Abstract
High-Throughput Biology
Human Primary Liver Cancer Organoids Reveal Intratumor and Interpatient Drug Response Heterogeneity
Liver cancer is the fourth leading cause of cancer-related mortality and is distinguished by a relative paucity of chemotherapy options. It has been hypothesized that intratumor genetic heterogeneity may contribute to the high failure rate of chemotherapy. Here, Li et al. evaluated functional heterogeneity in a cohort of primary human liver cancer organoid lines. Each primary human liver cancer surgical specimen was used to generate multiple cancer organoid lines, obtained from distinct regions of the tumor. A total of 27 liver cancer lines were established and tested with 129 cancer drugs, generating 3483 cell survival data points. The authors found a rich intratumor, functional (drug response) heterogeneity in their liver cancer patients. Furthermore, the authors established that most drugs were either ineffective or effective only in select organoid lines. In contrast, Li et al. found that a subset of drugs appeared pan-effective, displaying at least moderate activity in the majority of these cancer organoid lines. These drugs, which are FDA approved for indications other than liver cancers, deserve further consideration as either systemic or local therapeutics. Of note, molecular profiles, obtained for a reduced sample set, did not correlate with the drug response heterogeneity of liver cancer organoid lines. Taken together, these findings lay the foundation for in-depth studies of pan-effective drugs, as well as for functional personalized oncology approaches. Lastly, these functional studies demonstrate the utility of cancer organoid drug testing as part of a drug discovery pipeline. (Li, L.; et al. JCI Insight
Annotation of Gene Product Function from High-Throughput Studies Using the Gene Ontology
High-throughput studies constitute an essential and valued source of information for researchers. However, high-throughput experimental workflows are often complex, with multiple data sets that may contain large numbers of false positives. The representation of high-throughput data in the Gene Ontology (GO) therefore presents a challenging annotation problem, when the overarching goal of GO curation is to provide the most precise view of a gene’s role in biology. To address this, representatives from annotation teams within the GO Consortium reviewed high-throughput data annotation practices. Attrill et al. present an annotation framework for high-throughput studies that will facilitate good standards in GO curation and, using new high-throughput evidence codes, increase the visibility of these annotations to the research community. (Attrill, H.; et al. Database (Oxford)
Canvass: A Crowd-Sourced, Natural-Product Screening Library for Exploring Biological Space
Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, is outlined herein. One such highlight is the activity of (–)-2(S)-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening. (Kearney, S. E.; et al. ACS Cent. Sci.
An Automated Protoplast Transformation System
Efficient plant protoplast production from cell suspension cultures, leaf, and stem tissue allows for single-cell plant biology. Since protoplasts do not have cell walls, they can be readily transformed to enable rapid assessment of regulatory elements, synthetic constructs, gene expression, and more recently genome-editing tools and approaches. Historically, enzymatic cell wall digestion has been both expensive and laborious. Protoplast production, transformation, and analysis of fluorescence have recently been automated using an integrated robotic system. Here Lenaghan and Stewart describe its use for bulk protoplast isolation, counting, transformation, and analysis at very low cost for high-throughput experiments. (Lenaghan, S. C.; Stewart, N. C.; Jr. Methods Mol. Biol.
Genome-Wide Identification of Circulating-miRNA Expression Quantitative Trait Loci Reveals the Role of Several miRNAs in the Regulation of Cardiometabolic Phenotypes
Microfluidics
Advances in Isolation and Detection of Circulating Tumor Cells Based on Microfluidics
Circulating tumor cells (CTCs) are the cancer cells that circulate in the peripheral blood after escaping from the original or metastatic tumors. CTCs could be used as a noninvasive source of clinical information in the early diagnosis of cancer and evaluation of cancer development. In recent years, CTC research has become a hotspot field wherein many novel CTC detection technologies based on microfluidics have been developed. Great advances have been made that exhibit obvious technical advantages but cannot yet satisfy the current clinical requirements. In this study, Zou and Cui review the main advances in isolation and detection methods of CTC based on microfluidics research over several years, propose five technical indicators for evaluating these methods, and explore the application prospects. The authors also discuss the concepts, issues, approaches, advantages, limitations, and challenges with an aim of stimulating a broader interest in developing microfluidics-based CTC detection technology. (Zou, D.; Cui, D. Cancer Biol. Med.
