Immune-responsive biodegradable scaffolds for enhancing neutrophil regeneration.

Neutrophils are essential effector cells for mediating rapid host defense and their insufficiency arising from therapy-induced side-effects, termed neutropenia, can lead to immunodeficiency-associated complications. In autologous hematopoietic stem cell transplantation (HSCT), neutropenia is a complication that limits therapeutic efficacy. Here, we report the development and in vivo evaluation of an injectable, biodegradable hyaluronic acid (HA)-based scaffold, termed HA cryogel, with myeloid responsive degradation behavior. In mouse models of immune deficiency, we show that the infiltration of functional myeloid-lineage cells, specifically neutrophils, is essential to mediate HA cryogel degradation. Post-HSCT neutropenia in recipient mice delayed degradation of HA cryogels by up to 3 weeks. We harnessed the neutrophil-responsive degradation to sustain the release of granulocyte colony stimulating factor (G-CSF) from HA cryogels. Sustained release of G-CSF from HA cryogels enhanced post-HSCT neutrophil recovery, comparable to pegylated G-CSF, which, in turn, accelerated cryogel degradation. HA cryogels are a potential approach for enhancing neutrophils and concurrently assessing immune recovery in neutropenic hosts.

Matrix viscoelasticity controls spatiotemporal tissue organization.

Biomolecular and physical cues of the extracellular matrix environment regulate collective cell dynamics and tissue patterning. Nonetheless, how the viscoelastic properties of the matrix regulate collective cell spatial and temporal organization is not fully understood. Here we show that the passive viscoelastic properties of the matrix encapsulating a spheroidal tissue of breast epithelial cells guide tissue proliferation in space and in time. Matrix viscoelasticity prompts symmetry breaking of the spheroid, leading to the formation of invading finger-like protrusions, YAP nuclear translocation and epithelial-to-mesenchymal transition both in vitro and in vivo in a Arp2/3-complex-dependent manner. Computational modelling of these observations allows us to establish a phase diagram relating morphological stability with matrix viscoelasticity, tissue viscosity, cell motility and cell division rate, which is experimentally validated by biochemical assays and in vitro experiments with an intestinal organoid. Altogether, this work highlights the role of stress relaxation mechanisms in tissue growth dynamics, a fundamental process in morphogenesis and oncogenesis.

Active tissue adhesive activates mechanosensors and prevents muscle atrophy.

While mechanical stimulation is known to regulate a wide range of biological processes at the cellular and tissue levels, its medical use for tissue regeneration and rehabilitation has been limited by the availability of suitable devices. Here we present a mechanically active gel-elastomer-nitinol tissue adhesive (MAGENTA) that generates and delivers muscle-contraction-mimicking stimulation to a target tissue with programmed strength and frequency. MAGENTA consists of a shape memory alloy spring that enables actuation up to 40% strain, and an adhesive that efficiently transmits the actuation to the underlying tissue. MAGENTA activates mechanosensing pathways involving yes-associated protein and myocardin-related transcription factor A, and increases the rate of muscle protein synthesis. Disuse muscles treated with MAGENTA exhibit greater size and weight, and generate higher forces compared to untreated muscles, demonstrating the prevention of atrophy. MAGENTA thus has promising applications in the treatment of muscle atrophy and regenerative medicine.

Recent and Future Strategies of Mechanotherapy for Tissue Regenerative Rehabilitation.

Mechanotherapy, the application of various mechanical forces on injured or diseased tissue, is a viable option for tissue regenerative rehabilitation. Recent advances in tissue engineering (i.e., engineered materials and 3D printing) and soft-robotic technologies have enabled systematic and controlled studies to demonstrate the therapeutic impacts of mechanical stimulation on severely injured tissue. Along with innovation in actuation systems, improvements in analysis methods uncovering cellular and molecular landscapes during tissue regeneration under mechanical loading expand our understanding of how mechanical cues are translated into specific biological responses (i.e., stem cell self-renewal and differentiation, immune responses, etc.). Moving forward, the development of diversified actuation systems that are mechanically tissue friendly, easily scalable, and capable of delivering various modes of loading and monitoring functional biomarkers will facilitate systematic and controlled preclinical and clinical studies. Combining these future actuation systems with single-cell resolution analysis of cellular and molecular markers will enable detailed knowledge of underlying biological responses, and optimization of mechanotherapy protocols for specific tissues/injuries. These advancements will enable diverse mechanotherapy therapies in the future.

