Magnetically controlled cyclic microscale deformation of in vitro cancer invasion models.

Mechanical cues play an important role in the metastatic cascade of cancer. Three-dimensional (3D) tissue matrices with tunable stiffness have been extensively used as model systems of the tumor microenvironment for physiologically relevant studies. Tumor-associated cells actively deform these matrices, providing mechanical cues to other cancer cells residing in the tissue. Mimicking such dynamic deformation in the surrounding tumor matrix may help clarify the effect of local strain on cancer cell invasion. Remotely controlled microscale magnetic actuation of such 3D in vitro systems is a promising approach, offering a non-invasive means for in situ interrogation. Here, we investigate the influence of cyclic deformation on tumor spheroids embedded in matrices, continuously exerted for days by cell-sized anisotropic magnetic probes, referred to as µRods. Particle velocimetry analysis revealed the spatial extent of matrix deformation produced in response to a magnetic field, which was found to be on the order of 200 µm, resembling strain fields reported to originate from contracting cells. Intracellular calcium influx was observed in response to cyclic actuation, as well as an influence on cancer cell invasion from 3D spheroids, as compared to unactuated controls. Furthermore, RNA sequencing revealed subtle upregulation of certain genes associated with migration and stress, such as induced through mechanical deformation, for spheroids exposed to actuation vs. controls. Localized actuation at one side of a tumor spheroid tended to result in anisotropic invasion toward the µRods causing the deformation. In summary, our approach offers a strategy to test and control the influence of non-invasive micromechanical cues on cancer cell invasion and metastasis.

Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance.

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

Quantum Dot-Based Screening Identifies F3 Peptide and Reveals Cell Surface Nucleolin as a Therapeutic Target for Rhabdomyosarcoma.

Active drug delivery by tumor-targeting peptides is a promising approach to improve existing therapies for rhabdomyosarcoma (RMS), by increasing the therapeutic effect and decreasing the systemic toxicity, e.g., by drug-loaded peptide-targeted nanoparticles. Here, we tested 20 different tumor-targeting peptides for their ability to bind to two RMS cell lines, Rh30 and RD, using quantum dots Streptavidin and biotin-peptides conjugates as a model for nanoparticles. Four peptides revealed a very strong binding to RMS cells: NCAM-1-targeting NTP peptide, nucleolin-targeting F3 peptide, and two Furin-targeting peptides, TmR and shTmR. F3 peptide showed the strongest binding to all RMS cell lines tested, low binding to normal control myoblasts and fibroblasts, and efficient internalization into RMS cells demonstrated by the cytoplasmic delivery of the Saporin toxin. The expression of the nucleophosphoprotein nucleolin, the target of F3, on the surface of RMS cell lines was validated by competition with the natural ligand lactoferrin, by colocalization with the nucleolin-binding aptamer AS1411, and by the marked sensitivity of RMS cell lines to the growth inhibitory nucleolin-binding N6L pseudopeptide. Taken together, our results indicate that nucleolin-targeting by F3 peptide represents a potential therapeutic approach for RMS.

Achilles Tendons Display Region-Specific Transcriptomic Signatures Associated With Distinct Mechanical Properties.

BACKGROUND: Previous studies have examined the transcriptomes and mechanical properties of whole tendons in different regions of the body. However, less is known about these characteristics within a single tendon. PURPOSE: To develop a regional transcriptomic atlas and evaluate the region-specific mechanical properties of Achilles tendons. STUDY DESIGN: Descriptive laboratory study. METHODS: Achilles tendons from 2-month-old male Sprague Dawley rats were used. Tendons were isolated and divided into proximal, middle, and distal thirds for RNA sequencing (n = 5). For mechanical testing, the Achilles muscle-tendon-calcaneus unit was mounted in a custom-designed materials testing system with the unit clamped over the musculotendinous junction (MTJ) and the calcaneus secured at 90° of dorsiflexion (n = 9). Tendons were stretched to 20 N at a constant speed of 0.0167 mm/s. Cross-sectional area, strain, stress, and Young modulus were determined in each tendon region. RESULTS: An open-access, interactive transcriptional atlas was generated that revealed distinct gene expression signatures in each tendon region. The proximal and distal regions had the largest differences in gene expression, with 2596 genes significantly differentially regulated at least 1.5-fold (q < .01). The proximal tendon displayed increased expression of genes resembling a tendon phenotype and increased expression of nerve cell markers. The distal region displayed increases in genes involved in extracellular matrix synthesis and remodeling, immune cell regulation, and a phenotype similar to cartilage and bone. There was a 3.72-fold increase in Young modulus from the proximal to middle region (P < .01) and an additional 1.34-fold increase from the middle to distal region (P = .027). CONCLUSION: Within a single tendon, there are region-specific transcriptomic signatures and mechanical properties, and there is likely a gradient in the biological and functional phenotype from the proximal origin at the MTJ to the distal insertion at the enthesis. CLINICAL RELEVANCE: These findings improve our understanding of the underlying biological heterogeneity of tendon tissue and will help inform the future targeted use of regenerative medicine and tissue engineering strategies for patients with tendon disorders.

