Bone Endosteal Mimics Regulates Breast Cancer Development and Phenotype.

Bone is a frequent site for metastatic development in various cancer types, including breast cancer, with a grim prognosis due to the distinct bone environment. Despite considerable advances, our understanding of the underlying processes leading to bone metastasis progression remains elusive. Here, we applied a bioactive three-dimensional (3D) model capable of mimicking the endosteal bone microenvironment. MDA-MB-231 and MCF7 breast cancer cells were cultured on the scaffolds, and their behaviors and the effects of the biomaterial on the cells were examined over time. We demonstrated that close interactions between the cells and the biomaterial affect their proliferation rates and the expression of c-Myc, cyclin D, and KI67, leading to cell cycle arrest. Moreover, invasion assays revealed increased invasiveness within this microenvironment. Our findings suggest a dual role for endosteal mimicking signals, influencing cell fate and potentially acting as a double-edged sword, shuttling between cell cycle arrest and more active, aggressive states.

Chemical Antibody Mimics Inhibit Cadherin-Mediated Cell-Cell Adhesion: A Promising Strategy for Cancer Therapy.

One of the most promising strategies to treat cancer is the use of therapeutic antibodies that disrupt cell-cell adhesion mediated by dysregulated cadherins. The principal site where cell-cell adhesion occurs encompasses Trp2 found at the N-terminal region of the protein. Herein, we employed the naturally exposed highly conserved peptide Asp1-Trp2-Val3-Ile4-Pro5-Pro6-Ile7, as epitope to prepare molecularly imprinted polymer nanoparticles (MIP-NPs) to recognize cadherins. Since MIP-NPs target the site responsible for adhesion, they were more potent than commercially available therapeutic antibodies for inhibiting cell-cell adhesion in cell aggregation assays, and for completely disrupting three-dimensional tumor spheroids as well as inhibiting invasion of HeLa cells. These biocompatible supramolecular anti-adhesives may potentially be used as immunotherapeutic or sensitizing agents to enhance antitumor effects of chemotherapy.

Evaluation of biodegradable Zn-1%Mg and Zn-1%Mg-0.5%Ca alloys for biomedical applications.

Increasing interest in biodegradable metals (Mg, Fe, and Zn) as structural materials for orthopedic and cardiovascular applications mainly relates to their promising biocompatibility, mechanical properties and ability to self-remove. However, Mg alloys suffer from excessive corrosion rates associated with premature loss of mechanical integrity and gas embolism risks. Fe based alloys produce voluminous corrosion products that have a detrimental effect on neighboring cells and extracellular matrix. In contrast, Zn does not appear to exhibit a harmful mode of corrosion. Unfortunately, pure zinc possesses insufficient mechanical strength for biomedical structural applications. The present study aimed at examining the potential of two new zinc based alloys, Zn-1%Mg and Zn-1%Mg-0.5%Ca to serve as structural materials for biodegradable implants. This examination was carried out under in vitro conditions, including immersion testing, potentiodynamic polarization analysis, electrochemical impedance spectroscopy (EIS), and stress corrosion cracking (SCC) assessments in terms of slow strain rate testing (SSRT). In order to assess the cytotoxicity of the tested alloys, cell viability was evaluated indirectly using Saos-2 cells. The results demonstrate that both zinc alloys can be considered as potential candidates for biodegradable implants, with a relative advantage to the Zn-1%Mg alloy in terms of its corrosion resistance and SCC performance.

CD44 in Bone Metastasis Development: A Key Player in the Fate Decisions of the Invading Cells?

A participant in key developmental processes, the adhesion glycoprotein CD44 is also expressed in several types of malignancies and can promote metastasis. In addition, the expression of CD44 isoforms in different types of cancer such as prostate and breast cancers may facilitate bone metastases by enhancing tumorigenicity, osteomimicry, cell migration, homing to bone, and anchorage within the bone specialized domains. Moreover, there is evidence that the CD44-ICD fragments in breast cancer cells may promote the cells' osteolytic nature. Yet the mechanisms by which CD44 and its downstream effectors promote the establishment of these cells within the bone are not fully elucidated. In this review, we summarize the current data on the roles played by CD44 in cancer progression and bone metastasis and the possible effects of its interaction with the different components of the bone marrow milieu.

Single-cell mapping reveals several immune subsets associated with liver metastasis of pancreatic ductal adenocarcinoma.

BACKGROUND: Identifying a metastasis-correlated immune cell composition within the tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) will help to develop promising and innovative therapeutic strategies. However, the dynamics of immune cell lineages in the TME of advanced PDAC remains elusive. METHODS: Twenty-six samples from 11 patients (including 11 primary tumor tissues, 10 blood, and 5 lymph nodes) with different stages were used to develop a multiscale immune profile. High-dimensional single-cell analysis with mass cytometry was performed to search for metastasis-correlated immune changes in the microenvironment. The findings were further validated by published single-cell RNA sequencing (scRNA-seq) data and multiplex fluorescent immunohistochemistry. FINDINGS: High-dimensional single-cell profiling revealed that the three immune-relevant sites formed a distinct immune atlas. Interestingly, the PDAC microenvironment with the potential for metastatic spread to the liver was characterized by a decreased proportion of CD103+PD-1+CD39+ T cells with cytotoxic and exhausted functional status and an increased proportion of CD73+ macrophages. Analysis of scRNA-seq data of PDAC further confirmed the identified subsets and revealed strong potential interactions via various ligand-receptor pairs between the identified T subsets and the macrophages. Moreover, stratified patients with different immune compositions correlated with clinical outcomes of PDAC. CONCLUSIONS: Our study uncovered metastasis-correlated immune changes, suggesting that ecosystem-based patient classification in PDAC will facilitate the identification of candidates likely to benefit from immunotherapy. FUNDING: This work was supported by the National Key Research and Development Program of China, the Shanghai International Science and Technology Collaboration Program, the Shanghai Sailing Program, and the Key Laboratory of diagnosis and treatment of severe hepato-pancreatic diseases of Zhejiang Province.

Wnt Signaling in the Development of Bone Metastasis.

Wnt signaling occurs through evolutionarily conserved pathways that affect cellular proliferation and fate decisions during development and tissue maintenance. Alterations in these highly regulated pathways, however, play pivotal roles in various malignancies, promoting cancer initiation, growth and metastasis and the development of drug resistance. The ability of cancer cells to metastasize is the primary cause of cancer mortality. Bone is one of the most frequent sites of metastases that generally arise from breast, prostate, lung, melanoma or kidney cancer. Upon their arrival to the bone, cancer cells can enter a long-term dormancy period, from which they can be reactivated, but can rarely be cured. The activation of Wnt signaling during the bone metastasis process was found to enhance proliferation, induce the epithelial-to-mesenchymal transition, promote the modulation of the extracellular matrix, enhance angiogenesis and immune tolerance and metastasize and thrive in the bone. Due to the complexity of Wnt pathways and of the landscape of this mineralized tissue, Wnt function during metastatic progression within bone is not yet fully understood. Therefore, we believe that a better understanding of these pathways and their roles in the development of bone metastasis could improve our understanding of the disease and may constitute fertile ground for potential therapeutics.

Review old bone, new tricks.

Despite the significant progress made over the past decade with combination of molecular profiling data and the development of new clinical strategies, our understanding of metastasis remains elusive. Bone metastasis is a complex process and a major cause of mortality in breast and prostate cancer patients, for which there is no effective treatment to-date. The current review summarizes the routes taken by the metastatic cells and the interactions between them and the bone microenvironment. We emphasize the role of the specified niches and cues that promote cellular adhesion, colonization, prolonged dormancy, and reactivation. Understanding these mechanisms will provide better insights for future studies and treatment strategies for bone metastatic conditions.

Molecularly imprinted polymer nanogels targeting the HAV motif in cadherins inhibit cell-cell adhesion and migration.

