Next generation microfluidics: fulfilling the promise of lab-on-a-chip technologies.

Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise. We describe how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues. In this report, we discuss the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use. While our report focuses on using blood as the prototypical biofluid sample, the proposed ideas and research directions can be extrapolated to other areas of hematology, oncology, biology, and medicine.

CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon RB function in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue sarcomas with limited treatment options, and new effective therapeutic strategies are desperately needed. We observe antiproliferative potency of genetic depletion of PTPN11 or pharmacological inhibition using the SHP2 inhibitor (SHP2i) TNO155. Our studies into the signaling response to SHP2i reveal that resistance to TNO155 is partially mediated by reduced RB function, and we therefore test the addition of a CDK4/6 inhibitor (CDK4/6i) to enhance RB activity and improve TNO155 efficacy. In combination, TNO155 attenuates the adaptive response to CDK4/6i, potentiates its antiproliferative effects, and converges on enhancement of RB activity, with greater suppression of cell cycle and inhibitor-of-apoptosis proteins, leading to deeper and more durable antitumor activity in in vitro and in vivo patient-derived models of MPNST, relative to either single agent. Overall, our study provides timely evidence to support the clinical advancement of this combination strategy in patients with MPNST and other tumors driven by loss of NF1.

High-throughput quantification of red blood cell deformability and oxygen saturation to probe mechanisms of sickle cell disease.

The complex, systemic pathology of sickle cell disease is driven by multiple mechanisms including red blood cells (RBCs) stiffened by polymerized fibers of deoxygenated sickle hemoglobin. A critical step toward understanding the pathologic role of polymer-containing RBCs is quantifying the biophysical changes in these cells in physiologically relevant oxygen environments. We have developed a microfluidic platform capable of simultaneously measuring single RBC deformability and oxygen saturation under controlled oxygen and shear stress. We found that RBCs with detectable amounts of polymer have decreased oxygen affinity and decreased deformability. Surprisingly, the deformability of the polymer-containing cells is oxygen-independent, while the fraction of these cells increases as oxygen decreases. We also find that some fraction of these cells is present at most physiologic oxygen tensions, suggesting a role for these cells in the systemic pathologies. Additionally, the ability to measure these pathological cells should provide clearer targets for evaluating therapies.

Genetic reversal of the globin switch concurrently modulates both fetal and sickle hemoglobin and reduces red cell sickling.

We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications. Here we report results of exploratory single-cell analysis of patients in which BCL11A is targeted molecularly and compare results with cells of patients treated with hydroxyurea (HU), the current standard of care. We use single-cell assays to assess HbF, HbS, oxygen saturation, and hemoglobin polymer content in RBCs for nine gene therapy trial subjects (BCLshmiR, median HbF% = 27.9) and compare them to 10 HU-treated subjects demonstrating high and comparable levels of HbF (HU High Responders, median HbF% = 27.0). All BCL11A patients achieved the primary endpoint for NCT03282656, which was defined by an absolute neutrophil count greater than or equal to 0.5 × 109 cells/L for three consecutive days, achieved within 7 weeks following infusion. Flow cytometric assessment of single-RBC HbF and HbS shows fewer RBCs with high HbS% that would be most susceptible to sickling in BCLshmiR vs. HU High Responders: median 42% of RBCs with HbS%>70% in BCLshmiR vs. 61% in HU High Responders (p = 0.004). BCLshmiR subjects also demonstrate more RBCs resistant to HbS polymerization at lower physiologic oxygen tension: median 32% vs. 25% in HU High Responders (p = 0.006). Gene therapy-induced BCL11A down-regulation reverses the fetal-to-adult hemoglobin switch and induces RBCs with higher HbF%, lower HbS%, and greater resistance to deoxygenation-induced polymerization in clinical trial subjects compared with a cohort of highly responsive hydroxyurea-treated subjects.

iCLOTS: open-source, artificial intelligence-enabled software for analyses of blood cells in microfluidic and microscopy-based assays.

