Functional Test Platform Research Articles

Drug testing of monodisperse arrays of live microdissected tumors using a valved multiwell microfluidic platform.

Cancer drug testing in animals is an extremely poor predictor of the drug's safety and efficacy observed in humans. Hence there is a pressing need for functional testing platforms that better predict traditional and immunotherapy responses in human, live tumor tissue or tissue constructs, and at the same time are compatible with the use of mouse tumor tissue to facilitate building more accurate disease models. Since many cancer drug actions rely on mechanisms that depend on the tumor microenvironment (TME), such platforms should also retain as much of the native TME as possible. Additionally, platforms based on miniaturization technologies are desirable to reduce animal use and sensitivity to human tissue scarcity. Present high-throughput testing platforms that have some of these features, e.g. based on patient-derived tumor organoids, require a growth step that alters the TME. On the other hand, microdissected tumors (μDTs) or "spheroids" that retain an intact TME have shown promising responses to immunomodulators acting on native immune cells. However, difficult tissue handling after microdissection has reduced the throughput of drug testing on μDTs, thereby constraining the inherent advantages of producing numerous TME-preserving units of tissue for drug testing. Here we demonstrate a microfluidic 96-well platform designed for drug treatment of hundreds of similarly-sized, cuboidal μDTs ("cuboids") produced from a single tumor sample. The platform organizes a monodisperse array of four cuboids per well in 384 hydrodynamic traps. The microfluidic device, entirely fabricated in thermoplastics, features 96 microvalves that fluidically isolate each well after the cuboid loading step for straightforward multi-drug testing. Since our platform makes the most of scarce tumor tissue, it can potentially be applied to human biopsies that preserve the human TME while minimizing animal testing.

Microdissected "cuboids" for microfluidic drug testing of intact tissues.

As preclinical animal tests often do not accurately predict drug effects later observed in humans, most drugs under development fail to reach the market. Thus there is a critical need for functional drug testing platforms that use human, intact tissues to complement animal studies. To enable future multiplexed delivery of many drugs to one small biopsy, we have developed a multi-well microfluidic platform that selectively treats cuboidal-shaped microdissected tissues or "cuboids" with well-preserved tissue microenvironments. We create large numbers of uniformly-sized cuboids by semi-automated sectioning of tissue with a commercially available tissue chopper. Here we demonstrate the microdissection method on normal mouse liver, which we characterize with quantitative 3D imaging, and on human glioma xenograft tumors, which we evaluate after time in culture for viability and preservation of the microenvironment. The benefits of size uniformity include lower heterogeneity in future biological assays as well as facilitation of their physical manipulation by automation. Our prototype platform consists of a microfluidic circuit whose hydrodynamic traps immobilize the live cuboids in arrays at the bottom of a multi-well plate. Fluid dynamics simulations enabled the rapid evaluation of design alternatives and operational parameters. We demonstrate the proof-of-concept application of model soluble compounds such as dyes (CellTracker, Hoechst) and the cancer drug cisplatin. Upscaling of the microfluidic platform and microdissection method to larger arrays and numbers of cuboids could lead to direct testing of human tissues at high throughput, and thus could have a significant impact on drug discovery and personalized medicine.

A microfluidic platform for functional testing of cancer drugs on intact tumor slices.

Present approaches to assess cancer treatments are often inaccurate, costly, and/or cumbersome. Functional testing platforms that use live tumor cells are a promising tool both for drug development and for identifying the optimal therapy for a given patient, i.e. precision oncology. However, current methods that utilize patient-derived cells from dissociated tissue typically lack the microenvironment of the tumor tissue and/or cannot inform on a timescale rapid enough to guide decisions for patient-specific therapy. We have developed a microfluidic platform that allows for multiplexed drug testing of intact tumor slices cultured on a porous membrane. The device is digitally-manufactured in a biocompatible thermoplastic by laser-cutting and solvent bonding. Here we describe the fabrication process in detail, we characterize the fluidic performance of the device, and demonstrate on-device drug-response testing with tumor slices from xenografts and from a patient colorectal tumor.

Partial thermal imidization of polyelectrolyte multilayer cell tethering surfaces (TetherChip) enables efficient cell capture and microtentacle fixation for circulating tumor cell analysis.

