Modeling mechanical activation of macrophages during pulmonary fibrogenesis for targeted anti-fibrosis therapy.

Pulmonary fibrosis is an often fatal lung disease. Immune cells such as macrophages were shown to accumulate in the fibrotic lung, but their contribution to the fibrosis development is unclear. To recapitulate the involvement of macrophages in the development of pulmonary fibrosis, we developed a fibrotic microtissue model with cocultured human macrophages and fibroblasts. We show that profibrotic macrophages seeded on topographically controlled stromal tissues became mechanically activated. The resulting co-alignment of macrophages, collagen fibers, and fibroblasts promoted widespread fibrogenesis in micro-engineered lung tissues. Anti-fibrosis treatment using pirfenidone disrupts the polarization and mechanical activation of profibrotic macrophages, leading to fibrosis inhibition. Pirfenidone inhibits the mechanical activation of macrophages by suppressing integrin αMß2 and Rho-associated kinase 2. These results demonstrate a potential pulmonary fibrogenesis mechanism at the tissue level contributed by macrophages. The cocultured microtissue model is a powerful tool to study the immune-stromal cell interactions and the anti-fibrosis drug mechanism.

Tobacco and menthol flavored nicotine-free electronic cigarettes induced inflammation and dysregulated repair in lung fibroblast and epithelium.

BACKGROUND: Electronic cigarette (e-cig) vaping has increased in the past decade in the US, and e-cig use is misleadingly marketed as a safe cessation for quitting smoking. The main constituents in e-liquid are humectants, such as propylene glycol (PG) and vegetable glycerine (VG), but different flavoring chemicals are also used. However, the toxicology profile of flavored e-cigs in the pulmonary tract is lacking. We hypothesized that menthol and tobacco-flavored e-cig (nicotine-free) exposure results in inflammatory responses and dysregulated repair in lung fibroblast and epithelium. METHOD: We exposed lung fibroblast (HFL-1) and epithelium (BEAS-2B) to Air, PG/VG, menthol flavored, or tobacco-flavored e-cig, and determined the cytotoxicity, inflammation, and wound healing ability in 2D cells and 3D microtissue chip models. RESULTS: After exposure, HFL-1 showed decreased cell number with increased IL-8 levels in the tobacco flavor group compared to air. BEAS-2B also showed increased IL-8 secretion after PG/VG and tobacco flavor exposure, while menthol flavor exposure showed no change. Both menthol and tobacco-flavored e-cig exposure showed decreased protein abundance of type 1 collagen α 1 (COL1A1), α-smooth-muscle actin (αSMA), and fibronectin as well as decreased gene expression level of αSMA (Acta2) in HFL-1. After tobacco flavor e-cig exposure, HFL-1 mediated wound healing and tissue contractility were inhibited. Furthermore, BEAS-2B exposed to menthol flavor showed significantly decreased tight junction gene expressions, such as CDH1, OCLN, and TJP1. CONCLUSION: Overall, tobacco-flavored e-cig exposure induces inflammation in both epithelium and fibroblasts, and tobacco-flavored e-cig inhibits wound healing ability in fibroblasts.

Tobacco and Menthol flavored electronic cigarettes induced inflammation and dysregulated repair in lung fibroblast and epithelium.

Background: Electronic cigarette (e-cig) vaping has increased in the past decade in the US, and e-cig use is misleadingly marketed as a safe cessation for quitting smoking. The main constituents in e-liquid are humectants, such as propylene glycol (PG) and vegetable glycerine (VG), but different flavoring chemicals are also used. However, the toxicology profile of flavored e-cigs in the pulmonary tract is lacking. We hypothesized that menthol and tobacco-flavored e-cig (nicotine-free) exposure results in inflammatory responses and dysregulated repair in lung fibroblast and epithelium. Method: We exposed lung fibroblast (HFL-1) and epithelium (BEAS-2B) to Air, PG/VG, menthol flavored, or tobacco-flavored e-cig, and determined the cytotoxicity, inflammation, and wound healing ability of the cells in a microtissue chip model. Results: After exposure, HFL-1 showed decreased cell number with increased IL-8 levels in the tobacco flavor group compared to air. BEAS-2B also showed increased IL-8 secretion after PG/VG and tobacco flavor exposure, while menthol flavor exposure showed no change. Both menthol and tobacco-flavored e-cig exposure showed decreased protein abundance of type 1 collagen (COL1A1), α-smooth-muscle actin (αSMA), and fibronectin as well as decreased gene expression level of αSMA (Acta2) in HFL-1. After tobacco flavor e-cig exposure, HFL-1 mediated wound healing and tissue contractility were inhibited. Furthermore, BEAS-2B exposed to menthol flavor showed significantly decreased gene expression of CDH1, OCLN, and TJP1. Conclusion: Overall, tobacco-flavored e-cig exposure induces inflammation in both epithelium and fibroblasts, and tobacco-flavored e-cig inhibits wound healing ability in fibroblast.

