Advances, challenges, and future directions in the clinical translation of ECM biomaterials for regenerative medicine applications.

Extracellular Matrix (ECM) scaffolds and biomaterials have been widely used for decades across a variety of diverse clinical applications and have been implanted in millions of patients worldwide. ECM-based biomaterials have been especially successful in soft tissue repair applications but their utility in other clinical applications such as for regeneration of bone or neural tissue is less well understood. The beneficial healing outcome with the use of ECM biomaterials is the result of their biocompatibility, their biophysical properties and their ability to modify cell behavior after injury. As a consequence of successful clinical outcomes, there has been motivation for the development of next-generation formulations of ECM materials ranging from hydrogels, bioinks, powders, to whole organ or tissue scaffolds. The continued development of novel ECM formulations as well as active research interest in these materials ensures a wealth of possibilities for future clinical translation and innovation in regenerative medicine. The clinical translation of next generation formulations ECM scaffolds faces predictable challenges such as manufacturing, manageable regulatory pathways, surgical implantation, and the cost required to address these challenges. The current status of ECM-based biomaterials, including clinical translation, novel formulations and therapies currently under development, and the challenges that limit clinical translation of ECM biomaterials are reviewed herein.

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using 'Catch and Display' on Ultrathin Nanoporous Silicon Nitride Membranes.

Extracellular vesicles (EVs) are particles secreted by all cells that carry bioactive cargo and facilitate intercellular communication with roles in normal physiology and disease pathogenesis. EVs have tremendous diagnostic and therapeutic potential and accordingly, the EV field has grown exponentially in recent years. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are undermined by complicated detection schemes often coupled with prohibitive instrumentation. To address these issues, we propose a microfluidic technique for EV characterization called 'catch and display for liquid biopsy (CAD-LB)'. CAD-LB rapidly captures fluorescently labeled EVs in the similarly-sized pores of an ultrathin silicon nitride membrane. Minimally processed sample is introduced via pipette injection into a simple microfluidic device which is directly imaged using fluorescence microscopy for a rapid assessment of EV number and biomarker colocalization. In this work, nanoparticles were first used to define the accuracy and dynamic range for counting and colocalization by CAD-LB. Following this, the same assessments were made for purified EVs and for unpurified EVs in plasma. Biomarker detection was validated using CD9 in which Western blot analysis confirmed that CAD-LB faithfully recapitulated differing expression levels among samples. We further verified that CAD-LB captured the known increase in EV-associated ICAM-1 following the cytokine stimulation of endothelial cells. Finally, to demonstrate CAD-LB's clinical potential, we show that EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients undergoing immune checkpoint blockade.

Mitigation of influenza-mediated inflammation by immunomodulatory matrix-bound nanovesicles.

Cytokine storm describes a life-threatening, systemic inflammatory syndrome characterized by elevated levels of proinflammatory cytokines and immune cell hyperactivation associated with multi-organ dysfunction. Matrix-bound nanovesicles (MBV) are a subclass of extracellular vesicle shown to down-regulate proinflammatory immune responses. The objective of this study was to assess the efficacy of MBV in mediating influenza-induced acute respiratory distress syndrome and cytokine storm in a murine model. Intravenous administration of MBV decreased influenza-mediated total lung inflammatory cell density, proinflammatory macrophage frequencies, and proinflammatory cytokines at 7 and 21 days following viral inoculation. MBV decreased long-lasting alveolitis and the proportion of lung undergoing inflammatory tissue repair at day 21. MBV increased the proportion of activated anti-viral CD4+ and CD8+ T cells at day 7 and memory-like CD62L+ CD44+, CD4+, and CD8+ T cells at day 21. These results show immunomodulatory properties of MBV that may benefit the treatment of viral-mediated pulmonary inflammation with applicability to other viral diseases such as SARS-CoV-2.

Biocompatibility and biodistribution of matrix-bound nanovesicles in vitro and in vivo.