Response and resistance to BRAFV600E inhibition in gliomas: Roadblocks ahead?

BRAFV600E represents the most common BRAF mutation in all human cancers. Among central nervous system (CNS) tumors, BRAFV600E is mostly found in pediatric low-grade gliomas (pLGG, ~20%) and, less frequently, in pediatric high-grade gliomas (pHGG, 5-15%) and adult glioblastomas (GBM, ~5%). The integration of BRAF inhibitors (BRAFi) in the treatment of patients with gliomas brought a paradigm shift to clinical care. However, not all patients benefit from treatment due to intrinsic or acquired resistance to BRAF inhibition. Defining predictors of response, as well as developing strategies to prevent resistance to BRAFi and overcome post-BRAFi tumor progression/rebound growth are some of the main challenges at present in the field. In this review, we outline current achievements and limitations of BRAF inhibition in gliomas, with a special focus on potential mechanisms of resistance. We discuss future directions of targeted therapy for BRAFV600E mutated gliomas, highlighting how insights into resistance to BRAFi could be leveraged to improve outcomes.

Cells expressing PAX8 are the main source of homeostatic regeneration of adult mouse endometrial epithelium and give rise to serous endometrial carcinoma.

Humans and mice have cyclical regeneration of the endometrial epithelium. It is expected that such regeneration is ensured by tissue stem cells, but their location and hierarchy remain debatable. A number of recent studies have suggested the presence of stem cells in the mouse endometrial epithelium. At the same time, it has been reported that this tissue can be regenerated by stem cells of stromal/mesenchymal or bone marrow cell origin. Here, we describe a single-cell transcriptomic atlas of the main cell types of the mouse uterus and epithelial subset transcriptome and evaluate the contribution of epithelial cells expressing the transcription factor PAX8 to the homeostatic regeneration and malignant transformation of adult endometrial epithelium. According to lineage tracing, PAX8+ epithelial cells are responsible for long-term maintenance of both luminal and glandular epithelium. Furthermore, multicolor tracing shows that individual glands and contiguous areas of luminal epithelium are formed by clonal cell expansion. Inactivation of the tumor suppressor genes Trp53 and Rb1 in PAX8+ cells, but not in FOXJ1+ cells, leads to the formation of neoplasms with features of serous endometrial carcinoma, one of the most aggressive types of human endometrial malignancies. Taken together, our results show that the progeny of single PAX8+ cells represents the main source of regeneration of the adult endometrial epithelium. They also provide direct experimental genetic evidence for the key roles of the P53 and RB pathways in the pathogenesis of serous endometrial carcinoma and suggest that PAX8+ cells represent the cell of origin of this neoplasm.

Single-cell transcriptomic analysis identifies extensive heterogeneity in the cellular composition of mouse Achilles tendons.

Tendon is a dense connective tissue that stores and transmits forces between muscles and bones. Cellular heterogeneity is increasingly recognized as an important factor in the biological basis of tissue homeostasis and disease, yet little is known about the diversity of cell types that populate tendon. To address this, we determined the heterogeneity of cell populations within mouse Achilles tendons using single-cell RNA sequencing. In assembling a transcriptomic atlas of Achilles tendons, we identified 11 distinct types of cells, including three previously undescribed populations of tendon fibroblasts. Prior studies have indicated that pericytes, which are found in the vasculature of tendons, could serve as a potential source of progenitor cells for adult tendon fibroblasts. Using trajectory inference analysis, we provide additional support for the notion that pericytes are likely to be at least one of the progenitor cell populations for the fibroblasts that compose adult tendons. We also modeled cell-cell interactions and identified previously undescribed ligand-receptor signaling interactions involved in tendon homeostasis. Our novel and interactive tendon atlas highlights previously underappreciated heterogeneity between and within tendon cell populations. The atlas also serves as a resource to further the understanding of tendon extracellular matrix assembly and maintenance and in the design of therapies for tendinopathies.

