Prominent role of RAB39A-RXRB axis in cancer development and stemness

In this study, we found that RAB39A, a member of the RAS oncogene family, was selectively expressed in cancer cells of different histotypes, by analyzing gene expression in human osteosarcoma cells and the cancer stem cells (CSCs) and by comparing them with normal cells through global transcriptomics and principal component analyses. We further validated RAB39A as a therapeutic target, by silencing its expression. The silencing impaired cancer stemness and spherogenic ability in vitro, as well as tumorigenesis in vivo. RNA-seq analyses in the silenced spheres suggested that RAB39A is associated downstream with RXRB and KLF4. Notably, RXRB expression was inhibited in RAB39A-silenced CSCs. Induced overexpression of RXRB in RAB39A-silenced cells restored spherogenic ability and tumorigenesis, confirming RXRB as a major effector of RAB39A. Quantitative RT-PCR analysis of 3c400 human cancer tissues showed that RAB39A was highly expressed in sarcomas and in malignancies of lymphoid, adrenal and testicular tissues. Our data provide the rationale for targeting of the RAB39A-RXRB axis as a therapy for aggressive cancers

Tumor-activated mesenchymal stromal cells promote osteosarcoma stemness and migratory potential via IL-6 secretion

Osteosarcoma (OS) is an aggressive bone malignancy with a high relapse rate despite combined treatment with surgery and multiagent chemotherapy. As for other cancers, OS-associated microenvironment may contribute to tumor initiation, growth, and metastasis. We consider mesenchymal stromal cells (MSC) as a relevant cellular component of OS microenvironment, and have previously found that the interaction between MSC and tumor cells is bidirectional: tumor cells can modulate their peripheral environment that in turn becomes more favorable to tumor growth through metabolic reprogramming. Here, we determined the effects of MSC on OS stemness and migration, two major features associated with recurrence and chemoresistance. The presence of stromal cells enhanced the number of floating spheres enriched in cancer stem cells (CSC) of the OS cell population. Furthermore, the co-culturing with MSC stimulated the migratory capacity of OS via TGF\u3b21 and IL-6 secretion, and the neutralizing antibody anti-IL-6 impaired this effect. Thus, stromal cells in combination with OS spheres exploit a vicious cycle where the presence of CSC stimulates mesenchymal cytokine secretion, which in turn increases stemness, proliferation, migration, and metastatic potential of CSC, also through the increase of expression of adhesion molecules like ICAM-1. Altogether, our data corroborate the concept that a comprehensive knowledge of the interplay between tumor and stroma that also includes the stem-like fraction of tumor cells is needed to develop novel and effective anti-cancer therapies

FT-IR Spectral Signature of Sensitive and Multidrug-Resistant Osteosarcoma Cell-Derived Extracellular Nanovesicles

Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, the outcome is unsatisfactory, and multidrug resistance (MDR) is a pivotal process in OS treatment failure. OS-derived extracellular vesicles (EVs) promote drug resistance to chemotherapy and target therapy through different mechanisms. The aim of this study was to identify subpopulations of osteosarcoma-EVs by Fourier transform infrared spectroscopy (FT-IR) to define a specific spectral signature for sensitive and multidrug-resistant OS-derived EVs. EVs were isolated from sensitive and MDR OS cells as well as from mesenchymal stem cells by differential centrifugation and ultracentrifugation. EVs size, morphology and protein expression were characterized. FT-IR/ATR of EVs spectra were acquired in the region of 400-4000 cm(-1) (resolution 4 cm(-1), 128 scans). The FT-IR spectra obtained were consistently different in the EVs compared to cells from which they originate. A specific spectral signature, characterized by a shift and a new band (1601 cm(-1)), permitted to clearly distinguish EVs isolated by sensitive and multidrug-resistant OS cells. Our data suggest that FT-IR spectroscopy allows to characterize and define a specific spectral signature for sensitive and MDR OS-derived EVs

Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium.Preprin

Incorporation/Enrichment of 3D Bioprinted Constructs by Biomimetic Nanoparticles: Tuning Printability and Cell Behavior in Bone Models

Reproducing in vitro a model of the bone microenvironment is a current need. Preclinical in vitro screening, drug discovery, as well as pathophysiology studies may benefit from in vitro three-dimensional (3D) bone models, which permit high-throughput screening, low costs, and high reproducibility, overcoming the limitations of the conventional two-dimensional cell cultures. In order to obtain these models, 3D bioprinting offers new perspectives by allowing a combination of advanced techniques and inks. In this context, we propose the use of hydroxyapatite nanoparticles, assimilated to the mineral component of bone, as a route to tune the printability and the characteristics of the scaffold and to guide cell behavior. To this aim, both stoichiometric and Sr-substituted hydroxyapatite nanocrystals are used, so as to obtain different particle shapes and solubility. Our findings show that the nanoparticles have the desired shape and composition and that they can be embedded in the inks without loss of cell viability. Both Sr-containing and stoichiometric hydroxyapatite crystals permit enhancing the printing fidelity of the scaffolds in a particle-dependent fashion and control the swelling behavior and ion release of the scaffolds. Once Saos-2 cells are encapsulated in the scaffolds, high cell viability is detected until late time points, with a good cellular distribution throughout the material. We also show that even minor modifications in the hydroxyapatite particle characteristics result in a significantly different behavior of the scaffolds. This indicates that the use of calcium phosphate nanocrystals and structural ion-substitution is a promising approach to tune the behavior of 3D bioprinted constructs

Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells

The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma.The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma

Bone on-a-chip: a 3D dendritic network in a screening platform for osteocyte-targeted drugs

Age-related musculoskeletal disorders, including osteoporosis, are frequent and associated with long lasting morbidity, in turn significantly impacting on healthcare system sustainability. There is therefore a compelling need to develop reliable preclinical models of disease and drug screening to validate novel drugs possibly on a personalized basis, without the need of in vivo assay. In the context of bone tissue, although the osteocyte (Oc) network is a well-recognized therapeutic target, current in vitro preclinical models are unable to mimic its physiologically relevant and highly complex structure. To this purpose, several features are needed, including an osteomimetic extracellular matrix, dynamic perfusion, and mechanical cues (e.g. shear stress) combined with a three-dimensional (3D) culture of Oc. Here we describe, for the first time, a high throughput microfluidic platform based on 96-miniaturized chips for large-scale preclinical evaluation to predict drug efficacy. We bioengineered a commercial microfluidic device that allows real-time visualization and equipped with multi-chips by the development and injection of a highly stiff bone-like 3D matrix, made of a blend of collagen-enriched natural hydrogels loaded with hydroxyapatite nanocrystals. The microchannel, filled with the ostemimetic matrix and Oc, is subjected to passive perfusion and shear stress. We used scanning electron microscopy for preliminary material characterization. Confocal microscopy and fluorescent microbeads were used after material injection into the microchannels to detect volume changes and the distribution of cell-sized objects within the hydrogel. The formation of a 3D dendritic network of Oc was monitored by measuring cell viability, evaluating phenotyping markers (connexin43, integrin alpha V/CD51, sclerostin), quantification of dendrites, and responsiveness to an anabolic drug. The platform is expected to accelerate the development of new drug aimed at modulating the survival and function of osteocytes