Matrix-bound nanovesicles (MBV) are a distinct subtype of extracellular vesicles that are firmly embedded within biomaterials composed of extracellular matrix (ECM). MBV both store and transport a diverse, tissue specific portfolio of signaling molecules including proteins, miRNAs, and bioactive lipids. MBV function as a key mediator in ECM-mediated control of the local tissue microenvironment. One of the most important mechanisms by which MBV in ECM bioscaffolds support constructive tissue remodeling following injury is immunomodulation and, specifically, the promotion of an anti-inflammatory, pro-remodeling immune cell activation state. Recent in vivo studies have shown that isolated MBV have therapeutic efficacy in rodent models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV administered independent of the rest of the ECM, the in vitro and in vivo safety and biodistribution profile of MBV remain uncharacterized. The purpose of the present study was to thoroughly characterize the pre-clinical safety profile of MBV through a combination of in vitro cytotoxicity and MBV uptake studies and in vivo toxicity, immunotoxicity, and imaging studies. The results showed that MBV isolated from porcine urinary bladder are well-tolerated and are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive compared to the potent immunosuppressive drug cyclophosphamide. Furthermore, this safety profile was sustained across a wide range of MBV doses. STATEMENT OF SIGNIFICANCE: Matrix-bound nanovesicles (MBV) are a distinct subtype of bioactive extracellular vesicles that are embedded within biomaterials composed of extracellular matrix (ECM). Recent studies have shown therapeutic efficacy of MBV in models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV, the in vitro and in vivo biocompatibility and biodistribution profile of MBV remain uncharacterized. The results of the present study showed that MBV are a well-tolerated ECM-derived therapy that are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive. Collectively, these data highlight the translational feasibility of MBV therapeutics across a wide variety of clinical applications.

3D Bioprinted Patient-Specific Extracellular Matrix Scaffolds for Soft Tissue Defects.

Soft tissue injuries such as volumetric muscle loss (VML) are often too large to heal normally on their own, resulting in scar formation and functional deficits. Decellularized extracellular matrix (dECM) scaffolds placed into these wounds have shown the ability to modulate the immune response and drive constructive healing. This provides a potential solution for functional tissue regeneration, however, these acellular dECM scaffolds are challenging to fabricate into complex geometries. 3D bioprinting is uniquely positioned to address this, being able to create patient-specific scaffolds based on clinical 3D imaging data. Here, a process to use freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting and computed tomography (CT) imaging to build large volume, patient-specific dECM patches (≈12 × 8 × 2 cm) for implantation into canine VML wound models is developed. Quantitative analysis shows that these dECM patches are dimensionally accurate and conformally adapt to the surface of complex wounds. Finally, this approach is extended to a human VML injury to demonstrate the fabrication of clinically relevant dECM scaffolds with precise control over fiber alignment and micro-architecture. Together these advancements represent a step towards an improved, clinically translatable, patient-specific treatment for soft tissue defects from trauma, tumor resection, and other surgical procedures.

Matrix Bound Nanovesicles Have Tissue-Specific Characteristics That Suggest a Regulatory Role.

Recent studies have identified an extracellular vesicle population that is tightly anchored within the extracellular matrix (ECM) of tissues and organs until released by matrix turnover events. Evidence suggests that these matrix-bound nanovesicles (MBVs) are a ubiquitous component of the ECM, raising questions regarding their tissue-specific identity and their biologic function(s). The primary objective of this study was to examine MBVs isolated from six different tissues and compare their physical and compositional characteristics to determine the common and differentially expressed features. Accordingly, the results of this characterization show that while MBVs are a ubiquitous component of the ECM, they contain a protein and microRNA cargo that is tissue specific. The results furthermore suggest that MBVs have an important role in regulating tissue homeostasis.

Transcriptomic Regulation of Macrophages by Matrix-Bound Nanovesicle-Associated Interleukin-33.

