Mimicking the Tumor Niche: Methods for Isolation, Culture, and Characterization of Cancer Stem Cells and Multicellular Spheroids.

Cancer stem cells (CSCs) play a significant role in driving several tumor hallmarks. Their behavior and tumor progression are strictly related to the tumor microenvironment (TME). The dynamic interplay between CSCs and TME drives metastasis, chemoresistance, and disease relapse. In this chapter, we describe different techniques and protocols for isolating, culturing, and characterizing CSCs and we explain the methodology for the culture of multicellular spheroids comprising CSCs.

Toxic Metal and Essential Element Concentrations in the Blood and Tissues of Pancreatic Ductal Adenocarcinoma Patients.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal neoplasm, and it has an average 5-year survival rate of less than 10%. Although the factors that influence PDAC development remain unclear, exposure to toxic metals or the imbalance in essential elements may have a role in PDAC-associated metabolic pathways. METHODS: This study determined the concentrations of Cd, Cr, Cu, Fe, Mn, Ni, Pb, Se and Zn in whole blood, cancer and non-cancer tissues of patients affected by PDAC, and compared them with levels in healthy controls using inductively coupled plasma mass spectrometry. RESULTS: Results of the whole blood showed significantly higher levels of Cr, Cu and Cu/Zn ratio in PDAC patients compared to the controls. In addition, the concentrations of Cu, Se, Fe and Zn significantly increased in cancer tissue compared to the healthy counterparts. CONCLUSIONS: This study revealed evidence of altered metal levels in the blood and pancreatic tissues of PDAC patients with respect to healthy controls. These changes may contribute to multiple mechanisms involved in metal-induced carcinogenesis, including oxidative stress, DNA damage, genetic alteration, decreased antioxidant barriers and inflammatory responses. Thus, the analysis of metals can be used in the diagnosis and monitoring of PDAC neoplasms.

Human tissue lead (Pb) levels and amyotrophic lateral sclerosis: a systematic review and meta-analysis of case-control studies.

AIM: To combine the current scientific literature evidence and elucidate the differences of lead (Pb) bioaccumulation in human tissues by comparing amyotrophic lateral sclerosis (ALS) patients and healthy controls. METHODS: We systematically searched for case-control studies on the association of Pb levels with ALS, in human cells, tissues, and body fluids (nervous tissue, muscle, blood, cerebrospinal fluid, urine, skin appendages). Then, we performed a meta-analysis for all the tissues in which at least five case-control studies were available: whole blood (9 studies), serum/plasma (5 studies), and cerebrospinal fluid (CSF) (6 studies). Differences between cases and controls were evaluated using standardized mean difference, and combined estimates were derived using random effect maximum likelihood (REML) meta-analyses. RESULTS: Among 1734 records, we identified 46 full-text studies, of which 14 case-control studies met the meta-analysis inclusion criteria. We found higher Pb levels in ALS cases than controls in blood (standardized mean difference (SMD) = 0.61; 95% confidence interval (CI) 0.20, 1.01; p = 0.003), plasma/serum (SMD = 0.27; 95% CI - 0.16, 0.70; p = 0.26), and CSF (SMD = 0.53; 95% CI - 0.09, 1.15; p = 0.09). CONCLUSIONS: This work provides further evidence of the association between Pb bioaccumulation and ALS in body fluids. The lack of association studies in solid tissues did not allow a robust meta-analysis. Future prospective studies are needed to clarify the causality in the association of Pb bioaccumulation with ALS.

Aspiration Thrombectomy with the Indigo System for Acute Lower Limb Ischemia: Preliminary experience and analysis of parameters affecting the outcome.

BACKGROUND: The purpose of our study is to assess the short-term technical success and the safety of the Indigo System in a series of patients undergoing vacuum-assisted catheter direct thrombus aspiration (IS-CDTA) for acute lower limb ischemia (ALLI) and to evaluate which parameters may affect the outcome. METHODS: All procedures using the IS-CDTA for ALLI, performed in a single-centre Interventional Radiology Unit from February 2016 to March 2020, were retrospectively analysed. Technical success was defined as the achievement of nearly-complete or complete revascularization (TIPI grade 2/3) and considered as a good outcome. Variables potentially correlated with the IS-CDTA outcome were analysed. RESULTS: 33 procedures were performed in 29 patients. Mean age was 69 years old (range 47 - 88), 24 males (83%) and 5 females (18%). The technical success was 70%. Catheter-directed thrombolysis following IS-CDTA was performed in 23 cases and the overall technical success increased from 70% to 90%, afterwards. The median time between symptoms insurgency and IS-CDTA was significantly shorter in patients with good outcome (10 hours; IQR 2.75-48) compared to those with poor outcome (168 hours; IQR 36-336) (P = 0.003). No statistically significant differences were found between the two groups regarding ATK vs. BTK (P = 0.34), native vessel vs. graft (P = 0.25), occlusion nature P = 0.28) or Rutherford score (P = 0.80). CONCLUSION: IS-CDTA is a valid option for a rapid and percutaneous treatment of ALLI. Our experience indicates that the time elapsing from the symptoms insurgency and the endovascular procedure is the best positive predictor of the outcome.

The levels of trace elements in sputum as biomarkers for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a rare lung disease that quickly leads to death. This paper addressed the issue of whether the levels of trace elements in sputum samples are suitable biomarkers for IPF disease. The sputum Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn concentrations were measured by sector field inductively coupled plasma mass spectrometry in populations sampled in Sardinia Island (Italy) including 31 patients with IPF, 31 patients with other lung-related diseases and 30 age- and gender-matched healthy controls. Risk factors in the disease as gender, age, severity and duration of the disease were assessed. Results showed that IPF patients had significantly increased sputum levels of Cd, Cr, Cu and Pb respect to controls. In males, but not in females, sputum levels of Cd, Cr and Cu were significantly higher in IPF cases respect to controls. In addition, Cr and Pb were increased in male patients with IPF compared to male patients with other lung diseases. Regarding Zn, it was found higher with the more serious stage of disease. Moreover, the ratios Cu/Zn, Fe/Mn and Cu/Mn were significantly increased in IPF patients and in non-IPF patients than in control subjects. These data showed clear increases in the concentration of some trace elements in sputum from patients with IPF and patients with other lung-related diseases that may contribute to the injury. The non-invasiveness of the sputum analysis is beneficial for its use as biomarker of trace element status in diseased patients for both the researcher and the clinic.

An overview on amyotrophic lateral sclerosis and cadmium.

The present review represents an update about the knowledge of the possible role of Cadmium (Cd) in amyotrophic lateral sclerosis (ALS) initiation and its progression. ALS is a neurodegenerative disease that occurs in adulthood; its etiology is unknown and leads to death within a few years from its appearance. Among the various possible causes that can favor the development of the disease, heavy metals cannot be excluded. Cadmium is a heavy metal that does not play a biological role, but its neurotoxicity is well known. Numerous in vitro studies on cell and animal models confirm the toxicity of the metal on the nervous system, but these data are not accompanied by an epidemiological evidence, and, thus, an unclear correlation between Cd and the onset of the disease can be pointed out. On the other hand, a possible multifactorial and synergic mechanism in which Cd may have a role can explain the ALS onset. More efforts in new clinical, biochemical, and epidemiological studies are necessary to better elucidate the involvement of Cd in this lethal disease.

The Inhibitory Role of miR-486-5p on CSC Phenotype Has Diagnostic and Prognostic Potential in Colorectal Cancer.

Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cancer deaths. Increasing evidences supports the idea that the poor prognosis of patients is related to the presence of cancer stem cells (CSCs), a cell population able to drive cancer recurrence and metastasis. The deregulation of microRNAs (miRNAs) plays a role in the formation of CSC. We investigated the role of hsa-miR-486-5p (miR-486-5p) in CRC, CSCs, and metastasis, in order to reach a better understanding of the biomolecular and epigenetic mechanisms mir-486-5p-related. The expression of miR-486-5p was investigated in three different matrices from CRC patients and controls and in CSCs obtained from the CRC cell lines HCT-116, HT-29, and T-84. In the human study, miR-486-5p was up-regulated in serum and stool of CRC patients in comparison with healthy controls but down-regulated in tumor tissue when compared with normal mucosa. miR-486-5p was also down-regulated in the sera of metastatic patients. In vitro, miR-486-5p was down-regulated in CSC models and it induced an inhibitory effect on stem factors and oncogenes in the main pathways of CSCs. Our results provide a step forward in understanding the role of mir-486-5p in CRC and CSC, and suggest that further studies are needed to investigate its diagnostic and prognostic power, possibly in combination with other biomarkers.

Amyotrophic lateral sclerosis and lead: A systematic update.

Heavy metals are considered to be among the leading environmental factors that trigger amyotrophic lateral sclerosis (ALS). However, no convincing biopathological mechanism and therapeutic clinical implication of such metals in ALS pathogenesis have been established. This is partly attributable to the technical and scientific difficulties in demonstrating a direct and causative role of heavy metals in the onset of ALS in patients. However, a body of epidemiological, clinical and experimental evidences suggest that lead (Pb), more than other metals, could actually play a major role in the onset and progression of ALS. Here, to clarify the nature of the association and the causative role of Pb in ALS, we comprehensively reviewed the scientific literature of the last decade with objective database searches and the methods typically adopted in systematic reviews, critically analysing and summarising the various scientifically sound evidence on the relationship between ALS and Pb. From these tasks, we noted a number of multidisciplinary associations between ALS and Pb, and specifically the importance of occupational exposure to Pb in ALS development and/or progression. We also report the possible involvement of TAR DNA binding protein (TDP-43)-based molecular mechanism in Pb-mediated ALS, although these data rely on a single study, which included both in vitro experiments and an animal model, and are therefore still preliminary. Finally, we briefly examined whether this knowledge could inspire new targeted therapies and policies in the fight against ALS.

A pilot study to evaluate the levels of aqueous humor trace elements in open-angle glaucoma.

BACKGROUND: Primary open angle glaucoma (POAG) is the most common form of chronic, progressive optic neuropathies characterized by slow degeneration of the retinal ganglion cells and their axons, resulting in visual field loss. Risk factors for this disease are elevated intraocular pressure (IOP), increased age, European and African ethnicity, family history, myopia and decreased corneal thickness. In addition, studies indicated that levels of trace elements are also significantly related to the POAG. METHOD: The association between toxic and essential elements and POAG was explored in a population-based case-control study in the Sardinia Island (Italy). The aqueous humor levels of Al, Cd, Cu, Fe, Hg, Mn, Ni, Pb and Zn were measured in 25 POAG patients compared to 20 age- and gender-matched healthy controls by sector field inductively coupled mass spectrometry. Risk factors as gender, age and increased IOP were also explored. RESULTS: The concentrations of Fe, Hg and Zn were significantly higher in POAG patients than in control subjects, showing these elements as possible determinants in POAG development or degeneration. Other findings were the increased Cu and Fe levels in glaucomatous patients with age less than 70 years. Levels of Ni were found elevated in POAG females. Mercury accumulated more in POAG females, in patients over 70 years and in those with higher levels of IOP in the left eye. Moreover, the positive associations CuFe and Mn-Zn may indicate synergistic effects of elements. CONCLUSIONS: Altogether, these findings suggested a multifactorial role in the risk for POAG disease. The present study documented the levels of trace elements in aqueous humor of Sardinian POAG patients for the first time.

Deregulation of cancer-stem-cell-associated miRNAs in tissues and sera of colorectal cancer patients.

