Confinement controls the directional cell responses to fluid forces.

Our understanding of how fluid forces influence cell migration in confining environments remains limited. By integrating microfluidics with live-cell imaging, we demonstrate that cells in tightly-but not moderately-confined spaces reverse direction and move upstream upon exposure to fluid forces. This fluid force-induced directional change occurs less frequently when cells display diminished mechanosensitivity, experience elevated hydraulic resistance, or sense a chemical gradient. Cell reversal requires actin polymerization to the new cell front, as shown mathematically and experimentally. Actin polymerization is necessary for the fluid force-induced activation of NHE1, which cooperates with calcium to induce upstream migration. Calcium levels increase downstream, mirroring the subcellular distribution of myosin IIA, whose activation enhances upstream migration. Reduced lamin A/C levels promote downstream migration of metastatic tumor cells by preventing cell polarity establishment and intracellular calcium rise. This mechanism could allow cancer cells to evade high-pressure environments, such as the primary tumor.

Downregulation of YAP Activity Restricts P53 Hyperactivation to Promote Cell Survival in Confinement.

Cell migration through confining three dimensional (3D) topographies can lead to loss of nuclear envelope integrity, DNA damage, and genomic instability. Despite these detrimental phenomena, cells transiently exposed to confinement do not usually die. Whether this is also true for cells subjected to long-term confinement remains unclear at present. To investigate this, photopatterning and microfluidics are employed to fabricate a high-throughput device that circumvents limitations of previous cell confinement models and enables prolonged culture of single cells in microchannels with physiologically relevant length scales. The results of this study show that continuous exposure to tight confinement can trigger frequent nuclear envelope rupture events, which in turn promote P53 activation and cell apoptosis. Migrating cells eventually adapt to confinement and evade cell death by downregulating YAP activity. Reduced YAP activity, which is the consequence of confinement-induced YAP1/2 translocation to the cytoplasm, suppresses the incidence of nuclear envelope rupture and abolishes P53-mediated cell death. Cumulatively, this work establishes advanced, high-throughput biomimetic models for better understanding cell behavior in health and disease, and underscores the critical role of topographical cues and mechanotransduction pathways in the regulation of cell life and death.

Effects of low-intensity pulsed ultrasound stimulation on cell seeded 3D hybrid scaffold as a novel strategy for meniscus regeneration: An in vitro study.

Menisci are fibrocartilaginous structures in the knee joint with an inadequate regenerative capacity, which causes low healing potential and further leads to osteoarthritis. Recently, three-dimensional (3D) printing techniques and ultrasound treatment have gained plenty of attention for meniscus tissue engineering. The present study investigates the effectiveness of low-intensity pulsed ultrasound stimulations (LIPUS) on the proliferation, viability, morphology, and gene expression of the chondrocytes seeded on 3D printed polyurethane scaffolds dip-coated with gellan gum, hyaluronic acid, and glucosamine. LIPUS stimulation was performed at 100, 200, and 300 mW/cm2 intensities for 20 min/day. A faster gap closure (78.08 ± 2.56%) in the migration scratch assay was observed in the 200 mW/cm2 group after 24 h. Also, inverted microscopic and scanning electron microscopic images showed no cell morphology changes during LIPUS exposure at different intensities. The 3D cultured chondrocytes under LIPUS treatment revealed a promotion in cell proliferation rate and viability as the intensity doses increased. Additionally, LIPUS could stimulate chondrocytes to overexpress the aggrecan and collagen II genes and improve their chondrogenic phenotype. This study recommends that the combination of LIPUS treatment and 3D hybrid scaffolds can be considered as a valuable treatment for meniscus regeneration based on our in vitro data.

Fabrication and assessment of a novel hybrid scaffold consisted of polyurethane-gellan gum-hyaluronic acid-glucosamine for meniscus tissue engineering.

The meniscus has inadequate intrinsic regenerative capacity and its damage can lead to degeneration of articular cartilage. Meniscus tissue engineering aims to restore an injured meniscus followed by returning its normal function through bioengineered scaffolds. In the present study, the structural and biological properties of 3D-printed polyurethane (PU) scaffolds dip-coated with gellan gum (GG), hyaluronic acid (HA), and glucosamine (GA) were investigated. The optimum concentration of GG was 3% (w/v) with maintaining porosity at 88.1%. The surface coating of GG-HA-GA onto the PU scaffolds increased the compression modulus from 30.30 kPa to 59.10 kPa, the water uptake ratio from 27.33% to 60.80%, degradation rate from 5.18% to 8.84%, whereas the contact angle was reduced from 104.8° to 59.3°. MTT assay, acridine orange/ethidium bromide (AO/EB) fluorescent staining, and SEM were adopted to assess the behavior of the seeded chondrocytes on scaffolds, and it was found that the ternary surface coating stimulated the cell proliferation, viability, and adhesion. Moreover, the coated scaffolds showed higher expression levels of collagen II and aggrecan genes at day 7 compared to the control groups. Therefore, the fabricated PU-3% (w/v) GG-HA-GA scaffold can be considered as a promising scaffold for meniscus tissue engineering.

Integrating Pharmacogenomics Data-Driven Computational Drug Prediction with Single-Cell RNAseq to Demonstrate the Efficacy of a NAMPT Inhibitor against Aggressive, Taxane-Resistant, and Stem-like Cells in Lethal Prostate Cancer.

Metastatic prostate cancer/PCa is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and, therefore, androgen deprivation therapies/ADT-sensitive. However, eventual resistance to standard medical castration (AR-inhibitors) and secondary chemotherapies (taxanes) is nearly universal. Further, the presence of cancer stem-like cells (EMT/epithelial-to-mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/lethal/advanced variants of PCa (AVPC). In this study, we introduced a pharmacogenomics data-driven optimization-regularization-based computational prediction algorithm ("secDrugs") to predict novel drugs against lethal PCa. Integrating secDrug with single-cell RNA-sequencing/scRNAseq as a 'Double-Hit' drug screening tool, we demonstrated that single-cells representing drug-resistant and stem-cell-like cells showed high expression of the NAMPT pathway genes, indicating potential efficacy of the secDrug FK866 which targets NAMPT. Next, using several cell-based assays, we showed substantial impact of FK866 on clinically advanced PCa as a single agent and in combination with taxanes or AR-inhibitors. Bulk-RNAseq and scRNAseq revealed that, in addition to NAMPT inhibition, FK866 regulates tumor metastasis, cell migration, invasion, DNA repair machinery, redox homeostasis, autophagy, as well as cancer stemness-related genes, HES1 and CD44. Further, we combined a microfluidic chip-based cell migration assay with a traditional cell migration/'scratch' assay and demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using PCa patient datasets, we showed that FK866 is potentially capable of reversing the expression of several genes associated with biochemical recurrence, including IFITM3 and LTB4R. Thus, using FK866 as a proof-of-concept candidate for drug repurposing, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.