Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

Lung cancer is the leading cause of cancer-related deaths in the world, and non-small cell lung cancer (NSCLC) is the most common subset. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-Seq profiles of TIMs from several LUSC metastatic models with inflammatory monocytes (IMs) of non-tumor-bearing controls. We identified Spon1 as upregulated in TIMs and found that Spon1 expression in LUSC tumors corresponded with poor survival and enrichment of collagen extracellular matrix signatures. We observed SPON1+ TIMs mediate their effects directly through LRP8 on NSCLC cells, which resulted in TGF-ß1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrated that SPON1+ TIMs were sufficient to promote NSCLC metastases. Additionally, we found that Spon1 loss in the host, or Lrp8 loss in cancer cells, resulted in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirmed the relevance of the SPON1/LRP8/TGF-ß1 axis with collagen production and survival in patients with NSCLC. Taken together, our study describes how SPON1+ TIMs promote collagen remodeling and NSCLC metastases through an LRP8/TGF-ß1 signaling axis.

N-cadherin and ß1 integrin coordinately regulate growth plate cartilage architecture.

Spatial and temporal regulation of chondrocyte maturation in the growth plate drives growth of many bones. One essential event to generate the ordered cell array characterizing growth plate cartilage is the formation of chondrocyte columns in the proliferative zone via 90-degree rotation of daughter cells to align with the long axis of the bone. Previous studies have suggested crucial roles for cadherins and integrin ß1 in column formation. The purpose of this study was to determine the relative contributions of cadherin- and integrin-mediated cell adhesion in column formation. Here we present new mechanistic insights generated by application of live time-lapse confocal microscopy of cranial base explant cultures, robust genetic mouse models, and new quantitative methods to analyze cell behavior. We show that conditional deletion of either the cell-cell adhesion molecule Cdh2 or the cell-matrix adhesion molecule Itgb1 disrupts column formation. Compound mutants were used to determine a potential reciprocal regulatory interaction between the two adhesion surfaces and identified that defective chondrocyte rotation in a N-cadherin mutant was restored by a heterozygous loss of integrin ß1. Our results support a model for which integrin ß1, and not N-cadherin, drives chondrocyte rotation and for which N-cadherin is a potential negative regulator of integrin ß1 function.

PTEN-restoration abrogates brain colonisation and perivascular niche invasion by melanoma cells.

BACKGROUND: Melanoma brain metastases (MBM) continue to be a significant clinical problem with limited treatment options. Highly invasive melanoma cells migrate along the vasculature and perivascular cells may contribute to residual disease and recurrence. PTEN loss and hyperactivation of AKT occur in MBM; however, a role for PTEN/AKT in perivascular invasion has not been described. METHODS: We used in vivo intracranial injections of murine melanoma and bulk RNA sequencing of melanoma cells co-cultured with brain endothelial cells (brECs) to investigate brain colonisation and perivascular invasion. RESULTS: We found that PTEN-null melanoma cells were highly efficient at colonising the perivascular niche relative to PTEN-expressing counterparts. PTEN re-expression (PTEN-RE) in melanoma cells significantly reduced brain colonisation and migration along the vasculature. We hypothesised this phenotype was mediated through vascular-induced TGFß secretion, which drives AKT phosphorylation. Disabling TGFß signalling in melanoma cells reduced colonisation and perivascular invasion; however, the introduction of constitutively active myristolated-AKT (myrAKT) restored overall tumour size but not perivascular invasion. CONCLUSIONS: PTEN loss facilitates perivascular brain colonisation and invasion of melanoma. TGFß-AKT signalling partially contributes to this phenotype, but further studies are needed to determine the complementary mechanisms that enable melanoma cells to both survive and spread along the brain vasculature.

A Facile Strategy for the Fabrication of Cell-laden Porous Alginate Hydrogels Based on Two-phase Aqueous Emulsions.

