Cleanroom-Free Microfluidic Device for Natural Induction of Hypoxia in 2D and 3D Tumor Models.

Hypoxia is a common and critical feature of solid tumors that contributes to the plasticity and heterogeneity of the cancer cells. Cancer cell populations take on a region-specific adaptation induced by hypoxia, and each cancer cell population will show different levels of sensitivity and resistance to cancer therapeutics. Therefore, a faithful recapitulation of tumor hypoxia that allows for accurate assessments of hypoxia-induced adaptations, heterogeneity, and response to therapy is needed to develop new therapeutic approaches. The existing hypoxic tumor models rely on complex fabrication methods and external gas sources that make them unfavorable for the early-stage screening of new therapeutics. Here, we demonstrate how to establish a cleanroom-free microfluidic device that supports both 2D and 3D hypoxic tumor modeling through natural cancer cell metabolism and confirm the induction of the hypoxic gradient.

Hypoxic 3D Tumor Model for Evaluating of CAR-T Cell Therapy In Vitro.

Solid tumors contain abnormal physical and biochemical barriers that hinder chimeric antigen receptor (CAR) T cell therapies. However, there is a lack of understanding on how the solid tumor microenvironment (e.g. hypoxia) modulates CAR-T cell function. Hypoxia is a common feature of many advanced solid tumors that contributes to reprogramming of cancer and T cell metabolism as well as their phenotypes and interactions. To gain insights into the activities of CAR-T cells in solid tumors and to assess the effectiveness of new combination treatments involving CAR-T cells, in vitro models that faithfully reflect CAR-T cell-solid tumor interactions under physiologically relevant tumor microenvironment is needed. Here we demonstrate how to establish a hypoxic 3-dimensional (3-D) tumor model using a cleanroom-free, micromilling-based microdevice and assess the efficacy of the combination treatment with CAR-T cells and PD-1/PD-L1 inhibition.

Recapitulating Tumor Hypoxia in a Cleanroom-Free, Liquid-Pinning-Based Microfluidic Tumor Model.

In tumors, the metabolic demand of cancer cells often outpaces oxygen supply, resulting in a gradient of tumor hypoxia accompanied with heterogeneous resistance to cancer therapeutics. Models recapitulating tumor hypoxia are therefore essential for developing more effective cancer therapeutics. Existing in vitro models often fail to capture the spatial heterogeneity of tumor hypoxia or involve high-cost, complex fabrication/handling techniques. Here, we designed a highly tunable microfluidic device that induces hypoxia through natural cell metabolism and oxygen diffusion barriers. We adopted a cleanroom-free, micromilling-replica-molding strategy and a microfluidic liquid-pinning approach to streamline the fabrication and tumor model establishment. We also implemented a thin-film oxygen diffusion barrier design, which was optimized through COMSOL simulation, to support both two-dimensional (2-D) and three-dimensional (3-D) hypoxic models. We demonstrated that liquid-pinning enables an easy, injection-based micropatterning of cancer cells of a wide range of parameters, showing the high tunability of our design. Human breast cancer and prostate cancer cells were seeded and stained after 24 h of 2-D and 3-D culture to validate the natural induction of hypoxia. We further demonstrated the feasibility of the parallel microfluidic channel design to evaluate dual therapeutic conditions in the same device. Overall, our new microfluidic tumor model serves as a user-friendly, cost-effective, and highly scalable platform that provides spatiotemporal analysis of the hypoxic tumor microenvironments suitable for high-content biological studies and therapeutic discoveries.

A Pillar-Free Diffusion Device for Studying Chemotaxis on Supported Lipid Bilayers.

