Extracellular Vesicles from Highly Metastatic Osteosarcoma Cells Induce Pro-Tumorigenic Macrophage Phenotypes.

Metastasis is the principal factor in poor prognosis for individuals with osteosarcoma (OS). Understanding the events that lead to metastasis is critical to develop better interventions for this disease. Alveolar macrophages are potentially involved in priming the lung microenvironment for OS metastasis, yet the mechanisms involved in this process remain unclear. Since extracellular vesicles (EVs) are a known actor in primary tumor development, their potential role in OS metastagenesis through macrophage modulation is explored here. The interaction of EVs isolated from highly metastatic (K7M2) and less metastatic (K12) osteosarcoma cell lines is compared with a peritoneal macrophage cell line. An EV concentration that reproducibly induced macrophage migration is identified first, then used for later experiments. By confocal microscopy, both EV types associated with M0 or M1 macrophages; however, only K7M2-EVs are associated with M2 macrophages, an interaction that is abrogated by EV pre-treatment with anti-CD47 antibody. Interestingly, all interactions appeared to be surface binding, not internalized. In functional studies, K7M2-EVs polarized fewer macrophages to M1. Together, these data suggest that K7M2-EVs have unique interactions with macrophages that can contribute to the production of a higher proportion of pro-tumor type macrophages, thereby accelerating metastasis.

Engineered bone marrow as a clinically relevant ex vivo model for primary bone cancer research and drug screening.

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances, treatment options for OS have remained unchanged since the advent of standard chemotherapeutics and offer less than a 25% 5-y survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and necessitates the study of this disease in an innovative system. Here, we adapt a previously described engineered bone marrow (eBM) construct for use as a three-dimensional platform to study how microenvironmental and immune factors affect OS tumor progression. We form eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs after 8 wk for study. We interrogate the influence of the anatomical implantation site on eBM tissue quality, test ex vivo stability under normoxic (5% O2) and standard (21% O2) culture conditions, culture OS cells within these constructs, and compare them to human OS samples. We show that eBM stably recapitulates the composition of native bone marrow. OS cells exhibit differential behavior dependent on metastatic potential when cultured in eBM, thus mimicking in vivo conditions. Furthermore, we highlight the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment and show that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that is useful for mechanistic bone cancer research and drug screening.

Mesenchymal stromal cell spheroids in sulfated alginate enhance muscle regeneration.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is critically linked to the potency of the complex mixture of growth factors, cytokines, exosomes, and other biological cues that they secrete. The duration of cell-based approaches is limited by rapid loss of cells upon implantation, motivating the need to prolong cell viability and extend the therapeutic influence of the secretome. We and others demonstrated that the secretome is upregulated when MSCs are formed into spheroids. Although the efficacy of the MSC secretome has been characterized in the literature, no studies have reported the therapeutic benefit of in situ sequestration of the secretome within a wound site using engineered biomaterials. We previously demonstrated the capacity of sulfated alginate hydrogels to sequester components of the MSC secretome for prolonged presentation in vitro, yet the efficacy of this platform has not been evaluated in vivo. In this study, we used sulfated alginate hydrogels loaded with MSC spheroids to aid in the regeneration of a rat muscle crush injury. We hypothesized that the use of sulfated alginate to bind therapeutically relevant growth factors from the MSC spheroid secretome would enhance muscle regeneration by recruiting host cells into the tissue site. The combination of sulfated alginate and MSC spheroids resulted in decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions 2 weeks after injury. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreased fibrosis and increased functional regeneration of muscle. STATEMENT OF SIGNIFICANCE: The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is attributed to the complex diversity of the secretome. Cell-based approaches are limited by rapid cell death, motivating the need to extend the availability of the secretome. We previously demonstrated that sulfated alginate hydrogels sequester components of the MSC secretome for prolonged presentation in vitro, yet no studies have reported the in situ sequestration of the secretome. Herein, we transplanted MSC spheroids in sulfated alginate hydrogels to promote muscle regeneration. MSC spheroids in sulfated alginate decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreasing fibrosis and increasing functional muscle regeneration.