Musculoskeletal Consequences of COVID-19.

Coronavirus disease 2019 (COVID-19) is an emerging pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the majority of patients who become infected with SARS-CoV-2 are asymptomatic or have mild symptoms, some patients develop severe symptoms that can permanently detract from their quality of life. SARS-CoV-2 is closely related to SARS-CoV-1, which causes severe acute respiratory syndrome (SARS). Both viruses infect the respiratory system, and there are direct and indirect effects of this infection on multiple organ systems, including the musculoskeletal system. Epidemiological data from the SARS pandemic of 2002 to 2004 identified myalgias, muscle dysfunction, osteoporosis, and osteonecrosis as common sequelae in patients with moderate and severe forms of this disease. Early studies have indicated that there is also considerable musculoskeletal dysfunction in some patients with COVID-19, although long-term follow-up studies have not yet been conducted. The purpose of this article was to summarize the known musculoskeletal pathologies in patients with SARS or COVID-19 and to combine this with computational modeling and biochemical signaling studies to predict musculoskeletal cellular targets and long-term consequences of the SARS-CoV-2 infection.

A reference single-cell transcriptomic atlas of human skeletal muscle tissue reveals bifurcated muscle stem cell populations.

Single-cell RNA-sequencing (scRNA-seq) facilitates the unbiased reconstruction of multicellular tissue systems in health and disease. Here, we present a curated scRNA-seq dataset of human muscle samples from 10 adult donors with diverse anatomical locations. We integrated ~ 22,000 single-cell transcriptomes using Scanorama to account for technical and biological variation and resolved 16 distinct populations of muscle-resident cells using unsupervised clustering of the data compendium. These cell populations included muscle stem/progenitor cells (MuSCs), which bifurcated into discrete "quiescent" and "early-activated" MuSC subpopulations. Differential expression analysis identified transcriptional profiles altered in the activated MuSCs including genes associated with aging, obesity, diabetes, and impaired muscle regeneration, as well as long non-coding RNAs previously undescribed in human myogenic cells. Further, we modeled ligand-receptor cell-communication interactions and observed enrichment of the TWEAK-FN14 pathway in activated MuSCs, a characteristic signature of muscle wasting diseases. In contrast, the quiescent MuSCs have enhanced expression of the EGFR receptor, a recognized human MuSC marker. This work provides a new benchmark reference resource to examine human muscle tissue heterogeneity and identify potential targets in MuSC diversity and dysregulation in disease contexts.

Single-Cell Analysis of the Muscle Stem Cell Hierarchy Identifies Heterotypic Communication Signals Involved in Skeletal Muscle Regeneration.

Muscle regeneration relies on the regulation of muscle stem cells (MuSCs) through paracrine signaling interactions. We analyzed muscle regeneration in mice using single-cell RNA sequencing (scRNA-seq) and generated over 34,000 single-cell transcriptomes spanning four time-points. We identified 15 distinct cell types including heterogenous populations of muscle stem and progenitor cells. We resolved a hierarchical map of these myogenic cells by trajectory inference and observed stage-specific regulatory programs within this continuum. Through ligand-receptor interaction analysis, we identified over 100 candidate regeneration-associated paracrine communication pairs between MuSCs and non-myogenic cells. We show that myogenic stem/progenitor cells exhibit heterogeneous expression of multiple Syndecan proteins in cycling myogenic cells, suggesting that Syndecans may coordinate myogenic fate regulation. We performed ligand stimulation in vitro and confirmed that three paracrine factors (FGF2, TGFß1, and RSPO3) regulate myogenic cell proliferation in a Syndecan-dependent manner. Our study provides a scRNA-seq reference resource to investigate cell communication interactions in muscle regeneration.