A synthetic metastatic niche reveals antitumor neutrophils drive breast cancer metastatic dormancy in the lungs.

Biomaterial scaffolds mimicking the environment in metastatic organs can deconstruct complex signals and facilitate the study of cancer progression and metastasis. Here we report that a subcutaneous scaffold implant in mouse models of metastatic breast cancer in female mice recruits lung-tropic circulating tumor cells yet suppresses their growth through potent in situ antitumor immunity. In contrast, the lung, the endogenous metastatic organ for these models, develops lethal metastases in aggressive breast cancer, with less aggressive tumor models developing dormant lungs suppressing tumor growth. Our study reveals multifaceted roles of neutrophils in regulating metastasis. Breast cancer-educated neutrophils infiltrate the scaffold implants and lungs, secreting the same signal to attract lung-tropic circulating tumor cells. Second, antitumor and pro-tumor neutrophils are selectively recruited to the dormant scaffolds and lungs, respectively, responding to distinct groups of chemoattractants to establish activated or suppressive immune environments that direct different fates of cancer cells.

Maresin-2 promotes mucosal repair and has therapeutic potential when encapsulated in thermostable nanoparticles.

Resolution of inflammation and mucosal wound healing are crucial processes required to re-establish homeostasis following injury of mucosal tissues. Maresin-2 (MaR2), a lipid specialized pro-resolving mediator derived from omega-3 polyunsaturated fatty acid, has been reported to promote resolution of inflammation. However, a potential role for MaR2 in regulating mucosal repair remains undefined. Using lipidomic analyses, we demonstrate biosynthesis of MaR2 in healing intestinal mucosal wounds in vivo. Importantly, administration of exogenous MaR2 promoted mucosal repair following dextran sulfate sodium-induced colitis or biopsy-induced colonic mucosal injury. Functional analyses revealed that MaR2 promotes mucosal wound repair by driving intestinal epithelial migration through activation of focal cell-matrix adhesion signaling in primary human intestinal epithelial cells. Because of its labile nature, MaR2 is easily degradable and requires ultracold storage to maintain functionality. Thus, we created thermostable polylactic acid MaR2 nanoparticles that retain biological activity following extended storage at 4 °C or above. Taken together, these results establish MaR2 as a potent pro-repair lipid mediator with broad therapeutic potential for use in promoting mucosal repair in inflammatory diseases.

Pancreatic cancer is marked by complement-high blood monocytes and tumor-associated macrophages.

Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of C1qa, C1qb, Trem2, and Chil3 Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated C1qa, C1qb, and Trem2, the other with high Chil3, Ly6c2 and Plac8 In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of C1QA, C1QB, and TREM2 is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.

Engineered immunological niches to monitor disease activity and treatment efficacy in relapsing multiple sclerosis.

Relapses in multiple sclerosis can result in irreversible nervous system tissue injury. If these events could be detected early, targeted immunotherapy could potentially slow disease progression. We describe the use of engineered biomaterial-based immunological niches amenable to biopsy to provide insights into the phenotype of innate immune cells that control disease activity in a mouse model of multiple sclerosis. Differential gene expression in cells from these niches allow monitoring of disease dynamics and gauging the effectiveness of treatment. A proactive treatment regimen, given in response to signal within the niche but before symptoms appeared, substantially reduced disease. This technology offers a new approach to monitor organ-specific autoimmunity, and represents a platform to analyze immune dysfunction within otherwise inaccessible target tissues.

Engineered Niches to Analyze Mechanisms of Metastasis and Guide Precision Medicine.

Cancer metastasis poses a challenging problem both clinically and scientifically, as the stochastic nature of metastatic lesion formation introduces complexity for both early detection and the study of metastasis in preclinical models. Engineered metastatic niches represent an emerging approach to address this stochasticity by creating bioengineered sites where cancer can preferentially metastasize. As the engineered niche captures the earliest metastatic cells at a nonvital location, both noninvasive and biopsy-based monitoring of these sites can be performed routinely to detect metastasis early and monitor alterations in the forming metastatic niche. The engineered metastatic niche also provides a new platform technology that serves as a tunable site to molecularly dissect metastatic disease mechanisms. Ultimately, linking the engineered niches with advances in sensor development and synthetic biology can provide enabling tools for preclinical cancer models and fosters the potential to impact the future of clinical cancer care.

Metastatic Conditioning of Myeloid Cells at a Subcutaneous Synthetic Niche Reflects Disease Progression and Predicts Therapeutic Outcomes.

