Albert Folch - US grants

Objectives: The PI's research goal is to combine cultured living cells with microfluidic devices in order to 1) recreate, in vitro, the size range and variety of interactions between the cell and its environment as found in vivo, and 2) simultaneously analyze large numbers of cells and culture conditions at a single-cell level. As described in detail later, the long-term focus will be to answer central neurobiology questions which are unapproachable with present cell culture technology because they require the interrogation of large numbers of single cells and the microengineering of their environment.

His educational objective is to teach instrumentation and novel technologies to bioengineers and biologists with a strong emphasis on understanding the underlying scientific principles on which the instrument operates, on interactive laboratory classes, and on creatively measuring phenomena encountered in everyday life. Included in this educational endeavor will be a comprehensive introduction to microfabrication technology as an enabling tool for bioengineers and biologists at the graduate and undergraduate level, as well as a web-based K-12 outreach effort.

DESCRIPTION (Provided by Applicant): Our objective is to develop a technology that will allow us to probe, stimulate, and modify nanometer-scale areas of the cell membrane surface on large numbers of cultured cells simultaneously. The novelty of our approach, and its very engineering challenge, is to build an array of addressable submicron-sized fluidic channels which can be sealed against the cell membrane. The cells will rest on or be attached to a surface containing nanometric apertures, and each aperture will communicate with a dedicated microfluidic channel embedded under the cell culture surface. The cell membrane will be sealed against the aperture(s) by applying gentle suction to the channels. Thus, the microchannels will provide fluidic access only to the area of the cell membrane sealed against the aperture, thereby forming a "nanofluidic probe" that can be used to sample and stimulate the area just above the aperture. Optionally, stronger suction may be used to rupture the cell membrane while maintaining the seal and thus create a mechanically stable, fluidically-addressable window for sampling or modifying the intracellular milieu. Our approach represents a radical paradigm shift from presently-available technology for probing small areas of cell membranes, which is based on glass micropipettes. These "patch clamp" pipettes can indeed be sealed against the cell membrane surface and are routinely used to sample the activity of ion channels and stimulate receptors on a local scale. However, bringing the pipette in contact with the cell membrane involves the careful manipulation of a three-axis micropositioner by a skilled human operator under a microscope's visual field. Clearly, this procedure cannot be used to probe many cells simultaneously nor be repeated rapidly, limiting it to low-throughput, statistically-weak studies on a small number of cells. While the technique's throughput can be improved by increasing the number of resources (personnel, time, equipment), the number of cells that can be probed simultaneously remains fundamentally limited. Essentially, we propose to reverse the geometrical configuration of present electrophysiology setups, i.e. to embed the recording probes under the cell culture surface. Since the probes can be manufactured in an array format, the technology we propose to develop will allow for high-throughput, simultaneous probing of large numbers of cells. Consequently, it could have a significant impact in problems ranging from basic biology to drug discovery.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] A major goal in neuroscience is to understand the formation and development of synapses, the tiny membrane specializations that enable nerve cells to communicate with each other. The sequence of molecular signals leading to synapse formation ("synaptogenesis") is qualitatively well known for the more accessible neuromuscular synapse. It is well established that, immediately after contacting the muscle cell, the nerve terminal secretes agrin to induce the clustering of acetylcholine (ACh) receptors at the postsynaptic site. After a cascade of events, the nerve is able to depolarize the muscle cell by releasing pulses of ACh. However, very little is known of the quantities (concentration, duration, onset, etc.) of