The innate immune response, particularly the phenotype of responding macrophages, has significant clinical implications in the remodeling outcome following implantation of biomaterials and engineered tissues. In general, facilitation of an anti-inflammatory (M2-like) phenotype is associated with tissue repair and favorable outcomes, whereas pro-inflammatory (M1-like) activation can contribute to chronic inflammation and a classic foreign body response. Biologic scaffolds composed of extracellular matrix (ECM) and, more recently, matrix-bound nanovesicles (MBV) embedded within the ECM are known to direct macrophages toward an anti-inflammatory phenotype and stimulate a constructive remodeling outcome. The mechanisms of MBV-mediated macrophage activation are not fully understood, but interleukin-33 (IL-33) within the MBV appears critical for M2-like activation. Previous work has shown that IL-33 is encapsulated within the lumen of MBV and stimulates phenotypical changes in macrophages independent of its canonical surface receptor stimulation-2 (ST2). In the present study, we used next-generation RNA sequencing to determine the gene signature of macrophages following exposure to MBV with and without intraluminal IL-33. MBV-associated IL-33 instructed an anti-inflammatory phenotype in both wild-type and st2-/- macrophages by upregulating M2-like and downregulating M1-like genes. The repertoire of genes regulated by ST2-independent IL-33 signaling were broadly related to the inflammatory response and crosstalk between cells of both the innate and adaptive immune systems. These results signify the importance of the MBV intraluminal protein IL-33 in stimulating a pro-remodeling M2-like phenotype in macrophages and provides guidance for the designing of next-generation biomaterials and tissue engineering strategies. Impact statement The phenotype of responding macrophages is predictive of the downstream remodeling response to an implanted biomaterial. The clinical impact of macrophage phenotype has motivated studies to investigate the factors that regulate macrophage activation. Matrix-bound nanovesicles (MBV) embedded within the extracellular matrix direct macrophages toward an anti-inflammatory (M2)-like phenotype that is indicative of a favorable remodeling response. Although the mechanisms of MBV-mediated macrophage activation are not fully understood, the intraluminal protein interleukin-33 (IL-33) is clearly a contributing signaling molecule. The present study identifies those genes regulated by MBV-associated IL-33 that promote a pro-remodeling M2-like macrophage activation state and can guide future therapies in regenerative medicine.

Immunomodulatory matrix-bound nanovesicles mitigate acute and chronic pristane-induced rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and destruction of synovial joints affecting ~7.5 million people worldwide. Disease pathology is driven by an imbalance in the ratio of pro-inflammatory vs. anti-inflammatory immune cells, especially macrophages. Modulation of macrophage phenotype, specifically an M1 to M2, pro- to anti-inflammatory transition, can be induced by biologic scaffold materials composed of extracellular matrix (ECM). The ECM-based immunomodulatory effect is thought to be mediated in part through recently identified matrix-bound nanovesicles (MBV) embedded within ECM. Isolated MBV was delivered via intravenous (i.v.) or peri-articular (p.a.) injection to rats with pristane-induced arthritis (PIA). The results of MBV administration were compared to intraperitoneal (i.p.) administration of methotrexate (MTX), the clinical standard of care. Relative to the diseased animals, i.p. MTX, i.v. MBV, and p.a. MBV reduced arthritis scores in both acute and chronic pristane-induced arthritis, decreased synovial inflammation, decreased adverse joint remodeling, and reduced the ratio of synovial and splenic M1 to M2 macrophages (p < 0.05). Both p.a. and i.v. MBV reduced the serum concentration of RA and PIA biomarkers CXCL10 and MCP-3 in the acute and chronic phases of disease (p < 0.05). Flow-cytometry revealed the presence of a systemic CD43hi/His48lo/CD206+, immunoregulatory monocyte population unique to p.a. and i.v. MBV treatment associated with disease resolution. The results show that the therapeutic efficacy of MBV is equal to that of MTX for the management of acute and chronic pristane-induced arthritis and, further, this effect is associated with modulation of local synovial macrophages and systemic myeloid populations.

A liquid fraction of extracellular matrix inhibits glioma cell viability in vitro and in vivo.