Immunohistochemical evaluation of bone metastases

  Introduction. Metastases are the most common type of malignancy involving the bone, while bone is the third most frequent site for metastases, after the lung and liver. In some patients, medical history, physical and laboratory exami­nation are not conclusive to identify the primary tumor site. In such cases a bone biopsy and immunohistochemical analysis may contribute to the diagnosis, determination of appropriate treatment and evaluation of prognosis. In this study, we tried to evaluate the imunochistochemical expression in bone metastases. Material and methods. We reviewed 125 patients, with a mean age of 63 years, treated for bone metastases in our institution. All patients received palliative orthopaedic surgery for bone metastatic carcinoma. Fifty-eight patients had already an established diagnosis of the primary tumor, while 67 patients presented metastases with an unknown primary tumor origin. Immunohistochemical analysis was performed to intra-operative bone biopsy specimens. The expression of cytokeratine 7, cytokeratin 20 and the expression of a panel of other organ-specific markers were re­corded. In patients with a known primary tumor, we examined the relationship between the origin of metastases, as suggested by the cytokeratin phenotype, compared with the one indicated by the initial histological diagnosis. We also recorded the efficacy of organ-specific markers to identify the primary tumor origin in epithelial bone metastases and we evaluated the prognosis between patients with a immunohistologically determined primary tumor origin, with those with an undetermined one. Results. Associations of cytokeratine 7 and cytokeratine 20 expression confirmed diagnosis in 51 out of the 58 patients (88%) with a known primary tumor (Cohen’s K test 0.79 SE 0.80, P < 0.0005). Immunohistochemical analysis also contributed to establish the diagnosis of patients with an unknown primary tumor, yielding diagnosis in 35 out of the 67 cases (52%). Patients with an immunochistologically undetermined primary tumor site presented a statisti­cally significant poorer prognosis. Conclusions. Cytokeratine 7 and cytokeratine20 are useful immunochistochemical markers in determining a pre­liminary evaluation of bone metastases. Organ-specific immunohistochemical markers have a reliable role in either suggesting or confirming the possible origin of metastases. An indeterminate immunohistochemical phenotype seems to relate to a less differentiated lesion, with a worse prognosis

Immunohistochemical evaluation of bone metastases

  Introduction. Metastases are the most common type of malignancy involving the bone, while bone is the third most frequent site for metastases, after the lung and liver. In some patients, previous medical history, physical and laboratory examination are not conclusive to identify the primary tumor site. In such cases a bone biopsy and im­munohistochemical analysis may contribute to the diagnosis, determination of appropriate treatment and evaluation of prognosis. In this study, we tried to evaluate the imunochistochemical expression in bone metastases. Material and methods. We reviewed 125 patients, with a mean age of 63 years, treated for bone metastases in our institution. All patients received palliative orthopaedic surgery for bone metastatic carcinoma. Fifty-eight patients had already an established diagnosis of the primary tumor, while 67 patients presented metastases with an unknown primary tumor origin. Immunohistochemical analysis was performed to intra-operative bone biopsy specimens. The expression of cytokeratine 7, cytokeratin 20 and the expression of a panel of other organ-specific markers were re­corded. In patients with a known primary tumor, we examined the relationship between the origin of metastases, as suggested by the cytokeratin phenotype, compared with the one indicated by the initial histological diagnosis. We also recorded the efficacy of organ-specific markers to identify the primary tumor origin in epithelial bone metastases and we evaluated the prognosis between patients with a immunohistologically determined primary tumor origin, with those with an undetermined one. Results. Associations of cytokeratine 7 and cytokeratine 20 expression confirmed diagnosis in 51 out of the 58 patients (88%) with a known primary tumor (Cohen’s K test 0.79 SE 0.80, P < 0.0005). Immunohistochemical analysis also contributed to establish the diagnosis of patients with an unknown primary tumor, yielding diagnosis in 35 out of the 67 cases (52%). Patients with an immunochistologically undetermined primary tumor site presented a statisti­cally significant poorer prognosis. Conclusions. Cytokeratine 7 and cytokeratine20 are useful immunochistochemical markers in determining a pre­liminary evaluation of bone metastases. Organ-specific immunohistochemical markers have a reliable role in either suggesting or confirming the possible origin of metastases. An indeterminate immunohistochemical phenotype seems to relate to a less differentiated lesion, with a worse prognosis. Introduction. Metastases are the most common type of malignancy involving the bone, while bone is the third most frequent site for metastases, after the lung and liver. In some patients, previous medical history, physical and laboratory examination are not conclusive to identify the primary tumor site. In such cases a bone biopsy and im­munohistochemical analysis may contribute to the diagnosis, determination of appropriate treatment and evaluation of prognosis. In this study, we tried to evaluate the imunochistochemical expression in bone metastases. Material and methods. We reviewed 125 patients, with a mean age of 63 years, treated for bone metastases in our institution. All patients received palliative orthopaedic surgery for bone metastatic carcinoma. Fifty-eight patients had already an established diagnosis of the primary tumor, while 67 patients presented metastases with an unknown primary tumor origin. Immunohistochemical analysis was performed to intra-operative bone biopsy specimens. The expression of cytokeratine 7, cytokeratin 20 and the expression of a panel of other organ-specific markers were re­corded. In patients with a known primary tumor, we examined the relationship between the origin of metastases, as suggested by the cytokeratin phenotype, compared with the one indicated by the initial histological diagnosis. We also recorded the efficacy of organ-specific markers to identify the primary tumor origin in epithelial bone metastases and we evaluated the prognosis between patients with a immunohistologically determined primary tumor origin, with those with an undetermined one. Results. Associations of cytokeratine 7 and cytokeratine 20 expression confirmed diagnosis in 51 out of the 58 patients (88%) with a known primary tumor (Cohen’s K test 0.79 SE 0.80, P < 0.0005). Immunohistochemical analysis also contributed to establish the diagnosis of patients with an unknown primary tumor, yielding diagnosis in 35 out of the 67 cases (52%). Patients with an immunochistologically undetermined primary tumor site presented a statisti­cally significant poorer prognosis. Conclusions. Cytokeratine 7 and cytokeratine20 are useful immunochistochemical markers in determining a pre­liminary evaluation of bone metastases. Organ-specific immunohistochemical markers have a reliable role in either suggesting or confirming the possible origin of metastases. An indeterminate immunohistochemical phenotype seems to relate to a less differentiated lesion, with a worse prognosis