Modeling Mechanical Activation of Macrophages During Pulmonary Fibrogenesis for Targeted Anti-Fibrosis Therapy.

Pulmonary fibrosis, as seen in idiopathic pulmonary fibrosis (IPF) and COVID-induced pulmonary fibrosis, is an often-fatal lung disease. Increased numbers of immune cells such as macrophages were shown to accumulate in the fibrotic lung, but it is unclear how they contribute to the development of fibrosis. To recapitulate the macrophage mechanical activation in the fibrotic lung tissue microenvironment, we developed a fibrotic microtissue model with cocultured human macrophages and fibroblasts. We show that profibrotic macrophages seeded on topographically controlled stromal tissue constructs become mechanically activated. The resulting co-alignment of macrophages, collagen fibers and fibroblasts promote widespread fibrogenesis in micro-engineered lung tissues. Anti-fibrosis treatment using pirfenidone disrupts the polarization and mechanical activation of profibrotic macrophages, leading to fibrosis inhibition. Pirfenidone inhibits the mechanical activation of macrophages by suppressing integrin αMß2 (CD11b/CD18) and Rho-associated kinase 2, which is a previously unknown mechanism of action of the drug. Together, these results demonstrate a potential pulmonary fibrogenesis mechanism at the tissue level contributed by mechanically activated macrophages. We propose the coculture, force-sensing microtissue model as a powerful tool to study the complex immune-stromal cell interactions and the mechanism of action of anti-fibrosis drugs.

Bortezomib-loaded mixed micelles realize a "three-in-one" effect for enhanced breast cancer treatment.

Comprehensively regulating the TME is now regarded as a promising approach for cancer treatment. Herein, a novel "three-in-one" effect is presented for simultaneously killing tumor cells, inhibiting the EMT of CAFs, and improving immune responses. In this study, bortezomib (BTZ) is selected for the treatment of breast cancer; it has multiple pharmacological mechanisms for killing tumor cells through the NF-κB signaling pathway, inhibiting the activity of CAFs by activating caspase-3, and enhancing the function of CD8+ T cells by regulating the expression of immune-stimulating factors. To improve the druggability of BTZ in solid tumors, BTZ-loaded lipid/glycocholic acid mixed micelles (BTZ-LGs) were prepared to verify the "three-in-one" effect in killing tumor cells, inhibiting CAFs, and improving immune responses. In the present work, BTZ-LGs were verified to show enhanced in vitro cytotoxicity in both 4T1 cells and 4T1/NIH3T3 co-cultured cells, as well as a superior in vivo treatment effect in different tumor-bearing mouse models. Additionally, BTZ-LGs could regulate the expression of α-SMA, caspase-3, E-cadherin, and N-cadherin, indicating their good inhibiting ability on both tumor cells and CAFs. More importantly, immunological analysis revealed that BTZ-LGs promoted the expression of the immunostimulatory factor IL-2 in tumor tissues, activated anti-tumor T cells, and overcame tumor-induced CD8+ T cell dysfunction. All these findings suggest that BTZ-LGs can achieve a "three-in-one" effect in terms of killing tumor cells, suppressing CAFs, and improving immune responses. This simple and multi-effective therapeutic strategy offers a promising approach for cancer therapy.

Predicting the preclinical efficacy of anti-fibrosis agents using a force-sensing fibrosis on chip system.

