Aspirin Affects MDA-MB-231 Vesicle Production and Their Capacity to Induce Fibroblasts towards a Pro-Invasive State.

Long-term administration of aspirin (ASA, acetylsalicylic acid) in oncogenic patients has been related to a reduction in cancer risk incidence, but its precise mechanism of action is unclear. The activation of cancer-associated fibroblasts (CAFs) is a key element in tumor progression and can be triggered by cancer-derived extracellular vesicles (EVs). Targeting the communication between cancer cells and the surrounding tumor microenvironment (TME) may control cancer progression. Our aim was to investigate the effect of ASA on breast cancer cells, focusing on EV secretion and their effect on the biological properties of CAFs. As a result, ASA was shown to reduce the amount and alter the size distribution of EVs produced by MDA-MB-231 tumor cells. Fibroblasts stimulated with EVs derived from MDA-MB-231 treated with ASA (EV-ASA) showed a lower expression of alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP2) but not fibroblast activation protein (FAP) in respect to the ones stimulated with EVs from untreated breast cancer cells (EV-CTR). Furthermore, invasion assays using a three-dimensional (3D) fibroblast spheroid model showed reduced MDA-MB-231 invasion towards fibroblast spheroids pretreated with EV-ASA as compared to spheroids prepared with EV-CTR-stimulated fibroblasts. This suggests that ASA partially inhibits the ability of tumor EVs to stimulate CAFs to promote cancer invasion. In conclusion, ASA can interfere with tumor communication by reducing EV secretion by breast tumor cells as well as by interfering with their capacity to stimulate fibroblasts to become CAFs.

An association between successful engraftment of osteosarcoma patient-derived xenografts and clinicopathological findings.

Although osteosarcoma is a rare disease, with a global incidence rate estimated at 5.0/million/year, it is the most frequent primary bone sarcoma in children and adolescents. In translational research, the patient-derived xenograft (PDX) model is considered an authentic in vivo model for several types of cancer, as tumorgrafts faithfully retain the biological characteristics of the primary tumors. Our goal was to investigate the association between PDX formation and clinical findings of osteosarcoma patients and the ability of the model to preserve in immunocompromized mice the characteristics of the parental tumor. A fresh sample of the patient tumor obtained from a representative biopsy or from surgical resection was implanted into nude mice. When tumor outgrowths reached ~1,500mm³, fresh PDX fragments were re-transplanted into new hosts. Engraftment in mice was obtained after a latency period of 19-225 days (median 92 days) in 40.54% of the implanted samples. We confirmed the histopathological fidelity between the patient tumor and their respective established PDXs, including the expression of biomarkers. PDX take rate was higher in surgical resection samples, in post-chemotherapy surgical samples and in samples from patients with metastatic disease at presentation. In conclusion, we have shown that the osteosarcoma PDX model reliably recapitulates the morphological aspects of the human disease after serial passage in mice. The observation that more aggressive forms of osteosarcoma, including those with metastatic disease at presentation, have a higher efficiency to generate PDXs provides a promising scenario to address several unanswered issues in clinical oncology.

The Manufacture of GMP-Grade Bone Marrow Stromal Cells with Validated In Vivo Bone-Forming Potential in an Orthopedic Clinical Center in Brazil.

In vitro-expanded bone marrow stromal cells (BMSCs) have long been proposed for the treatment of complex bone-related injuries because of their inherent potential to differentiate into multiple skeletal cell types, modulate inflammatory responses, and support angiogenesis. Although a wide variety of methods have been used to expand BMSCs on a large scale by using good manufacturing practice (GMP), little attention has been paid to whether the expansion procedures indeed allow the maintenance of critical cell characteristics and potency, which are crucial for therapeutic effectiveness. Here, we described standard procedures adopted in our facility for the manufacture of clinical-grade BMSC products with a preserved capacity to generate bone in vivo in compliance with the Brazilian regulatory guidelines for cells intended for use in humans. Bone marrow samples were obtained from trabecular bone. After cell isolation in standard monolayer flasks, BMSC expansion was subsequently performed in two cycles, in 2- and 10-layer cell factories, respectively. The average cell yield per cell factory at passage 1 was of 21.93 ± 12.81 × 106 cells, while at passage 2, it was of 83.05 ± 114.72 × 106 cells. All final cellular products were free from contamination with aerobic/anaerobic pathogens, mycoplasma, and bacterial endotoxins. The expanded BMSCs expressed CD73, CD90, CD105, and CD146 and were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Most importantly, nine out of 10 of the cell products formed bone when transplanted in vivo. These validated procedures will serve as the basis for in-house BMSC manufacturing for use in clinical applications in our center.

