevelopmental disorders and negative exome sequencing results.

Characterization of a new set of monoclonal β-amyloid antibodies

β-Amyloid (Aβ) deposition is a commonly studied neuropathology in the human brain, occurring as compact and diffuse parenchymal plaques, cerebral amyloid angiopathy (CAA), and meningeal/subpial amyloidosis. Compact plaques associated with dystrophic neurites generally referred to as neuritic plaques, are a pathological hallmark of Alzheimer's disease (AD). We evaluated three recently developed monoclonal mouse antibodies against Aβ (A1, A2 and A3) using appropriate tissue samples and assay controls in the present study. In immunohistochemistry, antibodies A1, A2 and A3 displayed all forms of cerebral Aβ deposition in a range of dilutions in cryostat and paraffin sections. These labeled profiles appeared morphologically comparable to that visualized by two commercial Aβ antibody clones, 6E10 and D12B2. In immune-dot blotting assays, antibodies A1, A2 and A3 at highly diluted concentrations detected an increase of Aβ in neocortical lysates of AD samples compared to control. Moreover, these antibodies clearly labeled Aβ pathology in brain sections of three commonly used transgenic mouse models of AD, namely, APP/PS1 mice, 5XFAD mice, and 3XTg-AD mice. Taken together, these monoclonal mouse anti-Aβ antibodies can serve as new experimental tools for basic, translational, and diagnostic research into aging and AD-related cerebral Aβ neuropathology in both human and experimental animal brains.

Patient-derived intrafemoral orthotopic xenografts of peripheral blood or bone marrow from acute myeloid and acute lymphoblastic leukemia patients: clinical characterization, methodology, and validation

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are malignant clonal diseases of the hematopoietic system with an unsatisfactory overall prognosis. The main obstacle is the increased resistance of AML and ALL cells to chemotherapy. The development and validation of new therapeutic strategies for acute leukemia require preclinical models that accurately recapitulate the genetic, pathological, and clinical features of acute leukemia. A patient-derived orthotopic xenograft (PDOX) model is established using surgical orthotopic implantation. They closely resemble human tumor progression and microenvironment and are more reliable translational research tools than subcutaneous-transplant models. In this study, we established PDOX models by direct intrafemoral injection of bone marrow and peripheral blood cells from AML and ALL patients, characterized their pathology, cytology, and genetics, and compared the model's characteristics and drug responsiveness with those of the corresponding patients.

Combined Systemic Drug Treatment with Proton Therapy: Investigations on Patient-Derived Organoids.

Simple SummaryRadiotherapy is part of the standard of care for many solid tumors. In pancreatic ductal adenocarcinoma (PDAC), good responses to radiotherapy can only be observed in a minority of patients. In our study, we used PDAC patient-derived organoids (PDO) to investigate alternative radiotherapy approaches for PDAC, such as proton irradiation and combined radiochemotherapy (RCT). Although only very distinct differences in treatment response could be identified, we show the utility of PDOs in translational proton research and found synergistic effects of combined treatments with chemotherapy and proton irradiation in individual PDOs.To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.

Structural insights from G-protein-coupled receptor complexes enable the rational engineering of improved light-activated designer receptors

Engineered signaling proteins permit precise modulation of cell signaling networks and are valuable tools for basic and translational research. In this issue of Structure, Tichy and colleagues leverage high-resolution GPCR-G protein complex structures to rationally design improved light-activated chimeric GPCRs (termed OptoXRs) with increased sensitivity and tunable signaling features.