the various neurochemical signals involved in synaptogenesis. Importantly, all except for one of the axons innervating a given myotube at birth retract after a period of a week or so according to a synaptic competition process that remains, for lack of quantitative methods, poorly understood. Such quantitative description is lacking because present experimental setups for the study of the neuromuscular junction do not allow for a precise control over the many variables involved in synaptogenesis. Therefore, we propose a quantitative approach based on substituting the presynaptic neuron by an artificial mimic, a nanofluidic device that will stimulate the muscle cell in a physiologically relevant way. We hypothesize that the focal delivery of synaptogenic factors will recruit the synaptic machinery to the stimulated area of the membrane. The device will consist of a set of microfluidic channels buried underneath the cell culture surface and that will "communicate" with the cells through nanoholes. The cells will be sealed to the holes by applying suction to the microchannels. Unlike present experimental setups, our device will allow us to 1) confine the delivery of agrin/ACh to a submicron-diameter area of the cell membrane (as occurs in vivo); 2) interrogate the same area of the membrane with different factors sequentially; 3) stimulate several locations of the same cell simultaneously (with the same or dissimilar stimuli); and 4) experiment with high throughput (i.e. investigate large numbers of cells and stimulation conditions simultaneously). The proposed device has broad applicability to cell culture studies requiring nanometer-scale, focal exposure of the cells to a soluble factor. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): During embryonic development, neurons must project their axons along specific paths to make precise connections with their synaptic targets. A key challenge is to identify the involved factors and to understand their signaling mechanisms. The navigation of axonal tips ("growth cones"), both in vitro and in vivo, is guided by insoluble factors in the surrounding extracellular matrix (ECM) or cells and by concentration gradients of diffusing factors. Several families of guidance molecules have been identified and purified, such as netrins, semaphorins, ephrins and Slit proteins. The elucidation of pathfinding decisions in vivo is complicated by the simultaneous presence of different guidance factors in a dynamic environment. Recent studies suggest that the combined effects of these signals are often not obvious. For example, the growth cone attraction to netrin-1 can be converted to repulsion by laminin-1, a contact-attractant. The attractive effects of netrin-1 can also be silenced by the interaction between the cytoplasmic domains of netrin-1 receptor and Slit2 receptor while Slit2 alone did not induce a direct response. In addition, the interaction of netrin-1 and Slit2 pathways is developmentally regulated: older neurons become repelled by Slit2 or by a combination of netrin-1 and Slit2 but show no response to netrin-1 alone. Due to experimental complexity, most studies have only been able to investigate one or two signaling molecules at a time. A traditional in vitro assay records the growth cone responses to gradients of soluble factors delivered by a micro-positioned pipette. This assay provides a means to study single cells, but it generates gradients that are imprecise and evolve in time, and it yields results at very low throughput (-1 cell/hour). Here we propose to build a microfluidic device that will allow us to study multiple axon guidance factors simultaneously on cultured neurons. We will: a) design a combinatorial diffusive mixer that will generate multiple combinations of gradients of different axon guidance molecules, b) record the responses of neurons to each combination using time-lapse imaging, and c) employ automated analysis tools to process large amounts of images. The microfluidic device will allow us to: 1) generate stable concentration gradients, 2) precisely control spatiotemporal changes of the gradients, and 3) simultaneously study many different combinations of gradients at high throughput. Consequently this study will probe axon guidance factor interactions of unprecedented complexity and will significantly further our understanding of neural development. It may also shed light on new clinical therapies for certain neurological disorders and for nerve regeneration after injury.