The high attrition rate of drug candidates contributes to the long duration and high cost in modern drug development. A major barrier in drug development is the poor predicting power of the preclinical models. In the current study, a human pulmonary fibrosis on chip system was developed for the preclinical evaluation of anti-fibrosis drugs. Pulmonary fibrosis is a severe disease characterized by progressive tissue stiffening that leads to respiration failure. To recapitulate the unique biomechanical feature of the fibrotic tissues, we developed flexible micropillars that can serve as in-situ force sensors to detect the changes in the mechanical properties of engineered lung microtissues. Using this system, we modeled the fibrogenesis of the alveolar tissues including the tissue stiffening and the expression of α-smooth muscle actin (α-SMA) and pro-collagen. Two anti-fibrosis drug candidates that are currently under clinical trials (KD025 and BMS-986020) were tested for their potential anti-fibrosis efficacy and the results were compared to those of FDA-approved anti-fibrosis drugs pirfenidone and nintedanib. Both pre-approval drugs were effective in inhibiting transforming growth factor beta 1 (TGF-ß1) induced increases in tissue contractile force, stiffness and expressions of fibrotic biomarkers, which are similar to the effects of FDA-approved anti-fibrosis drugs. These results demonstrated the potential utility of the force-sensing fibrosis on chip system in the pre-clinical development of anti-fibrosis drugs.

Delivery of anti-microRNA-21 by lung-targeted liposomes for pulmonary fibrosis treatment.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder with a low survival rate. Pulmonary fibrosis is one of the complications of COVID-19 and has a high prevalence in COVID-19 patients. Currently, no effective therapies other than lung transplantation are available to cure IPF and post-COVID-19 pulmonary fibrosis. MicroRNAs are small non-coding RNAs that mediate the development and progression of pulmonary fibrosis, thus making them potent drug candidates for this serious disease. MicroRNA-21 (miR-21) promotes not only the differentiation of fibroblasts to myofibroblasts but also epithelial-mesenchymal transition, both of which have been proposed as fundamental processes in pulmonary fibrosis development. Delivery of anti-miR-21 to block the miR-21-associated fibrogenic pathways represents a promising therapy for pulmonary fibrosis. However, microRNA treatment is challenged by quick degradation of RNA in blood, poor cellular uptake, and off-target effects. To overcome these challenges, we developed a lung-targeted, cationic liposome formulation to encapsulate anti-miR-21, enhance its delivery efficiency, and improve the therapeutic efficacy. We optimized the liposome formulation and demonstrated the anti-fibrotic effects using both in vitro and in vivo lung fibrosis models. Our results showed that anti-miR-21 delivered by cationic liposomes suppressed myofibroblast differentiation, reduced the synthesis of extracellular matrix, and inhibited fibrosis progression.

Recent Advances in CXCL12/CXCR4 Antagonists and Nano-Based Drug Delivery Systems for Cancer Therapy.

Chemokines can induce chemotactic cell migration by interacting with G protein-coupled receptors to play a significant regulatory role in the development of cancer. CXC chemokine-12 (CXCL12) can specifically bind to CXC chemokine receptor 4 (CXCR4) and is closely associated with the progression of cancer via multiple signaling pathways. Over recent years, many CXCR4 antagonists have been tested in clinical trials; however, Plerixafor (AMD3100) is the only drug that has been approved for marketing thus far. In this review, we first summarize the mechanisms that mediate the physiological effects of the CXCL12/CXCR4 axis. Then, we describe the use of CXCL12/CXCR4 antagonists. Finally, we discuss the use of nano-based drug delivery systems that exert action on the CXCL12/CXCR4 biological axis.

Engineering tumor stromal mechanics for improved T cell therapy.

Adoptive cellular therapies (ACT), including the engineered T cell receptor (TCR) therapy and chimeric antigen receptor (CAR) T Cell Therapy, are currently at the forefront of cancer immunotherapy. However, their efficacy for the treatment of solid tumors has not been confirmed. The fibrotic stroma surrounding the solid tumor has been suggested as the main barrier in the disarmament and suppression of the engineered T cells. In this review, we will discuss the recent findings on the mechanism of T cell suppression by the tumor stroma with a special emphasis on the effect of stromal mechanics. We will also discuss the engineering approaches used to dissect the mechanism of the T cell suppression by the stromal mechanical factors. Finally, we will provide a future outlook on the strategies to improve the efficacy of T cell therapy through altering the tumor stromal fibrosis.

Compressive Buckling Fabrication of 3D Cell-Laden Microstructures.

