An Integrated Next-Generation Sequencing System for Analyzing DNA Mutations, Gene Fusions, and RNA Expression in Lung Cancer.

We developed and characterized a next-generation sequencing (NGS) technology for streamlined analysis of DNA and RNA using low-input, low-quality cancer specimens. A single-workflow, targeted NGS panel for non-small cell lung cancer (NSCLC) was designed covering 135 RNA and 55 DNA disease-relevant targets. This multiomic panel was used to assess 219 formalin-fixed paraffin-embedded NSCLC surgical resections and core needle biopsies. Mutations and expression phenotypes were identified consistent with previous large-scale genomic studies, including mutually exclusive DNA and RNA oncogenic driver events. Evaluation of a second cohort of low cell count fine-needle aspirate smears from the BATTLE-2 trial yielded 97% agreement with an independent, validated NGS panel that was used with matched surgical specimens. Collectively, our data indicate that broad, clinically actionable insights that previously required independent assays, workflows, and analyses to assess both DNA and RNA can be conjoined in a first-tier, highly multiplexed NGS test, thereby providing faster, simpler, and more economical results.

Design, Optimization, and Multisite Evaluation of a Targeted Next-Generation Sequencing Assay System for Chimeric RNAs from Gene Fusions and Exon-Skipping Events in Non-Small Cell Lung Cancer.

Lung cancer accounts for approximately 14% of all newly diagnosed cancers and is the leading cause of cancer-related deaths. Chimeric RNA resulting from gene fusions (RNA fusions) and other RNA splicing errors are driver events and clinically addressable targets for non-small cell lung cancer (NSCLC). The reliable assessment of these RNA markers by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We describe the development and verification of a system for targeted RNA sequencing for the analysis of challenging, low-input solid tumor biopsies that includes reagents for nucleic acid quantification and library preparation, run controls, and companion bioinformatics software. Assay development reconciled sequence discrepancies in public databases, created predictive formalin-fixed, paraffin-embedded RNA qualification metrics, and eliminated read misidentification attributable to index hopping events on the next-generation sequencing flow cell. The optimized and standardized system was analytically verified internally and in a multiphase study conducted at five independent laboratories. The results show accurate, reproducible, and sensitive detection of RNA fusions, alternative splicing events, and other expression markers of NSCLC. This comprehensive approach, combining sample quantification, quality control, library preparation, and interpretive bioinformatics software, may accelerate the routine implementation of targeted RNA sequencing of formalin-fixed, paraffin-embedded samples relevant to NSCLC.

Static and cyclic mechanical loading of mesenchymal stem cells on elastomeric, electrospun polyurethane meshes.

Biomaterial substrates composed of semi-aligned electrospun fibers are attractive supports for the regeneration of connective tissues because the fibers are durable under cyclic tensile loads and can guide cell adhesion, orientation, and gene expression. Previous studies on supported electrospun substrates have shown that both fiber diameter and mechanical deformation can independently influence cell morphology and gene expression. However, no studies have examined the effect of mechanical deformation and fiber diameter on unsupported meshes. Semi-aligned large (1.75 µm) and small (0.60 µm) diameter fiber meshes were prepared from degradable elastomeric poly(esterurethane urea) (PEUUR) meshes and characterized by tensile testing and scanning electron microscopy (SEM). Next, unsupported meshes were aligned between custom grips (with the stretch axis oriented parallel to axis of fiber alignment), seeded with C3H10T1/2 cells, and subjected to a static load (50 mN, adjusted daily), a cyclic load (4% strain at 0.25 Hz for 30 min, followed by a static tensile loading of 50 mN, daily), or no load. After 3 days of mechanical stimulation, confocal imaging was used to characterize cell shape, while measurements of deoxyribonucleic acid (DNA) content and messenger ribonucleic acid (mRNA) expression were used to characterize cell retention on unsupported meshes and expression of the connective tissue phenotype. Mechanical testing confirmed that these materials deform elastically to at least 10%. Cells adhered to unsupported meshes under all conditions and aligned with the direction of fiber orientation. Application of static and cyclic loads increased cell alignment. Cell density and mRNA expression of connective tissue proteins were not statistically different between experimental groups. However, on large diameter fiber meshes, static loading slightly elevated tenomodulin expression relative to the no load group, and tenascin-C and tenomodulin expression relative to the cyclic load group. These results demonstrate the feasibility of maintaining cell adhesion and alignment on semi-aligned fibrous elastomeric substrates under different mechanical conditions. The study confirms that cell morphology is sensitive to the mechanical environment and suggests that expression of select connective tissue genes may be enhanced on large diameter fiber meshes under static tensile loads.

Electrospun fibre diameter, not alignment, affects mesenchymal stem cell differentiation into the tendon/ligament lineage.

Efforts to develop engineered tendons and ligaments have focused on the use of a biomaterial scaffold and a stem cell source. However, the ideal scaffold microenvironment to promote stem cell differentiation and development of organized extracellular matrix is unknown. Through electrospinning, fibre scaffolds can be designed with tailorable architectures to mimic the intended tissue. In this study, the effects of fibre diameter and orientation were examined by electrospinning thin mats, consisting of small (< 1 µm), medium (1-2 µm) or large (> 2 µm) diameter fibres with either random or aligned fibre orientation. C3H10T1/2 model stem cells were cultured on the six different electrospun mats, as well as smooth spin-coated films, and the morphology, growth and expression of tendon/ligament genes were evaluated. The results demonstrated that fibre diameter affects cellular behaviour more significantly than fibre alignment. Initially, cell density was greater on the small fibre diameter mats, but similar cell densities were found on all mats after an additional week in culture. After 2 weeks, gene expression of collagen 1α1 and decorin was increased on all mats compared to films. Expression of the tendon/ligament transcription factor scleraxis was suppressed on all electrospun mats relative to spin-coated films, but expression on the large-diameter fibre mats was consistently greater than on the medium-diameter fibre mats. These results suggest that larger-diameter fibres (e.g. > 2 µm) may be more suitable for in vitro development of a tendon/ligament tissue.

Bioreactor system using noninvasive imaging and mechanical stretch for biomaterial screening.

Screening of biomaterial and tissue systems in vitro, for guidance of performance in vivo, remains a major requirement in the field of tissue engineering. It is critical to understand how culture stimulation affects both tissue construct maturation and function, with the goal of eliminating resource-intensive trial-and-error screening and better matching specifications for various in vivo needs. In this article a multifunctional and robust bioreactor design that addresses this need is presented. The design enables a range of mechanical inputs, durations, and frequencies to be applied in coordination with noninvasive optical assessments. A variety of biomaterial systems, including micro- and nano-fiber and porous sponge biomaterials, as well as cell-laden tissue engineering constructs were used in validation studies to demonstrate the versatility and utility of this new bioreactor design. The silk-based biomaterials highlighted in these studies offered several unique optical signatures for use in label-free nondestructive imaging that allowed for sequential profiling. Both short- and long-term culture studies were conducted to evaluate several practical scenarios of usage: on a short-term basis, the authors demonstrate that construct cellularity can be monitored by usage of nonpermanent dyes; on a more long-term basis, the authors show that cell ingrowth can be monitored by green-fluorescent protein (GFP)-labeling, and construct integrity probed with concurrent load/displacement data. The ability to nondestructively track cells, biomaterials, and new matrix formation without harvesting designated samples at each time point will lead to less resource-intensive studies and should enhance our understanding and the discovery of biomaterial designs related to functional tissue engineering.