Developing a translational triage research tool: part two—evaluating the tool through a Delphi study among experts

BackgroundThere are different prehospital triage systems, but no consensus on what constitutes the optimal choice. This heterogeneity constitutes a threat in a mass casualty incident in which triage is used during multiagency collaboration to prioritize casualties according to the injuries’ severity. A previous study has confirmed the feasibility of using a Translational Triage Tool consisting of several steps which translate primary prehospital triage systems into one. This study aims to evaluate and verify the proposed algorithm using a panel of experts who in their careers have demonstrated proficiency in triage management through research, experience, education, and practice.MethodSeveral statements were obtained from earlier reports and were presented to the expert panel in two rounds of a Delphi study.ResultsThere was a consensus in all provided statements, and for the first time, the panel of experts also proposed the manageable number of critical victims per healthcare provider appropriate for proper triage management.ConclusionThe feasibility of the proposed algorithm was confirmed by experts with some minor modifications. The utility of the translational triage tool needs to be evaluated using authentic patient cards used in simulation exercises before being used in actual triage scenarios.

Ultrahigh resolution whole body photon counting computed tomography as a novel versatile tool for translational research from mouse to man

X-ray computed tomography (CT) is a cardinal tool in clinical practice. It provides cross-sectional images within seconds. The recent introduction of clinical photon-counting CT allowed for an increase in spatial resolution by more than a factor of two resulting in a pixel size in the center of rotation of about 150 µm. This level of spatial resolution is in the order of dedicated preclinical micro-CT systems. However so far, the need for different dedicated clinical and preclinical systems often hinders the rapid translation of early research results to applications in men. This drawback might be overcome by ultra-high resolution (UHR) clinical photon-counting CT unifying preclinical and clinical research capabilities in a single machine. Herein, the prototype of a clinical UHR PCD CT (SOMATOM CounT, Siemens Healthineers, Forchheim, Germany) was used. The system comprises a conventional energy-integrating detector (EID) and a novel photon-counting detector (PCD). While the EID provides a pixel size of 0.6 mm in the centre of rotation, the PCD provides a pixel size of 0.25 mm. Additionally, it provides a quantification of photon energies by sorting them into up to four distinct energy bins. This acquisition of multi-energy data allows for a multitude of applications, e.g. pseudo-monochromatic imaging. In particular, we examine the relation between spatial resolution, image noise and administered radiation dose for a multitude of use-cases. These cases include ultra-high resolution and multi-energy acquisitions of mice administered with a prototype bismuth-based contrast agent (nanoPET Pharma, Berlin, Germany) as well as larger animals and actual patients. The clinical EID provides a spatial resolution of about 9 lp/cm (modulation transfer function at 10%, MTF10%) while UHR allows for the acquisition of images with up to 16 lp/cm allowing for the visualization of all relevant anatomical structures in preclinical and clinical specimen. The spectral capabilities of the system enable a variety of applications previously not available in preclinical research such as pseudo-monochromatic images. Clinical ultra-high resolution photon-counting CT has the potential to unify preclinical and clinical research on a single system enabling versatile imaging of specimens and individuals ranging from mice to man.

Microscopic modulation and analysis of islets of Langerhans in living zebrafish larvae.

Microscopic analysis of molecules and physiology in living cells and systems is a powerful tool in life sciences. While in vivo subcellular microscopic analysis of healthy and diseased human organs remains impossible, zebrafish larvae allow studying pathophysiology of many organs using in vivo microscopy. Here, we review the potential of the larval zebrafish pancreas in the context of islets of Langerhans and Type 1 diabetes. We highlight the match of zebrafish larvae with the expanding toolbox of fluorescent probes that monitor cell identity, fate and/or physiology in real time. Moreover, fast and efficient modulation and localization of fluorescence at a subcellular level, through fluorescence microscopy, including confocal and light sheet (single plane illumination) microscopes tailored to in vivo larval research, is addressed. These developments make the zebrafish larvae an extremely powerful research tool for translational research. We foresee that living larval zebrafish models will replace many cell line-based studies in understanding the contribution of molecules, organelles and cells to organ pathophysiology in whole organisms.