Tissue architecture is a prerequisite for its biological functions. Recapitulating the three-dimensional (3D) tissue structure represents one of the biggest challenges in tissue engineering. Two-dimensional (2D) tissue fabrication methods are currently in the main stage for tissue engineering and disease modeling. However, due to their planar nature, the created models only represent very limited out-of-plane tissue structure. Here compressive buckling principle is harnessed to create 3D biomimetic cell-laden microstructures from microfabricated planar patterns. This method allows out-of-plane delivery of cells and extracellular matrix patterns with high spatial precision. As a proof of principle, a variety of polymeric 3D miniature structures including a box, an octopus, a pyramid, and continuous waves are fabricated. A mineralized bone tissue model with spatially distributed cell-laden lacunae structures is fabricated to demonstrate the fabrication power of the method. It is expected that this novel approach will help to significantly expand the utility of the established 2D fabrication techniques for 3D tissue fabrication. Given the widespread of 2D fabrication methods in biomedical research and the high demand for biomimetic 3D structures, this method is expected to bridge the gap between 2D and 3D tissue fabrication and open up new possibilities in tissue engineering and regenerative medicine.

Engineered microenvironment for the study of myofibroblast mechanobiology.

Myofibroblasts are mechanosensitive cells and a variety of their behaviours including differentiation, migration, force production and biosynthesis are regulated by the surrounding microenvironment. Engineered cell culture models have been developed to examine the effect of microenvironmental factors such as the substrate stiffness, the topography and strain of the extracellular matrix (ECM) and the shear stress on myofibroblast biology. These engineered models provide well-mimicked, pathophysiologically relevant experimental conditions that are superior to those enabled by the conventional two-dimensional (2D) culture models. In this perspective, we will review the recent advances in the development of engineered cell culture models for myofibroblasts and outline the findings on the myofibroblast mechanobiology under various microenvironmental conditions. These studies have demonstrated the power and utility of the engineered models for the study of microenvironment-regulated cellular behaviours. The findings derived using these models contribute to a greater understanding of how myofibroblast behaviour is regulated in tissue repair and pathological scar formation.

Mechanosensitive expression of lamellipodin promotes intracellular stiffness, cyclin expression and cell proliferation.

Cell cycle control is a key aspect of numerous physiological and pathological processes. The contribution of biophysical cues, such as stiffness or elasticity of the underlying extracellular matrix (ECM), is critically important in regulating cell cycle progression and proliferation. Indeed, increased ECM stiffness causes aberrant cell cycle progression and proliferation. However, the molecular mechanisms that control these stiffness-mediated cellular responses remain unclear. Here, we address this gap and show good evidence that lamellipodin (symbol RAPH1), previously known as a critical regulator of cell migration, stimulates ECM stiffness-mediated cyclin expression and intracellular stiffening in mouse embryonic fibroblasts. We observed that increased ECM stiffness upregulates lamellipodin expression. This is mediated by an integrin-dependent FAK-Cas-Rac signaling module and supports stiffness-mediated lamellipodin induction. Mechanistically, we find that lamellipodin overexpression increased, and lamellipodin knockdown reduced, stiffness-induced cell cyclin expression and cell proliferation, and intracellular stiffness. Overall, these results suggest that lamellipodin levels may be critical for regulating cell proliferation. This article has an associated First Person interview with the first author of the paper.

Fibrosis on a Chip for Screening of Anti-Fibrosis Drugs.

Idiopathic pulmonary fibrosis (IPF) is a chronic pathological disorder that targets alveoli interstitial tissues and is characterized by the progressive stiffening of alveolar membrane. The median survival rate of the patients with IPF is less than 5 years. Currently, IPF has no cure and there are few options to alleviate the progress of this disease. A critical roadblock in developing new anti-fibrosis therapies is the absence of reliable cell based in vitro models that can recapitulate the progressive features of this disease. Here a novel fibrotic microtissue on a chip system is created to model the fibrotic transition of the lung interstitial tissue and the effect of anti-fibrosis drugs on such transitions. This system will not only help to expedite the efficacy analysis of anti-fibrotic therapies but also help to unveil their potential mode of action.

Fast Stereolithography Printing of Large-Scale Biocompatible Hydrogel Models.