Porous alginate hydrogels possess many advantages as cell carriers. However, current pore generation methods require either complex or harsh fabrication processes, toxic components, or extra purification steps, limiting the feasibility and affecting the cellular survival and function. In this study, a simple and cell-friendly approach to generate highly porous cell-laden alginate hydrogels based on two-phase aqueous emulsions is reported. The pre-gel solutions, which contain two immiscible aqueous phases of alginate and caseinate, are crosslinked by calcium ions. The porous structure of the hydrogel construct is formed by subsequently removing the caseinate phase from the ion-crosslinked alginate hydrogel. Those porous alginate hydrogels possess heterogeneous pores around 100 µm and interconnected paths. Human white adipose progenitors (WAPs) encapsulated in these hydrogels self-organize into spheroids and show enhanced viability, proliferation, and adipogenic differentiation, compared to non-porous constructs. As a proof of concept, this porous alginate hydrogel platform is employed to prepare core-shell spheres for coculture of WAPs and colon cancer cells, with WAP clusters distributed around cancer cell aggregates, to investigate cellular crosstalk. This efficacious approach is believed to provide a robust and versatile platform for engineering porous-structured alginate hydrogels for applications as cell carriers and in disease modeling.

The modes of angiogenesis: an updated perspective.

Following the process of vasculogenesis during development, angiogenesis generates new vascular structures through a variety of different mechanisms or modes. These different modes of angiogenesis involve, for example, increasing microvasculature density by sprouting of endothelial cells, splitting of vessels to increase vascular surface area by intussusceptive angiogenesis, fusion of capillaries to increase blood flow by coalescent angiogenesis, and the recruitment of non-endothelial cells by vasculogenic mimicry. The recent reporting on coalescent angiogenesis as a new mode of vessel formation warrants a brief overview of angiogenesis mechanisms to provide a more complete picture. The journal Angiogenesis is devoted to the delineation of the different modes and mechanisms that collectively dictate blood vessel formation, inhibition, and function in health and disease.

Periostin+ Stromal Cells Guide Lymphovascular Invasion by Cancer Cells.

Cancer cell dissemination to sentinel lymph nodes is associated with poor patient outcomes, particularly in breast cancer. The process by which cancer cells egress from the primary tumor upon interfacing with the lymphatic vasculature is complex and driven by dynamic interactions between cancer cells and stromal cells, including cancer-associated fibroblasts (CAF). The matricellular protein periostin can distinguish CAF subtypes in breast cancer and is associated with increased desmoplasia and disease recurrence in patients. However, as periostin is secreted, periostin-expressing CAFs are difficult to characterize in situ, limiting our understanding of their specific contribution to cancer progression. Here, we used in vivo genetic labeling and ablation to lineage trace periostin+ cells and characterize their functions during tumor growth and metastasis. Periostin-expressing CAFs were spatially found at periductal and perivascular margins, were enriched at lymphatic vessel peripheries, and were differentially activated by highly metastatic cancer cells versus poorly metastatic counterparts. Surprisingly, genetically depleting periostin+ CAFs slightly accelerated primary tumor growth but impaired intratumoral collagen organization and inhibited lymphatic, but not lung, metastases. Periostin ablation in CAFs impaired their ability to deposit aligned collagen matrices and inhibited cancer cell invasion through collagen and across lymphatic endothelial cell monolayers. Thus, highly metastatic cancer cells mobilize periostin-expressing CAFs in the primary tumor site that promote collagen remodeling and collective cell invasion within lymphatic vessels and ultimately to sentinel lymph nodes. SIGNIFICANCE: Highly metastatic breast cancer cells activate a population of periostin-expressing CAFs that remodel the extracellular matrix to promote escape of cancer cells into lymphatic vessels and drive colonization of proximal lymph nodes.

Pathological angiogenesis: mechanisms and therapeutic strategies.

In multicellular organisms, angiogenesis, the formation of new blood vessels from pre-existing ones, is an essential process for growth and development. Different mechanisms such as vasculogenesis, sprouting, intussusceptive, and coalescent angiogenesis, as well as vessel co-option, vasculogenic mimicry and lymphangiogenesis, underlie the formation of new vasculature. In many pathological conditions, such as cancer, atherosclerosis, arthritis, psoriasis, endometriosis, obesity and SARS-CoV-2(COVID-19), developmental angiogenic processes are recapitulated, but are often done so without the normal feedback mechanisms that regulate the ordinary spatial and temporal patterns of blood vessel formation. Thus, pathological angiogenesis presents new challenges yet new opportunities for the design of vascular-directed therapies. Here, we provide an overview of recent insights into blood vessel development and highlight novel therapeutic strategies that promote or inhibit the process of angiogenesis to stabilize, reverse, or even halt disease progression. In our review, we will also explore several additional aspects (the angiogenic switch, hypoxia, angiocrine signals, endothelial plasticity, vessel normalization, and endothelial cell anergy) that operate in parallel to canonical angiogenesis mechanisms and speculate how these processes may also be targeted with anti-angiogenic or vascular-directed therapies.