Skeletal muscle regeneration with robotic actuation-mediated clearance of neutrophils.

Mechanical stimulation (mechanotherapy) can promote skeletal muscle repair, but a lack of reproducible protocols and mechanistic understanding of the relation between mechanical cues and tissue regeneration limit progress in this field. To address these gaps, we developed a robotic device equipped with real-time force control and compatible with ultrasound imaging for tissue strain analysis. We investigated the hypothesis that specific mechanical loading improves tissue repair by modulating inflammatory responses that regulate skeletal muscle regeneration. We report that cyclic compressive loading within a specific range of forces substantially improves functional recovery of severely injured muscle in mice. This improvement is attributable in part to rapid clearance of neutrophil populations and neutrophil-mediated factors, which otherwise may impede myogenesis. Insights from this work will help advance therapeutic strategies for tissue regeneration broadly.

Viscoelastic surface electrode arrays to interface with viscoelastic tissues.

Living tissues are non-linearly elastic materials that exhibit viscoelasticity and plasticity. Man-made, implantable bioelectronic arrays mainly rely on rigid or elastic encapsulation materials and stiff films of ductile metals that can be manipulated with microscopic precision to offer reliable electrical properties. In this study, we have engineered a surface microelectrode array that replaces the traditional encapsulation and conductive components with viscoelastic materials. Our array overcomes previous limitations in matching the stiffness and relaxation behaviour of soft biological tissues by using hydrogels as the outer layers. We have introduced a hydrogel-based conductor made from an ionically conductive alginate matrix enhanced with carbon nanomaterials, which provide electrical percolation even at low loading fractions. Our combination of conducting and insulating viscoelastic materials, with top-down manufacturing, allows for the fabrication of electrode arrays compatible with standard electrophysiology platforms. Our arrays intimately conform to the convoluted surface of the heart or brain cortex and offer promising bioengineering applications for recording and stimulation.

Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates.

Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple "cryptic leading edges" within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as "cell polarization barriers," decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.

Generation of the Compression-induced Dedifferentiated Adipocytes (CiDAs) Using Hypertonic Medium.

Current methods to obtain mesenchymal stem cells (MSCs) involve sampling, culturing, and expanding of primary MSCs from adipose, bone marrow, and umbilical cord tissues. However, the drawbacks are the limited numbers of total cells in MSC pools, and their decaying stemness during in vitro expansion. As an alternative resource, recent ceiling culture methods allow the generation of dedifferentiated fat cells (DFATs) from mature adipocytes. Nevertheless, this process of spontaneous dedifferentiation of mature adipocytes is laborious and time-consuming. This paper describes a modified protocol for in vitro dedifferentiation of adipocytes by employing an additional physical stimulation, which takes advantage of augmenting the stemness-related Wnt/ß-catenin signaling. Specifically, this protocol utilizes a polyethylene glycol (PEG)-containing hypertonic medium to introduce extracellular physical stimulation to obtain higher efficiency and introduce a simpler procedure for adipocyte dedifferentiation.

Biomaterial-based scaffold for in situ chemo-immunotherapy to treat poorly immunogenic tumors.

Poorly immunogenic tumors, including triple negative breast cancers (TNBCs), remain resistant to current immunotherapies, due in part to the difficulty of reprogramming the highly immunosuppressive tumor microenvironment (TME). Here we show that peritumorally injected, macroporous alginate gels loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) for concentrating dendritic cells (DCs), CpG oligonucleotides, and a doxorubicin-iRGD conjugate enhance the immunogenic death of tumor cells, increase systemic tumor-specific CD8 + T cells, repolarize tumor-associated macrophages towards an inflammatory M1-like phenotype, and significantly improve antitumor efficacy against poorly immunogenic TNBCs. This system also prevents tumor recurrence after surgical resection and results in 100% metastasis-free survival upon re-challenge. This chemo-immunotherapy that concentrates DCs to present endogenous tumor antigens generated in situ may broadly serve as a facile platform to modulate the suppressive TME, and enable in situ personalized cancer vaccination.