Monitoring metastatic events in distal tissues is challenged by their sporadic occurrence in obscure and inaccessible locations within these vital organs. A synthetic biomaterial scaffold can function as a synthetic metastatic niche to reveal the nature of these distal sites. These implanted scaffolds promote tissue ingrowth, which upon cancer initiation is transformed into a metastatic niche that captures aggressive circulating tumor cells. We hypothesized that immune cell phenotypes at synthetic niches reflect the immunosuppressive conditioning within a host that contributes to metastatic cell recruitment and can identify disease progression and response to therapy. We analyzed the expression of 632 immune-centric genes in tissue biopsied from implants at weekly intervals following inoculation. Specific immune populations within implants were then analyzed by single-cell RNA-seq. Dynamic gene expression profiles in innate cells, such as myeloid-derived suppressor cells, macrophages, and dendritic cells, suggest the development of an immunosuppressive microenvironment. These dynamics in immune phenotypes at implants was analogous to that in the diseased lung and had distinct dynamics compared with blood leukocytes. Following a therapeutic excision of the primary tumor, longitudinal tracking of immune phenotypes at the implant in individual mice showed an initial response to therapy, which over time differentiated recurrence versus survival. Collectively, the microenvironment at the synthetic niche acts as a sentinel by reflecting both progression and regression of disease. SIGNIFICANCE: Immune dynamics at biomaterial implants, functioning as a synthetic metastatic niche, provides unique information that correlates with disease progression. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/602/F1.large.jpg.See related commentary by Wolf and Elisseeff, p. 377.

Elevated Thrombospondin 2 Contributes to Delayed Wound Healing in Diabetes.

Impaired wound healing is a major complication of diabetes, and despite the associated risks, treatment strategies for diabetic wounds remain limited. This is due, in part, to an incomplete understanding of the underlying pathological mechanisms, including the effects of hyperglycemia on components of the extracellular matrix (ECM). In the current study, we explored whether the expression of thrombospondin 2 (TSP2), a matricellular protein with a demonstrated role in response to injury, was associated with delayed healing in diabetes. First, we found that TSP2 expression was elevated in diabetic mice and skin from patients with diabetes. Then, to determine the contribution of TSP2 to impaired healing in diabetes, we developed a novel diabetic TSP2-deficient model. Though the TSP2-deficient mice developed obesity and hyperglycemia comparable with diabetic control mice, they exhibited significantly improved healing, characterized by accelerated reepithelialization and increased granulation tissue formation, fibroblast migration, and blood vessel maturation. We further found that hyperglycemia increased TSP2 expression in fibroblasts, the major cellular source of TSP2 in wounds. Mechanistically, high glucose increased activation of the hexosamine pathway and nuclear factor-κB signaling to elevate TSP2 expression. Our studies demonstrate that hyperglycemia-induced TSP2 expression contributes to impaired healing in diabetes.

LMO7 Is a Negative Feedback Regulator of Transforming Growth Factor ß Signaling and Fibrosis.

BACKGROUND: Vascular smooth muscle cells (SMCs) synthesize extracellular matrix (ECM) that contributes to tissue remodeling after revascularization interventions. The cytokine transforming growth factor ß (TGF-ß) is induced on tissue injury and regulates tissue remodeling and wound healing, but dysregulated signaling results in excess ECM deposition and fibrosis. The LIM (Lin11, Isl-1 & Mec-3) domain protein LIM domain only 7 (LMO7) is a TGF-ß1 target gene in hepatoma cells, but its role in vascular physiology and fibrosis is unknown. METHODS: We use carotid ligation and femoral artery denudation models in mice with global or inducible smooth muscle-specific deletion of LMO7, and knockout, knockdown, overexpression, and mutagenesis approaches in mouse and human SMC, and human arteriovenous fistula and cardiac allograft vasculopathy samples to assess the role of LMO7 in neointima and fibrosis. RESULTS: We demonstrate that LMO7 is induced postinjury and by TGF-ß in SMC in vitro. Global or SMC-specific LMO7 deletion enhanced neointimal formation, TGF-ß signaling, ECM deposition, and proliferation in vascular injury models. LMO7 loss of function in human and mouse SMC enhanced ECM protein expression at baseline and after TGF-ß treatment. TGF-ß neutralization or receptor antagonism prevented the exacerbated neointimal formation and ECM synthesis conferred by loss of LMO7. Notably, loss of LMO7 coordinately amplified TGF-ß signaling by inducing expression of Tgfb1 mRNA, TGF-ß protein, αv and ß3 integrins that promote activation of latent TGF-ß, and downstream effectors SMAD3 phosphorylation and connective tissue growth factor. Mechanistically, the LMO7 LIM domain interacts with activator protein 1 transcription factor subunits c-FOS and c-JUN and promotes their ubiquitination and degradation, disrupting activator protein 1-dependent TGF-ß autoinduction. Importantly, preliminary studies suggest that LMO7 is upregulated in human intimal hyperplastic arteriovenous fistula and cardiac allograft vasculopathy samples, and inversely correlates with SMAD3 phosphorylation in cardiac allograft vasculopathy. CONCLUSIONS: LMO7 is induced by TGF-ß and serves to limit vascular fibrotic responses through negative feedback regulation of the TGF-ß pathway. This mechanism has important implications for intimal hyperplasia, wound healing, and fibrotic diseases.