DESCRIPTION (provided by applicant): We owe our sense of smell to the ability of odorant compounds to activate odorant receptors present on the membrane surface of olfactory sensory neurons in our nose. Our present knowledge indicates that, in mammals, there are around 1,000 types of odorant receptors and that any given neuron only expresses one type of receptor, to which different odorants can bind with different affinities. Thus, any odorant can be thought of as being "encoded" by the olfactory system in the form of a set of affinities between itself and each of the odorant receptors. Conversely, odorant receptors may, in principle, be categorized by their pattern of activation by (some set of) odorants. Present approaches to detect matches between odorants and their receptors range from in-vivo patch-clamp recordings from the olfactory epithelium, bulb or cortex, to Ca2+ imaging of a small number of cultured neurons, in response to a small set of odorants. In addition, since the neuron's response is concentration-dependent, each odorant must be screened at various concentrations with isolated olfactory sensory neurons. Once the response of a given neuron by a given odorant at any concentration is detected, the receptor can be cloned by reverse-transcriptase polymerase chain reaction and subsequently sequenced. Although this approach allows for exquisite detail in characterizing the molecular structure of the receptor, it yields an incomplete functional description, i.e. it might bind to hundreds of other odorants that had to be missed in the experiment due to throughput limitations. Furthermore, the odorant receptor can only be probed once because the responsive cell is probed at random. Additionally, the approach is inadequate for performing an exhaustive search of all the odorant receptors that are activated by a given odorant from a library of hundreds of odorants; hence the encoding of the odorant within the olfactory system is unknown. We seek to obtain a functional description of all the odorant receptors present in the mouse olfactory epithelium. This functional description will not require knowledge of the receptors' molecular structure. We propose to develop a high-throughput experimental system that will allow for detecting matches between a large library of around 100 odorants and the complete set of interacting receptors (approximately 1,000), i.e. we will find the exact encoding of each member of the odorant library. The proposed system consists of 1) a computer-controlled microfluidic device that can perfuse a large microarray of olfactory sensory neurons with sequences of odorants; 2) an optics setup capable of performing Ca2+ imaging over large areas (>5,000 cells); and 3) simple image/data processing algorithms for automated extraction of odorant-receptor matches.

This IGERT integrates graduate research and education toward understanding and exploiting macroscopic manifestations of nanoscale phenomena. Research will focus on: i) nanoscale materials: assembling and engineering nanomaterials with integrated functionality; (ii) nanotechnology tools and devices: developing tools to integrate biological or synthetic nanoscale building blocks into devices; and (iii) nanomedical applications: studying, diagnosing, and treating the roots of diseases at the nanoscale. Education will focus on increasing the diversity of discipline, venue (e.g., academia, industry, government laboratories) and culture to which students are exposed, enhancing their ability to lead an increasingly diverse workforce. The goal is to produce highly qualified Ph.D. graduates who will make seamless transitions into productive careers as leaders in myriad aspects of nanotechnology. In addition the program will benefit many other students who use the educational programs created; these include a dual degree option enabling students in any of ten departments to obtain a Ph.D. in "Home Department" and Nanotechnology. Moreover, the program strengthens ties among participating departments in Natural Sciences, Engineering, and Medicine through encouraging student-centered interdisciplinary research projects. Coordination with other IGERTs and interdisciplinary programs on campus will focus on common issues of (i) overcoming institutional barriers to interdisciplinary education and (ii) recruiting a diverse student body. Impact will extend beyond the local campus through publication and public presentation of research and educational innovations, and collaboration with emerging regional undergraduate nanotechnology education programs. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

[unreadable] DESCRIPTION (provided by applicant):.We are requesting the acquisition of a photolithography system, a "suite" of pieces of equipment that altogether allow for producing micropatterns of photoresist on surfaces using UV light and photomasks. (We have obtained permission from the Program Official Dr. Marjorie Tingle to request a set of instruments, rather than a single one, because the instruments always go together - they are useless without each other.) The suite includes 1) a contact mask aligner (which aligns and presses the mask against a surface, and exposes the surface through the mask to collimated UV light), 2) a photoresist spinner (which allows for dispensing photoresist onto a surface while the surface is being spun at high speed, so as to form a thin, homogeneous film of photoresist); 3) a wet station (to develop the photoresist after