Suppressive effects of extracellular matrix (ECM) upon various cancers have been reported. Glioblastoma multiforme has poor prognosis and new therapies are desired. This work investigated the effects of a saline-soluble fraction of urinary bladder ECM (ECM-SF) upon glioma cells. Viability at 24 hours in 1, 5, or 10 mg/mL ECM-SF-spiked media was evaluated in primary glioma cells (0319, 1015, 1119), glioma cell lines (A172, T98G, U87MG, C6), and brain cell lines (HCN-2, HMC3). Viability universally decreased at 5 and 10 mg/mL with U87MG, HCN-2, and HCM3 being least sensitive. Apoptosis in 0319 and 1119 cells was confirmed via NucView 488. Bi-weekly intravenous injection of ECM-SF (120 mg/kg) for 10 weeks in Sprague-Dawley rats did not affect weight, temperature, complete blood count, or multi-organ histology (N = 5). Intratumoral injection of ECM-SF (10 uL of 30 mg/mL) at weeks 2-4 post C6 inoculation in Wistar rats increased median survival from 24.5 to 51 days (hazard ratio for death 0.22) and decreased average tumor volume at time of death from 349 mm3 to 90 mm3 over 10 weeks (N = 6). Mass spectrometry identified 2,562 protein species in ECM-SF, parent ECM, and originating tissue. These results demonstrate the suppressive effects of ECM on glioma and warrant further study.

The effect of normal, metaplastic, and neoplastic esophageal extracellular matrix upon macrophage activation.

INTRODUCTION: Macrophages are capable of extreme plasticity and their activation state has been strongly associated with solid tumor growth progression and regression. Although the macrophage response to extracellular matrix (ECM) isolated from normal tissue is reasonably well understood, there is a relative dearth of information regarding their response to ECM isolated from chronically inflamed tissues, pre-neoplastic tissues, and neoplastic tissues. Esophageal adenocarcinoma (EAC) is a type of neoplasia driven by chronic inflammation in the distal esophagus, and the length of the esophagus provides the opportunity to investigate macrophage behavior in the presence of ECM isolated from a range of disease states within the same organ. METHODS: Normal, metaplastic, and neoplastic ECM hydrogels were prepared from decellularized EAC tissue. The hydrogels were evaluated for their nanofibrous structure (SEM), biochemical profile (targeted and global proteomics), and direct effect upon macrophage (THP-1 cell) activation state (qPCR, ELISA, immunolabeling) and indirect effect upon epithelial cell (Het-1A) migration (Boyden chamber). RESULTS: Nanofibrous ECM hydrogels from the three tissue types could be formed, and normal and neoplastic ECM showed distinctive protein profiles by targeted and global mass spectroscopy. ECM proteins functionally related to cancer and tumorigenesis were identified in the neoplastic esophageal ECM including collagen alpha-1(VIII) chain (COL8A1), lumican, and elastin. Metaplastic and neoplastic esophageal ECM induce distinctive effects upon THP-1 macrophage signaling compared to normal esophageal ECM. These effects include activation of pro-inflammatory IFNγ and TNFα gene expression and anti-inflammatory IL1RN gene expression. Most notably, neoplastic ECM robustly increased macrophage TNFα protein expression. The secretome of macrophages pre-treated with metaplastic and neoplastic ECM increases the migration of normal esophageal epithelial cells, similar behavior to that shown by tumor cells. Metaplastic ECM shows similar but less pronounced effects than neoplastic ECM suggesting the abnormal signals also exist within the pre-cancerous state. CONCLUSION: A progressively diseased ECM, as exists within the esophagus exposed to chronic gastric reflux, can provide insights into novel biomarkers of early disease and identify potential therapeutic targets.

Role of 4-hydroxybutyrate in increased resistance to surgical site infections associated with surgical meshes.