Abstract 6373: Spheroid culture of ER+ breast cancer patient-derived tumor cells enriches cancer stem-likecells with EpCAM-/CD49f+and high ALDH activity and poor prognostic gene signatures

Abstract Background: Cancer stem-like cells (CSCs) represent a critical subset of the tumor population, which contributes to tumor recurrence, metastasis and therapy resistance. Spheroid culture is unique method for CSC enrichment. Patient-derived breast cancer cells (PDBCCs) has provided an invaluable tool in preclinical and translational research. The objective of this study is to characterize the immunophenotype and transcriptomes of PDBCCs grown in the adherent monolayer and spheroid condition. Methods: PDBCCs were harvested from residual tumor of fresh surgical specimens of patients with ER-positive subtype (8 cases). The adherent monolayer(M) and spheroid(S) culture of PDBCCs were applied on collagen I-coated plate and an ultra-low attachment polymer-X coated plate. Flow cytometry and immunocytochemistry was performed by using fluorescent-dye conjugated antibodies for EpCAM, CD49f, CD24, CD44, and cytokeratin. ALDEFLUOR® assay to detect ALDH activity was used. Total RNA-sequencing was applied to investigate differentially expressed genes (DEGs), followed by GO and KEGG pathway enrichment analysis. Real-time RT-PCR was performed to evaluate BCSC-related RNA levels. The Kaplan-Meier Plotter online database was used to evaluate the prognostic value of DEGs (2-fold change, p<0.05). Results: Under adherent monolayer condition, more than 95% of PDBCCs passaged for up to 5 passages on collagen I-coated plate sustained EpCAM+/cytokeratin+/fibroblast marker_ phenotype. On polymer-X coated plates, PDBCCs formed compact multicellular spheroids with a diameter of less than 300 µm. As compared with monolayer, CSCs with EpCAM-/CD49f+ phenotype (M vs S; 0.16±0.06 vs 2.34±0.65, p=0.015) and high ALDH activity (M vs S; 0.73±0.43 vs 7.05±1.48, p=0.038) were significantly increased by spheroid culture. In whole-transcriptome analysis (3 cases) between spheroid and monolayer, a total of 561 DEGs were identified, including 290 upregulated and 271 downregulated DEGs (2- fold change, p<0.05) in spheroid. The upregulated and downregulated DEGs in spheroids were enriched in 8 and 19 KEGG pathways, respectively. In recurrence-free survival analysis based on the Kaplan-Meier Plotter database of the genes corresponding to Oncotype Dx, Mammaprint, and Prosigna and top 30 ranked DEGs identified in spheroids, 15 upregulated and 14 downregulated DEGs were associated with poor prognosis of breast cancer patients. Conclusion: Spheroid culture of PDBCCs with ER+ subtype enriches CSCs and poor prognostic gene signatures. Future studies to explore the specific molecular mechanisms underlying these observations may provide new molecular targets for the development of therapies for targeting CSCs in breast cancer patient. Citation Format: Hoe Suk Kim, Seungyeon Ryu, So-Hyun Yoon, Sangeun Lee, Junhyuk Song, Yoonjung Choi, Moonjou Baek, Han-Byoel Lee, Sangyong Jon, Daeyoup Lee, Wonshik Han. Spheroid culture of ER+ breast cancer patient-derived tumor cells enriches cancer stem-likecells with EpCAM-/CD49f+and high ALDH activity and poor prognostic gene signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6373.

Abstract 3039: Decreased pH-sensitive potassium channel gene expressions in glioblastomas compared to oligodendrogliomas detected with house keeping genes may shift glioblastoma membrane potentials towards proton efflux to the microenvironment