Priming a vascular-selective cytokine response permits CD8+ T-cell entry into tumors.

Targeting DNA methyltransferase 1 (DNMT1) has immunomodulatory and anti-neoplastic activity, especially when paired with cancer immunotherapies. Here we explore the immunoregulatory functions of DNMT1 in the tumor vasculature of female mice. Dnmt1 deletion in endothelial cells (ECs) impairs tumor growth while priming expression of cytokine-driven cell adhesion molecules and chemokines important for CD8+ T-cell trafficking across the vasculature; consequently, the efficacy of immune checkpoint blockade (ICB) is enhanced. We find that the proangiogenic factor FGF2 promotes ERK-mediated DNMT1 phosphorylation and nuclear translocation to repress transcription of the chemokines Cxcl9/Cxcl10 in ECs. Targeting Dnmt1 in ECs reduces proliferation but augments Th1 chemokine production and extravasation of CD8+ T-cells, suggesting DNMT1 programs immunologically anergic tumor vasculature. Our study is in good accord with preclinical observations that pharmacologically disrupting DNMT1 enhances the activity of ICB but suggests an epigenetic pathway presumed to be targeted in cancer cells is also operative in the tumor vasculature.

The rising impact of angiogenesis research.

While inhibiting pathological angiogenesis has been long associated with the field of oncology, recent advances in angiogenesis research have impacted the progress of disease treatment for additional non-malignant diseases or chronic conditions in the fields of ophthalmology, cardiology, and gynecology. Moreover, stimulators of angiogenesis find application in ischemic diseases, while inhibitors of angiogenesis are being used to limit blood vessel formation, but in judicious ways that modify or "reprogram" the vasculature as a reinforcement for immunotherapy. We have noticed an increasing impact, as evidenced by increases in the total number of citations, in the literature surrounding the angiogenesis field suggesting that targeting angiogenesis per se is well established as a tractable approach for therapy in diverse conditions.

Extracellular mechanotransduction.

We highlight the force-sensing function of extracellular matrix and present a complementary mechanotransduction paradigm.

Knee joint unloading and daily physical activity associate with cartilage T2 relaxation times 1 month after ACL injury.

Osteoarthritis (OA) is prevalent after anterior cruciate ligament (ACL) injury, but mechanismsunderlying its development are poorly understood. The purpose of this study was to determine if gait biomechanics and daily physical activity (PA) associate with cartilage T2 relaxation times, a marker of collagen organization and water content, 1 month after ACL injury. Twenty-seven participants (15-35 years old) without chondral lesions completed magnetic resonance imaging, three-dimensional gait analysis, and 1 week of PA accelerometry. Interlimb differences and ratios were calculated for gait biomechanics and T2 relaxation times, respectively. Multiple linear regression models adjusted for age, sex, and concomitant meniscus injury were used to determine the association between gait biomechanics and PA with T2 relaxation times, respectively. Altered knee adduction moment (KAM) impulse, less knee flexion excursion (kEXC) and higher daily step counts accounted for 35.8%-65.8% of T2 relaxation time variation in the weightbearing and posterior cartilage of the medial and lateral compartment (all p ≤ .011). KAM impulse was the strongest factor for T2 relaxation times in all models (all p ≤ .001). Lower KAM impulse associated with longer T2 relaxation times in the injured medial compartment (ß = -.720 to -.901) and shorter T2 relaxation in the lateral compartment (ß = .713 to .956). At 1 month after ACL injury, altered KAM impulse, less kEXC, and higher PA associated with longer T2 relaxation times, which may indicate poorer cartilage health. Statement of Clinical Significance: Gait biomechanics and daily PA are modifiable targets that may improve cartilage health acutely after ACL injury and slow progression to OA.

Dynamic hyaluronic acid hydrogel with covalent linked gelatin as an anti-oxidative bioink for cartilage tissue engineering.

