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Metastasis is the leading cause of mortality in cancer patients. To migrate to distant sites, cancer cells would need to adapt their behaviour in response to different tissue environments. Thus, it is essential to study this process in models that can closely replicate the tumour microenvironment. Here, we evaluate the use of organotypic liver and brain slices to study cancer metastasis. Morphological and viability parameters of the slices were monitored daily over 3 days in culture to assess their stability as a realistic 3D tissue platform for in vitro metastatic assays. Using these slices, we evaluated the invasion of MDA-MB-231 breast cancer cells and of a subpopulation that was selected for increased motility. We show that the more aggressive invasion of the selected cells likely resulted not only from their lower stiffness, but also from their lower adhesion to the surrounding tissue. Different invasion patterns in the brain and liver slices were observed for both subpopulations. Cells migrated faster in the brain slices (with an amoeboid-like mode) compared to in the liver slices (where they migrated with mesenchymal or collective migration-like modes). Inhibition of the Ras/MAPK/ERK pathway increased cell stiffness and adhesion forces, which resulted in reduced invasiveness. These results illustrate the potential for organotypic tissue slices to more closely mimic in vivo conditions during cancer cell metastasis than most in vitro models.

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.

We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin–sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor–mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.

Mammalian nervous system function involves billions of neurons which are interconnected in a multitude of neural circuits. Here we describe a genetic approach to chart neural circuits. By using an olfactory-specific promoter, we selectively expressed barley lectin in sensory neurons in the olfactory epithelium and vomeronasal organ of transgenic mice. The lectin was transported through the axons of those neurons to the olfactory bulb, transferred to the bulb neurons with which they synapse, and transported through the axons of bulb neurons to the olfactory cortex. The lectin also was retrogradely transported from the bulb to neuromodulatory brain areas. No evidence could be obtained for adverse effects of the lectin on odorant receptor gene expression, sensory axon targeting in the bulb, or the generation or transmission of signals by olfactory sensory neurons. Transneuronal transfer was detected prenatally in the odor-sensing pathway, but only postnatally in the pheromone-sensing pathway, suggesting that odors, but not pheromones, may be sensed in utero . Our studies demonstrate that a plant lectin can serve as a transneuronal tracer when its expression is genetically targeted to a subset of neurons. This technology can potentially be applied to a variety of vertebrate and invertebrate neural systems and may be particularly valuable for mapping connections formed by small subsets of neurons and for studying the development of connectivity as it occurs in utero .

We studied modulation of current in human embryonic kidney tsA-201 cells coexpressing rat erg1 channels with M(1) muscarinic receptors. Maximal current was inhibited 30% during muscarinic receptor stimulation, with a small positive shift of the midpoint of activation. Inhibition was attenuated by coexpression of the regulator of G-protein signalling RGS2 or of a dominant-negative protein, G(q), but not by N-ethylmaleimide or C3 toxin. Overexpression of a constitutively active form of G(q) (but not of G(13) or of G(s)) abolished the erg current. Hence it is likely that G(q/11), and not G(i/o) or G(13), mediates muscarinic inhibition. Muscarinic suppression of erg was attenuated by chelating intracellular Ca(2+) to1 nm free Ca(2+) with 20 mm BAPTA in the pipette, but suppression was normal if internal Ca(2+) was strongly clamped to a 129 nm free Ca(2+) level with a BAPTA buffer and this was combined with numerous other measures to prevent intracellular Ca(2+) transients (pentosan polysulphate, preincubation with thapsigargin, and removal of extracellular Ca(2+)). Hence a minimum amount of Ca(2+) was necessary for the inhibition, but a Ca(2+) elevation was not. The ATP analogue AMP-PCP did not prevent inhibition. The protein kinase C (PKC) blockers staurosporine and bisindolylmaleimide I did not prevent inhibition, and the PKC-activating phorbol ester PMA did not mimic it. Neither the tyrosine kinase inhibitor genistein nor the tyrosine phosphatase inhibitor dephostatin prevented inhibition by oxotremorine-M. Hence protein kinases are not needed. Experiments with a high concentration of wortmannin were consistent with recovery being partially dependent on PIP(2) resynthesis. Wortmannin did not prevent muscarinic inhibition. Our studies of muscarinic inhibition of erg current suggest a role for phospholipase C, but not the classical downstream messengers, such as PKC or a calcium transient.

Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor-mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of Galphaq and Galpha11, but not Galpha13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPbetaS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPbetaS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPbetaS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein-stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.

Topographical image measures have been used to characterize the subnuclear distribution of DNA topoisomerase I in human tumor cell lines. This topographical analysis allowed a mathematical description of staining patterns to be produced that did not depend on subjective grading. The redistribution of topoisomerase I in response to increasing concentrations of topotecan was then monitored by this method. The cell lines were stained for topoisomerase I by indirect immunofluorescence methods. Digital imaging microscopy and image analysis were used to extract the nucleus from each cell, and nine parameters describing the topography of the distribution of topoisomerase I within the nucleus were computed for each. Use of multivariate analysis of variance enabled this nine-parameter set to be reduced to a single canonical variable, representing 60–90% of the observed internuclear variance. Plotting the canonical variable vs drug concentration resulted in dose–response curves that could be fitted well by a simpleEmaxmodel. From these curve fits, EC50andEmaxvalues for drug-induced redistribution of topoisomerase I were determined. Our results indicate that neither the maximum extent of topoisomerase I redistribution (Emax) nor the EC50for drug-induced redistribution correlated well with the growth inhibition produced by continuous exposure to topotecan in these cell lines. However, the EC50determined for the 1-h high-concentration exposure did reflect the growth inhibition produced in cells exposed to the drug for 1 h. The methodology described may also be generally applied to any antigen of interest.

The mammalian olfactory system is able to discriminate among tens of thousands of odorant molecules. In mice, each odorant is sensed by a small subset of the approximately 1,000 odorant receptor (OR) types, with one OR gene expressed by each olfactory sensory neuron (OSN). However, the sum of the large repertoire of OR/OSN types and difficulties with heterologous expression have made it almost impossible to analyze odorant responsiveness across all OR/OSN types. We have developed a microfluidic approach that allowed us to screen over 20,000 single cells at once in microwells. By using calcium imaging, we were able to detect and analyze odorant responses of about 2,900 OSNs simultaneously. Importantly, this technique allows for both the detection of rare responding OSNs as well as the identification of OSN populations broadly responsive to odorants of unrelated structures. This technique is generally applicable for screening large numbers of single cells and should help to characterize rare cell behaviors in fields such as toxicology, pharmacology, and cancer research.