Abstract Glial buffering of K+ prevents seizures, hence gliomas in patients on anticonvulsants may harbor potassium related anomalies. Cell membrane potentials are very sensitive to K+ and may link to tumor acidosis when they become more positive than the equilibrium potential for H+ ions (Vaupel, P, et al., Cancer Res, 1989). Kir5.1(KCNJ16) heterodimerizes with Kir4.1(KCNJ10), a major glial potassium channel, to stop K+ conductance in acidosis. Trek1&2(KCNK2&10) are also pH sensitive K+ channels. Glioblastomas (more malignant and invasive) versus oligodendrogliomas showed decreased KCNJ16 in patients on anticonvulsants (Beckner, ME, Proc AACR, A2319, 2021). However, loss of genomic integrity in tumors and potential variation between assays prompted house keeping gene (HKG) normalization to lessen confounding factors. REMBRANDT (12/31/2020, Georgetown Database of Cancer) had 220 glioblastomas and 67 oligodendrogliomas with microarray gene expression via medians of each reporter (1-8 per gene) in Adobe Flash readouts (ended 1/1/21). A subgroup, 75 glioblastomas and 24 oligodendroglioma patients, took anticonvulsants. Comparison of genes of interest (GOI) initially had t tests between types of gliomas in the subgroup. Here, differences between glioma types for each GOI reporter were compared to differences for HKGs (PPIA, RPLP0, YWHAZ, and B2M). In gliomas with anticonvulsants, paired 2-tailed t tests for a null difference of median reporters between tumor types were less sensitive than new comparisons of oligodendroglioma minus glioblastoma (OMG) differences for GOIs versus OMG differences in HKGs. For KCNJ16, the t test, p = 0.015, for a decrease in glioblastomas (versus oligodendrogliomas) in initial studies improved to p ≤ 0.0001 by using HKGs in comparisons. The KCNK2&10 decrease in glioblastomas became significant, p = 0.047. Among other glycolytic and acidosis related genes, CA12, GLO1&2, PFKFB1&3, showed stronger trends, with p values from 0.08 to 0.13 (decreases and increase) in comparisons using HKGs. Conclusion: HKG normalization presumably corrects for loss of genomic integrity, assay variations, etc. in comparisons using microarrays of different tumors. The pH-sensitive K+ channel genes studied here were expressed less in glioblastomas. The resulting pH-insensitive K+ distributions in glioblastomas putatively shift their cell membrane potentials to less negative values which are sufficient to allow passive H+ efflux that causes extracellular acidosis with accompanying activation of invasion. Acknowledgement: The data utilized in this study were provided by the Georgetown Database of Cancer (G-DOC), a project of the Georgetown Lombardi Comprehensive Cancer Center designed to provide translational research tools to the scientific community. Citation Format: Marie E. Beckner. Decreased pH-sensitive potassium channel gene expressions in glioblastomas compared to oligodendrogliomas detected with house keeping genes may shift glioblastoma membrane potentials towards proton efflux to the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3039.

Overall Survival in Phase 3 Clinical Trials and the Surveillance, Epidemiology, and End Results Database in Patients With Metastatic Colorectal Cancer, 1986-2016