Tunable Hydrogels Derived from Genetically Engineered Extracellular Matrix Accelerate Diabetic Wound Healing.

Hydrogels composed of solubilized decellularized extracellular matrix (ECM) are attractive materials because they combine the complexity of native ECM with injectability and ease of use. Nevertheless, these materials are typically only tunable by altering the concentration, which alters the ligand landscape, or by incorporating synthetic components, which can result in an unfavorable host response. Herein, we demonstrate the fabrication of genetically tunable ECM-derived materials, by utilizing wild type (WT) and (thrombospondin-2 knockout) TSP-2 KO decellularized skins to prepare hydrogels. The resulting materials exhibited distinct mechanical properties characterized by rheology and different concentrations of collagens when characterized by quantitative proteomics. Mixtures of the gels achieved intermediate effects between the WT and the KO, permitting tunability of the gel properties. In vivo, the hydrogels exhibited tunable cell invasion with a correlation between the content of TSP-2 KO hydrogel and the extent of cell invasion. Additionally, TSP-2 KO hydrogels significantly improved diabetic wound healing at 10 and 21 days. Furthermore, hydrogels derived from genetically engineered in vitro cell-derived matrix mimicked the trends observed for tissue-derived matrix, providing a platform for faster screening of novel manipulations and easier clinical translation. Overall, we demonstrate that genetic engineering approaches impart tunability to ECM-based hydrogels and can result in materials capable of enhanced regeneration.

Nanopatterned bulk metallic glass-based biomaterials modulate macrophage polarization.

Polarization of macrophages by chemical, topographical and mechanical cues presents a robust strategy for designing immunomodulatory biomaterials. Here, we studied the ability of nanopatterned bulk metallic glasses (BMGs), a new class of metallic biomaterials, to modulate murine macrophage polarization. Cytokine/chemokine analysis of IL-4 or IFNγ/LPS-stimulated macrophages showed that the secretion of TNF-α, IL-1α, IL-12, CCL-2 and CXCL1 was significantly reduced after 24-hour culture on BMGs with 55 nm nanorod arrays (BMG-55). Additionally, under these conditions, macrophages increased phagocytic potential and exhibited decreased cell area with multiple actin protrusions. These in vitro findings suggest that nanopatterning can modulate biochemical cues such as IFNγ/LPS. In vivo evaluation of the subcutaneous host response at 2 weeks demonstrated that the ratio of Arg-1 to iNOS increased in macrophages adjacent to BMG-55 implants, suggesting modulation of polarization. In addition, macrophage fusion and fibrous capsule thickness decreased and the number and size of blood vessels increased, which is consistent with changes in macrophage responses. Our study demonstrates that nanopatterning of BMG implants is a promising technique to selectively polarize macrophages to modulate the immune response, and also presents an effective tool to study mechanisms of macrophage polarization and function. STATEMENT OF SIGNIFICANCE: Implanted biomaterials elicit a complex series of tissue and cellular responses, termed the foreign body response (FBR), that can be influenced by the polarization state of macrophages. Surface topography can influence polarization, which is broadly characterized as either inflammatory or repair-like. The latter has been linked to improved outcomes of the FBR. However, the impact of topography on macrophage polarization is not fully understood, in part, due to a lack of high moduli biomaterials that can be reproducibly processed at the nanoscale. Here, we studied macrophage interactions with nanopatterned bulk metallic glasses (BMGs), a class of metallic alloys with amorphous microstructure and formability like polymers. We show that nanopatterned BMGs modulate macrophage polarization and transiently induce less fibrotic and more angiogenic responses. Overall, we demonstrate nanopatterning of BMG implants as a technique to polarize macrophages and modulate the FBR.

Decellularized materials derived from TSP2-KO mice promote enhanced neovascularization and integration in diabetic wounds.