In the past decade, cartilage tissue engineering has arisen as a promising therapeutic option for degenerative joint diseases, such as osteoarthritis, in the hope of restoring the structure and physiological functions. Hydrogels are promising biomaterials for developing engineered scaffolds for cartilage regeneration. However, hydrogel-delivered mesenchymal stem cells or chondrocytes could be exposed to elevated levels of reactive oxygen species (ROS) in the inflammatory microenvironment after being implanted into injured joints, which may affect their phenotype and normal functions and thereby hinder the regeneration efficacy. To attenuate ROS induced side effects, a multifunctional hydrogel with an innate anti-oxidative ability was produced in this study. The hydrogel was rapidly formed through a dynamic covalent bond between phenylboronic acid grafted hyaluronic acid (HA-PBA) and poly(vinyl alcohol) and was further stabilized through a secondary crosslinking between the acrylate moiety on HA-PBA and the free thiol group from thiolated gelatin. The hydrogel is cyto-compatible and injectable and can be used as a bioink for 3D bioprinting. The viscoelastic properties of the hydrogels could be modulated through the hydrogel precursor concentration. The presence of dynamic covalent linkages contributed to its shear-thinning property and thus good printability of the hydrogel, resulting in the fabrication of a porous grid construct and a meniscus like scaffold at high structural fidelity. The bioprinted hydrogel promoted cell adhesion and chondrogenic differentiation of encapsulated rabbit adipose derived mesenchymal stem cells. Meanwhile, the hydrogel supported robust deposition of extracellular matrix components, including glycosaminoglycans and type II collagen, by embedded mouse chondrocytesin vitro. Most importantly, the hydrogel could protect encapsulated chondrocytes from ROS induced downregulation of cartilage-specific anabolic genes (ACAN and COL2) and upregulation of a catabolic gene (MMP13) after incubation with H2O2. Furthermore, intra-articular injection of the hydrogel in mice revealed adequate stability and good biocompatibilityin vivo. These results demonstrate that this hydrogel can be used as a novel bioink for the generation of 3D bioprinted constructs with anti-ROS ability to potentially enhance cartilage tissue regeneration in a chronic inflammatory and elevated ROS microenvironment.

The effects of maturation and aging on the rotator cuff tendon-to-bone interface.

Rotator cuff tendon injuries often occur at the tendon-to-bone interface (i.e., enthesis) area, with a high prevalence for the elderly population, but the underlying reason for this phenomenon is still unknown. The objective of this study is to identify the histological, molecular, and biomechanical alterations of the rotator cuff enthesis with maturation and aging in a mouse model. Four different age groups of mice (newborn, young, adult, and old) were studied. Striking variations of the entheses were observed between the newborn and other matured groups, with collagen content, proteoglycan deposition, collagen fiber dispersion was significantly higher in the newborn group. The compositional and histological features of young, adult, and old groups did not show significant differences, except having increased proteoglycan deposition and thinner collagen fibers at the insertion sites in the old group. Nanoindentation testing showed that the old group had a smaller compressive modulus at the insertion site when compared with other groups. However, tensile mechanical testing reported that the old group demonstrated a significantly higher failure stress when compared with the young and adult groups. The proteomics analysis detected dramatic differences in protein content between newborn and young groups but minor changes among young, adult, and old groups. These results demonstrated: (1) the significant alterations of the enthesis composition and structure occur from the newborn to the young time period; (2) the increased risk of rotator cuff tendon injuries in the elderly population is not solely because of old age alone in the rodent model.

3D printed alginate bead generator for high-throughput cell culture.

Alginate hydrogel beads are a common platform for generating 3D cell cultures in biomedical research. Simple methods for bead generation using a manual pipettor or syringe are low-throughput and produce beads showing high variability in size and shape. To address these challenges, we designed a 3D printed bead generator that uses an airflow to cleave beads from a stream of hydrogel solution. The performance of the proposed alginate bead generator was evaluated by changing the volume flow rates of alginate (QAlg) and air (QA), the diameter of device nozzle (d) and the concentration of alginate gel solution (C). We identified that the diameter of beads (D = 0.9 -2.8 mm) can be precisely controlled by changing QA and d. Also the bead generation frequency (f) can be tuned by changing QAlg. Finally, we demonstrated that viability and biological function (pericellular matrix deposition) of chondrocytes were not adversely affected by high f using this bead generator. Because 3D printing is becoming a more accessible technique, our unique design will allow greater access to average biomedical research laboratories, STEM education and industries in cost- and time-effective manner.

Red panda: a novel method for detecting variants in single-cell RNA sequencing.