An increased resistance to surgical site infections has been associated with surgical meshes composed of naturally occurring materials, including poly-4-hydroxybutrate (4HB). 4HB is a naturally occurring short-chain fatty acid that has been shown to promote endogenous expression of the Cramp gene coding for the antimicrobial peptide (AMP) cathelicidin LL-37 in murine bone marrow-derived macrophages. The molecular pathways involved in the 4HB-induced cathelicidin LL-37 expression have not yet been identified. The present study showed that transcriptional activation of the Cramp gene by 4HB is independent of inhibition of histone deacetylase (HDAC) activity, and that upregulation of Cramp is modulated by the G-protein coupled receptor GPR109A. Furthermore, an intracellular signaling cascade that promotes the activation of the MAP kinases, p38 and JNK, and a subsequent NF-κB phosphorylation downstream from p38 is essential for the AMP transcriptional response in 4HB-stimulated macrophages. The findings provide a solid scientific basis and rationale for the decreased incidence of surgical site infections with the use of this type of surgical meshes. Further clinical significance is found in the fact that the 4HB activated molecular pathway includes common targets of frequently used nonsteroidal anti-inflammatory drugs (NSAIDs) and other FDA approved drugs recognizing G-protein coupled receptors.

Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection.

Alarmins, sequestered self-molecules containing damage-associated molecular patterns, are released during tissue injury to drive innate immune cell proinflammatory responses. Whether endogenous negative regulators controlling early immune responses are also released at the site of injury is poorly understood. Herein, we establish that the stromal cell-derived alarmin interleukin 33 (IL-33) is a local factor that directly restricts the proinflammatory capacity of graft-infiltrating macrophages early after transplantation. By assessing heart transplant recipient samples and using a mouse heart transplant model, we establish that IL-33 is upregulated in allografts to limit chronic rejection. Mouse cardiac transplants lacking IL-33 displayed dramatically accelerated vascular occlusion and subsequent fibrosis, which was not due to altered systemic immune responses. Instead, a lack of graft IL-33 caused local augmentation of proinflammatory iNOS+ macrophages that accelerated graft loss. IL-33 facilitated a metabolic program in macrophages associated with reparative and regulatory functions, and local delivery of IL-33 prevented the chronic rejection of IL-33-deficient cardiac transplants. Therefore, IL-33 represents what we believe is a novel regulatory alarmin in transplantation that limits chronic rejection by restraining the local activation of proinflammatory macrophages. The local delivery of IL-33 in extracellular matrix-based materials may be a promising biologic for chronic rejection prophylaxis.

Ultrasonic cavitation to prepare ECM hydrogels.

Hydrogels composed of extracellular matrix (ECM) have been used as a substrate for 3D organoid culture, and in numerous preclinical and clinical applications to facilitate repair and reconstruction of a variety of tissues. However, these ECM hydrogel materials are fabricated using lengthy methods that have focused on enzymatic digestion of the ECM with an acid protease in an acidic solution; or the use of chaotropic extraction buffers and dialysis procedures which can affect native protein structure and function. Herein we report a method to prepare hydrogels from ECM bioscaffolds using ultrasonic cavitation. The solubilized ECM can be induced to rapidly self-assemble into a gel by adjusting temperature, and the material properties of the gel can be tailored by adjusting ECM concentration and sonication parameters. The present study shows that ECM bioscaffolds can be successfully solubilized without enzymatic digestion and induced to repolymerize into a gel form capable of supporting cell growth. STATEMENT OF SIGNIFICANCE: ECM hydrogels have been used in numerous preclinical studies to facilitate repair of tissue following injury. However, there has been relatively little advancement in manufacturing techniques, thereby impeding progress in advancing this technology toward the clinic. Laboratory techniques for producing ECM hydrogels have focused on protease digestion methods, which require lengthy incubation times. The significance of this work lies in the development of a fundamentally different approach whereby an ECM hydrogel is rapidly formed without the need for acidic solutions or protease digestion. The ultrasonic cavitation method described herein represents a marked improvement in rheological properties and processing time over traditional enzymatic methods, and may lend itself as a platform for large-scale manufacturing of ECM hydrogels.

Lipidomics and RNA sequencing reveal a novel subpopulation of nanovesicle within extracellular matrix biomaterials.