exposure); and 4) hot plates and ovens (needed to evaporate the solvent from the photoresist prior to exposure and to complete the photochemical reaction after exposure). The photolithography suite will be primarily for use by the Bioengineering Department and in general for biologists on campus at the University of Washington in Seattle, and will be located in a HEPA-filtered room of the new Bioengineering Building (Foege Building, inaugurated March 2006). The room is ready to move the equipment in, being equipped with HEPA filters, vacuum outlets, fume exhaust, power lines, and Ethernet lines. The department is effectively cost-sharing in that it hasalready invested in reserving and conditioning the HEPA-filtered room for use as an eventual photolithography facility. A floor plan of the HEPA room is attached. A vented cabinet for chemicals is already built in. Relevance Given the growing role that microengineering plays in Bioengineering and Biology research (including microfluidic "Lab on a Chip" and cell culture systems, micropatterned substrates, microelectrodes, etc.), a small-scale approach to the use of photolithography facilities is critical for fostering innovation, speeding research, and reducing costs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Ion channels play key roles in the physiology of neuronal and non-neuronal cells, in information processing in the nervous system, in brain development, and in a broad spectrum of neurological and non-neurological disorders, such as cardiovascular disease and infertility conditions. To this day, the gold standard for studying ion channels is the patch clamp technique, a laborious technique based on carefully sealing the aperture of a pipette against the cell membrane (the gigaohm seal ). The technique is not easily amenable to automation, so the low throughput of pipette-based recordings is a serious bottleneck for pharmacological screening of ion channel-targeting compounds. Furthermore, recording from >2-3 cells simultaneously is not possible with pipettes;as a result, investigations of communication in complex mature and developing neural networks are limited to extracellular recordings or optical imaging of intracellular calcium activity, both of which are limited in their ability to provide detailed information about intracellular electrical activity. Several groups have recently reported the successful operation of various designs of patch clamp chips, all based on positioning the cell against a microfabricated aperture. We have recently developed a microfluidic patch clamp chip that allows for obtaining gigaohm seals with yields comparable to or surpassing those achievable with a pipette;the performance was evaluated on single rat basophilic leukemia cells during whole-cell recordings of the inward- rectifying potassium ion channel. Here we propose to extend our recent work to recordings from cultured embryonic cortical slices using a novel slice preparation that has a clean layer of cells on the surface of the slice. With the multi-unit patch clamp chip we will investigate the propagation of neuronal signals across developing cortical networks. PUBLIC HEALTH RELEVANCE The successful completion of this project could reveal neuronal communication mechanisms that underlie the propagation of activity waves seen in development (as part of the normal developmental program) as well as in epilepsy. Furthermore, the same technology would enable low-cost screening of ion channel-targeting compounds (which constitute ~25% of drugs) for their effects on single ion channel currents (approximately 50% of safety-related withdrawals of drugs from the market are due to undesired side effects on ion channels, so the FDA now recommends that all drug candidates usually pools of >10,000 compounds be patch clamp-tested for their effects on the hERG channel).Ion channels play key roles in all known brain functions. Using patch clamp chips, it is now possible to monitor the ion channel activity of large numbers of single dissociate cells (but not of brain slices). We propose to develop a patch clamp chip design for monitoring multiple cells on the surface of brain slices. We will use the device to investigate the propagation of neuronal signals across developing cortical networks.

DESCRIPTION (provided by applicant): We have recently developed a microfluidic perfusion and imaging platform for large-scale detection of the response of dissociated mouse olfactory sensory neurons (OSNs) to various odorants;we refer to this platform as "smell-on-a-chip platform". The overall goal of this project is to further develop the existing smell-on-a-chip platform to increase automation so as to improve data quality and increase overall experimental throughput. Our goal is to characterize the dynamics and the specificity of the responses of OSNs to a large variety of odorants, complex odors, and pheromones. Pheromones are volatile organic compounds that elicit or modulate innate behaviors such as mating, rearing of young, aggression and territory marking. In mammals, pheromones are primarily detected by the vomeronasal organ (VNO), but the olfactory epithelium (OE) also appears to play a role in pheromone detection because (in rodents) pheromone-associated behaviors and activation of the olfactory bulb (OB) are still present even if the VNO is removed. Although the VNO in humans has no detectable connection to the brain, the role of pheromone-sensitive OSNs in the mouse OE strongly suggests that