Decellularized biologic scaffolds are gaining popularity over synthetic biomaterials as naturally derived materials capable of promoting improved healing. Nevertheless, the most widely used biologic material - acellular dermal matrix (ADM) - exhibits slow repopulation and remodeling, which prevents integration. Additionally, engineering control of these materials is limited because they require a natural source for their production. In the current report, we demonstrate the feasibility of using genetically engineered animals to create decellularized biologic scaffolds with favorable extracellular matrix (ECM) properties. Specifically, we utilized skin from thrombospondin (TSP)-2 KO mice to derive various decellularized products. Scanning electron microscopy and mechanical testing showed that TSP-2 KO ADM exhibited an altered structure and a reduction in elastic modulus and ultimate tensile strength, respectively. When a powdered form of KO ADM was implanted subcutaneously, it was able to promote enhanced vascularization over WT. Additionally, when implanted subcutaneously, intact slabs of KO ADM were populated by higher number of host cells when compared to WT. In vitro studies confirmed the promigratory properties of KO ADM. Specifically, degradation products released by pepsin digestion of KO ADM induced greater cell migration than WT. Moreover, cell-derived ECM from TSP-2 null fibroblasts was more permissive to fibroblast migration. Finally, ADMs were implanted in a diabetic wound model to examine their ability to accelerate wound healing. KO ADM exhibited enhanced remodeling and vascular maturation, indicative of efficient integration. Overall, we demonstrate that genetic manipulation enables engineered ECM-based materials with increased regenerative potential.

Multicompartment Drug Release System for Dynamic Modulation of Tissue Responses.

Pharmacological modulation of responses to injury is complicated by the need to deliver multiple drugs with spatiotemporal resolution. Here, a novel controlled delivery system containing three separate compartments with each releasing its contents over different timescales is fabricated. Core-shell electrospun fibers create two of the compartments in the system, while electrosprayed spheres create the third. Utility is demonstrated by targeting the foreign body response to implants because it is a dynamic process resulting in implant failure. Sequential delivery of a drug targeting nuclear factor-κB (NF-κB) and an antifibrotic is characterized in in vitro experiments. Specifically, macrophage fusion and p65 nuclear translocation in the presence of releasate or with macrophages cultured on the surfaces of the constructs are evaluated. In addition, releasate from pirfenidone scaffolds is shown to reduce transforming growth factor-ß (TGF-ß)-induced pSMAD3 nuclear localization in fibroblasts. In vivo, drug eluting constructs successfully mitigate macrophage fusion at one week and fibrotic encapsulation in a dose-dependent manner at four weeks, demonstrating effective release of both drugs over different timescales. Future studies can employ this system to improve and prolong implant lifetimes, or load it with other drugs to modulate other dynamic processes.

The host response to naturally-derived extracellular matrix biomaterials.

Biomaterials based on natural materials including decellularized tissues and tissue-derived hydrogels are becoming more widely used for clinical applications. Because of their native composition and structure, these biomaterials induce a distinct form of the foreign body response that differs from that of non-native biomaterials. Differences include direct interactions with cells via preserved moieties as well as the ability to undergo remodeling. Moreover, these biomaterials could elicit adaptive immune responses due to the presence of modified native molecules. Therefore, these biomaterials present unique challenges in terms of understanding the progression of the foreign body response. This review covers this response to natural materials including natural polymers, decellularized tissues, cell-derived matrix, tissue derived hydrogels, and biohybrid materials. With the expansion of the fields of regenerative medicine and tissue engineering, the current repertoire of biomaterials has also expanded and requires continuous investigation of the responses they elicit.

Anisotropic engineered heart tissue made from laser-cut decellularized myocardium.

We have developed an engineered heart tissue (EHT) system that uses laser-cut sheets of decellularized myocardium as scaffolds. This material enables formation of thin muscle strips whose biomechanical characteristics are easily measured and manipulated. To create EHTs, sections of porcine myocardium were laser-cut into ribbon-like shapes, decellularized, and mounted in specialized clips for seeding and culture. Scaffolds were first tested by seeding with neonatal rat ventricular myocytes. EHTs beat synchronously by day five and exhibited robust length-dependent activation by day 21. Fiber orientation within the scaffold affected peak twitch stress, demonstrating its ability to guide cells toward physiologic contractile anisotropy. Scaffold anisotropy also made it possible to probe cellular responses to stretch as a function of fiber angle. Stretch that was aligned with the fiber direction increased expression of brain natriuretic peptide, but off-axis stretches (causing fiber shear) did not. The method also produced robust EHTs from cardiomyocytes derived from human embryonic stem cells and induced pluripotent stem cells (hiPSC). hiPSC-EHTs achieved maximum peak stress of 6.5 mN/mm(2) and twitch kinetics approaching reported values from adult human trabeculae. We conclude that laser-cut EHTs are a viable platform for novel mechanotransduction experiments and characterizing the biomechanical function of patient-derived cardiomyoctyes.