Colorectal cancer (CRC) is a deadly tumour in Western countries characterized by high cellular/molecular heterogeneity. Cancer stem cells (CSC) act in cancer recurrence, drug-resistance and in metastatic epithelial-to-mesenchymal transition. microRNAs (miRNAs) contribute to cancer is increasing, and miRNA roles in CSC phenotype and fate and their utility as CRC biomarkers have also been reported. Here, we investigated miR-21, miR-221, miR-18a, miR-210, miR-31, miR-34a, miR-10b and miR-16 expression in experimental ALDH+ and CD44+/CD326+ colorectal CSCs obtained from the human CRC cell lines HCT-116, HT-29 and T-84. Then, we moved our analysis in cancer tissue (CT), healthy tissue (HT) and serum (S) of adult CRC patients (n=12), determining relationships with clinical parameters (age, sex, metastasis, biochemical serum markers). Specific miRNA patterns were evident in vitro (normal, monolayers and CSCs) and in patients' samples stratified by TNM stage (LOW vs HIGH) or metastasis (Met vs no-Met). miR-21, miR-210, miR-34a upregulation ad miR-16 dowregulation associated with the CSCs phenotype. miR-31b robustly overexpressed in monolayers and CSCs, and in CT ad S of HIGH grade and Met patients, suggesting a role as marker of CRC progression and metastasis. miR-18a upregulated in all cancer models and associated to CSC phenotype, and to metastasis and age in patients. miR-10b downregulated in CT and S of LOW/HIGH grade and no-Met patients. Our results identify miRNAs useful as colorectal CSC biomarker and that miR-21, miR-210, miR-10b and miR-31b are promising markers of CRC. A specific role of miR-18a as metastatic CRC serum biomarker in adult patients was also highlighted.

MHO_0730 as a Surface-Exposed Calcium-Dependent Nuclease of Mycoplasma hominis Promoting Neutrophil Extracellular Trap Formation and Escape.

Mycoplasma lipoproteins play a relevant role in pathogenicity and directly interact with the host immune system. Among human mycoplasmas, Mycoplasma hominis is described as a commensal bacterium that can be associated with a number of genital and extragenital conditions. Mechanisms of M. hominis pathogenicity are still largely obscure, and only a limited number of proteins have been associated with virulence. The current study focused on investigating the role of MHO_0730 as a virulence factor and demonstrated that MHO_0730 is a surface lipoprotein, potentially expressed in vivo during natural infection, acting both as a nuclease with its amino acidic portion and as a potent inducer of Neutrophil extracellular trapsosis with its N-terminal lipid moiety. Evidence for M. hominis neutrophil extracellular trap escape is also presented. Results highlight the relevance of MHO_0730 in promoting infection and modulation and evasion of innate immunity and provide additional knowledge on M. hominis virulence and survival in the host.

Trace elements in ALS patients and their relationships with clinical severity.

An exploratory study of trace elements in ALS and their relationships with clinical severity was detected. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that causes irreversible damage in humans, with the consequent loss of function of motoneurons (MNs), with a prognosis up to 5 years after diagnosis. Except to genetic rare cases it is not known the etiology of the disorder. Aim of our research is to investigate the possible role of heavy metals in the severity of the disease. In this study, by the use of plasma mass (ICP-MS), we have analyzed the content of essential and heavy metals such: Pb, Cd, Al, Hg, Mn, Fe, Cu, Zn, Se, Mg, and Ca, in blood, urine and hair of ALS patients and controls; moreover we divided the patients in two groups for disease severity and analyzed the difference among the groups, in order to study a possible involvement of metals in the severity of the damage. Our results suggest a protective role of Selenium, involved in protective antioxidant mechanisms, and a risk factor in the case of presence of Lead in blood. The levels of the other metals are not easy to interpret, because these may be due to life style and for essential metals a consequence of the disease condition, not a cause.