Manuel Scimeca

Microcalcifications in breast cancer: an active phenomenon mediated by epithelial cells with mesenchymal characteristics

BackgroundMammary microcalcifications have a crucial role in breast cancer detection, but the processes that induce their formation are unknown. Moreover, recent studies have described the occurrence of the epithelial–mesenchymal transition (EMT) in breast cancer, but its role is not defined. In this study, we hypothesized that epithelial cells acquire mesenchymal characteristics and become capable of producing breast microcalcifications.MethodsBreast sample biopsies with microcalcifications underwent energy dispersive X-ray microanalysis to better define the elemental composition of the microcalcifications. Breast sample biopsies without microcalcifications were used as controls. The ultrastructural phenotype of breast cells near to calcium deposits was also investigated to verify EMT in relation to breast microcalcifications. The mesenchymal phenotype and tissue mineralization were studied by immunostaining for vimentin, BMP-2, β2-microglobulin, β-catenin and osteopontin (OPN).ResultsThe complex formation of calcium hydroxyapatite was strictly associated with malignant lesions whereas calcium-oxalate is mainly reported in benign lesions. Notably, for the first time, we observed the presence of magnesium-substituted hydroxyapatite, which was frequently noted in breast cancer but never found in benign lesions. Morphological studies demonstrated that epithelial cells with mesenchymal characteristics were significantly increased in infiltrating carcinomas with microcalcifications and in cells with ultrastructural features typical of osteoblasts close to microcalcifications. These data were strengthened by the rate of cells expressing molecules typically involved during physiological mineralization (i.e. BMP-2, OPN) that discriminated infiltrating carcinomas with microcalcifications from those without microcalcifications.ConclusionsWe found significant differences in the elemental composition of calcifications between benign and malignant lesions. Observations of cell phenotype led us to hypothesize that under specific stimuli, mammary cells, which despite retaining a minimal epithelial phenotype (confirmed by cytokeratin expression), may acquire some mesenchymal characteristics transforming themselves into cells with an osteoblast-like phenotype, and are able to contribute to the production of breast microcalcifications.

Assessment of metal contaminants in non-small cell lung cancer by EDX microanalysis

Human cardio-respiratory diseases are strongly correlated to concentrations of atmospheric elements. Bioaccumulation of heavy metals is strictly monitored, because of its possible toxic effects. In this work, we utilized the EDX microanalysis in order to identify the potential heavy metal accumulation in the lung tissue. To this aim, we enrolled 45 human lung biopsies: 15 non-small cell lung cancers, 15 lung benign lesions and 15 control biopsies. Lung samples were both paraffin embedded for light microscopy study and epon-epoxid embedded for transmission electron microscopy. EDX microanalysis was performed on 100 nm thick unstained ultrathin-sections placed on specific copper grids. Our results demonstrated that the EDX technology was particularly efficient in the study of elemental composition of lung tissues, where we found heavy metals, such as Cobalt (Co), Chromium (Cr), Manganese (Mn) and Lead (Pb). Furthermore, in malignant lesions we demonstrated the presence of multiple bio-accumulated elements. In fact, a high rate of lung cancers was associated with the presence of 3 or more bio-accumulated elements compared to benign lesions and control tissue (91.7%, 0%, 8.3%, respectively). The environmental impact on pulmonary carcinogenesis could be better clarified by demonstrating the presence of polluting agents in lung tissues. The application of EDX microanalysis on biological tissues could shed new light in the study of the possible bioaccumulation of polluting agents in different human organs and systems.

Emerging prognostic markers related to mesenchymal characteristics of poorly differentiated breast cancers.

Despite the screening program, breast cancer is the commonest cause of cancer death in women in the industrialized world. In this study, we investigate the correlation among poorly differentiated carcinoma, epithelial to mesenchymal transition (EMT) phenomenon, and expression of NF-kB, Sonic Hedgehog (SHH), K-RAS, and PTX3 in breast cancer in 100 breast biopsies. Samples were classified as follows: 30 benign lesions (BL), 30 ductal infiltrating carcinomas low grade (MLG1), and 40 ductal infiltrating carcinomas high grade (MLG3). Expression of vimentin, CD44, β-catenin, NF-kB, SHH, K-RAS, CD44, and PTX3 was studied by immunohistochemistry. The different rate of cells with vimentin, nuclear β-catenin, and CD44 expression in MLG3 as compared with MLG1 and BL suggested that the process of de-differentiation of breast cancer cells could be related to the EMT. Our results showed a significant increase in NF-kB signal in MLG3 (2.33 ± 0.77) with respect to MLG1 (1.26 ± 0.55) and BL (0.86 ± 0.52). SHH expression appeared low in BL (1.00 ± 0.41) and homogenously widespread in MLG1 (1.23 ± 0.63) and MLG3 (1.56 ± 0.54). An important increase in K-RAS signal was observed in MLG3 compared to that in BL (2.20 ± 0.69 vs 0.82 ± 0.59). As regards PTX3, we observed a strong expression in MLG3 (2.00 ± 0.78) with respect to BL (0.58 ± 0.55) and MLG1 (1.53 ± 0.76). The recurring expression of NF-kB, SHH, K-RAS, and PTX3 in vimentin- and CD44-positive breast cancer cells allows to speculate that breast cells acquire the ability to express these molecules in concomitance to EMT phenomenon.