pheromone signaling in humans occurs through the OE. OB activation is clearly a proof that there are olfactory sensory neurons (OSNs) in the OE that participate in pheromonal detection, yet the detection of these OSNs by traditional methods has proven elusive, likely because they exist in very low numbers. Hence, identification of these "pheromone-specialist" OSNs in the OE will require high-throughput detection methods. In this proposal we will utilize our smell-on-a-chip platform to detect the responses of dissociated mouse OSNs to known odorants, odors and pheromones. By imaging thousands of OSNs simultaneously we will be able to find rarely-occurring OSN responses. The detection and isolation of pheromone-specialist mouse OSNs would open the way for single-cell gene expression studies in mice towards a deeper, molecular-level understanding of pheromonal-induced behaviors in humans. The characterization of pheromonal responses at the cellular and molecular level is of paramount importance for understanding a large number of innate behaviors (such as sexual attraction, mother-child bonding, and menstrual cycle synchronization, to name only a few), and are of vital interest to the perfume and food industries. The successful completion of this project would also provide a platform of general applicability in a variety of fields for measuring the behavior of a large number of single cells, such as in toxicity studies, drug testing, and small-molecule screening (e.g. for cancer biomarkers), and in finding cells with rare, pathological behaviors from a large population of normally-behaving cells (e.g. the presence of tumorigenic cells in the bloodstream). Obtaining rich, single-cell statistics allows for discerning uniquely-responsive sub-populations of cells and for determining intrinsic cell behavior variability. In the proposed study, we will apply the smell-on-a-chip platform to screen OSNs, but the platform has broad applicability to any imaging-based, high-throughput screen of single dissociated cells in large numbers. Furthermore, rare cells (amongst an array of >28,000 microwells, only one cell per well) can be singled out and manually retrieved for further analysis (e.g. PCR amplification) after characterizing their response to known compounds. PUBLIC HEALTH RELEVANCE: In this proposal we will further develop our smell-on-a-chip platform (successfully prototyped under previous R21 support) to detect and characterize the dynamics, the specificity and the adaptation of the responses of dissociated mouse OSNs to odorants, complex odors, and pheromones. By imaging thousands of OSNs simultaneously we will be able to find rarely-occurring OSN responses. The detection and isolation of pheromone-specialist mouse OSNs would open the way for single-cell gene expression studies in mice towards a deeper, molecular-level understanding of pheromonal-induced behaviors in humans.

DESCRIPTION (provided by applicant): A major goal in neuroscience is to understand the formation and development of synapses, the tiny membrane specializations that enable nerve cells to communicate with each other. The sequence of molecular signals leading to synapse formation (synaptogenesis) is qualitatively well known for the more accessible neuromuscular junction (NMJ) [2]. However, very little is known of the quantities (concentration, duration, onset, etc.) of the various neurochemical signals involved in synaptogenesis. Intriguingly, all but one of the axons innervating a given myotube at birth retract after a period of ~1 week as a result of a synaptic competition process that remains, for lack of quantitative methods, poorly understood. Our overall objective is to uncover some of the rules governing the formation and elimination of synapses at the NMJ using a microfluidic cell culture system developed under a previous R01 (which we seek to renew for the first time). Our approach is based on substituting the presynaptic neuron by an artificial microfluidic device that delivers known doses of various synaptogenic neurochemicals to micrometer-scale areas of the membrane of cultured myotubes. We will focus on the three key factors - agrin, neuregulin, and the neurotransmitter acetylcholine (ACh) - secreted by the nerve tip during synaptogenesis. We will measure muscle cell responses that are specific to ACh receptors (AChRs), such as AChR aggregation/disaggregation, degradation/synthesis, insertion, co-localization with other receptors and cytoskeletal proteins, intracellular signaling pathways, etc. Under previous support, we have developed a microfluidic mimic of the innervation process that allows for focally stimulating >80 single, isolated (microengineered) myotubes using laminar flow streams (orthogonal to the myotubes). We have found that a) focal application of agrin entices myotubes to recruit new AChRs to the stimulated area; b) when the microtracks are formed with Matrigel, a basal lamina extract, the microengineered myotubes display AChR clusters of complex, in-vivo-like morphologies even before agrin is applied, similarly to what happens in vivo; and c) when agrin is focally applied to those agrin-predating clusters, AChRs are degraded at reduced rates, suggesting that a putative role for agrin in vivo is to help stabilize AChRs. We seek to continue these investigations by studying the dynamics and spatial patterns of various AChR-specific responses upon (competitive, synergistic, or combinatorial) stimulation with agrin, neuregulin, and ACh.