Phase 3 trials for patients with metastatic colorectal cancer (mCRC) have been conducted with varying designs and often with surrogate end points for overall survival (OS). To critically examine the factors associated with clinically relevant improvement in OS (defined as ≥2 months) in these trials and to evaluate their association with outcomes reflected in Surveillance, Epidemiology, and End Results (SEER) registry data. Medline, EMBASE, Cochrane, Web of Science, ClinicalTrials.gov, EU Clinical Trials Register, and the International Clinical Trials Registry Platform were searched for phase 3 trials of systemic therapy for patients with mCRC by decade (1986-1996, 1997-2006, and 2007-2016), excluding early or pilot studies, studies that did not involve an anticancer drug, studies on cancer screening and prevention, reports of pooled data from multiple trials, and studies with nonpharmaceutical approaches. The association of drug development with OS outside the clinical trial setting was evaluated using data from the SEER registry, including adult patients with a primary cancer site in the colon or rectum, including adenocarcinoma, mucinous adenocarcinoma, or signet ring cell carcinoma; a distant stage; and receipt of chemotherapy as first-line therapy. Kaplan-Meier curves and log-rank tests were used to assess OS. The literature search identified 150 phase III clinical trials with 77 494 total enrollments, and 67 126 patients with mCRC were identified from the SEER database. Significant increases in survival were noted over time, best reflected in the experimental arm of first-line therapy (OS increased by 5.7 months per 10 years; 95% CI, 4.7-6.6 months; progression-free survival increased by 1.4 months per 10 years; 95% CI, 0.7-2.1 months). Although 69 of 148 trials (46.6%) met their predefined primary end point (including 20 of 44 trials [45.5%] with OS as the primary end point), only 35 of 132 trials (26.5%) resulted in improvement in OS by 2 months or more (including 13 of 42 trials [31.0%] with OS as the primary end point). Multivariable logistic regression showed that third-line therapies or later (odds ratio, 0.57; 95% CI, 0.51-0.63) and funding by pharmaceutical companies (odds ratio, 0.57; 95% CI, 0.54-0.60) were less often associated with improvement in OS. Furthermore, there was a decrease in the novelty of targets and agents over time, with trials that evaluated regimens composed entirely of previously approved drugs for mCRC increasing from 28% to 50%. Data from the SEER database showed that median OS increased from 12 months (95% CI, 12-13 months) (1986-1996) to 21 months (95% CI, 21-22 months) (2007-2015) (P < .001), but the 5-year OS continued to be low at 12.2% in 2011. In this systematic review, OS for patients with mCRC appeared to improve significantly in trials, translating into meaningful benefits outside the clinical trial setting; however, these advances, although significant cumulatively, are largely incremental individually. These data should be a call to aim for larger gains from future trials with novel drugs, building on the increasing understanding of the biology of mCRC and sophisticated translational research tools.

Endometrial Cancer Patient-Derived Xenograft Models: A Systematic Review.

Background: Because patient-derived xenograft (PDX) models resemble the original tumors, they can be used as platforms to find target agents for precision medicine and to study characteristics of tumor biology such as clonal evolution and microenvironment interactions. The aim of this review was to identify articles on endometrial cancer PDXs (EC-PDXs) and verify the methodology and outcomes. Methods: We used PubMed to research and identify articles on EC-PDX. The data were analyzed descriptively. Results: Post literature review, eight studies were selected for the systematic review. Eighty-five EC-PDXs were established from 173 patients with EC, with a total success rate of 49.1%. A 1–10 mm3 fragment was usually implanted. Fresh-fragment implantation had higher success rates than using overnight-stored or frozen fragments. Primary tumors were successfully established with subcutaneous implantation, but metastasis rarely occurred; orthotopic implantation via minced tumor cell injection was better for metastatic models. The success rate did not correspond to immunodeficiency grades, and PDXs using nude mice reduced costs. The tumor growth period ranged from 2 weeks to 13 months. Similar characteristics were observed between primary tumors and PDXs, including pathological findings, gene mutations, and gene expression. Conclusion: EC-PDXs are promising tools for translational research because they closely resemble the features of tumors in patients and retain molecular and histological features of the disease.

Making connections: using anatomy to guide tissue engineering approaches at the enthesis.

The enthesis demonstrates a distinct highly ordered zonal microanatomy at the osteotendinous/osteoligamentous tissue connection that allows for the smooth transmission of mechanical forces between tissues. Interfacial tissue engineering (ITE), a subset of the interdisciplinary field of tissue engineering, is directed at replicating this complex transitional anatomy of the enthesis in vitro. Yet, the limited understanding of tissue boundaries, gradients and structural relationships at specific anatomical locations hampers the development of novel therapeutic strategies for bespoke enthesis regeneration, thus reducing their direct clinical applicability. This review provides an overview of ITE approaches for repair of the osteotendinous/osteoligamentous junction and highlights the importance of complementary inclusion of direct anatomical research. The cross-disciplinary collaboration across an array of experts, including anatomists, involved in the design, development and utilisation of bioengineered tissues will enhance the properties of such tissues and improve their clinical relevance. More specifically, a detailed anatomical analysis of the region of interest should drive the in vitro design and enable researchers to develop anatomically and clinically relevant tissue-engineered replacement tissues for human implantation. Finally, the present review discusses the challenges and future directions of the ITE field and highlights the importance of anatomically driven tissue engineering as an emerging tool in clinical translational research.