The technical challenges of imaging non-adherent tumor cells pose a critical barrier to understanding tumor cell responses to the non-adherent microenvironments of metastasis, like the bloodstream or lymphatics. In this study, we optimized a microfluidic device (TetherChip) engineered to prevent cell adhesion with an optically-clear, thermal-crosslinked polyelectrolyte multilayer nanosurface and a terminal lipid layer that simultaneously tethers the cell membrane for improved spatial immobilization. Thermal imidization of the TetherChip nanosurface on commercially-available microfluidic slides allows up to 98% of tumor cell capture by the lipid tethers. Importantly, time-lapse microscopy demonstrates that unique microtentacles on non-adherent tumor cells are rapidly destroyed during chemical fixation, but tethering microtentacles to the TetherChip surface efficiently preserves microtentacle structure post-fixation and post-blood isolation. TetherChips remain stable for more than 6 months, enabling shipment to distant sites. The broad retention capability of TetherChips allows comparison of multiple tumor cell types, revealing for the first time that carcinomas beyond breast cancer form microtentacles in suspension. Direct integration of TetherChips into the Vortex VTX-1 CTC isolation instrument shows that live CTCs from blood samples are efficiently captured on TetherChips for rapid fixation and same-day immunofluorescence analysis. Highly efficient and unbiased label-free capture of CTCs on a surface that allows rapid chemical fixation also establishes a streamlined clinical workflow to stabilize patient tumor cell samples and minimize analytical variables. While current studies focus primarily on CTC enumeration, this microfluidic device provides a novel platform for functional phenotype testing in CTCs with the ultimate goal of identifying anti-metastatic, patient-specific therapies.

Novel Stenotic Microchannels to Study Thrombus Formation in Shear Gradients: Influence of Shear Forces and Human Platelet-Related Factors.

Thrombus formation in hemostasis or thrombotic disease is initiated by the rapid adhesion, activation, and aggregation of circulating platelets in flowing blood. At arterial or pathological shear rates, for example due to vascular stenosis or circulatory support devices, platelets may be exposed to highly pulsatile blood flow, while even under constant flow platelets are exposed to pulsation due to thrombus growth or changes in vessel geometry. The aim of this study is to investigate platelet thrombus formation dynamics within flow conditions consisting of either constant or variable shear. Human platelets in anticoagulated whole blood were exposed ex vivo to collagen type I-coated microchannels subjected to constant shear in straight channels or variable shear gradients using different stenosis geometries (50%, 70%, and 90% by area). Base wall shears between 1800 and 6600 s−1, and peak wall shears of 3700 to 29,000 s−1 within stenoses were investigated, representing arterial-pathological shear conditions. Computational flow-field simulations and stenosis platelet thrombi total volume, average volume, and surface coverage were analysed. Interestingly, shear gradients dramatically changed platelet thrombi formation compared to constant base shear alone. Such shear gradients extended the range of shear at which thrombi were formed, that is, platelets became hyperthrombotic within shear gradients. Furthermore, individual healthy donors displayed quantifiable differences in extent/formation of thrombi within shear gradients, with implications for future development and testing of antiplatelet agents. In conclusion, here, we demonstrate a specific contribution of blood flow shear gradients to thrombus formation, and provide a novel platform for platelet functional testing under shear conditions.

Molecular and Functional Diagnostic Tools in Precision Oncology for Urological Malignancies.

Urological malignancies, represented mainly by prostate, bladder, and renal cancers, are some of the leading causes of cancer-related mortalities worldwide. Despite various efforts over decades to develop early detection tests and effective therapeutic paradigms, the response rate to the existing treatments remains low for both primary and late stage/recurrent phases of these cancers. The evolving landscape of molecular diagnostics, aiming to make the diagnosis and treatment more patient-driven, underpins precision oncology and particularly intends to rationally profile individual tumors and highlight the mechanistic insight and complexity of tumor microenvironment in order to develop biomarkers of toxicity risks and response prediction in a clinically oriented dynamical setting. The present review is an effort to capture some of the recent developments in the area of molecular diagnostics and functional testing platforms and their potential application in clinical decision making in the premises of precision oncology of urological malignancies.

