Environmentally adaptive automated recognition of underwater mines with synthetic aperture sonar imagery.

This work demonstrates that automated mine countermeasure (MCM) tasks are greatly facilitated by characterizing the seafloor environment in which the sensors operate as a first step within a comprehensive strategy for how to exploit information from available sensors, multiple detector types, measured features, and target classifiers, depending on the specific seabed characteristics present within the high-frequency synthetic aperture sonar (SAS) imagery used to perform MCM tasks. This approach is able to adapt as environmental characteristics change and includes the ability to recognize novel seabed types. Classifiers are then adaptively retrained through active learning in these unfamiliar seabed types, resulting in improved mitigation of challenging environmental clutter as it is encountered. Further, a segmentation constrained network algorithm is introduced to enable enhanced generalization abilities for recognizing mine-like objects from underrepresented environments within the training data. Additionally, a fusion approach is presented that allows the combination of multiple detectors, feature types spanning both measured expert features and deep learning, and an ensemble of classifiers for the particular seabed mixture proportions measured around each detected target. The environmentally adaptive approach is demonstrated to provide the best overall performance for automated mine-like object recognition.

Micromanipulation of Live Microdissected Tissues with a Low-Cost Integrated Robotic Platform.

The small amount of human tissue available for testing is a paramount challenge in cancer drug development, cancer disease models, and personalized oncology. Technologies that combine the microscale manipulation of tissues with fluid handling offer the exciting possibility of miniaturizing and automating drug evaluation workflows. This approach minimizes animal testing and enables inexpensive, more efficient testing of samples with high clinical biomimicry using scarce materials. We have developed an inexpensive platform based on an off-the-shelf robot that can manipulate microdissected tissues (µDTs) into user-programmed positions without using intricate microfluidic designs nor any other accessories such as a microscope or a pneumatic controller. The robot integrates complex functions such as vision and fluid actuation by incorporating simple items including a USB camera and a rotary pump. Through the robot's camera, the platform software optically recognizes randomly-seeded µDTs on the surface of a petri dish and positions a mechanical arm above the µDTs. Then, a custom rotary pump actuated by one of the robot's motors generates enough microfluidic lift to hydrodynamically pick and place µDTs with a pipette at a safe distance from the substrate without requiring a proximity sensor. The platform's simple, integrated construction is cost-effective and compact, allowing placement inside a tissue culture hood for sterile workflows. The platform enables users to select µDTs based on their size, place them in user-programmed arrays, such as multi-well plates, and control various robot motion parameters. As a case application, we use the robotic system to conduct semi-automated drug testing of mouse and human µDTs in 384-well plates. Our user-friendly platform promises to democratize microscale tissue research to clinical and biological laboratories worldwide.

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.

Label-Free, Real-Time Monitoring of Cytochrome C Responses to Drugs in Microdissected Tumor Biopsies with a Multi-Well Aptasensor Platform.

Functional assays on intact tumor biopsies can potentially complement and extend genomics-based approaches for precision oncology, drug testing, and organs-on-chips cancer disease models by capturing key determinants of therapeutic response, such as tissue architecture, tumor heterogeneity, and the tumor microenvironment. Currently, most of these assays rely on fluorescent labeling, a semi-quantitative method best suited to be a single-time-point terminal assay or labor-intensive terminal immunostaining analysis. Here, we report integrated aptamer electrochemical sensors for on-chip, real-time monitoring of increases of cytochrome C, a cell death indicator, from intact microdissected tissues with high affinity and specificity. The platform features a multi-well sensor layout and a multiplexed electronic setup. The aptasensors measure increases in cytochrome C in the supernatant of mouse or human microdissected tumors after exposure to various drug treatments. Since the aptamer probe can be easily exchanged to recognize different targets, the platform could be adapted for multiplexed monitoring of various biomarkers, providing critical information on the tumor and its microenvironment. This approach could not only help develop more advanced cancer disease models but also apply to other complex in vitro disease models, such as organs-on-chips and organoids.

Microdissected tumor cuboids: a microscale cancer model that retains a complex tumor microenvironment.

Present cancer disease models - typically based on cell cultures and animal models that lack the human tumor microenvironment (TME) - are extremely poor predictors of human disease outcomes. Microscale cancer models that combine the micromanipulation of tissues and fluids offer the exciting possibility of miniaturizing the drug testing workflow, enabling inexpensive, more efficient tests of high clinical biomimicry that maximize the use of scarce human tissue and minimize animal testing. Critically, these microscale models allow for precisely addressing the impact of the structural features of the heterogeneous TME to properly target and understand the contributions of these unique zones to therapeutic response. We have recently developed a precision slicing method that yields large numbers of cuboidal micro-tissues ("cuboids", ∼ (400 µm) 3 ) from a single tumor biopsy. Here we evaluate cuboids from syngeneic mouse tumor models and human tumors, which contain native immune cells, as models for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as their cellular architecture (immune cells and vasculature), cytokine secretion, proteomics profiles, and their response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong functional responses such as cytokine and drug responses. Overall, our results suggest that cuboids make an excellent model for applications that require the TME, such as immunotherapy drug evaluations, including for clinical trials and personalized oncology approaches.