MyCTC chip: microfluidic-based drug screen with patient-derived tumour cells from liquid biopsies

Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns, often leading to ineffective treatment. Current clinical practice for treatment decision-making is commonly based on primary or secondary tumour biopsies, yet when disease progression accelerates, tissue biopsies are not performed on a regular basis. It is in this context that liquid biopsies may offer a unique window to uncover key vulnerabilities, providing valuable information about previously underappreciated treatment opportunities. Here, we present MyCTC chip, a novel microfluidic device enabling the isolation, culture and drug susceptibility testing of cancer cells derived from liquid biopsies. Cancer cell capture is achieved through a label-free, antigen-agnostic enrichment method, and it is followed by cultivation in dedicated conditions, allowing on-chip expansion of captured cells. Upon growth, cancer cells are then transferred to drug screen chambers located within the same device, where multiple compounds can be tested simultaneously. We demonstrate MyCTC chip performance by means of spike-in experiments with patient-derived breast circulating tumour cells, enabling >95% capture rates, as well as prospective processing of blood from breast cancer patients and ascites fluid from patients with ovarian, tubal and endometrial cancer, where sensitivity to specific chemotherapeutic agents was identified. Together, we provide evidence that MyCTC chip may be used to identify personalized drug response patterns in patients with advanced metastatic disease and with limited treatment opportunities

The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts

Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species’ threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project – and avert – future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups – including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems – www.predicts.org.uk).We make site-level summary data available alongside this article. The full database will be publicly available in 2015

The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts

Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species’ threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project – and avert – future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups – including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems – www.predicts.org.uk). We make site-level summary data available alongside this article. The full database will be publicly available in 2015

Microfluidics for Functional Analysis of Circulating Tumor Cells

Microfluidic technologies use micrometer-sized channel networks to handle and manipulate fluids and biological samples. This has made it possible to manipulate and analyze individual cells and led to a huge improvement in our understanding of cell-to-cell heterogeneities. Single-cell analysis is especially important in cancer research, as individual circulating tumor cells (CTCs) promote metastasis and a single surviving cell can cause relapse after treatment. A variety of microfluidic devices has been developed to compartmentalize individual cells for subsequent analysis. These devices have proven extremely useful to elucidate cell behavior and helped to answer many fundamental biological questions. However, their diagnostic use for analysis of CTCs is limited as these platforms do not match the high capture efficiency, selectivity, and throughput demands for isolation of CTCs from whole blood samples. In contrast, platforms that were developed for efficient CTCs isolation from whole blood do not provide the means for single-cell compartmentalization necessary for functional studies. To advance our understanding of cancer progression, there is a major demand for a platform that performs efficient CTCs isolation in a format compatible with functional single-cell analysis. To address this unmet analytical need, a microfluidic platform for the extraction and functional analysis of CTCs from whole blood was developed. This thesis is describing the gradual improvement, which started from an existing microchamber technology and finally resulted in a highly efficient and versatile platform for CTC analysis. At the beginning, a microfluidic device for single cell drug response testing with high throughput is described and evaluated. The device is capable of performing more than 600 single-cell experiments and the results are similar to traditional bulk assays in well plate format. This platform was adapted for multiplexed analysis of three different intracellular proteins on multiple cancer cell lines through the use of magnetic forces and barcoded beads. By combining magnetic magnetic bead traps with high throughput single-cell isolation, we were finally able to realize a device for functional analysis of CTCs from blood samples. The platform was characterized with CTCs from a mouse model to determine G-CSF secretion levels together with immunostaining for HER2, EpCAM, and CD45. We additionally tested our device with samples from several late stage breast cancer patients, where we found that current culturing conditions are insufficient for functional tests on patient-derived CTCs. Furthermore, a compact system that allows fluorescence analysis of many microfluidic chips with a smartphone to enable point-of-care testing is presented. Currently, research groups around the world are working on improved CTC culture conditions. Thus, direct functional tests on CTCs using devices such as the presented platform will become commonplace in the near future. It promises to offer unique insight to cancer biology and opportunities in personalized medicine

Microbial factories: monitoring vitamin B-2 production by Escherichia coli in microfluidic cultivation chambers