PATIENT-DERIVED XENOGRAFTS AS A PRECLINICAL MODEL FOR BONE SARCOMAS.

OBJECTIVE: The purpose of this study was to reproduce a mouse model of bone sarcomas for use in cancer research. METHODS: A fresh sample of the tumor tissue was implanted subcutaneously into nude mice. When the patient-derived xenograft (PDX) reached a volume of 1500 mm3, it was harvested for re-implantation into additional mice. Histology was used to compare the morphological characteristics of different generations of sarcoma xenografts with the primary tumor. RESULTS: Sixteen sarcoma tissue samples were engrafted into nude mice. Nine patients were diagnosed with osteosarcoma, two with chondrosarcoma, two with malignant peripheral nerve sheath tumor, one with synovial sarcoma, one with pleomorphic sarcoma, and one with Ewing's sarcoma. PDX tumors were generated in 11 of the 16 implanted specimens (69% success rate in P1). Six P1 tumors grew sufficiently for transfer into additional mice, producing the P2 generation, and three P2 tumors established the P3 generation. CONCLUSION: PDX tumors generated from bone sarcomas were successfully established in immunodeficient mice and reproduced the characteristics of the primary tumor with a high degree of fidelity. The preclinical PDX model described herein may represent an important tool for translational oncology research and for evaluating therapeutic strategies for bone sarcomas. Level of Evidence I; Experimental study.

OBJETIVO: O propósito deste estudo foi reproduzir em camundongos um modelo de sarcomas ósseos para uso em pesquisa oncológica. MÉTODO: Amostras frescas de tecido tumoral foram implantadas por via subcutânea em camundongos Nude. Quando o xenoenxerto derivado do paciente (PDX) alcançava 1500 mm3, ele era retirado do animal e reimplantado em outros camundongos. Estudos histológicos foram realizados para comparar as características morfológicas de diferentes gerações de xenoenxertos com o tumor primário. RESULTADOS: Dezesseis amostras de tecido sarcomatoso foram enxertadas em camundongos. Nove pacientes foram diagnosticados com osteossarcoma, dois com condrossarcoma, dois com tumor maligno de bainha de nervo periférico, um com sarcoma sinovial, um com sarcoma pleomórfico e um com sarcoma de Ewing. Foram gerados tumores PDX em 11 das 16 amostras enxertadas (taxa de sucesso de 69% em P1). Destes, seis tumores P1 cresceram o suficiente para serem transferidos para outros camundongos, dando origem à geração P2 e três dos tumores P2 estabeleceram a geração P3. CONCLUSÕES: Os tumores PDX de sarcomas ósseos foram estabelecidos com sucesso em camundongos imunodeficientes e reproduziram com alta precisão as características do tumor primário. O modelo pré-clínico de PDX descrito pode representar uma ferramenta importante para a pesquisa oncológica translacional e para avaliar estratégias terapêuticas para sarcomas ósseos. Nível de Evidência I; Estudo experimental.

Patient-derived xenografts as a preclinical model for bone sarcomas / Xenoenxertos derivados de pacientes como modelo pré-clínico de sarcomas ósseos

ABSTRACT Objective: The purpose of this study was to reproduce a mouse model of bone sarcomas for use in cancer research. Methods: A fresh sample of the tumor tissue was implanted subcutaneously into nude mice. When the patient-derived xenograft (PDX) reached a volume of 1500 mm3, it was harvested for re-implantation into additional mice. Histology was used to compare the morphological characteristics of different generations of sarcoma xenografts with the primary tumor. Results: Sixteen sarcoma tissue samples were engrafted into nude mice. Nine patients were diagnosed with osteosarcoma, two with chondrosarcoma, two with malignant peripheral nerve sheath tumor, one with synovial sarcoma, one with pleomorphic sarcoma, and one with Ewing's sarcoma. PDX tumors were generated in 11 of the 16 implanted specimens (69% success rate in P1). Six P1 tumors grew sufficiently for transfer into additional mice, producing the P2 generation, and three P2 tumors established the P3 generation. Conclusion: PDX tumors generated from bone sarcomas were successfully established in immunodeficient mice and reproduced the characteristics of the primary tumor with a high degree of fidelity. The preclinical PDX model described herein may represent an important tool for translational oncology research and for evaluating therapeutic strategies for bone sarcomas. Level of Evidence I; Experimental study.