Biomaterials composed of extracellular matrix (ECM) provide both mechanical support and a reservoir of constructive signaling molecules that promote functional tissue repair. Recently, matrix-bound nanovesicles (MBVs) have been reported as an integral component of ECM bioscaffolds. Although liquid-phase extracellular vesicles (EVs) have been the subject of intense investigation, their similarity to MBV is limited to size and shape. Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics and redox lipidomics were used to conduct a detailed comparison of liquid-phase EV and MBV phospholipids. Combined with comprehensive RNA sequencing and bioinformatic analysis of the intravesicular cargo, we show that MBVs are a distinct and unique subpopulation of EV and a distinguishing feature of ECM-based biomaterials. The results begin to identify the differential biologic activities mediated by EV that are secreted by tissue-resident cells and deposited within the ECM.

Matrix bound nanovesicle-associated IL-33 activates a pro-remodeling macrophage phenotype via a non-canonical, ST2-independent pathway.

The regenerative healing response of injured skeletal muscle is dependent upon an appropriately timed switch from a local type-I to a type-II immune response. Biologic scaffolds derived from extracellular matrix (ECM) have been shown to facilitate a macrophage phenotype transition that leads to downstream site-appropriate functional tissue deposition and myogenesis. However, the mechanisms by which ECM directs the switching of immune cell phenotype are only partially understood. Herein, we provide the first evidence that matrix bound nanovesicles (MBV) embedded within ECM-scaffolds are a rich and stable source of interleukin-33 (IL-33), an alarmin/cytokine with emerging reparative properties. We show that IL-33 encapsulated within MBV bypass the classical IL33/ST2 receptor signaling pathway to direct macrophage differentiation into the reparative, pro-remodeling M2 phenotype, which in turn facilitates myogenesis of skeletal muscle progenitor cells. Our results suggest the potential of IL-33+ MBV as a clinical therapy to augment the restorative efficacy of existing ECM-based and non-ECM based approaches.

Author Correction: Matrix-bound nanovesicles prevent ischemia-induced retinal ganglion cell axon degeneration and death and preserve visual function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

4-Hydroxybutyrate Promotes Endogenous Antimicrobial Peptide Expression in Macrophages.

IMPACT STATEMENT: This study evaluated the biological activity of hydroxylated derivatives of butyrate as inductors of antimicrobial peptides (AMPs) in murine bone marrow-derived macrophages in vitro. A differential modulation of AMP expression by the hydroxylated derivatives of butyrate is shown. The ability of sodium 4-hydroxybutyrate to upregulate AMP expression through a histone deacetylase inhibitory-independent mechanism, and to promote increased resistance to bacterial contamination in vivo are also shown. The findings provide an alternative for prevention of bacterial contamination of implanted biomaterials. Functionalization of biomaterials with hydroxylated derivatives of butyrate can enhance the endogenous antimicrobial activity of the immune system through increased production of AMPs by host cells, thus providing protection against bacterial contamination.

Matrix-bound nanovesicles prevent ischemia-induced retinal ganglion cell axon degeneration and death and preserve visual function.

Injury to retinal ganglion cells (RGC), central nervous system neurons that relay visual information to the brain, often leads to RGC axon degeneration and permanently lost visual function. Herein this study shows matrix-bound nanovesicles (MBV), a distinct class of extracellular nanovesicle localized specifically to the extracellular matrix (ECM) of healthy tissues, can neuroprotect RGCs and preserve visual function after severe, intraocular pressure (IOP) induced ischemia in rat. Intravitreal MBV injections attenuated IOP-induced RGC axon degeneration and death, protected RGC axon connectivity to visual nuclei in the brain, and prevented loss in retinal function as shown by histology, anterograde axon tracing, manganese-enhanced magnetic resonance imaging, and electroretinography. In the optic nerve, MBV also prevented IOP-induced decreases in growth associated protein-43 and IOP-induced increases in glial fibrillary acidic protein. In vitro studies showed MBV suppressed pro-inflammatory signaling by activated microglia and astrocytes, stimulated RGC neurite growth, and neuroprotected RGCs from neurotoxic media conditioned by pro-inflammatory astrocytes. Thus, MBV can positively modulate distinct signaling pathways (e.g., inflammation, cell death, and axon growth) in diverse cell types. Since MBV are naturally derived, bioactive factors present in numerous FDA approved devices, MBV may be readily useful, not only experimentally, but also clinically as immunomodulatory, neuroprotective factors for treating trauma or disease in the retina as well as other CNS tissues.