Chemotactic cell migration plays a crucial role in physiological and pathophysiological processes. In tissues, cells can migrate not only through extracellular matrix (ECM), but also along stromal cell surfaces via membrane-bound receptor-ligand interactions to fulfill critical functions. However, there remains a lack of models recapitulating chemotactic migration mediated through membrane-bound interactions. Here, using micro-milling, we engineered a multichannel diffusion device that incorporates a chemoattractant gradient and a supported lipid bilayer (SLB) tethered with membrane-bound factors that mimics stromal cell membranes. The chemoattractant channels are separated by hydrogel barriers from SLB in the cell loading channel, which enable precise control of timing and profile of the chemokine gradients applied on cells interacting with SLB. The hydrogel barriers are formed in pillar-free channels through a liquid pinning process, which eliminates complex cleanroom-based fabrications and distortion of chemoattractant gradient by pillars in typical microfluidic hydrogel barrier designs. As a proof-of-concept, we formed an SLB tethered with ICAM-1, and demonstrated its lateral mobility and different migratory behavior of Jurkat T cells on it from those on immobilized ICAM-1, under a gradient of chemokine CXCL12. Our platform can thus be widely used to investigate membrane-bound chemotaxis such as in cancer, immune, and stem cells.

Engineering a Vascularized Hypoxic Tumor Model for Therapeutic Assessment.

Solid tumors in advanced cancer often feature a structurally and functionally abnormal vasculature through tumor angiogenesis, which contributes to cancer progression, metastasis, and therapeutic resistances. Hypoxia is considered a major driver of angiogenesis in tumor microenvironments. However, there remains a lack of in vitro models that recapitulate both the vasculature and hypoxia in the same model with physiological resemblance to the tumor microenvironment, while allowing for high-content spatiotemporal analyses for mechanistic studies and therapeutic evaluations. We have previously constructed a hypoxia microdevice that utilizes the metabolism of cancer cells to generate an oxygen gradient in the cancer cell layer as seen in solid tumor sections. Here, we have engineered a new composite microdevice-microfluidics platform that recapitulates a vascularized hypoxic tumor. Endothelial cells were seeded in a collagen channel formed by viscous fingering, to generate a rounded vascular lumen surrounding a hypoxic tumor section composed of cancer cells embedded in a 3-D hydrogel extracellular matrix. We demonstrated that the new device can be used with microscopy-based high-content analyses to track the vascular phenotypes, morphology, and sprouting into the hypoxic tumor section over a 7-day culture, as well as the response to different cancer/stromal cells. We further evaluated the integrity/leakiness of the vascular lumen in molecular delivery, and the potential of the platform to study the movement/trafficking of therapeutic immune cells. Therefore, our new platform can be used as a model for understanding tumor angiogenesis and therapeutic delivery/efficacy in vascularized hypoxic tumors.

Eupatilin Inhibits Reactive Oxygen Species Generation via Akt/NF-κB/MAPK Signaling Pathways in Particulate Matter-Exposed Human Bronchial Epithelial Cells.

BACKGROUND: Eupatilin is an active flavon extracted from the Artemisia species and has properties such as antioxidant, anti-inflammatory, and anti-cancer. We examined the effect of eupatilin using fine particulate matter (FPM) and human bronchial epithelial cell line (BEAS-2B) to confirm the potential of eupatilin as a therapeutic agent for respiratory diseases caused by FPM. METHODS: Reactive oxygen species (ROS) levels were checked by flow cytometry to identify if FPM and eupatilin affect ROS production. Western blotting was performed to identify the mechanism of action of eupatilin in FPM-exposed BEAS-2B cells. RESULTS: When cells were exposed to FPM above 12.5 µg/mL concentration for 24 h, ROS production increased significantly compared to the control. When eupatilin was added to cells exposed to FPM, the ROS level decreased proportionally with the eupatilin dose. The phosphorylation of Akt, NF-κB p65, and p38 MAPK induced by FPM was significantly reduced by eupatilin, respectively. CONCLUSION: FPM cause respiratory disease by producing ROS in bronchial epithelial cells. Eupatilin has been shown to inhibit ROS production through altering signaling pathways. The ROS inhibiting property of eupatilin can be exploited in FPM induced respiratory disorders.

Factors influencing posttraumatic growth in ovarian cancer survivors.