DESCRIPTION (provided by applicant): During development of the nervous system the response of growing axons to their environment is critical to the formation of the complex wiring pattern between neurons. Growth and guidance factors combined with extracellular matrices influence the speed and direction of axonal growth. Although much progress has been made in identifying the factors that influence axonal growth, as well as how axons respond to these factors individually, much less is known about how axons behave in response to the combined effects of multiple factors. As a complementary approach to present in vivo molecular imaging approaches, we propose to develop an in vitro environment that potentially mimics some of the complexity found in vivo, in particular the development of the anterior visual pathway. In this system, the axon trajectories are simple, multiple relevant guidance molecules have been identified already (many tested with explants in vitro), and a common cause of blindness (Optic Nerve Hypoplasia) is associated with defects in this process. Additionally, the patterns of guidance molecules found on the flat anatomy of the retina are ideally suited to mimicking by micropatterning and microfluidics techniques. This mimicry will be accomplished by combining microfluidics patterning of diffusible gradients and laser patterning of substrate-bound axon pathfinding cues, including axon guidance factors and extracellular matrix molecules. As a source of highly homogeneous cell populations, we will isolate mouse retinal ganglion cells (RGCs), a cell type that responds to Netrin-1 gradients. For experiments designed to maximize the integrity of the cells (isolation procedures are damaging to cells), we will use retinal explants and we will microfluidically isolate the axons from their somas. RGCs (or their axons) will be exposed to various soluble factors that have previously been shown to affect their axon growth in vivo. The new microfluidic systems will allow us to test the combinatorial effects of multiple factors on the direction and speed of axonal growth of RGCs. These experiments will allow us to quantitatively examine the basic principles that govern axon pathfinding in the development of the anterior visual pathway. This information will help to better understand the basis of developmental defects in axon growth that alter the organization and function of the nervous system. PUBLIC HEALTH RELEVANCE: The study of axon guidance has been limited to a single signal gradient. In vivo, neurons encounter multiple signals (both bound to substrate and in solution) and must make choices in an information rich environment. The proposed study will probe axon guidance interactions of unprecedented complexity as well as with unprecedented measurement precision, which will significantly further our understanding of axon growth on a cellular and molecular level, neural development as a whole, and may provide insight on treating neurological disorders, nerve regeneration, and vascular pathologies.

DESCRIPTION: Goal: We propose to develop a therapeutic screening platform that rapidly determines the response of intact human tumor samples to many potential therapies in parallel. The aim is to identify the subset of therapies of greatest potential value to individual patients, n a timescale rapid enough to guide therapeutic decision- making. Innovation: We have developed a microfluidic perfusion system that maintains intact primary tumor slice tissue in culture and enables the arrayed delivery of large numbers of different drugs, drug combinations or drug regimens to anatomically-defined regions of the tumor. Our system improves upon existing models for screening chemotherapeutic drug activity (e.g. tumor cells in culture, mouse xenografts, or genetically engineered mouse models): it uses intact tumor samples that retain the human tumor microenvironment and allows the generation of response data in a time frame that can guide decision-making for the initial phases of therapy. Focus/Aims: Our proposed research focuses on human brain tumors, specifically the malignant gliomas that comprise the majority (70%) of primary adult brain tumors. These include Grade III malignant gliomas and the more common Grade IV glioblastoma multiforme (GBM). Patients diagnosed with GBM have a median survival of ~1 year and an overall 5-year survival of <5 %, despite decades of effort to improve treated outcomes. In this proposal we argue that an important way to make progress in treating these lethal, refractory tumors is to develop a way to rapidly test many potential therapeutic agents or regimens in parallel on intact GBM specimens from individual patients. Tumor response to drugs will be measured by imaging of markers for cell death and for cell viability. As individual tumor samples are often heterogeneous, it is critical that our microfluidi system enables multiplexed drug testing across different regions of the sample. Positive drug responses should be consistent and specific across the tumor. The multiplexed platform will also enable investigation of specific drug combinations and dosages that might be therapeutically useful. Impact: This work addresses the urgent need to develop approaches and devices to rapidly and reliably assess the response of often heterogeneous, intact primary tumor specimens to a range of possible therapies, with the goal of identifying the most effective subset of therapies for individual tumors and patients.