Inadequate Processing of Decellularized Dermal Matrix Reduces Cell Viability In Vitro and Increases Apoptosis and Acute Inflammation In Vivo.

Decellularized tissue scaffolds are commonly used in the clinic because they can be used as substitutes for more traditional biomaterials, while imparting additional physiological effects. Nevertheless, reports of complications associated with their use are widespread and poorly understood. This study probes possible causes of these complications by examining cell viability and apoptosis in response to eluents from decellularized dermis. Using multiple sources of decellularized dermis, this study shows that typical decellularized scaffolds (prepared with commonly used laboratory techniques, as well as purchased from commercial sources) contain soluble components that are cytotoxic and that these components can be removed by extensive washes in cell culture media. In addition, this study demonstrates that these observed in vitro phenotypes correlate with increased apoptosis and acute inflammation when implanted subcutaneously in mice.

Microvascular Perfusion Changes following Transarterial Hepatic Tumor Embolization.

PURPOSE: To quantify changes in tumor microvascular (< 1 mm) perfusion relative to commonly used angiographic endpoints. MATERIALS AND METHODS: Rabbit Vx2 liver tumors were embolized with 100-300-µm LC Bead particles to endpoints of substasis or complete stasis (controls were not embolized). Microvascular perfusion was evaluated by delivering two different fluorophore-conjugated perfusion markers (ie, lectins) through the catheter before embolization and 5 min after reaching the desired angiographic endpoint. Tumor microvasculature was labeled with an anti-CD31 antibody and analyzed with fluorescence microscopy for perfusion marker overlap/mismatch. Data were analyzed by analysis of variance and post hoc test (n = 3-5 per group; 18 total). RESULTS: Mean microvascular density was 70 vessels/mm(2) ± 17 (standard error of the mean), and 81% ± 1 of microvasculature (ie, CD31(+) structures) was functionally perfused within viable Vx2 tumor regions. Embolization to the extent of substasis eliminated perfusion in 37% ± 9 of perfused microvessels (P > .05 vs baseline), whereas embolization to the extent of angiographic stasis eliminated perfusion in 56% ± 8 of perfused microvessels. Persistent microvascular perfusion following embolization was predominantly found in the tumor periphery, adjacent to normal tissue. Newly perfused microvasculature was evident following embolization to substasis but not when embolization was performed to complete angiographic stasis. CONCLUSIONS: Nearly half of tumor microvasculature remained patent despite embolization to complete angiographic stasis. The observed preservation of tumor microvasculature perfusion with angiographic endpoints of substasis and stasis may have implications for tumor response to embolotherapy.

Molecular beacon-based detection and isolation of working-type cardiomyocytes derived from human pluripotent stem cells.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide a potential source of cells to repair injured ventricular myocardium. CM differentiation cultures contain non-cardiac cells and CMs of both nodal and working subtypes. Direct application of such cultures in clinical studies could induce arrhythmias; thus, further purification of working-type CMs from heterogeneous cultures is desirable. Here, we designed 10 molecular beacons (MBs) targeting NPPA mRNA, a marker associated with working-type CMs and highly up-regulated during differentiation. We examined these MBs by solution assays and established their specificity using NPPA-overexpressing CHO cells as well as hPSC-CMs. We selected one MB for subsequent CM subtype isolation using fluorescence-activated cell sorting because the signal-to-background ratio was the highest for this MB in solution assays and a linear correlation was observed between MB signals and the CM purity in differentiation cultures. Compared with cells with low MB signals, cells positively selected based on MB signal had higher expression levels of genes associated with working-type CMs and lower expression levels of genes associated with nodal-type CMs. Therefore, the MB-based method is capable of separating working-type CMs from nodal-type CMs with high specificity and throughput, potentially providing working-type CMs for biomedical applications.

Matricellular proteins and biomaterials.

Biomaterials are essential to modern medicine as components of reconstructive implants, implantable sensors, and vehicles for localized drug delivery. Advances in biomaterials have led to progression from simply making implants that are nontoxic to making implants that are specifically designed to elicit particular functions within the host. The interaction of implants and the extracellular matrix during the foreign body response is a growing area of concern for the field of biomaterials, because it can lead to implant failure. Expression of matricellular proteins is modulated during the foreign body response and these proteins interact with biomaterials. The design of biomaterials to specifically alter the levels of matricellular proteins surrounding implants provides a new avenue for the design and fabrication of biomimetic biomaterials.