PURPOSE: The purpose of this study was to examine posttraumatic growth (PTG), cancer coping, posttraumatic stress, and genetics knowledge among ovarian cancer survivors and to identify factors affecting PTG. METHODS: This cross-sectional study included 148 outpatient ovarian cancer survivors at a cancer center. Data were collected between February 25 and April 11, 2019, and were analyzed using t tests, ANOVA, Pearson-correlations, and multiple regression. RESULTS: On average, the ovarian cancer survivors scored 68.09 ± 20.17 in PTG, 59.75 ± 13.37 in cancer coping, 29.30 ± 17.25 in posttraumatic stress, and 9.42 ± 3.33 in genetics knowledge. There were significant differences in PTG according to religion (t = - 2.92, p = .004), marital status (F = 3.06, p = .050), and family history of cancer (t = 2.00, p = .047). In the final analysis, the statistically significant factors influencing PTG were religion (ß = .170, p = .004) and cancer coping (ß = .691, p < .001), and posttraumatic stress had borderline statistical significance (ß = - .107, p < .068). These factors explained 52.2% of the variance in PTG. CONCLUSIONS: Ovarian cancer survivors showed a moderate level of genetics knowledge while having a high risk for posttraumatic stress. Overall, this study showed that cancer coping was a powerful factor that influenced PTG in ovarian cancer survivors. Religion was found to positively affect PTG, and posttraumatic stress had a small negative effect. Spiritual nursing interventions and improving cancer coping while reducing posttraumatic stress are necessary to increase the PTG of ovarian cancer survivors.

Urban particulate matter regulates tight junction proteins by inducing oxidative stress via the Akt signal pathway in human nasal epithelial cells.

Recent studies have revealed that increased reactive oxidative stress (ROS) induced by particulate matter (PM) affects tight junction (TJ) functions; however, the molecular mechanisms underlying this effect have not been evaluated fully. Cultured human epithelial cells obtained from inferior turbinate tissues were exposed to an urban PM (UPM) standard reference material (SRM 1648a). Intracellular ROS level and expression of proinflammatory cytokines and TJ proteins were examined. Expression level of phosphorylated (p)-Akt, p38, p65 were compared between exposed and unexposed cells. Cells were pretreated with the ROS scavenger N-acetylcysteine (NAC) or Akt inhibitor MK-2206 before exposure to determine whether the changes in cellular ROS and TJ protein expression could be reversed. Exposure to UPM significantly increased ROS levels and inflammatory cytokine expression levels, and decreased expression of TJ proteins zonula occludins (ZO)-1, occludin, claudin-1, and E-cadherin. UPM exposure increased p-Akt, p-p38, and p65 expression levels, and NAC pretreatment reversed these effects. Akt inhibition decreased UPM-induced ROS formation and p38 and p65 protein phosphorylation, and restored the decreased ZO-1 and E-cadherin expression. Akt inhibition and ROS scavenging may provide targets for maintaining epithelial integrity by restoring decreased TJ protein expression during exposure to UPM.

Stem cell-loaded adhesive immiscible liquid for regeneration of myocardial infarction.

Myocardial infarction (MI) causes serious loss of cardiac muscle and dysfunction. To restore MI, exogenous stem cells should be efficiently delivered. However, due to severe physical and physiological cardiac environment, recent strategies have faced challenges, including low cell persistence, low integration, and delayed therapeutic effects. Herein, we proposed mesenchymal stem cell (MSC) therapeutic platform using adhesive protein-based immiscible condensed liquid system (APICLS) derived from bioengineered mussel adhesive protein (MAP). With high encapsulation efficiency and survival rate of encapsulated MSCs, APICLS was successfully grafted by intramyocardial injection and distributed throughout the scarred myocardium. Its underwater adhesiveness and biocompatibility fostered integration with damaged tissue, resulting in high cell persistence and maximized paracrine effects. Bioactive molecules released from APICLS with MSCs induced angiogenesis and cardioprotection, delayed cardiac remodeling, reduced fibrosis, and recovered contractive force. Thus, our proposed strategy represents an innovative approach for recovering infarcted cardiac tissues with damaged structural and contractive function.