Collagen microarchitecture mechanically controls myofibroblast differentiation.

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

Compression-induced dedifferentiation of adipocytes promotes tumor progression.

Dysregulated physical stresses are generated during tumorigenesis that affect the surrounding compliant tissues including adipocytes. However, the effect of physical stressors on the behavior of adipocytes and their cross-talk with tumor cells remain elusive. Here, we demonstrate that compression of cells, resulting from various types of physical stresses, can induce dedifferentiation of adipocytes via mechanically activating Wnt/ß-catenin signaling. The compression-induced dedifferentiated adipocytes (CiDAs) have a distinct transcriptome profile, long-term self-renewal, and serial clonogenicity, but do not form teratomas. We then show that CiDAs notably enhance human mammary adenocarcinoma proliferation both in vitro and in a xenograft model, owing to myofibrogenesis of CiDAs in the tumor-conditioned environment. Collectively, our results highlight unique physical interplay in the tumor ecosystem; tumor-induced physical stresses stimulate de novo generation of CiDAs, which feedback to tumor growth.

Treating ischemia via recruitment of antigen-specific T cells.

Ischemic diseases are a leading cause of mortality and can result in autoamputation of lower limbs. We explored the hypothesis that implantation of an antigen-releasing scaffold, in animals previously vaccinated with the same antigen, can concentrate TH2 T cells and enhance vascularization of ischemic tissue. This approach may be clinically relevant, as all persons receiving childhood vaccines recommended by the Centers for Disease Control and Prevention have vaccines that contain aluminum, a TH2 adjuvant. To test the hypothesis, mice with hindlimb ischemia, previously vaccinated with ovalbumin (OVA) and aluminum, received OVA-releasing scaffolds. Vaccinated mice receiving OVA-releasing scaffolds locally concentrated antigen-specific TH2 T cells in the surrounding ischemic tissue. This resulted in local angiogenesis, increased perfusion in ischemic limbs, and reduced necrosis and enhanced regenerating myofibers in the muscle. These findings support the premise that antigen depots may provide a treatment for ischemic diseases in patients previously vaccinated with aluminum-containing adjuvants.

Multiscale characterization of the mineral phase at skeletal sites of breast cancer metastasis.

Skeletal metastases, the leading cause of death in advanced breast cancer patients, depend on tumor cell interactions with the mineralized bone extracellular matrix. Bone mineral is largely composed of hydroxyapatite (HA) nanocrystals with physicochemical properties that vary significantly by anatomical location, age, and pathology. However, it remains unclear whether bone regions typically targeted by metastatic breast cancer feature distinct HA materials properties. Here we combined high-resolution X-ray scattering analysis with large-area Raman imaging, backscattered electron microscopy, histopathology, and microcomputed tomography to characterize HA in mouse models of advanced breast cancer in relevant skeletal locations. The proximal tibial metaphysis served as a common metastatic site in our studies; we identified that in disease-free bones this skeletal region contained smaller and less-oriented HA nanocrystals relative to ones that constitute the diaphysis. We further observed that osteolytic bone metastasis led to a decrease in HA nanocrystal size and perfection in remnant metaphyseal trabecular bone. Interestingly, in a model of localized breast cancer, metaphyseal HA nanocrystals were also smaller and less perfect than in corresponding bone in disease-free controls. Collectively, these results suggest that skeletal sites prone to tumor cell dissemination contain less-mature HA (i.e., smaller, less-perfect, and less-oriented crystals) and that primary tumors can further increase HA immaturity even before secondary tumor formation, mimicking alterations present during tibial metastasis. Engineered tumor models recapitulating these spatiotemporal dynamics will permit assessing the functional relevance of the detected changes to the progression and treatment of breast cancer bone metastasis.

Timed Delivery of Therapy Enhances Functional Muscle Regeneration.