RESUMO Objetivo: O propósito deste estudo foi reproduzir em camundongos um modelo de sarcomas ósseos para uso em pesquisa oncológica. Método: Amostras frescas de tecido tumoral foram implantadas por via subcutânea em camundongos Nude. Quando o xenoenxerto derivado do paciente (PDX) alcançava 1500 mm3, ele era retirado do animal e reimplantado em outros camundongos. Estudos histológicos foram realizados para comparar as características morfológicas de diferentes gerações de xenoenxertos com o tumor primário. Resultados: Dezesseis amostras de tecido sarcomatoso foram enxertadas em camundongos. Nove pacientes foram diagnosticados com osteossarcoma, dois com condrossarcoma, dois com tumor maligno de bainha de nervo periférico, um com sarcoma sinovial, um com sarcoma pleomórfico e um com sarcoma de Ewing. Foram gerados tumores PDX em 11 das 16 amostras enxertadas (taxa de sucesso de 69% em P1). Destes, seis tumores P1 cresceram o suficiente para serem transferidos para outros camundongos, dando origem à geração P2 e três dos tumores P2 estabeleceram a geração P3. Conclusões: Os tumores PDX de sarcomas ósseos foram estabelecidos com sucesso em camundongos imunodeficientes e reproduziram com alta precisão as características do tumor primário. O modelo pré-clínico de PDX descrito pode representar uma ferramenta importante para a pesquisa oncológica translacional e para avaliar estratégias terapêuticas para sarcomas ósseos. Nível de Evidência I; Estudo experimental.

Biological function of unique sulfated glycosaminoglycans in primitive chordates.

Glycosaminoglycans with unique sulfation patterns have been identified in different species of ascidians (sea squirts), a group of marine invertebrates of the Phylum Chordata, sub-phylum Tunicata (or Urochordata). Oversulfated dermatan sulfate composed of [4-α-L-IdoA-(2-O-SO3)-1 â†’ 3-ß-D-GalNAc(4-OSO3)-1]n repeating disaccharide units is found in the extracellular matrix of several organs, where it seems to interact with collagen fibers. This dermatan sulfate co-localizes with a decorin-like protein, as indicated by immunohistochemical analysis. Low sulfated heparin/heparan sulfate-like glycans composed mainly of [4-α-L-IdoA-(2-OSO3)-1 â†’ 4-α-D-GlcN(SO3)-1 (6-O-SO3)-1]n and [4-α-L-IdoA-(2-O-SO3)-1 â†’ 4-α-D-GlcN(SO3)-1]n have also been described in ascidians. These heparin-like glycans occur in intracellular granules of oocyte assessory cells, named test cells, in circulating basophil-like cells in the hemolymph, and at the basement membrane of different ascidian organs. In this review, we present an overview of the structure, distribution, extracellular and intracellular localization of the sulfated glycosaminoglycans in different species and tissues of ascidians. Considering the phylogenetic position of the subphylum Tunicata in the phylum Chordata, a careful analysis of these data can reveal important information about how these glycans evolved from invertebrate to vertebrate animals.

PS1/γ-Secretase-Mediated Cadherin Cleavage Induces ß-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells.

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of ß-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce ß-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced ß-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling ß-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

Extracellular galectin-3 in tumor progression and metastasis.

Galectin-3, the only chimera galectin found in vertebrates, is one of the best-studied galectins. It is expressed in several cell types and is involved in a broad range of physiological and pathological processes, such as cell adhesion, cell activation and chemoattraction, cell cycle, apoptosis, and cell growth and differentiation. However, this molecule raises special interest due to its role in regulating cancer cell activities. Galectin-3 has high affinity for ß-1,6-N-acetylglucosamine branched glycans, which are formed by the action of the ß1,6-N-acetylglucosaminyltransferase V (Mgat5). Mgat5-related changes in protein/lipid glycosylation on cell surface lead to alterations in the clustering of membrane proteins through lattice formation, resulting in functional advantages for tumor cells. Galectin-3 presence enhances migration and/or invasion of many tumors. Galectin-3-dependent clustering of integrins promotes ligand-induced integrin activation, leading to cell motility. Galectin-3 binding to mucin-1 increases transendothelial invasion, decreasing metastasis-free survival in an experimental metastasis model. Galectin-3 also affects endothelial cell behavior by regulating capillary tube formation. This lectin is found in the tumor stroma, suggesting a role for microenvironmental galectin-3 in tumor progression. Galectin-3 also seems to be involved in the recruitment of tumor-associated macrophages, possibly contributing to angiogenesis and tumor growth. This lectin can be a relevant factor in turning bone marrow in a sanctuary for leukemia cells, favoring resistance to therapy. Finally, galectin-3 seems to play a relevant role in orchestrating distinct cell events in tumor microenvironment and for this reason, it can be considered a target in tumor therapies. In conclusion, this review aims to describe the processes of tumor progression and metastasis involving extracellular galectin-3 and its expression and regulation.