The analysis of circulating tumor cells (CTCs) in blood is a powerful noninvasive alternative to conventional tumor biopsy. Inertial-based separation is a promising high-throughput, marker-free sorting strategy for the enrichment and isolation of CTCs. Here, we present and validate a double spiral microfluidic device that efficiently isolates CTCs with a fine-tunable cut-off value of 9 mu m and a separation range of 2 mu m. We designed the device based on computer simulations that introduce a novel, customized inertial force term, and provide practical fabrication guidelines. We validated the device using calibration beads, which allowed us to refine the simulations and redesign the device. Then we validated the redesigned device using blood samples and a murine model of metastatic breast cancer. Finally, as a proof of principle, we tested the device using peripheral blood from a patient with hepatocellular carcinoma, isolating more than 17 CTCs/ml, with purity/removal values of 96.03% and 99.99% of white blood cell and red blood cells, respectively. These results confirm highly efficient CTC isolation with a stringent cut-off value and better separation results than the state of the art.

CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-kappa B signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans. Costimulation has been shown to be required for optimal activation of T cells and it could be delivered either in trans with respect to the source of CD3-TCR ligation or in cis on the same cell. Here the authors show that CD137 costimulation is more effective when delivered in cis to enhance T cell proliferation and activation.

Motivation: Recent advances in multiplex immunostaining and multispectral cytometry have opened the door to simultaneously visualizing an unprecedented number of biomarkers both in liquid and solid samples. Properly unmixing fluorescent emissions is a challenging task, which normally requires the characterization of the individual fluorochromes from control samples. As the number of fluorochromes increases, the cost in time and use of reagents becomes prohibitively high. Here, we present a fully unsupervised blind spectral unmixing method for the separation of fluorescent emissions in highly mixed spectral data, without the need for control samples. To this end, we extend an existing method based on non-negative Matrix Factorization, and introduce several critical improvements: initialization based on the theoretical spectra, automated selection of 'sparse' data and use of a re-initialized multilayer optimizer. Results: Our algorithm is exhaustively tested using synthetic data to study its robustness against different levels of colocalization, signal to noise ratio, spectral resolution and the effect of errors in the initialization of the algorithm. Then, we compare the performance of our method to that of traditional spectral unmixing algorithms using novel multispectral flow and image cytometry systems. In all cases, we show that our blind unmixing algorithm performs robust unmixing of highly spatially and spectrally mixed data with an unprecedently low computational cost. In summary, we present the first use of a blind unmixing method in multispectral flow and image cytometry, opening the door to the widespread use of our method to efficiently pre-process multiplex immunostaining samples without the need of experimental controls.

3D cell culture models consisting of self-assembled tumour cells in suspension, commonly known as tumour spheroids, are becoming mainstream for high-throughput anticancer drug screening. A usual measurable outcome of screening studies is the growth rate of the spheroids in response to treatment. This is commonly quantified on images obtained using complex, expensive, optical microscopy systems, equipped with high-quality optics and customized electronics. Here we present a novel, portable, miniaturized microscope made of low-cost, mass-producible parts, which produces both fluorescence and phase-gradient contrast images. Since phase-gradient contrast imaging is based on oblique illumination, epi-illumination is used for both modalities, thus simplifying the design of the system. We describe the system, characterize its performance on synthetic samples and show proof-of-principle applications of the system consisting in imaging and monitoring the formation and growth of lung and pancreas cancer tumour spheroids within custom made microfluidic devices.

The migration of cancer cells is highly regulated by the biomechanical properties of their local microenvironment. Using 3D scaffolds of simple composition, several aspects of cancer cell mechanosensing (signal transduction, EMC remodeling, traction forces) have been separately analyzed in the context of cell migration. However, a combined study of these factors in 3D scaffolds that more closely resemble the complex microenvironment of the cancer ECM is still missing. Here, we present a comprehensive, quantitative analysis of the role of cell-ECM interactions in cancer cell migration within a highly physiological environment consisting of mixed Matrigel-collagen hydrogel scaffolds of increasing complexity that mimic the tumor microenvironment at the leading edge of cancer invasion. We quantitatively show that the presence of Matrigel increases hydrogel stiffness, which promotes beta 1 integrin expression and metalloproteinase activity in H1299 lung cancer cells. Then, we show that ECM remodeling activity causes matrix alignment and compaction that favors higher tractions exerted by the cells. However, these traction forces do not linearly translate into increased motility due to a biphasic role of cell adhesions in cell migration: at low concentration Matrigel promotes migration-effective tractions exerted through a high number of small sized focal adhesions. However, at high Matrigel concentration, traction forces are exerted through fewer, but larger focal adhesions that favor attachment yielding lower cell motility.

We present a novel method to characterize the morphology of semicircular canals of the inner ear. Previous experimental works have a common nexus, the human-operator subjectivity. Although these methods are mostly automatic, they rely on a human decision to determine some particular anatomical positions. We implement a systematic analysis where there is no human subjectivity. Our approach is based on a specific magnetic resonance study done in a group of 20 volunteers. From the raw data, the proposed method defines the centerline of all three semicircular canals through a skeletonization process and computes the angle of the functional pair and other geometrical parameters. This approach allows us to assess the inter-operator effect on other methods. From our results, we conclude that, although an average geometry can be defined, the inner ear anatomy cannot be reduced to a single geometry as seen in previous experimental works. We observed a relevant variability of the geometrical parameters in our cohort of volunteers that hinders this usual simplification.

The main objective of this work is the study and analysis of non-linearities forced through oscillating magnetic fields in a conducting fluid where the instabilities are triggered due to magnetohydrodynamic forces. Different geometries have been studied and different surface patterns that break the symmetries have been observed. First, an InGaSn drop of fluid where the system breaks the azimuthal and radial symmetries depending on the volume is observed. Second, we extend the study to an InGaSn annular configuration where the presence of patterns opens the door to discuss the possibility to extend these results to other configurations as biological systems, where the conducting fluid is an electrolyte. This configuration has an added interest, as it has been proposed that the vertigoes triggered on patients in an MRI test could be generated by the interaction of the magnetic field with the electrolyte present in the inner ear.

We present the experimental analysis of the instabilities generated on a large drop of liquid metal by a time-dependent magnetic field. The study is done exploring the range of tiny values of the control parameter (the ratio between the Lorentz forces and inertia) avoiding nonlinear effects. Two different instabilities break the symmetries generating spatial patterns that appear without a threshold for some specific frequencies (up to the experimental precision) and have been observed for parameter values two orders of magnitude lower than in previously published experiments [J. Fluid Mech. 239, 383 (1992)]. One of the instabilities corresponds to a boundary condition oscillation that generates surface waves and breaks the azimuthal symmetry. The other corresponds to a parametric forcing through a modulation of the Lorentz force. The competition between these two mechanisms produces time-dependent patterns near codimension-2 points.

We compare the dynamics obtained in two intermediate aspect ratio (diameter over height) experiments. These systems have rotational symmetry and consist of fluid layers that are destabilized using two different methods. The first one is a classical Bénard Marangoni experiment, where the destabilizing forces, buoyancy and surface tension, are created by temperature gradients. The second system consists of a large drop of liquid metal destabilized using oscillating magnetic fields. In this configuration, the instability is generated by a radial Lorentz force acting on the conducting fluid. Although there are many important differences between the two configurations, the dynamics are quite similar: the patterns break the rotational symmetry, and different azimuthal and radial wavenumbers appear depending on the experimental control parameters. These patterns in most cases are stationary, but for some parameters they exhibit different dynamical behaviours: rotations, transitions between different solutions or cyclic connections between different patterns