Large size cell-laden hydrogel models hold great promise for tissue repair and organ transplantation, but their fabrication using 3D bioprinting is limited by the slow printing speed that can affect the part quality and the biological activity of the encapsulated cells. Here a fast hydrogel stereolithography printing (FLOAT) method is presented that allows the creation of a centimeter-sized, multiscale solid hydrogel model within minutes. Through precisely controlling the photopolymerization condition, low suction force-driven, high-velocity flow of the hydrogel prepolymer is established that supports the continuous replenishment of the prepolymer solution below the curing part and the nonstop part growth. The rapid printing of centimeter-sized hydrogel models using FLOAT is shown to significantly reduce the part deformation and cellular injury caused by the prolonged exposure to the environmental stresses in conventional 3D printing methods. Embedded vessel networks fabricated through multiscale printing allows media perfusion needed to maintain the high cellular viability and metabolic functions in the deep core of the large-sized models. The endothelialization of this vessel network allows the establishment of barrier functions. Together, these studies demonstrate a rapid 3D hydrogel printing method and represent a first step toward the fabrication of large-sized engineered tissue models.

Bioengineered Skeletal Muscle as a Model of Muscle Aging and Regeneration.

With age, adult skeletal muscle (SkM) is known to decrease in muscle mass, strength, and functional capacity, a state known as sarcopenia. Here we developed an in vitro three-dimensional (3D) bioengineered senescent SkM tissue using primary human myoblasts. These tissues exhibited the characteristics of atrophied muscle, including expression of senescent genes, decreased number of satellite cells, reduced number and size of myofibers, and compromised metabolism and calcium flux. As a result, senescent SkM tissues showed impaired ability to generate force in response to electrical stimulation compared with young tissues. Furthermore, in contrast to young SkM tissues, senescent tissues failed to regenerate in response to injury, possibly as a result of persistent apoptosis and failure to initiate a proliferation program. Our findings suggest that 3D senescent SkM may provide a powerful model for studying aging and a platform for drug testing and discovery of therapeutic compounds to improve the function of sarcopenic muscle. Impact statement Skeletal muscle (SkM) plays important physiological roles and has significant regenerative capacity. However, aged SkM lose their functionality and regeneration ability. In this article, we present a senescent human bioengineering SkM tissue model that can be used to investigate senescence, metabolic or genetic diseases that inflict SkM, and to test various strategies including novel small molecules that restore muscle function and promote regeneration. One key limitation of two-dimensional cell culture system is the detachment of contractile myotubes from the surface over time, thereby limiting the evaluation of myogenic function. Here we use primary human myoblasts, which exhibit all major hallmarks of aging to mimic the organization and function of native muscle. Using this system, we were able to measure the contractile function, calcium transients, and regeneration capacity of SkM tissues. We also evaluated the response of senescent SkM tissues to injury and their ability to regenerate and recover, compared with "young" tissues. Our results suggest that three-dimensional constructs enable organization of contractile units including myosin and actin filaments, thereby providing a powerful platform for the quantitative assessment of muscle myotubes in response to injury, genetic or metabolic disorders, or pharmacological testing.

Progress on the Application of Bortezomib and Bortezomib-Based Nanoformulations.

Bortezomib (BTZ) is the first proteasome inhibitor approved by the Food and Drug Administration. It can bind to the amino acid residues of the 26S proteasome, thereby causing the death of tumor cells. BTZ plays an irreplaceable role in the treatment of mantle cell lymphoma and multiple myeloma. Moreover, its use in the treatment of other hematological cancers and solid tumors has been investigated in numerous clinical trials and preclinical studies. Nevertheless, the applications of BTZ are limited due to its insufficient specificity, poor permeability, and low bioavailability. Therefore, in recent years, different BTZ-based drug delivery systems have been evaluated. In this review, we firstly discussed the functions of proteasome inhibitors and their mechanisms of action. Secondly, the properties of BTZ, as well as recent advances in both clinical and preclinical research, were reviewed. Finally, progress in research regarding BTZ-based nanoformulations was summarized.

Cyclic Stretching of Fibrotic Microtissue Array for Evaluation of Anti-Fibrosis Drugs.