Microbial cells represent a standard production host for various important biotechnological products. Production yields can be increased by optimising strains and growth conditions and understanding deviations in production rates over time or within the microbial population. We introduce here microfluidic cultivation chambers for highly parallel studies on microbial cultures, enabling continuous biosynthesis monitoring of the industrially relevant product by Escherichia coli cells. The growth chambers are defined by ring-valves that encapsulate a volume of 200 pL when activated. Bacterial cells, labelled with magnetic beads, are inoculated in a small magnetic trap, positioned in the centre of each chamber. Afterwards, the ring-valves are partially activated, allowing for exchange reagents, such as the addition of fresh media or specific inducers of biosynthesis, while the bacterial cells and their progeny are maintained inside. On this platform, we monitor the production of riboflavin (vitamin B-2). We used different variants of a riboflavin-overproducing bacterial strain with different riboflavin production levels and could distinguish them on the level of individual micro-colonies. In addition, we could also observe differences in the bacterial morphology with respect to the production. The presented platform represents a flexible microfluidic tool for further studies of microbial cell factories.ISSN:1473-0197ISSN:1473-018

Identifying extracellular vesicle populations from long-term cultured single cells using multi-color TIRFM

We here report a double-layer microfluidic device for detecting and identifying secreted extracellular vesicle populations, among them exosomes, from single long-term cultured cells using multi-color Total Internal Reflection Fluorescence Microscopy (TIRFM). Using a custom-developed MATLAB script for image processing we achieved quantitative population analysis of vesicles secreted from single Michigan Cancer Foundation (MCF)-7 cells cultivated for 2-5 day

Single-cell protein profiling in microchambers with barcoded beads

Single-cell profiling provides insights into cellular behaviour that macroscale cell cultures and bulk measurements cannot reveal. In the context of personalized cancer treatment, the profiling of individual tumour cells may lead to higher success rates for therapies by rapidly selecting the most efficacious drugs. Currently, genomic analysis at the single-cell level is available through highly sensitive sequencing approaches. However, the identification and quantification of intracellular or secreted proteins or metabolites remains challenging. Here, we introduce a microfluidic method that facilitates capture, automated data acquisition and the multiplexed quantification of proteins from individual cells. The microfluidic platform comprises 1026 chambers with a volume of 152 pL each, in which single cells and barcoded beads are co-immobilized. We demonstrated multiplexed single-cell protein quantification with three different mammalian cell lines, including two model breast cancer cell lines. We established on-chip immunoassays for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), galectin-3 (Gal-3) and galectin-3 binding protein (Gal-3bp) with detection limits as low as 7.0 × 10^4, 2.3 × 10^5 and 1.8 × 10^3 molecules per cell, respectively. The three investigated cell types had high cytosolic levels of GAPDH and could be clearly differentiated by their expression levels of Gal-3 and Gal-3bp, which are important factors that contribute to cancer metastasis. Because it employed commercially available barcoded beads for this study, our platform could be easily used for the single-cell protein profiling of several hundred different targets. Moreover, this versatile method is applicable to the analysis of bacteria, yeast and mammalian cells and nanometre-sized lipid vesicles.ISSN:2096-1030ISSN:2055-743

Microfluidic system for cultivation and monitoring of individual riboflavin overproducing Escherichia coli cells

We present a microfluidic platform allowing real-time observation of heterogeneous bacterial populations for basic studies of microbial growth and production. We succeeded in batch culturing and monitoring Escherichia coli cells overexpressing the riboflavin (vitamin B2) biosynthetic gene cluster which enables them to secret preparative amounts of riboflavin. Production in cultures with and without induction of the gene cluster were compared

Co-capture of magnetic beads and cells for single-cell analysis in microfluidic chambers

We present a microfluidic chip for the capture of magnetic beads and magnetically labeled cells in more than 1000 microchambers of 0.15 nL size. The platform allows for immobilizing magnetic beads and labeled cells independent of their size for subsequent single cell analysis. Using a bead-based immunoassay we furthermore quantify secretion of Galectin-3 binding protein (Gal-3bp) from MCF-7 and SkBr3 cells with detection limits below 2000 molecules