ABSTRACT The goal of this project is to perform high-content analysis of drug and immunotherapy responses on hundreds of intact, live cultured fragments isolated from a single live tumor biopsy. In recent years, patient-derived tumor ?organoids? have shown great promise to predict drug responses for personalized cancer treatment. Immunotherapy, including cellular immunotherapy, represents the next generation of cancer therapy, and many of the relevant drugs act on the local tumor microenvironment (TME). There is a pressing need for functional testing platforms that use human, intact and live tumor tissue to better predict traditional and immunotherapy responses. Such platforms should also retain as much of the native TME as possible. 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, the micro-dissection of tumor tissue into ?spheroids? that contain the TME intact has shown promising responses to immunomodulators on native immune cells. We propose a microfluidic platform that enables drug treatment, exogenous T cell therapy, and high-content analysis using hundreds to thousands of similarly sized, precision-sliced cuboidal micro-tissues (CµTs) produced from a single tumor sample. Here we propose a combination of two methodologies to demonstrate the feasibility of our approach: 1) precision slicing methodology that will produce large numbers of cuboidal micro-tissues (CµTs) from a single tumor biopsy; and 2) microfluidic trapping of the CµTs in a multi-well platform, allowing for drug application to each individual CµT or groups of CµTs. We will be able to obtain several hundred patient-derived CµTs from each tumor resection. The size of the CµTs (initially 400 µm×400 µm×400 µm) will be reproducible and chosen to optimize viability and retention of the TME. As the CµTs are cultured, their cuboidal shape will relax into a more rounded one. We will study the viability of the CµTs and their TME composition as a function of size in various culture conditions, including collagen gels. We will focus on breast cancer immunotherapy using a syngeneic mouse breast tumor model. For this Aim, we will deliver various concentrations and combinations of immunomodulatory drugs, including antibody-based drugs, to breast tumor CµTs in the microfluidic device, and examine the effects on the resident immune system. We will assess cytokine production and use high-content immunohistochemistry and bioinformatics analysis to assess immune cell engagement with different cell types as well as cell death. We will apply the platform to deliver immune checkpoint inhibitors (CTLA4, PD-L1, PD-1) and other immunomodulators (such as IL-10) and examine the effect on the immune state, cell death, and the behavior of resident T cells (activation and localization). In the R33 phase we plan on applying our microfluidic platform to CµTs obtained from breast tumor patients (ongoing collaboration with Dr. V.K. Gadi, Fred Hutch).