Cell transplantation is a promising therapeutic strategy for the treatment of traumatic muscle injury in humans. Previous investigations have typically focused on the identification of potent cell and growth factor treatments and optimization of spatial control over delivery. However, the optimal time point for cell transplantation remains unclear. Here, this study reports how myoblast and morphogen delivery timed to coincide with specific phases of the inflammatory response affects donor cell engraftment and the functional repair of severely injured muscle. Delivery of a biomaterial-based therapy timed with the peak of injury-induced inflammation leads to potent early and long-term regenerative benefits. Diminished inflammation and fibrosis, enhanced angiogenesis, and increased cell engraftment are seen during the acute stage following optimally timed treatment. Over the long term, treatment during peak inflammation leads to enhanced functional regeneration, as indicated by reduced chronic inflammation and fibrosis along with increased tissue perfusion and muscle contractile force. Treatments initiated immediately after injury or after inflammation had largely resolved provided more limited benefits. These results demonstrate the importance of appropriately timing the delivery of biologic therapy in the context of muscle regeneration. Biomaterial-based timed delivery can likely be applied to other tissues and is of potential wide utility in regenerative medicine.

Breast cancer cells alter the dynamics of stromal fibronectin-collagen interactions.

Breast cancer cells recruit surrounding stromal cells, such as cancer-associated fibroblasts (CAFs), to remodel their extracellular matrix (ECM) and promote invasive tumor growth. Two major ECM components, fibronectin (Fn) and collagen I (Col I), are known to interact with each other to regulate cellular behavior. In this study, we seek to understand how Fn and Col I interplay and promote a dysregulated signaling pathway to facilitate tumor progression. Specifically, we investigated the evolution of tumor-conditioned stromal ECM composition, structure, and relaxation. Furthermore, we assessed how evolving Fn-Col I interactions gradually affected pro-angiogenic signaling. Our data first indicate that CAFs initially assembled a strained, viscous, and unfolded Fn matrix. This early altered Fn matrix was later remodeled into a thick Col I-rich matrix that was characteristic of a dense tumor mass. Next, our results suggest that this ECM remodeling was primarily mediated by matrix metalloproteinases (MMPs). This MMP activity caused profound structural and mechanical changes in the developing ECM, which then modified vascular endothelial growth factor (VEGF) secretion by CAFs and matrix sequestration. Collectively, these findings enhance our understanding of the mechanisms by which Fn and Col I synergistically interplay in promoting a sustained altered signaling cascade to remodel the breast tumor stroma for invasive breast tumor growth.

Influencing the Tumor Microenvironment: A Phase II Study of Copper Depletion Using Tetrathiomolybdate in Patients with Breast Cancer at High Risk for Recurrence and in Preclinical Models of Lung Metastases.

PURPOSE: Bone marrow-derived progenitor cells, including VEGFR2+ endothelial progenitor cells (EPCs) and copper-dependent pathways, model the tumor microenvironment. We hypothesized that copper depletion using tetrathiomolybdate would reduce EPCs in high risk for patients with breast cancer who have relapsed. We investigated the effect of tetrathiomolybdate on the tumor microenvironment in preclinical models. EXPERIMENTAL DESIGN: Patients with stage II triple-negative breast cancer (TNBC), stage III and stage IV without any evidence of disease (NED), received oral tetrathiomolybdate to maintain ceruloplasmin (Cp) between 8 and 17 mg/dL for 2 years or until relapse. Endpoints were effect on EPCs and other biomarkers, safety, event-free (EFS), and overall survival (OS). For laboratory studies, MDA-LM2-luciferase cells were implanted into CB17-SCID mice and treated with tetrathiomolybdate or water. Tumor progression was quantified by bioluminescence imaging (BLI), copper depletion status by Cp oxidase levels, lysyl oxidase (LOX) activity by ELISA, and collagen deposition. RESULTS: Seventy-five patients enrolled; 51 patients completed 2 years (1,396 cycles). Most common grade 3/4 toxicity was neutropenia (3.7%). Lower Cp levels correlated with reduced EPCs (P = 0.002) and LOXL-2 (P < 0.001). Two-year EFS for patients with stage II-III and stage IV NED was 91% and 67%, respectively. For patients with TNBC, EFS was 90% (adjuvant patients) and 69% (stage IV NED patients) at a median follow-up of 6.3 years, respectively. In preclinical models, tetrathiomolybdate decreased metastases to lungs (P = 0.04), LOX activity (P = 0.03), and collagen crosslinking (P = 0.012). CONCLUSIONS: Tetrathiomolybdate is safe, well tolerated, and affects copper-dependent components of the tumor microenvironment. Biomarker-driven clinical trials in high risk for patients with recurrent breast cancer are warranted. Clin Cancer Res; 23(3); 666-76. ©2016 AACR.