Asia-Inclusive Clinical Research and Development Enabled by Translational Science and Quantitative Clinical Pharmacology: Toward a Culture That Challenges the Status Quo.

Access lag to innovative therapies in Asian populations continues to present a challenge to global health. Recent progressive changes in the global regulatory landscape, including newer guidelines, are enabling simultaneous global drug development and near-simultaneous global drug registration. The International Conference on Harmonization (ICH) E17 guideline outlines general principles for the design and analysis of multiregional clinical trials (MRCTs). We posit that translational research and quantitative clinical pharmacology tools are core enablers for Asia-inclusive global drug development aligned with ICH E17 principles. Assessment of ethnic sensitivity should be initiated early in the development lifecycle to inform the need for, and extent of, Asian phase I ethno-bridging data. Relevant ethno-bridging data may be generated as standalone Asian phase I trials, as part of Western First-In-Human trials, or under accelerated development settings as a lead-in phase in an MRCT. Quantitative understanding of human clearance mechanisms and pharmacogenetic factors is vital to forecasting ethnic sensitivity in drug exposure using physiologically-based pharmacokinetic models. Stratification factors to control heterogeneity in MRCTs can be identified by reverse translational research incorporating pharmacometric disease models and model-based meta-analyses. Because epidemiological variations can extend to the molecular level, quantitative systems pharmacology models may be useful in forecasting how molecular variation in therapeutic targets or pathway proteins across populations might impact treatment outcomes. Through prospective evaluation of conservation in drug- and disease-related intrinsic and extrinsic factors, a pooled East Asian region can be implemented in Asia-inclusive MRCTs to maximize efficiency in substantiating evidence of benefit-risk for the region at-large with a Totality of Evidence approach.

Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates.

Summary Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field‐based model species for genetics and epigenetics. The availability of a high‐quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome‐level genome assembly of var. MN106‐Ref with improved gene annotation and use it to investigate gene structure differences between two accessions (MN108 and Spring32‐10) that are highly amenable to genetic transformation. We describe non‐coding RNAs, pseudogenes and transposable elements, and highlight tissue‐specific expression and methylation patterns. Resequencing of forty wild accessions provided insights into genome‐wide genetic variation, and QTL regions were identified for a seedling colour phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

Human bronchospheres – A mirror of bronchiolar surfaces?

BackgroundFor translational research on lung diseases it is desirable to have human organoids which resemble features of human tissue. Adult stem cells obtained from human bronchial epithelium can be differentiated to little tissue follicles, so-called bronchospheres. Aim of the study was to characterize structural details of bronchospheres treated with control media, lipopolysaccharide, and flagellin during the differentiation phase. Basic proceduresHuman bronchial epithelial stem cells were prepared from surgical specimens and differentiated in Matrigel to bronchospheres. Histology and transmission electron microscopy revealed the cellular structure of the bronchospheres. Main findingsFollicles with a diameter of about 120 µm were confined by a bilayered epithelium with surface differentiation towards the inner lumen of the follicle and junctional complexes. Microvilli, kinocilia as well as secretory cells were found. Treatment with the bacterial flagellin resulted in the formation of secretory granules and inhibited the occurrence of kinocilia. Principal conclusionsDespite clear limitations such as lacking of smooth muscles and columnar epithelium human bronchospheres are an interesting tool for translational research. They can be useful for studies addressing developmental processes, infections, drug testing and toxicology.