Cadherins are cell-surface proteins that mediate cell-cell adhesion. By regulating their grip formation and strength, cadherins play a pivotal role during normal tissue morphogenesis and homeostasis of multicellular organisms. However, their dysfunction is associated with cell migration and proliferation, cancer progression and metastasis. The conserved amino acid sequence His-Ala-Val (HAV) in the extracellular domain of cadherins is implicated in cadherin-mediated adhesion and migration. Antagonists of cadherin adhesion such as monoclonal antibodies and small molecule inhibitors based on HAV peptides, are of high therapeutic value in cancer treatment. However, antibodies are not stable outside their natural environment and are expensive to produce, while peptides have certain limitations as a drug as they are prone to proteolysis. Herein, we propose as alternative, a synthetic antibody based on molecularly imprinted polymer nanogels (MIP-NGs) to target the HAV domain. The MIP-NGs are biocompatible, have high affinity for N-cadherin and inhibit cell adhesion and migration of human cervical adenocarcinoma (HeLa) cells, as demonstrated by cell aggregation and Matrigel invasion assays, respectively. The emergence of MIPs as therapeutics for fighting cancer is still in its infancy and this novel demonstration reinforces the fact that they have a rightful place in cancer treatment.

Characteristics of the cancer stem cell niche and therapeutic strategies.

Distinct regions harboring cancer stem cells (CSCs) have been identified within the microenvironment of various tumors, and as in the case of their healthy counterparts, these anatomical regions are termed "niche." Thus far, a large volume of studies have shown that CSC niches take part in the maintenance, regulation of renewal, differentiation and plasticity of CSCs. In this review, we summarize and discuss the latest findings regarding CSC niche morphology, physical terrain, main signaling pathways and interactions within them. The cellular and molecular components of CSCs also involve genetic and epigenetic modulations that mediate and support their maintenance, ultimately leading to cancer progression. It suggests that the crosstalk between CSCs and their niche plays an important role regarding therapy resistance and recurrence. In addition, we updated diverse therapeutic strategies in different cancers in basic research and clinical trials in this review. Understanding the complex heterogeneity of CSC niches is a necessary pre-requisite for designing superior therapeutic strategies to target CSC-specific factors and/or components of the CSC niche.

The structural, compositional and mechanical features of the calcite shell of the barnacle Tetraclita rufotincta.

The microstructure and chemical composition of the calcite shell of the sea barnacle Tetraclita rufotincta (Pilsbry, 1916) were investigated using microscopic and analytical methods. The barnacle shell was separated mechanically into its three substructural units: outer, interior, and inner layers. The organic matrices of these structural parts were further separated into soluble and insoluble constituents and their characteristic functional groups were studied by FTIR. Investigation of the mechanical properties of the interior mass of the shell reveals remarkable viscoelastic behavior. In general, the mechanical behavior of the shell is a function of its geometry as well as of the material, of which it is constructed. In the case of T. rufotincta, as calcite is a brittle material, the elastic behavior of the shell is apparently related to its micro- and macroarchitecture. The latter enables the shell to fulfill its primary function which is to protect the organism from a hostile environment and enables its survival. Our detailed identification of the similarities and differences between the various structural components of the shell in regard to the composition and properties of the organic component will hopefully throw light on the role of organic matrices in biomineralization processes.

Evaluating the Prospects of Ti-Base Lattice Infiltrated with Biodegradable Zn-2%Fe Alloy as a Structural Material for Osseointegrated Implants-In Vitro Study.