Collagen I hydrogel microstructure and composition conjointly regulate vascular network formation.

UNLABELLED: Neovascularization is a hallmark of physiological and pathological tissue remodeling that is regulated in part by the extracellular matrix (ECM). Collagen I hydrogels or Matrigel are frequently used to study vascular network formation; however, in isolation these materials do not typically mimic the integrated effects of ECM structure and composition that may influence endothelial cells in vivo. Here, we have utilized microfabricated 3D culture models to control collagen I microstructure in the presence and absence of Matrigel and tested the effect of these variations on vascular network formation by human cerebral microvascular endothelial cells (hCMECs). Varied collagen microarchitecture was achieved by adjusting the gelation temperature and subsequently confirmed by structural analysis. Casting at colder temperature increased collagen fiber thickness and length, and inclusion of Matrigel further pronounced these differences. Interestingly, the presence of Matrigel affected vascular network formation by modulating hCMEC growth, whereas altered collagen fiber structure impacted the morphology and maturity of the developed vascular network. These differences were related to substrate-dependent changes in interleukin-8 (IL-8) secretion and were functionally relevant as vascular networks preformed in more fibrillar, Matrigel-containing hydrogels promoted angiogenic sprouting. Our studies indicate that collagen hydrogel microstructure and composition conjointly regulate vascular network formation with implications for translational and basic science approaches. STATEMENT OF SIGNIFICANCE: Neovascularization is a hallmark of both tissue homeostasis and disease and is in part regulated by cell remodeling that occurs in the extracellular matrix (ECM). The use of bio-mimetic hydrogel cell culture systems has been used to study the effects of the ECM on cell behavior. Here, we employ a hydrogel system that enables control over both the structure and composition of the ECM and subsequently investigated the effects that these have on blood vessel dynamics. Finally, we linked these differences to changes in protein secretion and the implications that this may play in scientific translation.

Fibronectin Mechanobiology Regulates Tumorigenesis.

Fibronectin (Fn) is an essential extracellular matrix (ECM) glycoprotein involved in both physiological and pathological processes. The structure-function relationship of Fn has been and is still being studied, as changes in its molecular structure are integral in regulating (or dysregulating) its biological activities via its cell, matrix component, and growth factor binding sites. Fn comprises three types of repeating modules; among them, FnIII modules are mechanically unstable domains that may be extended/unfolded upon cell traction and either uncover cryptic binding sites or disrupt otherwise exposed binding sites. Cells assemble Fn into a fibrillar network; its conformational flexibility implicates Fn as a critical mechanoregulator of the ECM. Fn has been shown to contribute to altered stroma remodeling during tumorigenesis. This review will discuss (i) the significance of the structure-function relationship of Fn at both the molecular and the matrix scales, (ii) the role of Fn mechanobiology in the regulation of tumorigenesis, and (iii) Fn-related advances in cancer therapy development.

Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis.

Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic factors for breast cancer. Whether they are functionally linked is uncertain. We investigated the hypothesis that obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness. Indeed, mammary fat of both diet- and genetically induced mouse models of obesity were enriched for myofibroblasts and stiffness-promoting ECM components. These differences were related to varied adipose stromal cell (ASC) characteristics because ASCs isolated from obese mice contained more myofibroblasts and deposited denser and stiffer ECMs relative to ASCs from lean control mice. Accordingly, decellularized matrices from obese ASCs stimulated mechanosignaling and thereby the malignant potential of breast cancer cells. Finally, the clinical relevance and translational potential of our findings were supported by analysis of patient specimens and the observation that caloric restriction in a mouse model reduces myofibroblast content in mammary fat. Collectively, these findings suggest that obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies.