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Many cancers metastasize to the bones, particularly in cases of breast and prostate cancers. Due to the "vicious cycle" of cancer cells inducing bone resorption, which promotes further tumor growth, they are difficult to treat and may lead to extreme pain. These factors increase the urgency for emerging therapeutics that target bone metastases more specifically and effectively. Animal studies are essential to the development of any therapeutics, but also require robust animal models of human diseases. Robust animal models are often challenging to develop in the case of bone metastasis studies. Previous methods to induce bone metastasis include intracardiac, intravenous, subcutaneous via mammary fat pad, and intraosseous cancer cell injections, but these methods all have limitations. By contrast, the caudal artery route of injection offers more robust bone metastasis, while also resulting in a lower rate of vital organ metastases than that of other routes of tumor implantation. A syngeneic animal model of bone metastasis is necessary in many cancer studies, because it allows the use of immunocompetent animals, which more accurately mimic cancer development observed in immunocompetent humans. Here we present a detailed method to generate robust and easily monitored 4T1-CLL1 syngeneic bone metastases with over 95% occurrence in BALB/c mice, within two weeks. This method can potentially increase consistency between animals in bone cancer metastasis studies and reduce the number of animals needed for studying bone metastases in mice.

Rheumatoid arthritis (RA) is an inflammatory disease of the joints, which causes severe pain and excessive systemic circulation of harmful inflammatory cytokines. Current treatments are limited, with some patients not responding well, and some experiencing severe and detrimental side effects. Mesenchymal stem cells (MSC) are cell-based therapeutics being evaluated as potent immunomodulators in RA and may provide relief to patients not responding well to drug-based treatments. We evaluated the safety and efficacy of BX-U001 human umbilical cord tissue-derived mesenchymal stem cells (hUC-MSC) to treat RA, in support of a successful investigational new drug application. A collagen-induced arthritis (CIA) mouse model of RA was established in DBA/1 J mice. Mice from the treatment assessment group were given a tail vein infusion of hUC-MSC 24 days after primary RA induction, while control assessment (CA) group mice were given cell-free carrier solution. All animals were evaluated daily for RA symptoms via clinical scoring, blood was taken periodically for cytokine analysis, and mice were dissected at end point for histological analysis. A linear mixed model was used to compare the rate of change among groups. The clinical scores of TA group were significantly reduced compared with CA group (P < 0.01), indicating therapeutic effects. The histological scores of the joints in TA group were significantly lower than those in the CA group (P < 0.05), but had no significant difference compared with Healthy groups (P > 0.05). The concentration of (interleukin) IL-6 in TA group was significantly reduced by 80.0% (P < 0.0001) 2 days after treatment and by 93.4% at the experimental endpoint compared with levels prior to hUC-MSC injection. A single intravenous infusion of hUC-MSC (2 × 106 cells/mouse), to CIA-induced DBA/1 J mice, resulted in significant alleviation of RA symptoms and may provide significant therapeutic benefits in humans.

Background The prevalence of synthetic drug abuse is high. In China, synthetic drugs have become the most commonly abused drugs characterized by strong addiction and great difficulty in quitting. The addicts are easily prone to serious psychological dependence and other psychological problems, which leading to relapse after withdrawal treatment. Therefore, exploring the influencing factors of mental health status in individuals addicted to new synthetic drugs has clinical and social significance for the prevention and management of synthetic drug addicts. Objective To explore the associated factors of mental health status and their intercorrelations in individuals addicted to new synthetic drugs. Methods One hundred and ninety-six people getting addicted to new synthetic drugs with inpatient withdrawal treatment were selected from Ankang Hospital of Tianjin Municipal Public Security Bureau from January 2018 to April 2019. Sociodemographic data and drug abuse-related clinical data were collected. The Symptom Checklist-90 (SCL-90) , Eysenck Personality Questionaire-Revised, Short Scale For Chinese (EPQ-RSC) , and Simplified Coping Style Questionnaire (SCSQ) were used to investigate the mental health. Spearman rank correlation analysis was used to analyze the inercorrelations of the influencing factors of mental health. SPSS 22.0 software was used for statistical analysis, and AMOS 22.0 was used to build a model for path analysis of data. Results The average total score of SCL-90 rated by the participants was (206.62±26.66) , which was higher than that rated by Chinese norm (P

BackgroundThis study aims to evaluate the quality of preclinical data, determine the effect sizes, and identify experimental measures that inform efficacy using mesenchymal stromal (or stem) cells (MSC) therapy in animal models of rheumatoid arthritis (RA).MethodsLiterature searches were performed on MSC preclinical studies to treat RA. MSC treatment effect sizes were determined by the most commonly used outcome measures, including paw thickness, clinical score, and histological score.FindingsA total of 48 studies and 94 treatment arms were included, among which 42 studies and 79 treatment arms reported that MSC improved outcomes. The effect sizes of RA treatments using MSC, when compared to the controls, were: paw thickness was ameliorated by 53.6% (95% confidence interval (CI): 26.7% -80.4%), histological score was decreased by 44.9% (95% CI: 33.3% -56.6%), and clinical score was decreased by 29.9% (95% CI: 16.7% -43.0%). Specifically, our results indicated that human umbilical cord derived MSC led to large improvements of the clinical score (-42.1%) and histological score (-51.4%).InterpretationTo the best of our knowledge, this meta-analysis is to quantitatively answer whether MSC represent a robust RA treatment in animal models. It suggests that in preclinical studies, MSC have consistently exhibited therapeutic benefits. The findings demonstrate a need for considering variations in different animal models and treatment protocols in future studies using MSC to treat RA in humans to maximise the therapeutic gains in the era of precision medicine.FundsNIH [1DP2CA195763], Baylx Inc.: BI-206512, NINDS/NIH Training Grant [Award# NS082174].

BackgroundBone metastases are common and devastating to cancer patients. Existing treatments do not specifically target the disease sites and are therefore ineffective and systemically toxic. Here we present a new strategy to treat bone metastasis by targeting both the cancer cells ("the seed"), and their surrounding niche ("the soil"), using stem cells engineered to home to the bone metastatic niche and to maximise local delivery of multiple therapeutic factors.MethodsWe used mesenchymal stem cells engineered using mRNA to simultaneously express P-selectin glycoprotein ligand-1 (PSGL-1)/Sialyl-Lewis X (SLEX) (homing factors), and modified versions of cytosine deaminase (CD) and osteoprotegerin (OPG) (therapeutic factors) to target and treat breast cancer bone metastases in two mouse models, a xenograft intratibial model and a syngeneic model of spontaneous bone metastasis.FindingsWe first confirmed that MSC engineered using mRNA produced functional proteins (PSGL-1/SLEX, CD and OPG) using various in vitro assays. We then demonstrated that mRNA-engineered MSC exhibit enhanced homing to the bone metastatic niche likely through interactions between PSGL-1/SLEX and P-selectin expressed on tumour vasculature. In both the xenograft intratibial model and syngeneic model of spontaneous bone metastasis, engineered MSC can effectively kill tumour cells and preserve bone integrity. The engineered MSC also exhibited minimal toxicity in vivo, compared to its non-targeted chemotherapy counterpart (5-fluorouracil).InterpretationOur combinatorial targeting of both the cancer cells and the niche represents a simple, safe and effective way to treat metastatic bone diseases, otherwise difficult to manage with existing strategies. It can also be applied to other cell types (e.g., T cells) and cargos (e.g., genome editing components) to treat a broad range of cancer and other complex diseases. FUND: National Institutes of Health, National Cancer Institute of the National Institutes of Health, Department of Defense, California Institute of Regenerative Medicine, National Science Foundation, Baylx Inc., and Fondation ARC pour la recherche sur le cancer.

Probing the biophysical properties of the tumor niche offers a new perspective in cancer mechanobiology, and supports the development of next-generation diagnostics and therapeutics for cancer, in particular for metastasis.

BackgroundMechanical and biophysical properties of the cellular microenvironment regulate cell fate decisions. Mesenchymal stem cell (MSC) fate is influenced by past mechanical dosing (memory), but the mechanisms underlying this process have not yet been well defined. We have yet to understand how memory affects specific cell fate decisions, such as the differentiation of MSCs into neurons, adipocytes, myocytes, and osteoblasts.ResultsWe study a minimal gene regulatory network permissive of multi-lineage MSC differentiation into four cell fates. We present a continuous model that is able to describe the cell fate transitions that occur during differentiation, and analyze its dynamics with tools from multistability, bifurcation, and cell fate landscape analysis, and via stochastic simulation. Whereas experimentally, memory has only been observed during osteogenic differentiation, this model predicts that memory regions can exist for each of the four MSC-derived cell lineages. We can predict the substrate stiffness ranges over which memory drives differentiation; these are directly testable in an experimental setting. Furthermore, we quantitatively predict how substrate stiffness and culture duration co-regulate the fate of a stem cell, and we find that the feedbacks from the differentiating MSC onto its substrate are critical to preserve mechanical memory. Strikingly, we show that re-seeding MSCs onto a sufficiently soft substrate increases the number of cell fates accessible.ConclusionsControl of MSC differentiation is crucial for the success of much-lauded regenerative therapies based on MSCs. We have predicted new memory regions that will directly impact this control, and have quantified the size of the memory region for osteoblasts, as well as the co-regulatory effects on cell fates of substrate stiffness and culture duration. Taken together, these results can be used to develop novel strategies to better control the fates of MSCs in vitro and following transplantation.

Despite decades of effort, little progress has been made to improve the treatment of cancer metastases. To leverage the central role of the mechanoenvironment in cancer metastasis, we present a mechanoresponsive cell system (MRCS) to selectively identify and treat cancer metastases by targeting the specific biophysical cues in the tumor niche in vivo. Our MRCS uses mechanosensitive promoter-driven mesenchymal stem cell (MSC)-based vectors, which selectively home to and target cancer metastases in response to specific mechanical cues to deliver therapeutics to effectively kill cancer cells, as demonstrated in a metastatic breast cancer mouse model. Our data suggest a strong correlation between collagen cross-linking and increased tissue stiffness at the metastatic sites, where our MRCS is specifically activated by the specific cancer-associated mechano-cues. MRCS has markedly reduced deleterious effects compared to MSCs constitutively expressing therapeutics. MRCS indicates that biophysical cues, specifically matrix stiffness, are appealing targets for cancer treatment due to their long persistence in the body (measured in years), making them refractory to the development of resistance to treatment. Our MRCS can serve as a platform for future diagnostics and therapies targeting aberrant tissue stiffness in conditions such as cancer and fibrotic diseases, and it should help to elucidate mechanobiology and reveal what cells "feel" in the microenvironment in vivo.

The delivery of therapeutics to the central nervous system (CNS) remains a major challenge in part due to the presence of the blood-brain barrier (BBB). Recently, cell-derived vesicles, particularly exosomes, have emerged as an attractive vehicle for targeting drugs to the brain, but whether or how they cross the BBB remains unclear. Here, we investigated the interactions between exosomes and brain microvascular endothelial cells (BMECs) in vitro under conditions that mimic the healthy and inflamed BBB in vivo. Transwell assays revealed that luciferase-carrying exosomes can cross a BMEC monolayer under stroke-like, inflamed conditions (TNF-α activated) but not under normal conditions. Confocal microscopy showed that exosomes are internalized by BMECs through endocytosis, co-localize with endosomes, in effect primarily utilizing the transcellular route of crossing. Together, these results indicate that cell-derived exosomes can cross the BBB model under stroke-like conditions in vitro. This study encourages further development of engineered exosomes as drug delivery vehicles or tracking tools for treating or monitoring neurological diseases.

Systemic administration of mesenchymal stem cells (MSCs) affords the potential to ameliorate the symptoms of Multiple Sclerosis (MS) in both preclinical and clinical studies. However, the efficacy of MSC-based therapy for MS likely depends on the number of cells that home to inflamed tissues and on the controlled production of paracrine and immunomodulatory factors. Previously, we reported that engineered MSCs expressing P-selectin glycoprotein ligand-1 (PSGL-1) and Sialyl-Lewis(x) (SLeX) via mRNA transfection facilitated the targeted delivery of anti-inflammatory cytokine interleukin-10 (IL-10) to inflamed ear. Here, we evaluated whether targeted delivery of MSCs with triple PSGL1/SLeX/IL-10 engineering improves therapeutic outcomes in mouse experimental autoimmune encephalomyelitis (EAE), a murine model for human MS. We found PSGL-1/SLeX mRNA transfection significantly enhanced MSC homing to the inflamed spinal cord. This is consistent with results from in vitro flow chamber assays in which PSGL-1/SleX mRNA transfection significantly increased the percentage of rolling and adherent cells on activated brain microvascular endothelial cells, which mimic the inflamed endothelium of blood brain/spinal cord barrier in EAE. In addition, IL-10-transfected MSCs show significant inhibitory activity on the proliferation of CD4(+) T lymphocytes from EAE mice. In vivo treatment with MSCs engineered with PSGL-1/SLeX/IL-10 in EAE mice exhibited a superior therapeutic function over native (unmodified) MSCs, evidenced by significantly improved myelination and decreased lymphocytes infiltration into the white matter of the spinal cord. Our strategy of targeted delivery of performance-enhanced MSCs could potentially be utilized to increase the effectiveness of MSC-based therapy for MS and other central nervous system (CNS) disorders.