Cancer-associated mesenchymal stroma fosters the stemness of osteosarcoma cells in response to intratumoral acidosis via NF-κB activation

The role of mesenchymal stem cells (MSC) in osteosarcoma (OS), the most common primary tumor of bone, has not been extensively elucidated. We have recently shown that OS is characterized by interstitial acidosis, a microenvironmental condition that is similar to a wound setting, in which mesenchymal reactive cells are activated to release mitogenic and chemotactic factors. We therefore intended to test the hypothesis that, in OS, acid-activated MSC influence tumor cell behavior. Conditioned media or co-culture with normal MSC previously incubated with short-term acidosis (pH 6.8 for 10 hr, H+-MSC) enhanced OS clonogenicity and invasion. This effect was mediated by NF-κB pathway activation. In fact, deep-sequencing analysis, confirmed by Real-Time PCR and ELISA, demonstrated that H+-MSC differentially induced a tissue remodeling phenotype with increased expression of RelA, RelB and NF-κB1, and downstream, of CSF2/GM-CSF, CSF3/G-CSF and BMP2 colony-promoting factors, and of chemokines (CCL5, CXCL5 and CXCL1), and cytokines (IL6 and IL8), with an increased expression of CXCR4. An increased expression of IL6 and IL8 were found only in normal stromal cells, but not in OS cells, and this was confirmed in tumor-associated stromal cells isolated from OS tissue. Finally, H+-MSC conditioned medium differentially promoted OS stemness (sarcosphere number, stem-associated gene expression), and chemoresistance also via IL6 secretion. Our data support the hypothesis that the acidic OS microenvironment is a key factor for MSC activation, in turn promoting the secretion of paracrine factors that influence tumor behavior, a mechanism that holds the potential for future therapeutic interventions aimed to target OS