TGFß promotes breast cancer stem cell self-renewal through an ILEI/LIFR signaling axis.

FAM3C/Interleukin-like EMT Inducer (ILEI) is an oncogenic member of the FAM3 cytokine family and serves essential roles in both epithelial-mesenchymal transition (EMT) and breast cancer metastasis. ILEI expression levels are regulated through a non-canonical TGFß signaling pathway by 3'-UTR-mediated translational silencing at the mRNA level by hnRNP E1. TGFß stimulation or silencing of hnRNP E1 increases ILEI translation and induces an EMT program that correlates with enhanced invasion and migration. Recently, EMT has been linked to the formation of breast cancer stem cells (BCSCs) that confer both tumor cell heterogeneity as well as chemoresistant properties. Herein, we demonstrate that hnRNP E1 knockdown significantly shifts normal mammary epithelial cells to mesenchymal BCSCs in vitro and in vivo. We further validate that modulating ILEI protein levels results in the abrogation of these phenotypes, promoting further investigation into the unknown mechanism of ILEI signaling that drives tumor progression. We identify LIFR as the receptor for ILEI, which mediates signaling through STAT3 to drive both EMT and BCSC formation. Reduction of either ILEI or LIFR protein levels results in reduced tumor growth, fewer tumor initiating cells and reduced metastasis within the hnRNP E1 knock-down cell populations in vivo. These results reveal a novel ligand-receptor complex that drives the formation of BCSCs and represents a unique target for the development of metastatic breast cancer therapies.

Cytocompatibility and mechanical properties of surgical sealants for cardiovascular applications.

OBJECTIVES: The present study compared physical, mechanical, and biologic characteristics of 4 clinically available surgical sealants for cardiovascular repair. METHODS: BioGlue (Cryolife Inc, Kennesaw, Ga), PreveLeak (Mallinckrodt Pharmaceuticals, St Louis, Mo), Tridyne VS (BD, Franklin Lakes, NJ), and Coseal (Baxter Healthcare Corporation, Westlake Village, Calif) were compared for the following properties: hydrated swelling, cytocompatibility, burst strength, biaxial stretching (elasticity), and in vitro degradation. RESULTS: Sealants showed a wide range of swelling upon hydration. By gravimetric and volumetric measurement, swelling was greatest for Coseal followed by Tridyne VS, BioGlue, and PreveLeak. Tridyne VS was the most cytocompatible based on Alamar Blue assay results, supporting 85% cell survival compared with 36% to 39% survival with the other sealants. All sealants withstood pressure above mean arterial pressure (70-110 mm Hg) and physiologic systolic blood pressure (90-140 mm Hg) in an ex vivo arterial flow burst model; lowest peak pressure at failure was PreveLeak at 235 ± 48 mm Hg, and highest peak pressure at failure was BioGlue at 596 ± 72 mm Hg. Biaxial tensile testing showed no differences in elasticity between ex vivo porcine aorta and carotid arteries and Tridyne VS or Coseal, and BioGlue and PreveLeak were significantly stiffer. In vitro degradation time for Coseal was 6 days and 21 days for Tridyne VS. No degradation was observed in BioGlue or PreveLeak for 30 days. CONCLUSIONS: Although all sealants withstood supraphysiologic arterial pressure, there were differences in characteristics that may be important in clinical outcome. Coseal degradation time was short compared with other sealants, whereas BioGlue and PreveLeak showed a significant compliance mismatch with native porcine carotid artery. Tridyne VS was significantly more cytocompatible than the other 3 sealants.