INTRODUCTION: Progression of pulmonary fibrosis, characterized by the deterioration of lung tissue's mechanical properties, is affected by respiratory motion-induced dynamic loading. Since the development of anti-fibrosis drugs faces major hurdles in animal tests and human clinical trials, preclinical models that can recapitulate fibrosis progression under physiologically-relevant cyclic loading hold great promise. However, the integration of these two functions has not been achieved in existing models. METHODS: Recently we developed static human lung microtissue arrays that recapitulate the progressive changes in tissue mechanics during lung fibrogenesis. In the current study, we integrate the lung microtissue array with a membrane stretching system to enable dynamic loading to the microtissues. The effects of a pro-fibrotic agent and anti-fibrosis drugs were tested under cyclic stretching. RESULTS: Cyclic stretching that mimics respiratory motion was shown to affect the cytoskeletal organization and cellular orientation in the microtissue and cause the increase in microtissue contractility and stiffness. Fibrosis induction using TGF-ß1 further promoted fibrosis-related mechanical activity of the lung microtissues. Using this system, we examined the therapeutic effects of two FDA approved anti-fibrotic drugs. Our results showed that Nintedanib was able to fully inhibit TGF-ß1 induced force increase but only partially inhibited stretching induced force increase. In contrast, Pirfenidone was able to fully inhibit both TGF-ß1 induced force increase and stretching-induced force increase. CONCLUSIONS: Together, these results highlight the pathophysiologically-relevant modeling capability of the current fibrotic microtissue system and demonstrated the potential of this system to be used for anti-fibrosis drug screening.

Microclot array elastometry for integrated measurement of thrombus formation and clot biomechanics under fluid shear.

Blood clotting at the vascular injury site is a complex process that involves platelet adhesion and clot stiffening/contraction in the milieu of fluid flow. An integrated understanding of the hemodynamics and tissue mechanics regulating this process is currently lacking due to the absence of an experimental system that can simultaneously model clot formation and measure clot mechanics under shear flow. Here we develop a microfluidic-integrated microclot-array-elastometry system (clotMAT) that recapitulates dynamic changes in clot mechanics under physiological shear. Treatments with procoagulants and platelet antagonists and studies with diseased patient plasma demonstrate the ability of the system to assay clot biomechanics associated with common antiplatelet treatments and bleeding disorders. The changes of clot mechanics under biochemical treatments and shear flow demonstrate independent yet equally strong effects of these two stimulants on clot stiffening. This microtissue force sensing system may have future research and diagnostic potential for various bleeding disorders.

Dispersible hydrogel force sensors reveal patterns of solid mechanical stress in multicellular spheroid cultures.

Understanding how forces orchestrate tissue formation requires technologies to map internal tissue stress at cellular length scales. Here, we develop ultrasoft mechanosensors that visibly deform under less than 10 Pascals of cell-generated stress. By incorporating these mechanosensors into multicellular spheroids, we capture the patterns of internal stress that arise during spheroid formation. We experimentally demonstrate the spontaneous generation of a tensional 'skin', only a few cell layers thick, at the spheroid surface, which correlates with activation of mechanobiological signalling pathways, and balances a compressive stress profile within the tissue. These stresses develop through cell-driven mechanical compaction at the tissue periphery, and suggest that the tissue formation process plays a critically important role in specifying mechanobiological function. The broad applicability of this technique should ultimately provide a quantitative basis to design tissues that leverage the mechanical activity of constituent cells to evolve towards a desired form and function.

Engineered Tissue Development in Biofabricated 3D Geometrical Confinement-A Review.

Living tissue is a complex, heterogeneous structure where spatially organized ECMs present embedded cells with a variety of biochemical and mechanical signals. These signals are important to the formation of tissue structures and maintaining tissue homeostasis and physiological functions. Recent advances in biofabrication technologies have allowed the creation of 3D geometrical patterns that can guide the dynamic interaction between cells and ECM, leading to the formation of morphologically controlled engineered tissues that recapitulate the structure and function of native tissues. In this work, we first review advanced biofabrication technologies including lithography-based microfabrication and bioprinting that have been adopted to create a variety of geometrical confinements such as microgrooves and microribs, microwells, micropillar arrays, and microfibers. For each confinement type, we review geometrically guided formation and maturation of a variety of tissue types, including skeletal and cardiac muscles, epithelial tissue, endothelial tissue, and fibrous tissue. Geometrical confinements are important microenvironmental cues that can be utilized to promote the formation of biomimetic structures in engineered tissues.