The term "osseointegrated implants" mainly relates to structural systems that contain open spaces, which enable osteoblasts and connecting tissue to migrate during natural bone growth. Consequently, the coherency and bonding strength between the implant and natural bone can be significantly increased, for example in operations related to dental and orthopedic applications. The present study aims to evaluate the prospects of a Ti-6Al-4V lattice, produced by selective laser melting (SLM) and infiltrated with biodegradable Zn2%Fe alloy, as an OI-TiZn system implant in in vitro conditions. This combined material structure is designated by this study as an osseointegrated implant (OI-TiZn) system. The microstructure of the tested alloys was examined both optically and using scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis. The mechanical properties were assessed in terms of compression strength, as is commonly acceptable in cases of lattice-based structures. The corrosion performance was evaluated by immersion tests and electrochemical analysis in terms of potentiodynamic polarization and electrochemical impedance spectroscopy (EIS), all in simulated physiological environments in the form of phosphate buffered saline (PBS) solution. The cytotoxicity was evaluated in terms of indirect cell viability. The results obtained demonstrate the adequate performance of the OI-TiZn system as a non-cytotoxic structural material that can maintain its mechanical integrity under compression, while presenting acceptable corrosion rate degradation.

Aggressiveness of 4T1 breast cancer cells hampered by Wnt production-2 inhibitor nanoparticles: An in vitro study.

Polymeric nanoparticles may enable delivery of drugs with lower systemic toxicity to solid tumors. Wnt signaling are evolutionary conserved pathways, involved in proliferation and fate decisions. Alterations in Wnt signaling play a pivotal role in various cancer types that promote cancer initiation, growth, metastasis and drug resistance. We designed a new strategy to allow an efficient targeting of both the canonical and the non-canonical Wnt pathways using nanoparticles loaded with inhibitor of Wnt productions-2 (IWP-2). This hydrophobic drug was successfully co-assembled into NPs composed of poly gamma-glutamic acid and a cationic and amphiphilic b-sheet peptide. Aggressive 4T1 breast cancer cells that were treated with IWP-2 loaded NPs gained a significant decrease in tumorigenic capacities attributed to improved IWP solubility, cellular uptake and efficacy.

In vitro behavior of bioactive hybrid implant composed of additively manufactured titanium alloy lattice infiltrated with Mg-based alloy.

We have developed a novel bioactive hybrid metallic implant that integrates the beneficial characteristics of a permanent matrix and a biodegradable substance. Such a combination may generate a material system that evolves into a porous structure within weeks to months following implantation and can be used to form strong interfacial bonding and osseointegration for orthopedic and dental applications. Presently, traditional technologies such as casting, powder metallurgy and plastic forming have limited ability to produce the complex bioactive implant structures that are required in practical applications. The present study aimed to develop an innovative bioactive TiMg (BTiMg) hybrid system using a Ti-lattice (Ti-6Al-4 V) produced by an additive manufacturing (AM) process, in combination with a new Mg-based alloy (Mg-2.4%Nd -0.6%Y -0.3%Zr) as a biodegradable filling material. We evaluated the in-vitro behavior of the BTiMg system in a simulated physiological environment, along with cytotoxicity assessment. The microstructure was evaluated by scanning electron microscopy and X-ray diffraction, mechanical properties were examined in terms of compressive strength, environmental performance analysis was conducted by electrochemical testing using potentiodynamic polarization and impedance spectroscopy (EIS), and cytotoxicity characteristics were assessed by indirect cell viability analysis. The results demonstrated the feasibility to produce geometrically complex implants by AM technology, as well as the strength and non-cytotoxic effects of the BTiMg system. Benefits included a relatively high ultimate compressive strength (UCS) and a high yield point (YP), along with an adequate cell viability response in the range between 70 and 120%.

Characteristics of pre-metastatic niche: the landscape of molecular and cellular pathways.

Metastasis is a major contributor to cancer-associated deaths. It involves complex interactions between primary tumorigenic sites and future metastatic sites. Accumulation studies have revealed that tumour metastasis is not a disorderly spontaneous incident but the climax of a series of sequential and dynamic events including the development of a pre-metastatic niche (PMN) suitable for a subpopulation of tumour cells to colonize and develop into metastases. A deep understanding of the formation, characteristics and function of the PMN is required for developing new therapeutic strategies to treat tumour patients. It is rapidly becoming evident that therapies targeting PMN may be successful in averting tumour metastasis at an early stage. This review highlights the key components and main characteristics of the PMN and describes potential therapeutic strategies, providing a promising foundation for future studies.