Effect of fluticasone propionate on human nasal fibroblasts exposed to urban particulate matter.

OBJECTIVE: Particulate matter (PM), which contains organic compounds and toxic metals, is the major cause of air pollution. PM enters the body, causing various health problems. Although the effects of PM on the lower respiratory tract have been extensively investigated, the effects on the upper respiratory tract (including the nasal cavity) require further evaluation. To investigate the effect of fluticasone propionate (FP) on nasal fibroblasts exposed to UPM. METHODS: Samples of inferior turbinate tissue were obtained from six patients. The fibroblasts isolated from these samples were exposed to UPM and/or FP. The expression of interleukin (IL)-6, CXC chemokine ligand (CXCL) 1, IL-1ß, and tumour necrosis factor-alpha (TNF-α) in nasal fibroblasts was analysed using real-time PCR and enzyme-linked immunosorbent assays. The protein levels of nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) were analysed by western blotting. RESULTS: FP reversed the UPM-induced reduction in cell viability. The mRNA and protein levels of IL-6, CXCL1, IL-1ß, and TNF-α were significantly increased by UPM. FP reversed the UPM-induced increases in the protein levels of NF-κB and phosphorylated-STAT3 in a dose-dependent manner. In addition, TNF-α, an inducer of NF-κB, reversed the FP-induced reduction in the levels of signalling molecules. CONCLUSION: UPM induces the expression of IL-6, CXCL1, IL-1ß, and TNF-α in nasal fibroblasts and this effect is reversed by FP via the STAT3 and NF-κB signalling pathways. These results suggest that FP has therapeutic potential for nasal diseases related to UPM, such as allergic and chronic rhinitis.

Microphysiological Engineering of Self-Assembled and Perfusable Microvascular Beds for the Production of Vascularized Three-Dimensional Human Microtissues.

The vasculature is an essential component of the circulatory system that plays a vital role in the development, homeostasis, and disease of various organs in the human body. The ability to emulate the architecture and transport function of blood vessels in the integrated context of their associated organs represents an important requirement for studying a wide range of physiological processes. Traditional in vitro models of the vasculature, however, largely fail to offer such capabilities. Here we combine microfluidic three-dimensional (3D) cell culture with the principle of vasculogenic self-assembly to engineer perfusable 3D microvascular beds in vitro. Our system is created in a micropatterned hydrogel construct housed in an elastomeric microdevice that enables coculture of primary human vascular endothelial cells and fibroblasts to achieve de novo formation, anastomosis, and controlled perfusion of 3D vascular networks. An open-top chamber design adopted in this hybrid platform also makes it possible to integrate the microengineered 3D vasculature with other cell types to recapitulate organ-specific cellular heterogeneity and structural organization of vascularized human tissues. Using these capabilities, we developed stem cell-derived microphysiological models of vascularized human adipose tissue and the blood-retinal barrier. Our approach was also leveraged to construct a 3D organotypic model of vascularized human lung adenocarcinoma as a high-content drug screening platform to simulate intravascular delivery, tumor-killing effects, and vascular toxicity of a clinical chemotherapeutic agent. Furthermore, we demonstrated the potential of our platform for applications in nanomedicine by creating microengineered models of vascular inflammation to evaluate a nanoengineered drug delivery system based on active targeting liposomal nanocarriers. These results represent a significant improvement in our ability to model the complexity of native human tissues and may provide a basis for developing predictive preclinical models for biopharmaceutical applications.

ß-Catenin-RAS interaction serves as a molecular switch for RAS degradation via GSK3ß.