The Role of UBR5 Mutations in the Pathogenesis of Mantle CELL Lymphoma

Intro: Mantle cell lymphoma (MCL) accounts for 6% of non-Hodgkin lymphomas and represents a particularly challenging disease with patient outcomes inferior to most other lymphoma subtypes. Using targeted capture sequencing of MCL biopsy samples, we recently reported frequent mutations (18%) in UBR5, a gene encoding an E3 ubiquitin-protein ligase that has not been previously implicated in lymphomagenesis. All mutations were clustered within 100bp in or around exon 58 of UBR5 and truncate the reading frame or change a key lysine residue. These mutations are predicted to result in the loss of the conserved cysteine residue in the HECT-domain, which is responsible for binding the ubiquitin co-factor. The recurrence and clustering of UBR5 mutations suggest their critical pathogenic involvement in a subgroup of MCL that might be therapeutically targetable. The aim of this study is to determine UBR5 mutation-associated proteome changes and altered cell signaling.Methods: As seen in MCL patients, mutations in exon 58 of UBR5 were introduced to three MCL cell lines (Granta-519, Jeko-1, and Mino) using the CRISPR-Cas9 genome engineering tool. First, mass spectrometry-based immunoprecipitation proteomics (IP-MS) was employed to identify differences in UBR5 interacting partners between UBR5 mutant and wildtype (WT) cells. Candidate UBR5 interacting proteins were validated by flow cytometry, western blotting, co-immunoprecipitation, and immunofluorescence. Next, global proteomes of UBR5 mutants and WT were analyzed by Tandem Mass Tag (TMT)-based mass spectrometry quantification to identify proteins with differential expression due to the UBR5 mutations.Results: The IP-MS analysis of WT vs UBR5 mutants revealed histone and cell cycle control proteins as candidate differential UBR5 interacting proteins (p<0.05). Particularly, histones H1, H4, and H2AFX, as well as the cell cycle genes CDC5L, BUB3, MAP4, RAD50 and CDK11B were identified as candidate UBR5 interacting partners. The global proteome analysis identified a set of differentially expressed genes (mutant vs wt; p<0.05) that are common among the MCL cell lines with the same direction of change. Gene ontology analysis of this set revealed DNA damage response, chromosome organization, and cell cycle response pathways as the predominant pathways affected. Moreover, our preliminary functional studies indicate constitutive phosphorylation of H2AFX in UBR5 mutants vs WT in line with the role of UBR5 in DNA damage response.Conclusions: Our results are consistent with UBR5 functioning as a key regulator of cell signalling and strongly suggest UBR5 as a novel regulator of histone modifications and DNA damage response. These findings provide an experimentally valid platform for further functional investigation and testing of target therapies for MCL harbouring UBR5 mutations. DisclosuresNo relevant conflicts of interest to declare.

Complementation of Yeast Genes with Human Genes as an Experimental Platform for Functional Testing of Human Genetic Variants.

While the pace of discovery of human genetic variants in tumors, patients, and diverse populations has rapidly accelerated, deciphering their functional consequence has become rate-limiting. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to fill this gap and serve as a platform for testing human genetic variants. To this end, we performed two parallel screens, a one-to-one complementation screen for essential yeast genes implicated in chromosome instability and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. We chose four human cDNAs (hLIG1, hSSRP1, hPPP1CA, and hPPP1CC) for which their yeast gene counterparts function in chromosome stability and assayed in yeast 35 tumor-specific missense mutations for growth defects and sensitivity to DNA-damaging agents. This resulted in a set of human–yeast gene complementation pairs that allow human genetic variants to be readily characterized in yeast, and a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. These data establish the utility of this cross-species experimental approach.

A functional test platform for the Community Land Model

The realistic representation of key biogeophysical and biogeochemical functions is the fundamental of process-based ecosystem models. A functional test platform is designed to create direct linkages between site measurements and the process-based ecosystem model within the Community Earth System Models (CESM). The platform consists of three major parts: 1) interactive user interfaces, 2) functional test models and 3) observational datasets. It provides much needed integration interfaces for both field experimentalists and ecosystem modelers to improve the model's representation of ecosystem processes within the CESM framework without large software overhead.

Universal platform for functional testing of printed circuit boards

Universal platform for functional testing of printed circuit boards

High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana.

To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps optimized for high throughput, this in planta expression platform is particularly suited for testing large panels of candidate genes in all possible permutations.

Mobility in an RF-isolated test platform

Understanding the practical impact of mobility in a wireless network is essential for the wireless networks of tomorrow. Mobility influences the network performance, behavior, and ability to provide seamless service across a wide area. Testing and evaluating the real impact of mobility is difficult in the field with so many variables such as interference, fading, and so on. Experimental wireless evaluation in a test environment must correspond to an actual deployment. Furthermore, it is important to achieve repeatability without sacrificing realism. This study presents a functional test platform that uses real IEEE 802.11 equipment, providing repeatability and reliability. The platform is used to test the practical impact of device movement in a WLAN cell while voice and data applications are running. The mobility characteristics of wireless devices are based on individual models used by researchers with the addition of real aspects of mobility from empirical studies to improve realism.