Sulfated Alginate Hydrogels Prolong the Therapeutic Potential of MSC Spheroids by Sequestering the Secretome.

Cell-based approaches to tissue repair suffer from rapid cell death upon implantation, limiting the window for therapeutic intervention. Despite robust lineage-specific differentiation potential in vitro, the function of transplanted mesenchymal stromal cells (MSCs) in vivo is largely attributed to their potent secretome comprising a variety of growth factors (GFs). Furthermore, GF secretion is markedly increased when MSCs are formed into spheroids. Native GFs are sequestered within the extracellular matrix (ECM) via sulfated glycosaminoglycans, increasing the potency of GF signaling compared to their unbound form. To address the critical need to prolong the efficacy of transplanted cells, alginate hydrogels are modified with sulfate groups to sequester endogenous heparin-binding GFs secreted by MSC spheroids. The influence of crosslinking method and alginate modification is assessed on mechanical properties, degradation rate, and degree of sulfate modification. Sulfated alginate hydrogels sequester a mixture of MSC-secreted endogenous biomolecules, thereby prolonging the therapeutic effect of MSC spheroids for tissue regeneration. GFs are sequestered for longer durations within sulfated hydrogels and retain their bioactivity to regulate endothelial cell tubulogenesis and myoblast infiltration. This platform has the potential to prolong the therapeutic benefit of the MSC secretome and serve as a valuable tool for investigating GF sequestration.

Hydrogel mechanics are a key driver of bone formation by mesenchymal stromal cell spheroids.

Mesenchymal stromal cells (MSCs) can promote tissue repair in regenerative medicine, and their therapeutic potential is further enhanced via spheroid formation. Stress relaxation of hydrogels has emerged as a potent stimulus to enhance MSC spreading and osteogenic differentiation, but the effect of hydrogel viscoelasticity on MSC spheroids has not been reported. Herein, we describe a materials-based approach to augment the osteogenic potential of entrapped MSC spheroids by leveraging the mechanical properties of alginate hydrogels. Compared to spheroids entrapped in covalently crosslinked elastic alginate, calcium deposition of MSC spheroids was consistently increased in ionically crosslinked, viscoelastic hydrogels. We previously demonstrated that intraspheroidal presentation of Bone Morphogenetic Protein-2 (BMP-2) on hydroxyapatite (HA) nanoparticles resulted in more spatially uniform MSC osteodifferentiation, providing a method to internally influence spheroid phenotype. In these studies, we observed significant increases in calcium deposition by MSC spheroids loaded with BMP-2-HA in viscoelastic gels compared to soluble BMP-2, which was greater than spheroids entrapped in all elastic alginate gels. Upon implantation in critically sized calvarial bone defects, bone formation was greater in all animals treated with viscoelastic hydrogels. Increases in bone formation were evident in viscoelastic gels, regardless of the mode of presentation of BMP-2 (i.e., soluble delivery or HA nanoparticles). These studies demonstrate that the dynamic mechanical properties of viscoelastic alginate are an effective strategy to enhance the therapeutic potential of MSC spheroids for bone formation and repair.

Tissue engineered platforms for studying primary and metastatic neoplasm behavior in bone.

Cancer is the second leading cause of death in the United States, claiming more than 560,000 lives each year. Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and young adults, while bone is a common site of metastasis for tumors initiating from other tissues. The heterogeneity, continual evolution, and complexity of this disease at different stages of tumor progression drives a critical need for physiologically relevant models that capture the dynamic cancer microenvironment and advance chemotherapy techniques. Monolayer cultures have been favored for cell-based research for decades due to their simplicity and scalability. However, the nature of these models makes it impossible to fully describe the biomechanical and biochemical cues present in 3-dimensional (3D) microenvironments, such as ECM stiffness, degradability, surface topography, and adhesivity. Biomaterials have emerged as valuable tools to model the behavior of various cancers by creating highly tunable 3D systems for studying neoplasm behavior, screening chemotherapeutic drugs, and developing novel treatment delivery techniques. This review highlights the recent application of biomaterials toward the development of tumor models, details methods for their tunability, and discusses the clinical and therapeutic applications of these systems.