Elemental analysis of histological specimens: a method to unmask nano asbestos fibers

There is an increasing amount of evidence that nanoparticles may enhance toxicological potential in comparison to the same material in the bulk form. The aim of this study was to develop a new method to unmask asbestos nanofibers from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. For the first time, in this study we applied Energy Dispersive X-ray (EDX) microanalysis through transmission electron microscopy to demonstrate the presence of asbestos nanofibers in histological specimens of patients with possible occupational exposure to asbestos. The diagnostic protocol was applied to 10 randomly selected lung cancer patients with no history of previous asbestos exposure. We detected asbestos nanofibers in close contact with lung cancer cells in two lung cancer patients with previous possible occupational exposure to asbestos. We were also able to identify the specific asbestos iso-type, which in one of the cases was the same rare variety used in the workplace of the affected patient. By contrast, asbestos nanofibers were not detected in lung cancer patients with no history of occupational asbestos exposure. The proposed technique can represent a potential useful tool for linking the disease to previous workplace exposure in uncertain cases. Furthermore, Formalin-Fixed Paraffin-Embedded (FFPE) tissues stored in the pathology departments might be re-evaluated for possible etiological attribution to asbestos in the case of plausible exposure. Since diseases acquired through occupational exposure to asbestos are generally covered by workers’ insurance in most countries, the application of the protocol used in this study may have also relevant social and economic implications.

Silver nanoparticles inhaled during pregnancy reach and affect the placenta and the foetus

Abstract Recently, interest for the potential impact of consumer-relevant engineered nanoparticles on pregnancy has dramatically increased. This study investigates whether inhaled silver nanoparticles (AgNPs) reach and cross mouse placental barrier and induce adverse effects. Apart from their relevance for the growing use in consumer products and biomedical applications, AgNPs are selected since they can be unequivocally identified in tissues. Pregnant mouse females are exposed during the first 15 days of gestation by nose-only inhalation to a freshly produced aerosol of 18–20 nm AgNPs for either 1 or 4 h, at a particle number concentration of 3.80 × 107 part./cm−3 and at a mass concentration of 640 μg/m³. AgNPs are identified and quantitated in maternal tissues, placentas and foetuses by transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy and single-particle inductively coupled plasma mass spectrometry. Inhalation of AgNPs results in increased number of resorbed foetuses associated with reduced oestrogen plasma levels, in the 4 h/day exposed mothers. Increased expression of pregnancy-relevant inflammatory cytokines is also detected in the placentas of both groups. These results prove that NPs are able to reach and cross the mouse placenta and suggest that precaution should be taken with respect to acute exposure to nanoparticles during pregnancy.

The role of sarcopenia with and without fracture

INTRODUCTION Bone and muscle tissues are in a close relationship. They are linked from a biological and functional point of view and both are related to an increased fracture risk in the elderly. The aging process is involved in the loss of functionality of both bones and muscles. In particular, aging-induced decline in muscle size and quality accompanies catabolic alterations in bone tissue; furthermore, age-related changes in bone alter its response to muscle-derived stimulation. The increased fracture risk in individuals with sarcopenia and osteoporosis is due to the decline of muscle mass and strength, the decrease in bone mineral density (BMD) and limited mobility. In this study, we investigated the role of sarcopenia and the main age-related bone diseases, osteoporosis (OP) and osteoarthritis (OA). METHODS Muscular performance status was evaluated using the Physical Activity Scale for the Elderly (PASE) test in 27 female patients with OP who underwent total hip arthroplasty for hip fracture, and in 27 age-matched female patients with OA who underwent total hip arthroplasty. Dual-energy X-ray absorptiometry (DEXA) was performed and the T-score values were used to discriminate between OP and OA patients. Body Mass Index (BMI) was calculated. As part of a multiparametric model of evaluation, biopsies of vastus lateralis muscle were analysed by immunohistochemical reaction to find a correlation with the above mentioned functional index. RESULTS The PASE test showed that the OP patients had a low or moderate level of physical activity before fracture occurred, whereas the OA patients had more intensive pre-fracture physical performances. Histological analysis showed that osteoporosis is characterised by a preferential type II fibre atrophy; in particular, data correlation showed that lower PASE test scores were related to lower diameter of type II fibres. No correlation was found between bone mineral density (BMD) and PASE test results. DISCUSSION AND CONCLUSION Osteoporosis is closely related to sarcopenia before and after fracture. Bone remodelling is influenced by muscle morphological and functional impairment and sarcopenia is considered one of the major factors for functional limitation and motor dependency in elderly osteoporotic individuals. Therefore, physical activity should be strongly recommended for OP patients at diagnosis.