The application of intestinal organoids and their co-culture systems in the study of gastrointestinal diseases

The 3-dimensional culture of intestinal organoids provides insights into the phenotype and physiology of the intestine. Intestinal organoids comprise intestinal stem cells and differentiated intestinal epithelial cells; they can be stably cultured over the long term and can be genetically manipulated. Current strategies for intestinal organoid co-culture with other cells existing in the intestinal tract and gut microbiota have been established to mimic the intestinal microenvironment and study host-microbial interactions. Therefore, intestinal organoids are promising tools for basic and translational research in gastroenterology. Gastrointestinal diseases are disorders of the intestinal tract that result in a reduced quality of life, and a deep understanding of these diseases would be effective for their treatment. In this review, we discuss how intestinal organoids and intestinal organoids integrated with cellular and microbiota niche components are biologically and physiologically relevant tools for the investigation of gastrointestinal diseases.

CRISPR DNA- and RNP-Mediated Genome Editing via Nicotiana benthamiana Protoplast Transformation and Regeneration.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated system) has become the multipurpose tool to manipulate plant genome via their programmable sequence recognition, binding, and cleavage activities. Efficient plant genome modification often requires robust plant transformation. For most plant species, the CRISPR/Cas reagents are delivered into plants as plasmids by Agrobacterium-mediated T-DNA transfer or biolistic approaches. However, these methods are generally inefficient, heavily genotype dependent, and low throughput. Among the alternative plant transformation approaches, the protoplast-based transformation holds the potential to directly deliver DNA, RNA, or protein molecules into plant cells in an efficient and high-throughput manner. Here, we presented a robust and simplified protocol for protoplast-based DNA/ribonucleoprotein (RNP )-mediated genome editing in the model species Nicotiana benthamiana. Using this protocol, we have achieved the gene editing efficiency at 30-60% in protoplasts and 50-80% in regenerated calli and plants. The edited protoplasts can be readily regenerated without selection agents owing to highly efficient DNA or preassembled RNP transformation frequency. Lastly, this protocol utilized an improved culture media regime to overcome the complex media composition used in the previous studies. It offers quick turnaround time and higher throughput to facilitate the development of new genetic engineering technologies and holds the promise to combine with other genetic and genomic tools for fundamental and translational plant research.

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice. Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo. In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

TB-8 Genetic and molecular properties of long-term proliferating tumorsphere -forming glioma derived cells

Long-term proliferating tumorsphere-forming glioma derived cells (LTP-TS-GDCs) and patient derived xenografts (PDXs) are essential tools for translational research for glioma. However, only small subsets of glioma samples are established as LTP-TS and/or PDXs and little is known about the genetics and molecular properties of LTP-TS -forming GDCs and PDX. In this study, we aim to analyze the characteristics of LTP-TS -forming GDCs and PDXs. We tried primary sphere cultures from 56 glioma patient-derived samples and established 11 LTP-TS-GDCs out of 45 glioblastoma samples and no long-term sphere culture was isolated from grade3 and grade 2 gliomas. LTP-TS-GDCs had self-renewal ability and possessed certain multipotency. However, they significantly less expressed SOX1 FOXG1 and TUBB3, whereas they expressed LGALS1 and EN1 significantly higher than normal neural stem/progenitor cells. In addition, we found that LTP-TS-GDCs shared the same genetic profiles with original patients’ tumors. Furthermore, we investigated the genetic differences between the glioma tissues which were successfully established as LTP-TS-GDCs and those which were not. We found that glioma tissues with TERT promotor mutations and triple copy number alteration (CNA) [EGFR, CDKN2A, and PTEN loci] are significantly established as LTP-TS-GDCs. Lastly, we next investigated in vivo characteristics of glioma PDXs. We have injected glioma PDXs lines into immunodeficient mice brains and histopathologically analyzed the characteristics of xenografts. Each xenograft well recapitulated histological features of original patients’ tumors and tumor cells remarkably invade through subventricular zone. In conclusion, each LTP-TS-GDCs and PDXs had various gene expression profiles, reflecting intratumoral and interpatient heterogeneities of glioma. In addition, TERT promotor mutations and triple CNA significantly correlated with success rate of LTP-TS-GDCs. These findings will be of use and advance the preclinical and translational researches of glioma.