While microscopy-based cellular assays, including microfluidics, have significantly advanced over the last several decades, there has not been concurrent development of widely-accessible techniques to analyze time-dependent microscopy data incorporating phenomena such as fluid flow and dynamic cell adhesion. As such, experimentalists typically rely on error-prone and time-consuming manual analysis, resulting in lost resolution and missed opportunities for innovative metrics. We present a user-adaptable toolkit packaged into the open-source, standalone Interactive Cellular assay Labeled Observation and Tracking Software (iCLOTS). We benchmark cell adhesion, single-cell tracking, velocity profile, and multiscale microfluidic-centric applications with blood samples, the prototypical biofluid specimen. Moreover, machine learning algorithms characterize previously imperceptible data groupings from numerical outputs. Free to download/use, iCLOTS addresses a need for a field stymied by a lack of analytical tools for innovative, physiologically-relevant assays of any design, democratizing use of well-validated algorithms for all end-user biomedical researchers who would benefit from advanced computational methods.

Robust microtubule dynamics facilitate low-tension kinetochore detachment in metaphase.

During mitosis, sister chromatids are stretched apart at their centromeres via their attachment to oppositely oriented kinetochore microtubules. This stretching generates inwardly directed tension across the separated sister centromeres. The cell leverages this tension signal to detect and then correct potential errors in chromosome segregation, via a mechanical tension signaling pathway that detaches improperly attached kinetochores from their microtubules. However, the sequence of events leading up to these detachment events remains unknown. In this study, we used microfluidics to sustain and observe low-tension budding yeast metaphase spindles over multiple hours, allowing us to elucidate the tension history prior to a detachment event. We found that, under conditions in which kinetochore phosphorylation weakens low-tension kinetochore-microtubule connections, the mechanical forces produced via the dynamic growth and shortening of microtubules is required to efficiently facilitate detachment events. Our findings underscore the critical role of robust kinetochore microtubule dynamics in ensuring the fidelity of chromosome segregation during mitosis.

Ex vivo to in vivo model of malignant peripheral nerve sheath tumors for precision oncology.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1). To address the critical need for novel therapeutics in MPNST, we aimed to establish an ex vivo 3D platform that accurately captured the genomic diversity of MPNST and could be utilized in a medium-throughput manner for drug screening studies to be validated in vivo using patient-derived xenografts (PDX). METHODS: Genomic analysis was performed on all PDX-tumor pairs. Selected PDX were harvested for assembly into 3D microtissues. Based on prior work in our labs, we evaluated drugs (trabectedin, olaparib, and mirdametinib) ex vivo and in vivo. For 3D microtissue studies, cell viability was the endpoint as assessed by Zeiss Axio Observer. For PDX drug studies, tumor volume was measured twice weekly. Bulk RNA sequencing was performed to identify pathways enriched in cells. RESULTS: We developed 13 NF1-associated MPNST-PDX and identified mutations or structural abnormalities in NF1 (100%), SUZ12 (85%), EED (15%), TP53 (15%), CDKN2A (85%), and chromosome 8 gain (77%). We successfully assembled PDX into 3D microtissues, categorized as robust (>90% viability at 48 h), good (>50%), or unusable (<50%). We evaluated drug response to "robust" or "good" microtissues, namely MN-2, JH-2-002, JH-2-079-c, and WU-225. Drug response ex vivo predicted drug response in vivo, and enhanced drug effects were observed in select models. CONCLUSIONS: These data support the successful establishment of a novel 3D platform for drug discovery and MPNST biology exploration in a system representative of the human condition.

CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon restoration of RB function in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft tissue sarcomas with limited treatment options, and novel effective therapeutic strategies are desperately needed. We observe anti-proliferative efficacy of genetic depletion or pharmacological inhibition using the clinically available SHP2 inhibitor (SHP2i) TNO155. Our studies into the signaling response to SHP2i reveal that resistance to TNO155 is partially mediated by reduced RB function, and we therefore test the addition of a CDK4/6 inhibitor (CDK4/6i) to enhance RB activity and improve TNO155 efficacy. In combination, TNO155 attenuates the adaptive response to CDK4/6i, potentiates its anti-proliferative effects, and converges on enhancement of RB activity, with greater suppression of cell cycle and inhibitor-of-apoptosis proteins, leading to deeper and more durable anti-tumor activity in in vitro and in vivo patient-derived models of MPNST, relative to either single agent. Overall, our study provides timely evidence to support the clinical advancement of this combination strategy in patients with MPNST and other tumors driven by loss of NF1.

JAK-STAT inhibition reduces endothelial prothrombotic activation and leukocyte-endothelial proadhesive interactions.

BACKGROUND: Vascular activation is characterized by increased proinflammatory, pro thrombotic, and proadhesive signaling. Several chronic and acute conditions, including Bcr-abl-negative myeloproliferative neoplasms (MPNs), graft-vs-host disease, and COVID-19 have been noted to have increased activation of the janus kinase (JAK)-signal transducer and downstream activator of transcription (STAT) pathways. Two notable inhibitors of the JAK-STAT pathway are ruxolitinib (JAK1/2 inhibitor) and fedratinib (JAK2 inhibitor), which are currently used to treat MPN patients. However, in some conditions, it has been noted that JAK inhibitors can increase the risk of thromboembolic complications. OBJECTIVES: We sought to define the anti-inflammatory and antithrombotic effects of JAK-STAT inhibitors in vascular endothelial cells. METHODS: We assessed endothelial activation in the presence or absence of ruxolitinib or fedratinib by using immunoblots, immunofluorescence, qRT-PCR, and function coagulation assays. Finally, we used endothelialized microfluidics perfused with blood from normal and JAK2V617F+ individuals to evaluate whether ruxolitinib and fedratinib changed cell adhesion. RESULTS: We found that both ruxolitinib and fedratinib reduced endothelial cell phospho-STAT1 and STAT3 signaling and attenuated nuclear phospho-NK-κB and phospho-c-Jun localization. JAK-STAT inhibition also limited secretion of proadhesive and procoagulant P-selectin and von Willebrand factor and proinflammatory IL-6. Likewise, we found that JAK-STAT inhibition reduced endothelial tissue factor and urokinase plasminogen activator expression and activity. CONCLUSIONS: By using endothelialized microfluidics perfused with whole blood samples, we demonstrated that endothelial treatment with JAK-STAT inhibitors prevented rolling of both healthy control and JAK2V617F MPN leukocytes. Together, these findings demonstrate that JAK-STAT inhibitors reduce the upregulation of critical prothrombotic pathways and prevent increased leukocyte-endothelial adhesion.

Simultaneous quantification of blood rheology and oxygen saturation to evaluate affinity-modifying therapies in sickle cell disease.

Sickle cell blood demonstrates oxygen-dependent flow behavior as a result of HbS polymerization during hypoxia, and these rheological changes provide a biophysical metric that can be used to quantify the pathological behavior of the blood. Relating these rheological changes directly to hemoglobin oxygen saturation would improve our understanding of SCD pathogenesis and the potential effects of therapeutic drugs. Towards this end, we have developed a microfluidic platform capable of spectrophotometric quantification of Hb-O2 saturation and simultaneous evaluation of the accompanying rheological changes in SCD blood flow. We demonstrated the ability to measure changes in Hb-O2 affinity and a restoration of oxygen-independent blood flow behavior after incubation with voxelotor, an oxygen affinity modifying drug approved for use in SCD. We also identified regimes in Hb-O2 saturation where the effects of HbS polymerization begin to take effect in contributing to pathological flow behavior, independent of voxelotor treatment. In contrast, incubation with voxelotor recovered oxygen-dependent blood flow over a range of PO2, providing a framework for understanding voxelotor's therapeutic effect in lowering the PO2 at which the requisite Hb-O2 saturation is reached to observe pathological blood flow. These results help explain the mechanistic effects of voxelotor and show the potential of this platform to identify affinity-modifying compounds and evaluate their therapeutic effect on blood flow.

Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.

The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α2ß2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.

Microfluidic methods to advance mechanistic understanding and translational research in sickle cell disease.

Sickle cell disease (SCD) is caused by a single point mutation in the ß-globin gene of hemoglobin, which produces an altered sickle hemoglobin (HbS). The ability of HbS to polymerize under deoxygenated conditions gives rise to chronic hemolysis, oxidative stress, inflammation, and vaso-occlusion. Herein, we review recent findings using microfluidic technologies that have elucidated mechanisms of oxygen-dependent and -independent induction of HbS polymerization and how these mechanisms elicit the biophysical and inflammatory consequences in SCD pathophysiology. We also discuss how validation and use of microfluidics in SCD provides the opportunity to advance development of numerous therapeutic strategies, including curative gene therapies.

Feature tracking microfluidic analysis reveals differential roles of viscosity and friction in sickle cell blood.

Characterization of blood flow rheology in hematological disorders is critical for understanding disease pathophysiology. Existing methods to measure blood rheological parameters are limited in their physiological relevance, and there is a need for new tools that focus on the microcirculation and extract properties at finer resolution than overall flow resistance. Herein, we present a method that combines microfluidic systems and powerful object-tracking computational technologies with mathematical modeling to separate the red blood cell flow profile into a bulk component and a wall component. We use this framework to evaluate differential contributions of effective viscosity and wall friction to the overall resistance in blood from patients with sickle cell disease (SCD) under a range of oxygen tensions. Our results demonstrate that blood from patients with SCD exhibits elevated frictional and viscous resistances at all physiologic oxygen tensions. Additionally, the viscous resistance increases more rapidly than the frictional resistance as oxygen tension decreases, which may confound analyses that extract only flow velocities or overall flow resistances. Furthermore, we evaluate the impact of transfusion treatments on the components of the resistance, revealing patient variability in blood properties that may improve our understanding of the heterogeneity of clinical responses to such treatments. Overall, our system provides a new method to analyze patient-specific blood properties and can be applied to a wide range of hematological and vascular disorders.

Extracellular Vesicles Mediate the Intercellular Exchange of Nanoparticles.

To exert their therapeutic effects, nanoparticles (NPs) often need to travel into the tissues composed of multilayered cells. Accumulative evidence has revealed the crucial role of transcellular transport route (entry into one cell, exocytosis, and re-entry into another) in this process. While NP endocytosis and subcellular transport are intensively characterized, the exocytosis and re-entry steps are poorly understood, which becomes a barrier for NP delivery into complex tissues. Here, the authors term the exocytosis and re-entry steps together as intercellular exchange. A collagen-based three-dimension assay is developed to specifically quantify the intercellular exchange of NPs, and distinguish the contributions of several potential mechanisms. The authors show that NPs can be exocytosed freely or enclosed inside extracellular vesicles (EVs) for re-entry, while direct cell-cell contact is hardly involved. EVs account for a significant fraction of NP intercellular exchange, and its importance in NP transport is demonstrated in vitro and in vivo. While freely released NPs engage with the same receptors for re-entry, EV-enclosed ones bypass this dependence. These studies provide an easy and precise system to investigate the intercellular exchange stage of NP delivery, and shed the first light in the importance of EVs in NP transport between cells and into complex tissues.

Ionophore-mediated swelling of erythrocytes as a therapeutic mechanism in sickle cell disease.