A Rapid Methods Development Workflow for High-Throughput Quantitative Proteomic Applications
Recent improvements in the speed and sensitivity of liquid chromatography–mass spectrometry systems have driven significant progress toward system-wide characterization of the proteome of many species. These efforts create large proteomic datasets that provide insight into biological processes and identify diagnostic proteins whose abundance changes significantly under different experimental conditions. Yet, these system-wide experiments are typically the starting point for hypothesis-driven, follow-up experiments to elucidate the extent of the phenomenon or the utility of the diagnostic marker, wherein many samples must be analyzed. Transitioning from a few discovery experiments to quantitative analyses on hundreds of samples requires significant resources to both develop sensitive and specific methods and analyze them in a high-throughput manner. To aid these efforts, Chen et al. developed a workflow using data acquired from discovery proteomic experiments, retention time prediction, and standard-flow chromatography to rapidly develop targeted proteomic assays. The authors demonstrated this workflow by developing MRM assays to quantify proteins of multiple metabolic pathways from multiple microbes under different experimental conditions. With this workflow, one can also target peptides in scheduled/dynamic acquisition methods from a shotgun proteomic dataset downloaded from online repositories, validate with appropriate control samples or standard peptides, and begin analyzing hundreds of samples in only a few minutes. (Chen, Y.; et al. PLOS One
Engineering Microfluidic Organoid-on-a-Chip Platforms
In vitro cell culture models are emerging as promising tools to understand human development and disease progression and provide reliable, rapid, and cost-effective results for drug discovery and screening. In recent years, an increasing number of in vitro models with complex organization and controlled microenvironment have been developed to mimic the in vivo organ structure and function. The invention of organoids, self-organized organ-like cell aggregates that originate from multipotent stem cells, has allowed a whole new level of biomimicry to be achieved. Microfluidic organoid-on-a-chip platforms can facilitate better nutrient and gas exchange and recapitulate 3D tissue architecture and physiology. They have the potential to transform the landscape of drug development and testing. In this review, Yu et al. discuss the challenges in the current organoid models and describe the recent progress in the field of organoid-on-a-chip. (Yu, F.; et al. Micromachines (Basel)
New Dimensions in Cancer and Synthetic Biology
Revolutionizing Cancer Immunology: The Power of Next-Generation Sequencing Technologies
It has long been appreciated that tumors are diverse, varying in mutational status, composition of cellular infiltrate, and organizational architecture. For the most part, the information embedded in this diversity has gone untapped due to the limited resolution and dimensionality of assays for analyzing nucleic acid expression in cells. The advent of high-throughput, next-generation sequencing (NGS) technologies that measure nucleic acids, particularly at the single-cell level, is fueling the characterization of the many components that comprise the tumor microenvironment (TME), with a strong focus on immune composition. Understanding the immune and nonimmune components of the TME, how they interact, and how this shapes their functional properties requires the development of novel computational methods and, eventually, the application of systems-based approaches. The continued development and application of NGS technologies holds great promise for accelerating discovery in the cancer immunology field. (Singer, M.; Anderson, A. C. Cancer Immunol. Res.
Chromatin Reprogramming as an Adaptation Mechanism in Advanced Prostate Cancer
Tumor evolution is based on the ability to constantly mutate and activate different pathways under the selective pressure of targeted therapies. Epigenetic alterations including those of the chromatin structure are associated with tumor initiation, progression, and drug resistance. Many cancers, including prostate cancer, present enlarged nuclei, and chromatin appears altered and irregular. These phenotypic changes are likely to result from epigenetic dysregulation. High-throughput sequencing applied to bulk samples and now to single cells has made it possible to study these processes in unprecedented detail. It is therefore timely to review the impact of chromatin relaxation and increased DNA accessibility on prostate cancer growth and drug resistance, and their effects on gene expression. The authors focus on the contribution of chromatin-associated proteins such as the bromodomain-containing proteins to chromatin relaxation. The authors discuss the consequence of this for androgen receptor transcriptional activity and briefly summarize wider gain-of-function effects on other oncogenic transcription factors and implications for more effective prostate cancer treatment. (Braadland, P. R.; Urbanucci, A. Endocr. Relat. Cancer
Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii
Microalgae constitute a highly diverse group of eukaryotic and photosynthetic microorganisms that have developed extremely efficient systems for harvesting and transforming solar energy into energy-rich molecules such as lipids. Although microalgae are one of the most promising platforms for the sustainable production of liquid oil, the oil content of these organisms is naturally low, and algal oil production is currently not economically viable. Chlamydomonas reinhardtii (Chlamydomonas) is an established algal model due to its fast growth, high transformation efficiency, and well-understood physiology and to the availability of detailed genome information and versatile molecular tools for this organism. In this review, Kong et al. summarize recent advances in the development of genetic manipulation tools for Chlamydomonas, from gene delivery methods to state-of-the-art genome-editing technologies and fluorescent dye-based high-throughput mutant screening approaches. Furthermore, the authors discuss practical strategies and toolkits that enhance transgene expression, such as choice of expression vector and background strain. Kong et al. then provide examples of how advanced genetic tools have been used to increase oil content in Chlamydomonas. Collectively, the current literature indicates that microalgal oil content can be increased by overexpressing key enzymes that catalyze lipid biosynthesis, blocking lipid degradation, silencing metabolic pathways that compete with lipid biosynthesis, and modulating redox state. The tools and knowledge generated through metabolic engineering studies should pave the way for developing a synthetic biological approach to enhance lipid productivity in microalgae. (Kong, F.; et al. Plant Cell Physiol.
