New mechanisms involved in the pathogenesis of osteosarcoma

Osteosarcoma is the most common and most often fatal primary bone tumour, especially affecting children and adolescents. It is a highly aggressive tumor that develops mainly in the long bones and metastasizes primarily to the lung. Osteosarcoma cells are known to establish a crosstalk with resident bone cells leading to a deleterious vicious cycle. In this context, we hypothesized that osteosarcoma cells can release in the bone microenvironment transforming extracellular vesicles (EVs) involved in regulating the bone cell proliferation and differentiation, thereby promoting the tumour growth. EVs are small, intact and heterogeneous membrane vesicles that exchange nucleic acids and proteins between cells, inducing phenotypic, biochemical and genetic changes in target cells. Aim of this thesis is to investigate the role of extracellular vesicles as mediators between cancer cells and resident bone cells. We used human osteosarcoma cell lines to set protocols aimed at isolating, visualizing and characterizing EVs. Our results showed that osteosarcoma cell lines produce EVs that are able to induce tumour phenotype in recipient murine fibroblasts NIH3T3, as already described for other cancer cell-derived EVs [1,2]. Indeed, in so treated NIH3T3, we observed an enhanced survival capability under low-serum conditions, high levels of activated survival pathways, an increased migration, the acquired capability to grow in an anchorage-independent manner and a de novo expression of osteoblastic and tumorigenic markers. As regarding bone cells, we used as target cells human osteoblasts, their precursors, mesenchymal stromal cells, and human monocytes as osteoclast precursors. The treatment with osteosarcoma-derived EVs induced an increase of MSC differentiation into mature ALP+ and Alizarin red+ cells. EVs derived from osteosarcoma cells promote the migration and the anchorage-independent growth in normal osteoblasts suggesting a transformation toward a tumour-like phenotype. Finally in monocytes, treated with osteosarcoma EVs, we observed an increased proliferation and the formation of TRAP+ multinucleated osteoclasts, because of presence in EVs of cytokines involved in osteoclast activation and differentiation. These findings highlight the key role of EVs in the crosstalk between the osteosarcoma and bone microenvironment cells, suggesting that further investigation are needed to better define this new field of research. [1] Antonyak MA, et al. Proc Natl Acad Sci U S A. (2011)4852-7 [2] Li B, et al. Oncogene. (2012) 4740-

A Principal component analysis to detect cancer cell line aggressiveness

In this paper, we propose the use of Principal Component Analysis (PCA) as a new post-processing method for the detection of breast and bone cancer cell lines cultured in vitro using a microwave biosensor. MDA-MB-231 and MCF7 breast cancer cell lines and SaOS-2 and 143B osteosarcoma cell lines were characterized using a circular patch resonator in the 1 MHz – 3 GHz frequency range. The return loss of each cancer cell line was analyzed, and the differences among each other were determined through Principal Component Analysis according to a protocol previously proposed mainly for electrocardiogram processing and X-ray photoelectron spectroscopy. Our results showed that the four cancer cell lines analyzed exhibited peculiar dielectric properties when compared to each other and the growth medium, confirming that PCA could be employed as an alternative methodology to analyze microwave characterization of cancer cell lines which, in turn, may be deeply exploited as a tool for the detection of cancer cells in healthy tissues

Nuclear Lamins and Emerin Are Differentially Expressed in Osteosarcoma Cells and Scale with Tumor Aggressiveness

The nuclear lamina is essential for the maintenance of nuclear shape and mechanics. Mutations in lamin genes have been identified in a heterogeneous spectrum of human diseases known as “laminopathies” associated with nuclear envelope defects and deregulation of cellular functions. Interestingly, osteosarcoma is the only neoplasm described in the literature in association with laminopathies. This study aims characterized the expression of A-type and B-type lamins and emerin in osteosarcoma, revealing a higher percentage of dysmorphic nuclei in osteosarcoma cells in comparison to normal osteoblasts and all the hallmarks of laminopathic features. Both lamins and emerin were differentially expressed in osteosarcoma cell lines in comparison to normal osteoblasts and correlated with tumor aggressiveness. We analysed lamin A/C expression in a tissue-microarray including osteosarcoma samples with different prognosis, finding a positive correlation between lamin A/C expression and the overall survival of osteosarcoma patients. An inefficient MKL1 nuclear shuttling and actin depolymerization, as well as a reduced expression of pRb and a decreased YAP nuclear content were observed in A-type lamin deficient 143B cells. In conclusion, we described for the first time laminopathic nuclear phenotypes in osteosarcoma cells, providing evidence for an altered lamins and emerin expression and a deregulated nucleoskeleton architecture of this tumor