Sickle cell disease (SCD) is characterized by sickle hemoglobin (HbS) which polymerizes under deoxygenated conditions to form a stiff, sickled erythrocyte. The dehydration of sickle erythrocytes increases intracellular HbS concentration and the propensity of erythrocyte sickling. Prevention of this mechanism may provide a target for potential SCD therapy investigation. Ionophores such as monensin can increase erythrocyte sodium permeability by facilitating its transmembrane transport, leading to osmotic swelling of the erythrocyte and decreased hemoglobin concentration. In this study, we treated 13 blood samples from patients with SCD with 10 nM of monensin ex vivo. We measured changes in cell volume and hemoglobin concentration in response to monensin treatment, and we perfused treated blood samples through a microfluidic device that permits quantification of blood flow under controlled hypoxia. Monensin treatment led to increases in cell volume and reductions in hemoglobin concentration in most blood samples, though the degree of response varied across samples. Monensin-treated samples also demonstrated reduced blood flow impairment under hypoxic conditions relative to untreated controls. Moreover, there was a significant correlation between the improvement in blood flow and the decrease in hemoglobin concentration. Thus, our results demonstrate that a reduction in intracellular HbS concentration by osmotic swelling improves blood flow under hypoxic conditions. Although the toxicity of monensin will likely prevent it from being a viable clinical treatment, these results suggest that osmotic swelling should be investigated further as a potential mechanism for SCD therapy.

Stromal architecture directs early dissemination in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an extremely metastatic and lethal disease. Here, in both murine and human PDA, we demonstrate that extracellular matrix architecture regulates cell extrusion and subsequent invasion from intact ductal structures through tumor-associated collagen signatures (TACS). This results in early dissemination from histologically premalignant lesions and continual invasion from well-differentiated disease, and it suggests TACS as a biomarker to aid in the pathologic assessment of early disease. Furthermore, we show that pancreatitis results in invasion-conducive architectures, thus priming the stroma prior to malignant disease. Analysis in potentially novel microfluidic-derived microtissues and in vivo demonstrates decreased extrusion and invasion following focal adhesion kinase (FAK) inhibition, consistent with decreased metastasis. Thus, data suggest that targeting FAK or strategies to reengineer and normalize tumor microenvironments may have roles not only in very early disease, but also for limiting continued dissemination from unresectable disease. Likewise, it may be beneficial to employ stroma-targeting strategies to resolve precursor diseases such as pancreatitis in order to remove stromal architectures that increase risk for early dissemination.

The effect of rigid cells on blood viscosity: linking rheology and sickle cell anemia.

Sickle cell anemia (SCA) is a disease that affects red blood cells (RBCs). Healthy RBCs are highly deformable objects that under flow can penetrate blood capillaries smaller than their typical size. In SCA there is an impaired deformability of some cells, which are much stiffer and with a different shape than healthy cells, and thereby affect regular blood flow. It is known that blood from patients with SCA has a higher viscosity than normal blood. However, it is unclear how the rigidity of cells is related to the viscosity of blood, in part because SCA patients are often treated with transfusions of variable amounts of normal RBCs and only a fraction of cells will be stiff. Here, we report systematic experimental measurements of the viscosity of a suspension varying the fraction of rigid particles within a suspension of healthy cells. We also perform systematic numerical simulations of a similar mixed suspension of soft RBCs, rigid particles, and their hydrodynamic interactions. Our results show that there is a rheological signature within blood viscosity to clearly identify the fraction of rigidified cells among healthy deformable cells down to a 5% volume fraction of rigidified cells. Although aggregation of RBCs is known to affect blood rheology at low shear rates, and our simulations mimic this effect via an adhesion potential, we show that such adhesion, or aggregation, is unlikely to provide a physical rationalization for the viscosity increase observed in the experiments at moderate shear rates due to rigidified cells. Through numerical simulations, we also highlight that most of the viscosity increase of the suspension is due to the rigidity of the particles rather than their sickled or spherical shape. Our results are relevant to better characterize SCA, provide useful insights relevant to rheological consequences of blood transfusions, and, more generally, extend to the rheology of mixed suspensions having particles with different rigidities, as well as offering possibilities for developments in the field of soft material composites.