Next-Generation Sequencing-Based Gene Panel Tests for the Management of Solid Tumors
Next-generation sequencing (NGS) has been an invaluable tool to put genomic sequencing into clinical practice. The incorporation of clinically relevant target sequences into NGS-based gene panel tests has generated practical diagnostic tools that enable individualized cancer-patient care. The clinical utility of gene panel testing includes investigation of the genetic basis for an individual’s response to therapy, such as signaling pathways associated with a response to specific therapies, microsatellite instability and a hypermutated phenotype, and deficiency in the DNA double-strand break repair pathway. In this review, the authors describe the concept of precision cancer medicine using target sequences in gene panel tests, as well as the importance of the control of sample quality in routine NGS-based genomic testing. Nagahashi et al. describe geographic and ethnic differences in cancer genomes and discuss issues that need to be addressed in the future based on their experiences in Japan. (Nagahashi, M.; et al. Cancer Sci.
Deubiquitinases Maintain Protein Homeostasis and Survival of Cancer Cells upon Glutathione Depletion
Cells are subjected to oxidative stress during the initiation and progression of tumors, and this imposes selective pressure for cancer cells to adapt mechanisms to tolerate these conditions. Here, Harris et al. examined the dependency of cancer cells on glutathione (GSH), the most abundant cellular antioxidant. While cancer cell lines displayed a broad range of sensitivities to inhibition of GSH synthesis, the majority were resistant to GSH depletion. To identify cellular pathways required for this resistance, the authors carried out genetic and pharmacologic screens. Both approaches revealed that inhibition of deubiquitinating enzymes (DUBs) sensitizes cancer cells to GSH depletion. Inhibition of GSH synthesis, in combination with DUB inhibition, led to an accumulation of polyubiquitinated proteins, induction of proteotoxic stress, and cell death. These results indicate that depletion of GSH renders cancer cells dependent on DUB activity to maintain protein homeostasis and cell viability and reveal a potentially exploitable vulnerability for cancer therapy. (Harris, I. S.; et al. Cell Metab.
Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization
Several general principles of global 3D genome organization have recently been established, including nonrandom positioning of chromosomes and genes in the cell nucleus, distinct chromatin compartments, and topologically associating domains (TADs). However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are still poorly characterized. Here, the authors systematically probe heterogeneity in genome organization. High-throughput optical mapping of several hundred intrachromosomal interactions in individual human fibroblasts demonstrates low association frequencies, which are determined by genomic distance, higher-order chromatin architecture, and chromatin environment. The structure of TADs is variable between individual cells, and inter-TAD associations are common. Furthermore, single-cell analysis reveals independent behavior of individual alleles in single nuclei. The authors’ observations reveal extensive variability and heterogeneity in genome organization at the level of individual alleles and demonstrate the coexistence of a broad spectrum of genome configurations in a cell population. (Finn, E. H.; et al. Cell
An Automated Microfluidic Gene-Editing Platform for Deciphering Cancer Genes
Gene-editing techniques such as RNA-guided endonuclease systems are becoming increasingly popular for phenotypic screening. Such screens are normally conducted in arrayed or pooled formats. There has been considerable interest in recent years to find new technological methods for conducting these gene-editing assays. Sinha et al. report here the first digital microfluidic method that can automate arrayed gene editing in mammalian cells. Specifically, this method was useful in culturing lung cancer cells for up to 6 days, as well as implementing automated gene transfection and knockout procedures. In addition, a standardized imaging pipeline to analyze fluorescently labeled cells was also designed and implemented during these procedures. A gene-editing assay for interrogating the MAPK/ERK pathway was performed to show the utility of the platform and to determine the effects of knocking out the RAF1 gene in lung cancer cells. In addition to gene knockout, the authors also treated the cells with an inhibitor, sorafenib tosylate, to determine the effects of enzymatic inhibition. The combination of enzymatic inhibition and guide targeting on the device resulted in lower drug concentrations for achieving half-inhibitory effects (IC50) compared with cells treated only with the inhibitor, confirming that lung cancer cells are being successfully edited on the device. The authors propose that this system will be useful for other types of gene-editing assays and applications related to personalized medicine. (Sinha, H.; et al. Lab Chip
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.