IntroductionMesenchymal stem cells (MSCs) are adult multipotent stem cells that possess regenerative and immunomodulatory properties. They have been widely investigated as therapeutic agents for a variety of disease conditions, including tissue repair, inflammation, autoimmunity, and organ transplantation. Importantly, systemically infused MSCs selectively home to primary and metastatic tumors, though the molecular mechanisms of tumor tropism of MSCs remain incompletely understood. We have exploited the active and selective MSCs homing to cancer microenvironments to develop a rapid and selective blood test for the presence of cancer.MethodsWe tested the concept of using transplanted MSCs as the basis for a simple cancer blood test. MSCs were engineered to express humanized Gaussia luciferase (hGluc). In a minimally invasive fashion, hGluc secreted by MSCs into circulation as a reporter for cancer presence, was assayed to probe whether MSCs co-localize with and persist in cancerous tissue.ResultsIn vitro, hGluc secreted by engineered MSCs was detected stably over a period of days in the presence of serum. In vivo imaging showed that MSCs homed to breast cancer lung metastases and persisted longer in tumor-bearing mice than in tumor-free mice (P < 0.05). hGluc activity in blood of tumor-bearing mice was significantly higher than in their tumor-free counterparts (P < 0.05).ConclusionsBoth in vitro and in vivo data show that MSCs expressing hGluc can identify and report small tumors or metastases in a simple blood test format. Our novel and simple stem cell-based blood test can potentially be used to screen, detect, and monitor cancer and metastasis at early stages and during treatment.

Quantification of miRNAs in blood can be potentially used for early disease detection, surveillance monitoring and drug response evaluation. However, quantitative and robust measurement of miRNAs in blood is still a major challenge in large part due to their low concentration and complicated sample preparation processes typically required in conventional assays. Here, we present the 'Integrated Comprehensive Droplet Digital Detection' (IC 3D) system where the plasma sample containing target miRNAs is encapsulated into microdroplets, enzymatically amplified and digitally counted using a novel, high-throughput 3D particle counter. Using Let-7a as a target, we demonstrate that IC 3D can specifically quantify target miRNA directly from blood plasma at extremely low concentrations ranging from 10s to 10 000 copies per mL in ≤3 hours without the need for sample processing such as RNA extraction. Using this new tool, we demonstrate that target miRNA content in colon cancer patient blood is significantly higher than that in healthy donor samples. Our IC 3D system has the potential to introduce a new paradigm for rapid, sensitive and specific detection of low-abundance biomarkers in biological samples with minimal sample processing.

Cell surface proteins are an important class of biomarkers for fundamental biological research and for disease diagnostics and treatment. In this communication, we report a universal strategy to construct sensors that can achieve rapid imaging of cell surface proteins without any separation by using binding-induced dynamic DNA assembly. As a proof-of-principle, we developed a real-time and wash-free sensor for an important breast cancer biomarker, human epidermal growth factor receptor-2 (HER2). We then demonstrated that this sensor could be used for imaging and sensing HER2 on both fixed and live breast cancer cells. Additionally, we have also incorporated toehold-mediated DNA strand displacement reactions into the HER2 sensor, which allows for reiterating (switching on/off) fluorescence signals for HER2 from breast cancer cells in real-time.

Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.

We report a simple, versatile, multivalent ligand system that is capable of specifically and efficiently modulating cell-surface receptor clustering and function. The multivalent ligand is made of a polymeric DNA scaffold decorated with biorecognition ligands (i.e., antibodies) to interrogate and modulate cell receptor signaling and function. Using CD20 clustering-mediated apoptosis in B-cell cancer cells as a model system, we demonstrated that our multivalent ligand is significantly more effective at inducing apoptosis of target cancer cells than its monovalent counterpart. This multivalent DNA material approach represents a new chemical biology tool to interrogate cell receptor signaling and functions and to potentially manipulate such functions for the development of therapeutics.

INTRODUCTION: Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition. METHODS AND RESULTS: Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T→A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells. CONCLUSIONS: We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway.

Systemically infused mesenchymal stem cells (MSCs) are emerging therapeutics for treating stroke, acute injuries, and inflammatory diseases of the central nervous system (CNS), as well as brain tumors due to their regenerative capacity and ability to secrete trophic, immune modulatory, or other engineered therapeutic factors. It is hypothesized that transplanted MSCs home to and engraft at ischemic and injured sites in the brain in order to exert their therapeutic effects. However, whether MSCs possess the ability to migrate across the blood-brain barrier (BBB) that separates the blood from the brain remains unresolved. This review analyzes recent advances in this area in an attempt to elucidate whether systemically infused MSCs are able to actively transmigrate across the CNS endothelium, particularly under conditions of injury or stroke. Understanding the fate of transplanted MSCs and their CNS trafficking mechanisms will facilitate the development of more effective stem-cell-based therapeutics and drug delivery systems to treat neurological diseases and brain tumors.