The Paradox of Nuclear Lamins in Pathologies: Apparently Controversial Roles Explained by Tissue-Specific Mechanobiology

The nuclear lamina is a complex meshwork of intermediate filaments (lamins) that is located beneath the inner nuclear membrane and the surrounding nucleoplasm. The lamins exert both structural and functional roles in the nucleus and, by interacting with several nuclear proteins, are involved in a wide range of nuclear and cellular activities. Due their pivotal roles in basic cellular processes, lamin gene mutations, or modulations in lamin expression, are often associated with pathological conditions, ranging from rare genetic diseases, such as laminopathies, to cancer. Although a substantial amount of literature describes the effects that are mediated by the deregulation of nuclear lamins, some apparently controversial results have been reported, which may appear to conflict with each other. In this context, we herein provide our explanation of such “controversy”, which, in our opinion, derives from the tissue-specific expression of nuclear lamins and their close correlation with mechanotransduction processes, which could be very different, or even opposite, depending on the specific mechanical conditions that should not be compared (a tissue vs. another tissue, in vivo studies vs. cell cultures on glass/plastic supports, etc.). Moreover, we have stressed the relevance of considering and reproducing the “mechano-environment” in in vitro experimentation. Indeed, when primary cells that are collected from patients or donors are maintained in a culture, the mechanical signals deriving from canonical experimental procedures of cell culturing could alter the lamin expression, thereby profoundly modifying the assessed cell type, in some cases even too much, compared to the cell of origin

Involvement of the FAK Network in Pathologies Related to Altered Mechanotransduction

Mechanotransduction is a physiological process in which external mechanical stimulations are perceived, interpreted, and translated by cells into biochemical signals. Mechanical stimulations exerted by extracellular matrix stiffness and cell–cell contacts are continuously applied to living cells, thus representing a key pivotal trigger for cell homeostasis, survival, and function, as well as an essential factor for proper organ development and metabolism. Indeed, a deregulation of the mechanotransduction process consequent to gene mutations or altered functions of proteins involved in perceiving cellular and extracellular mechanics can lead to a broad range of diseases, from muscular dystrophies and cardiomyopathies to cancer development and metastatization. Here, we recapitulate the involvement of focal adhesion kinase (FAK) in the cellular conditions deriving from altered mechanotransduction processes

The measurement of global shortening as a new parameter to evaluate bone specimen response to uniaxial loading

Mechanical load is nowadays considered one of the factor mainly affecting bone tissue properties, both as architecture and functionality. Mechanotransduction is the capability of cells to translate mechanical stresses into biochemical signals, and several studies performed on mouse models demonstrated that also bone cells show a high responsiveness to mechanical stimuli. To date, bone cells mechanotransduction is mainly investigated in animal models, by the use of organ cultures or directly in vivo, and the actual strains induced by the external loads are measured through the use of micro strain gauges placed on the tibia mid-diaphysis. With the aim of proposing a new parameter to come along with the measurement of the actual strains, we exploited the capability of tibial global shortening to return useful information. We employed an experimental system based on a dual mode actuator/transducer with an adequate force range and a high length resolution to retrieve the small shortening of the bone specimens subjected to uniaxially load. Preliminary results showed that the tibia global shortening has a linear relationship with the increasing load, in the range of force usually used in these studies. In addition, the tibia global shortening showed the capability of gathering the changes occurring in the bone tissue mechanical properties when subjecting the specimens to loading signals of different frequencies. When tested with load signals of a frequency equal or higher than 1 Hz, in fact, the bone specimens showed a more rigid behavior. At 9 N of load, for example, the average value of tibia global shortening measured at 0.1 Hz is, on average, 18 % higher than when measured at all the other tested frequencies

Development and mechanical validation of an in vitro system for bone cell vibration loading