Visualization of the ultrastructural interface of cells with the outer and inner-surface of coral skeletons.

Crystalline, porous biomaterials, such as marine invertebrate skeletons, have been widely used for functional reconstruction of human tissues like bone and dental implants. Since in such an abrasive microenvironment adequate cell-material interactions are crucial for a successful treatment, it is of great importance to improve the means to examine these interactions. We developed a method that reveals the ultrastructure of the interface between coral skeletons and cultured neural cells to a higher quality than do traditional methods as it does not include damaging procedures like decalcification or sectioning non-decalcified skeletons. It is rather based on generating two electron opacity distinct Araldite masks, of the skeleton and its surrounding, by polymerizing them to different durations. The contrast created at the border of the two masks outlined the fine and fragile crystals of the coral skeleton's outer and inner surfaces and their contact sites with the cells. The skeleton's internal structure contains a mesh of narrow (few microns wide) and large channel-shaped gaps interrupted by irregular-shaped crystalline material. Neural cells grew on the skeleton surface by stretching between crystal tips, with occasional rearrangements of cytoskeletal fibers located near the anchorage focal adherence points. Cell processes infiltrated the skeleton interior by stretching between inter-surface crystals and by adjusting their volume to the space of the conduits they grew into. The technique advances the study of coral biology and of neural cells-hard biomaterial interaction; it can be applied to other biomaterials and cell types and open new ways for studying tissue development and engineering.

Cnidarians biomineral in tissue engineering: a review.

Biomineralization is the process by which organisms precipitate minerals. Crystals formed in this way are exploited by the organisms for a variety of purposes, including mechanical support and protection of soft tissue. Skeletal precipitation, via millions of years of evolution, has produced a wide variety of architectural configurations and material properties. It is exactly these properties that now attract the attention of researchers searching for new materials for a variety of biomedical applications.

Superior survival and durability of neurons and astrocytes on 3-dimensional aragonite biomatrices.

Current needs of central nervous system therapy urge for the identification of scaffolds supporting the generation and long-term maintenance of healthy and functional neuronal tissue. We compared for the first time the viability of hippocampal neurons and astrocytes grown on conventional 2-dimensional (2D) conditions with that of cells grown on an aragonite bioactive 3-dimensional (3D) scaffold prepared from coralline exoskeleton. Cultures in 3D showed significantly lower mortality rate and higher neurons/astrocytes ratio than 2D cultures. Moreover, whereas cell survival in 2D was arrested in the absence of the supporting substrates poly-D-lysine and laminin, these substrates had negligible effect on the 3D cultures. Furthermore, aragonite matrices supported cell survival and growth under conditions of calcium and nutrients deprivation, whereas in 2D such treatments led to death of all neurons and of almost all astrocytes. To show that the aragonite matrices are permissive for neural cells also in vivo, aragonite matrices having no substrate coating grafted into postnatal rat cortex were invaded by neurons growing on the surface and in multilayer structures resembling those seen in the 3D culture in vitro. Hence, culture of neurons and astrocytes on 3D aragonite coralline matrices is a novel mean for production of stable neuronal tissue, with significant implication to the field of neuronal tissue restoration.

Responses of mesenchymal stem cell to chitosan-coralline composites microstructured using coralline as gas forming agent.