BACKGROUND: Single-cell sequencing enables us to better understand genetic diseases, such as cancer or autoimmune disorders, which are often affected by changes in rare cells. Currently, no existing software is aimed at identifying single nucleotide variations or micro (1-50 bp) insertions and deletions in single-cell RNA sequencing (scRNA-seq) data. Generating high-quality variant data is vital to the study of the aforementioned diseases, among others. RESULTS: In this study, we report the design and implementation of Red Panda, a novel method to accurately identify variants in scRNA-seq data. Variants were called on scRNA-seq data from human articular chondrocytes, mouse embryonic fibroblasts (MEFs), and simulated data stemming from the MEF alignments. Red Panda had the highest Positive Predictive Value at 45.0%, while other tools-FreeBayes, GATK HaplotypeCaller, GATK UnifiedGenotyper, Monovar, and Platypus-ranged from 5.8-41.53%. From the simulated data, Red Panda had the highest sensitivity at 72.44%. CONCLUSIONS: We show that our method provides a novel and improved mechanism to identify variants in scRNA-seq as compared to currently existing software. However, methods for identification of genomic variants using scRNA-seq data can be still improved.

Steered molecular dynamic simulations reveal Marfan syndrome mutations disrupt fibrillin-1 cbEGF domain mechanosensitive calcium binding.

Marfan syndrome (MFS) is a highly variable genetic connective tissue disorder caused by mutations in the calcium binding extracellular matrix glycoprotein fibrillin-1. Patients with the most severe form of MFS (neonatal MFS; nMFS) tend to have mutations that cluster in an internal region of fibrillin-1 called the neonatal region. This region is predominantly composed of eight calcium-binding epidermal growth factor-like (cbEGF) domains, each of which binds one calcium ion and is stabilized by three highly conserved disulfide bonds. Crucially, calcium plays a fundamental role in stabilizing cbEGF domains. Perturbed calcium binding caused by cbEGF domain mutations is thus thought to be a central driver of MFS pathophysiology. Using steered molecular dynamics (SMD) simulations, we demonstrate that cbEGF domain calcium binding decreases under mechanical stress (i.e. cbEGF domains are mechanosensitive). We further demonstrate the disulfide bonds in cbEGF domains uniquely orchestrate protein unfolding by showing that MFS disulfide bond mutations markedly disrupt normal mechanosensitive calcium binding dynamics. These results point to a potential mechanosensitive mechanism for fibrillin-1 in regulating extracellular transforming growth factor beta (TGFB) bioavailability and microfibril integrity. Such mechanosensitive "smart" features may represent novel mechanisms for mechanical hemostasis regulation in extracellular matrix that are pathologically activated in MFS.

Reporter mice for isolating and auditing cell type-specific extracellular vesicles in vivo.

Extracellular vesicles (EVs) are abundant, lipid-enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type-specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR-Cas9-mediated genome editing to generate mice bearing a CD63-emGFPloxP/stop/loxP knock-in cassette that enables the specific labeling of circulating CD63+ vesicles from any cell type when crossed with lineage-specific Cre recombinase driver mice. As proof-of-principle, we have crossed these mice with Cdh5-CreERT2 mice to generate CD63emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma-purified EVs contain a subpopulation of emGFP+ vesicles that are derived from the endothelium, co-express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR-126, miR-30c, and miR-125b). This new mouse strain should be a useful genetic tool for generating cell type-specific, CD63+ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.

Models and molecular mechanisms of blood vessel co-option by cancer cells.

Cancer cells have diverse mechanisms for utilizing the vasculature; they can initiate the formation of new blood vessels from preexisting ones (sprouting angiogenesis) or they can form cohesive interactions with the abluminal surface of preexisting vasculature in the absence of sprouting (co-option). The later process has received renewed attention due to the suggested role of blood vessel co-option in resistance to antiangiogenic therapies and the reported perivascular positioning and migratory patterns of cancer cells during tumor dormancy and invasion, respectively. However, only a few molecular mechanisms have been identified that contribute to the process of co-option and there has not been a formal survey of cell lines and laboratory models that can be used to study co-option in different organ microenvironments; thus, we have carried out a comprehensive literature review on this topic and have identified cell lines and described the laboratory models that are used to study blood vessel co-option in cancer. Put into practice, these models may help to shed new light on the molecular mechanisms that drive blood vessel co-option during tumor dormancy, invasion, and responses to different therapies.