A scalable 3D tissue culture pipeline to enable functional therapeutic screening for pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease targeting the alveolar gas exchange apparatus, leading to death by asphyxiation. IPF progresses on a tissue scale through aberrant matrix remodeling, enhanced cell contraction, and subsequent microenvironment densification. Although two pharmaceuticals modestly slow progression, IPF patient survival averages less than 5 years. A major impediment to therapeutic development is the lack of high-fidelity models that account for the fibrotic microenvironment. Our goal is to create a three-dimensional (3D) platform to enable lung fibrosis studies and recapitulate IPF tissue features. We demonstrate that normal lung fibroblasts encapsulated in collagen microspheres can be pushed toward an activated phenotype, treated with FDA-approved therapies, and their fibrotic function quantified using imaging assays (extracellular matrix deposition, contractile protein expression, and microenvironment compaction). Highlighting the system's utility, we further show that fibroblasts isolated from IPF patient lungs maintain fibrotic phenotypes and manifest reduced fibrotic function when treated with epigenetic modifiers. Our system enables enhanced screening due to improved predictability and fidelity compared to 2D systems combined with superior tractability and throughput compared to 3D systems.

An irradiated marrow niche reveals a small noncollagenous protein mediator of homing, dermatopontin.

Hematopoietic cell homing after hematopoietic cell transplant (HCT) is governed by several pathways involving marrow niche cells that are evoked after pre-HCT conditioning. To understand the factors that play a role in homing, we performed expression analysis on zebrafish marrow niche cells following conditioning. We determined that the noncollagenous protein extracellular matrix related protein dermatopontin (Dpt) was upregulated sevenfold in response to irradiation. Studies in mice revealed DPT induction with radiation and lipopolysaccharide exposure. Interestingly, we found that coincubation of zebrafish or murine hematopoietic cells with recombinant DPT impedes hematopoietic stem and progenitor cell homing by 50% and 86%, respectively. Similarly, this translated into a 24% reduction in long-term engraftment (vs control; P = .01). We found DPT to interact with VLA-4 and block hematopoietic cell-endothelial cell adhesion and transendothelial migration. Finally, a DPT-knockout mouse displayed a 60% increase in the homing of hematopoietic cells vs wild-type mice (P = .03) with a slight improvement in long-term lin-SCA1+cKIT+-SLAM cell engraftment (twofold; P = .04). These data show that the extracellular matrix-related protein DPT increases with radiation and transiently impedes the transendothelial migration of hematopoietic cells to the marrow.

GPCR-mediated YAP/TAZ inactivation in fibroblasts via EPAC1/2, RAP2C, and MAP4K7.

Yes-associated protein (YAP) and PDZ-binding motif (TAZ) have emerged as important regulators of pathologic fibroblast activation in fibrotic diseases. Agonism of Gαs-coupled G protein coupled receptors (GPCRs) provides an attractive approach to inhibit the nuclear localization and function of YAP and TAZ in fibroblasts that inhibits or reverses their pathological activation. Agonism of the dopamine D1 GPCR has proven effective in preclinical models of lung and liver fibrosis. However, the molecular mechanisms coupling GPCR agonism to YAP and TAZ inactivation in fibroblasts remain incompletely understood. Here, using human lung fibroblasts, we identify critical roles for the cAMP effectors EPAC1/2, the small GTPase RAP2c, and the serine/threonine kinase MAP4K7 as the essential elements in the downstream signaling cascade linking GPCR agonism to LATS1/2-mediated YAP and TAZ phosphorylation and nuclear exclusion in fibroblasts. We further show that this EPAC/RAP2c/MAP4K7 signaling cascade is essential to the effects of dopamine D1 receptor agonism on reducing fibroblast proliferation, contraction, and extracellular matrix production. Targeted modulation of this cascade in fibroblasts may prove a useful strategy to regulate YAP and TAZ signaling and fibroblast activities central to tissue repair and fibrosis.