RAS proteins play critical roles in various cellular processes, including growth and transformation. RAS proteins are subjected to protein stability regulation via the Wnt/ß-catenin pathway, and glycogen synthase kinase 3 beta (GSK3ß) is a key player for the phosphorylation-dependent RAS degradation through proteasomes. GSK3ß-mediated RAS degradation does not occur in cells that express a nondegradable mutant (MT) ß-catenin. Here, we show that ß-catenin directly interacts with RAS at the α-interface region that contains the GSK3ß phosphorylation sites, threonine 144 and threonine 148 residues. Exposure of these sites by prior ß-catenin degradation is required for RAS degradation. The introduction of a peptide that blocks the ß-catenin-RAS interaction by binding to ß-catenin rescues the GSK3ß-mediated RAS degradation in colorectal cancer (CRC) cells that express MT ß-catenin. The coregulation of ß-catenin and RAS stabilities by the modulation of their interaction provides a mechanism for Wnt/ß-catenin and RAS-ERK pathway cross-talk and the synergistic transformation of CRC by both APC and KRAS mutations.

The effect of urban particulate matter on cultured human nasal fibroblasts.

BACKGROUND: Exposure to urban particulate matter (UPM) has been linked to aggravation of various health problems. Although the effects of UPM on the lower respiratory tract have been extensively studied, more research is required on the impact of UPM on the upper respiratory tract and the underlying mechanisms. Thus, we investigated the cytotoxic effects of UPM on cultured human nasal fibroblasts, the underlying signaling pathways involved, and changes in cytokine levels. METHODS: Human turbinate tissue specimens were collected during partial turbinectomies performed on 6 patients, and then cultured. The effect of UPM on nasal fibroblast viability was explored. Real-time reverse transcription-polymerase chain reaction was used to measure the mRNA levels of genes encoding cytokines and chemokines (interleukin [IL]-4, IL-6, IL-8, and tumor necrosis factor-α) before and after 24 hours of UPM treatment. Enzyme-linked immunosorbent assays were employed to measure IL-6 and IL-8 levels. The status of the p38 and nuclear factor (NF)-κB signaling pathways was analyzed by Western blotting. RESULTS: UPM reduced cell viability in a dose-dependent manner and increased IL-6 and IL-8 expression at both the mRNA and protein levels. UPM induced the phosphorylation of p38 and NF-κB p65; inhibitors of the actions of these proteins repressed phosphorylation and the expression of IL-6 and IL-8. CONCLUSION: UPM induced IL-6 and IL-8 expression by fibroblasts via p38 and NF-κB classical signaling, suggesting that UPM can induce or aggravate allergic and/or chronic rhinitis.

Paricalcitol Pretreatment Attenuates Renal Ischemia-Reperfusion Injury via Prostaglandin E2 Receptor EP4 Pathway.

The protective mechanism of paricalcitol remains unclear in renal ischemia-reperfusion (IR) injury. We investigated the renoprotective effects of paricalcitol in IR injury through the prostaglandin E2 (PGE2) receptor EP4. Paricalcitol was injected into IR-exposed HK-2 cells and mice subjected to bilateral kidney ischemia for 23 min and reperfusion for 24 hr. Paricalcitol prevented IR-induced cell death and EP4 antagonist cotreatment offset these protective effects. Paricalcitol increased phosphorylation of Akt and cyclic AMP responsive element binding protein (CREB) and suppressed nuclear factor-κB (NF-κB) in IR-exposed cells and cotreatment of EP4 antagonist or EP4 small interfering RNA blunted these signals. In vivo studies showed that paricalcitol improved renal dysfunction and tubular necrosis after IR injury and cotreatment with EP4 antagonist inhibited the protective effects of paricalcitol. Phosphorylation of Akt was increased and nuclear translocation of p65 NF-κB was decreased in paricalcitol-treated mice with IR injury, which was reversed by EP4 blockade. Paricalcitol decreased oxidative stress and apoptosis in renal IR injury. Paricalcitol also attenuated the infiltration of inflammatory cells and production of proinflammatory cytokines after IR injury. EP4 antagonist abolished these antioxidant, anti-inflammatory, and antiapoptotic effects. The EP4 plays a pivotal role in the protective effects of paricalcitol in renal IR injury.