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.

Satellite Cells CD44 Positive Drive Muscle Regeneration in Osteoarthritis Patients

Age-related bone diseases, such as osteoarthritis and osteoporosis, are strongly associated with sarcopenia and muscle fiber atrophy. In this study, we analyzed muscle biopsies in order to demonstrate that, in osteoarthritis patients, both osteophytes formation and regenerative properties of muscle stem cells are related to the same factors. In particular, thanks to immunohistochemistry, transmission electron microscopy, and immunogold labeling we investigated the role of BMP-2 in muscle stem cells activity. In patients with osteoarthritis both immunohistochemistry and transmission electron microscopy allowed us to note a higher number of CD44 positive satellite muscle cells forming syncytium. Moreover, the perinuclear and cytoplasmic expression of BMP-2 assessed by in situ molecular characterization of satellite cells syncytia suggest a very strict correlation between BMP-2 expression and muscle regeneration capability. Summing up, the higher BMP-2 expression in osteoarthritic patients could explain the increased bone mineral density as well as decreased muscle atrophy in osteoarthrosic patients. In conclusion, our results suggest that the control of physiological BMP-2 balance between bone and muscle tissues may be considered as a potential pharmacological target in bone-muscle related pathology.

Breast Osteoblast-like Cells: A Reliable Early Marker for Bone Metastases From Breast Cancer

&NA; The development of bone metastasis from breast cancer results from a functional interaction between tumor cells and osteoclasts or osteoblasts. The finding of breast osteoblast‐like cells in primary breast lesions could represent a precursor (and hence an early predictor) of the formation of osteolytic bone metastasis. The identification of breast cancer cells with high affinity for bone environment opens new perspectives on prevention and therapy of bone metastases from breast. Background: The development of bone metastasis from breast cancer results from a functional interaction between tumor cells and osteoclasts or osteoblasts. The main aim of this study was therefore to test the hypothesis that the appearance of breast osteoblast‐like cells (BOLCs) in primary mammary lesions is a precursor (and hence an early predictor) of the formation of breast cancer metastases to bone. Patients and Methods: In this study, we collected 64 breast infiltrating carcinomas, 50 breast benignant lesions, and 10 biopsies of bone metastasis selected from patients with infiltrated carcinoma. Immunohistochemical, western blot, and ultrastructural analysis allowed us to investigate the presence of BOLCs in breast cancer lesions and metastatic sites. Results: We established the presence of a high amount of breast cancer cells that underwent mesenchymal transformation in infiltrating carcinomas. In addition, our results demonstrated that the microenvironment of breast cancer is very similar to the microenvironment of bone. We noted a significantly higher expression of BMP‐2/4 and PTX3 in breast‐infiltrating carcinomas compared with benign lesions. Moreover, we also identified numerous BOLCs positive to RANKL and Vitamin D receptor. Thanks to ultrastructural analysis, we also revealed the presence of BOLCs at the metastatic site. Conclusions: The identification of breast cancer cells with high affinity for a bone environment opens new perspectives on prevention and therapy of bone metastases from breast.

Impairment of PTX3 expression in osteoblasts: A key element for osteoporosis

Pentraxin 3 (PTX3) is a multifunctional glycoprotein regulating inflammatory response, cell proliferation and migration and deposition and remodelling of the extracellular matrix by a variety of cells. In this study, we investigated the possible role of PTX3 in bone homeostasis. To this end, we compared the expression and function of PTX3 in human osteoblasts of osteoporotic, osteoarthritic patients and young subjects not affected by bone diseases. Immunohistochemical analysis performed on bone head biopsies showed a close association between bone health and the number of osteoblasts expressing PTX3. Noteworthy, the proportion of PTX3-positive osteoblasts resulted to be significantly lower in osteoporotic patients compared with both young patients and osteoarthritic patients of the same age. Ex vivo culture of osteoblasts isolated from the three groups of patients confirmed in vivo observation. Specifically, we observed rare runt-related transcription factor 2 (RUNX2) immunopositive osteoblasts expressing PTX3 in cell cultures derived from osteoporotic patients and western blotting analysis showed 80% reduction of PTX3 in the corresponding culture extracts compared with young and osteoarthritic patients. The treatment of human osteoblast primary cultures derived from young patients with anti-PTX3 antibody dramatically affected osteoblast behaviour. Indeed, they lost the morphological and molecular features typical of mature osteoblasts, acquiring fibroblast-like shape and highly decreasing nuclear factor kappa-B ligand (RANKL) and RUNX2 expression. Also, the inhibition of PTX3 negatively affected osteoblast proliferation and their ability to form cell clusters and microhydroxyapatite crystals. Altogether, these results suggest a central role of PTX3 in bone homeostasis showing its involvement in osteoblast proliferation, differentiation and function.