Macroporous composites made of coralline:chitosan with new microstructural features were studied for their scaffolding potential in in vitro bone regeneration. By using different ratios of natural coralline powder, as in situ gas forming agent and reinforcing phase, followed by freeze-drying, scaffolds with controlled porosity and pore structure were prepared and cultured with mesenchymal stem cells (MSCs). Their supportive activity of cellular attachment, proliferation and differentiation were assessed through cell morphology studies, DNA content, alkaline phosphatase (ALP) activity and osteocalcin (OC) release. The coralline scaffolds showed by far the highest evaluation of cell number and ALP activity over all the other chitosan-based scaffolds. They were the only material on which the OC protein was released throughout the study. When used as a component of the chitosan composite scaffolds, these coralline's favourable properties seemed to improve the overall performance of the chitosan. Distinct cell morphology and osteoblastic phenotype expression were observed depending on the coralline-to-chitosan ratios composing the scaffolds. The coralline-chitosan composite scaffolds containing high coralline ratios generally showed higher total cell number, ALP activity and OC protein expression comparing to chitosan scaffolds. The results of this study strongly suggest that coralline:chitosan composite, especially those having a high coralline content, may enhance adhesion, proliferation and osteogenic differentiation of MSCs in comparison with pure chitosan. Coralline:chitosan composites could therefore be used as attractive scaffolds for developing new strategies for in vitro tissue engineering.

Use of natural coralline biomaterials as reinforcing and gas-forming agent for developing novel hybrid biomatrices: microarchitectural and mechanical studies.

This paper describes the first attempt in fabrication of three-dimensional macroporous composites of chitosan and natural coralline material with pore sizes of 300-400 microm, exceeding the upper pore size limit of 250 microm obtained with freeze-dried chitosan-based scaffolds. Natural coral particulates of less than 20 microm, which is mainly composed of calcium carbonate (CaCO3), was simultaneously used as reinforcing phase and gas-forming agent to obtain a structure with large pores and improved mechanical and biological properties. The reaction between the coralline material and the acidic chitosan polymer solvent, which produced carbon dioxide, was rapidly stopped by the subsequent thermally induced phase separation technique, leaving coralline particulates in the polymeric structure. Scaffolds containing five different proportions of coralline material (0, 25, 50, 75, and 100 wt%) were investigated. The coralline-chitosan weight ratio was studied for its effects on the physical properties of the scaffolds. The relation between scaffold microarchitecture and mechanical properties was assessed with scanning electron microscope (SEM), along with micro-CT imaging and compression testing. The scaffolds were used in bone marrow cell culturing experiments to assess the effect of composition on cell behavior through cell-material interaction and morphological observation by SEM. Higher coralline concentration increased the pore wall thickness and favored large pore formation. Varying the coralline particulate to chitosan polymer ratio from 0 to 75 wt% increased the average pore size from 80 microm to 400 microm while the porosity decreased from 91% to 78%. The compressive modulus was improved proportionally with the coralline content, and the 75 wt% composites had a significantly higher modulus than other chitosan-based scaffold groups. More cells were observed on scaffolds with higher coralline content. The cell culture experiments indicated that the scaffolds containing coralline material might have a high cell affinity, since it allowed fast cell attachment and spreading.

Biofabricated marine hydrozoan: a bioactive crystalline material promoting ossification of mesenchymal stem cells.

This study introduces a novel three-dimensional biomatrix obtained from the marine hydrocoral Millepora dichotoma as a scaffold for hard tissue engineering. Millepora dichotoma was biofabricated under field and laboratory conditions. Three-dimensional biomatrices were made in order to convert mesenchymal stem cells (MSCs) to exemplify osteoblastic phenotype. We investigated the effect of the biomatrices on MSCs proliferation and differentiation at 2, 3, 4, 7, 10, 14, 21, 28, and 42 days. Different analyses were made: light microscopy, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), calcium incorporation to newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregate formation (oil red O), collagen type I immunofluorescence, DNA concentrations, alkaline phosphatase (ALP) activity, and osteocalcin concentrations. MSCs seeded on Millepora dichotoma biomatrices showed higher levels of calcium and phosphate incorporation and higher type I collagen levels than did control Porites lutea biomatrices. ALP activity revealed that MSCs seeded on M. dichotoma biomatrices are highly osteogenic compared to those on control biomatrices. The osteocalcin content of MSCs seeded on M. dichotoma remained constant up to 2 weeks before rising to surpass that of seeded P. lutea biomatrices after 28 days. Our study thus showed that M. dichotoma biomatrices enhance the differentiation of MSCs into osteoblast and hence have excellent potential as bioscaffold for hard tissue engineering.