Vibration loading, both low magnitude and high magnitude at high frequency, has been demonstrated to have an anabolic effect on bone cells. The study of the mechanotransduction, the process by which mechanical loadings are detected by cells and converted in a chemical signal, is made accessible through the use of in vitro loading system. The aim of an in vitro loading system is to recreate the forces acting in the cell microenvironment. The goal of this study was to develop and mechanically validate a vibration loading system able to engender sinusoidal vertical vibration at different combinations of magnitude (0.3 g, 1 g, and 3 g) and frequency (30 Hz, 60 Hz and 90 Hz). A system like this can be therefore employed to study cell response to high and low magnitudes at high frequencies, thus providing a comprehensive evaluation of bone cell mechanotransduction. The mechanical validation, that is the characterization of the right loading input to the system to obtain the desired stimulation on cell culture, was performed in two different methods: open-loop and closed-loop mode. The results obtained in the open-loop mode showed a good intra-day repeatability of the measurements with values of index of dispersion always lower than 0.6%. While in the closed-loop mode a systematic search was implemented to reach the optimal amplitude stimulation. The vibration signals acquired on long-term test following the systematic search showed a good stability with index of dispersion always lower than 1%. Following the mechanical validation, the system was used to stimulate osteoblast like cells (Saos-2) with vibration loading of nine combinations of magnitude and frequency and the cell proliferation was studied 24h after the treatment by cell counting. Our preliminary results showed that no alterations in the proliferation were induced by 90 Hz vibration loading. On the other hand, small modulations in the proliferation were reported for lower stimulation frequency, being statistically significant when using 0.3 g of amplitude at 30 Hz

A novel microwave resonant sensor for measuring cancer cell line aggressiveness

The measurement of biological tissues’ dielectric properties plays a crucial role in determining the state of health, and recent studies have reported microwave biosensing to be an innovative method with great potential in this field. Research has been conducted from the tissue level to the cellular level but, to date, cellular adhesion has never been considered. In addition, conventional systems for diagnosing tumor aggressiveness, such as a biopsy, are rather expensive and invasive. Here, we propose a novel microwave approach for biosensing adherent cancer cells with different malignancy degrees. A circular patch resonator was designed adjusting its structure to a standard Petri dish and a network analyzer was employed. Then, the resonator was realized and used to test two groups of different cancer cell lines, based on various tumor types and aggressiveness: low- and high-aggressive osteosarcoma cell lines (SaOS-2 and 143B, respectively), and low- and high-aggressive breast cancer cell lines (MCF-7 and MDA-MB-231, respectively). The experimental results showed that the sensitivity of the sensor was high, in particular when measuring the resonant frequency. Finally, the sensor showed a good ability to distinguish low-metastatic and high-metastatic cells, paving the way to the development of more complex measurement systems for noninvasive tissue diagnosis

Detection of the strains induced in murine tibias by ex vivo uniaxial loading with different sensors

In this paper, the characterization of the main techniques and transducers employed to measure local and global strains induced by uniaxial loading of murine tibiae is presented. Micro strain gauges and digital image correlation (DIC) were tested to measure local strains, while a moving coil motor-based length transducer was employed to measure relative global shortening. Local strain is the crucial parameter to be measured when dealing with bone cell mechanotransduction, so we characterized these techniques in the experimental conditions known to activate cell mechanosensing in vivo. The experimental tests were performed using tibia samples excised from twenty-two C57BL/6 mice. To evaluate measurement repeatability we computed the standard deviation of ten repetitive compressions to the mean value. This value was lower than 3% for micro strain gauges, and in the range of 7%-10% for DIC and the length transducer. The coefficient of variation, i.e., the standard deviation to the mean value, was about 35% for strain gauges and the length transducer, and about 40% for DIC. These results provided a comprehensive characterization of three methodologies for local and global bone strain measurement, suggesting a possible field of application on the basis of their advantages and limitations

Circulating Extracellular Vesicles Impair Mesenchymal Stromal Cell Differentiation Favoring Adipogenic Rather than Osteogenic Differentiation in Adolescents with Obesity

Excess body weight has been considered beneficial to bone health because of its anabolic effect on bone formation; however, this results in a poor quality bone structure. In this context, we evaluated the involvement of circulating extracellular vesicles in the impairment of the bone phenotype associated with obesity. Circulating extracellular vesicles were collected from the plasma of participants with normal weight, as well as overweight and obese participants, quantified by flow cytometry analysis and used to treat mesenchymal stromal cells and osteoblasts to assess their effect on cell differentiation and activity. Children with obesity had the highest amount of circulating extracellular vesicles compared to controls. The treatment of mesenchymal stromal cells with extracellular vesicles from obese participants led to an adipogenic differentiation in comparison to vesicles from controls. Mature osteoblasts treated with extracellular vesicles from obese participants showed a reduction in differentiation markers in comparison to controls. Children with obesity who regularly performed physical exercise had a lower circulating extracellular vesicle amount in comparison to those with a sedentary lifestyle. This pilot study demonstrates how the high amount of circulating extracellular vesicles in children with obesity affects the bone phenotype and that physical activity can partially rescue this phenotype