ABSTRACT The complexity of cell-cell interactions in the tumor represents one of the biggest barriers to understanding cancer and to developing effective therapeutics. Cancer cells constantly interact with fibroblast cells, endothelial cells, immune cells, signaling molecules and the extracellular matrix in the tumor microenvironment (TME). The interactions between host and cancer cells are complex, with effects that may be tumor-suppressive or tumor-promoting. For example, macrophages are first recruited to fight cancer; however, interactions with cancer cells can render them tumor-supportive. Thus, enabling a greater understanding of the complexities of the interaction between cancer cells and their microenvironment can lead to a better understanding of mechanisms of drug resistance, identification of new molecular targets and help address the large unmet needs in treating cancer. Quantitative technologies, such as ours, that integrate ex vivo drug treatments and assess pharmacological responses with cellular and molecular phenotypes in native tissues, should accelerate the discovery and development of novel therapeutics. Yet present tools to study drug responses and the TME have not kept up with drug testing needs. Given the nearly infinite number of potential combinations and limited resources to actually test them, there is a great need for testing platforms that use human, intact tumor tissue ex vivo to predict in vivo responses to combination immunotherapies in miniaturized, multiplexed formats that retain as much of the TME as possible. In our first R01 cycle, we developed a microfluidic platform (called Oncoslice) that allows for selective spatiotemporal exposure of organotypic cultures to dozens of drug conditions, and we demonstrated its utility with cell death assays on xenograft and patient GBM slices. In this second R01 cycle, our goal is to apply our Oncoslice platform to evaluate combination immunotherapies and their interaction with the TME. The platform will be applied to two difficult-to-treat solid tumor types ? pancreatic cancer (PCa, mouse, and human) and triple-negative breast cancer (TNBC, mouse). Our recent finding that intratumoral migration of CD8+ T cells is a necessary precursor to anti-tumor activity in response to immune checkpoint inhibitor therapy in pancreatic cancer lends credence to our assertion that developing a micro-scale understanding of cell-cell interactions in the TME is critical. Both PCa and TNBC are extremely heterogeneous and respond poorly to current immune checkpoint inhibitors. New developments to our platform will include live imaging of dynamic changes in the immune TME, tissue and cytokine sampling, selective transcriptomics, and protein pathway profiling. We will integrate phenotypic responses in slices with molecular data to build predictive statistical models. Together, these approaches will establish an innovative platform for immuno-modulatory drug discovery designed to provide insights into the drug's mechanism of action and the TME's role in cancer treatment.

PROJECT SUMMARY / ABSTRACT Tissue Chips ? microfluidic devices containing human cells in 3D architectures that attempt to recapitulate the physiology and pathophysiology of human tissues and organs ? contain advanced designs that critically require 3D/modular fabrication, the incorporation of multiple materials and functionalities, and fluidic automation. The vast majority of Tissue Chips are still prototyped in poly(dimethylsiloxane) (PDMS). However, difficult barriers remain for PDMS as a Tissue Chip material. The surface of PDMS is porous and hydrophobic, so both absorption into PDMS and adsorption onto PDMS can potentially alter experimental outcomes by changing the target concentrations and by partitioning molecules in undesired regions of a microfluidic device. 3D-Printing holds an obvious potential for Tissue Chips. Stereolithography (SL), in particular, has been an excellent choice for modulating shapes in 3D at high resolution but modulating material composition is still a challenge. There is a critical need for more advanced, high-resolution and multi-material SL-printing approaches to building future Tissue Chips that can integrate the structural components of a device (channels and valves) with biofabrication (cells and scaffolds). Yet there is no off-the-shelf solution to SL-print multi-material microfluidic devices of wide applicability. This application proposes the synthesis and SL-printing of cyclo-octyne methacrylate (COMA) resin, which will enable the immobilization of any biomolecule-azide of choice onto the COMA-printed surfaces. COMA for derivatizing printed parts via straightforward copper-free (biocompatible) click chemistry, in this case by conjugation to an azide group which spontaneously and specifically reacts with the COMA group in aqueous solutions. Using biotin-azide (commercially available) and an avidin linkage, we will be able to immobilize any biotinylated biomolecule of choice onto the COMA-printed surfaces. We will also use a baseline acrylate resin composed of poly(ethylene glycol) diacrylate (MW~258) (PEG-DA-258), which has successfully been used in microfluidics by several labs, and will experiment with blending PEG-DA-258 with other diacrylates and/or monoacrylates to obtain resins with differing properties, such as higher flexibility. To print devices that are made partially with PEG-DA-258 resin (or blends) and partially with COMA resin (or blends), we will utilize a strategy for co-printing multiple acrylate resins recently utilized by the Folch lab that consists of pausing the print and exchanging the resins in the vat. This scheme will have wide applicability to 3D-print microfluidic devices with multiple regions bearing molecular functionalities (e.g. biomolecular detection, cell capture) and/or elements with distinct sensing/actuating properties (e.g. microvalves, force sensors, etc.). Examples include 3D-printed multiplexed immunosensors based on COMA-derivatized regions, cell trapping devices for drug screening, and organoid-on-a-chip automated platforms, among others.