Multicancer screening test based on the detection of circulating non haematological proliferating atypical cells.

BACKGROUND: the problem in early diagnosis of sporadic cancer is understanding the individual's risk to develop disease. In response to this need, global scientific research is focusing on developing predictive models based on non-invasive screening tests. A tentative solution to the problem may be a cancer screening blood-based test able to discover those cell requirements triggering subclinical and clinical onset latency, at the stage when the cell disorder, i.e. atypical epithelial hyperplasia, is still in a subclinical stage of proliferative dysregulation. METHODS: a well-established procedure to identify proliferating circulating tumor cells was deployed to measure the cell proliferation of circulating non-haematological cells which may suggest tumor pathology. Moreover, the data collected were processed by a supervised machine learning model to make the prediction. RESULTS: the developed test combining circulating non-haematological cell proliferation data and artificial intelligence shows 98.8% of accuracy, 100% sensitivity, and 95% specificity. CONCLUSION: this proof of concept study demonstrates that integration of innovative non invasive methods and predictive-models can be decisive in assessing the health status of an individual, and achieve cutting-edge results in cancer prevention and management.

Kinetic Rate Constants of Gold Nanoparticle Deposition on Silicon.

We fabricated gold nanoparticles on nanoporous silicon microparticles using electroless deposition in a hydrofluoric acid solution containing gold chloride. The reaction was followed by UV spectrometer analysis of the absorbance of the solution (proportional to the nanoparticle concentration) for two temperatures (20 and 50 °C). The results indicate that the process is autocatalytic, described by a pseudo-first-order reaction, the apparent rate constant kobs of which was determined by utilizing UV spectrometer data. We found that the reaction rate constant at 20 °C is 7 × 10-3 s-1 and that at 50 °C is 2.9 × 10-2 s-1. Scanning electron microscope (SEM) analysis of samples and diffusion-limited aggregation (DLA) simulations were used to validate the results. This study aims to resolve the kinetics of the electroless deposition of gold on silicon at the nanoscale, in the present state of art missing a quantitative characterization, for certain conditions of growth and given values of temperature and concentration of the reagents. Results may have applications to the synthesis of gold nanoparticles and their use as nanosensors, drug delivery systems, or metal nanometamaterials with advanced optical properties.

Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes.

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression.

Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers.

In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels- where the cells can flow one-by-one -, allowing single cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm of the each cell. Experiments are performed on red blood cells (RBCs), peripheral blood lymphocytes (PBLs) and myelogenous leukemia tumor cells (K562).

Lipid droplets: a new player in colorectal cancer stem cells unveiled by spectroscopic imaging.

The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs.

Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells.

Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor's stadiation, therapy, and early relapsing lesions. Within surface's bio-functionalization and cell's isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient's blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.

Direct determination of the resonance properties of metallic conical nanoantennas.

We present a simple method that is able to predict the resonant frequencies of a metallic conical nanoantenna. The calculation is based on an integral relation that takes into account the dependence of the effective refractive index of the plasmonic mode on the cone radius. Numerical simulations retrieving the near field properties of nanocones with different lengths are also performed for comparison. The fine agreement between the two approaches demonstrates the validity of our method.

Electroless deposition and nanolithography can control the formation of materials at the nano-scale for plasmonic applications.

The new revolution in materials science is being driven by our ability to manipulate matter at the molecular level to create structures with novel functions and properties. The aim of this paper is to explore new strategies to obtain plasmonic metal nanostructures through the combination of a top down method, that is electron beam lithography, and a bottom up technique, that is the chemical electroless deposition. This technique allows a tight control over the shape and size of bi- and three-dimensional metal patterns at the nano scale. The resulting nanostructures can be used as constituents of Surface Enhanced Raman Spectroscopy (SERS) substrates, where the electromagnetic field is strongly amplified. Our results indicate that, in electroless growth, high quality metal nanostructures with sizes below 50 nm may be easily obtained. These findings were explained within the framework of a diffusion limited aggregation (DLA) model, that is a simulation model that makes it possible to decipher, at an atomic level, the rules governing the evolution of the growth front; moreover, we give a description of the physical mechanisms of growth at a basic level. In the discussion, we show how these findings can be utilized to fabricate dimers of silver nanospheres where the size and shape of those spheres is controlled with extreme precision and can be used for very large area SERS substrates and nano-optics, for single molecule detection.

Microfluidic biofunctionalisation protocols to form multi-valent interactions for cell rolling and phenotype modification investigations.

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro-fluidic protocols. Many available processes either require expensive and time-consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi-valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin-biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 µL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC-I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC-I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC-I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC-I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.

Raman database of amino acids solutions: a critical study of extended multiplicative signal correction.

The Raman spectra of biological materials always exhibit complex profiles, constituting several peaks and/or bands which arise due to the large variety of biomolecules. The extraction of quantitative information from these spectra is not a trivial task. While qualitative information can be retrieved from the changes in peaks frequencies or from the appearance/disappearance of some peaks, quantitative analysis requires an examination of peak intensities. Unfortunately in biological samples it is not easy to identify a reference peak for normalizing intensities, and this makes it very difficult to study the peak intensities. In the last decades a more refined mathematical tool, the extended multiplicative signal correction (EMSC), has been proposed for treating infrared spectra, which is also capable of providing quantitative information. From the mathematical and physical point of view, EMSC can also be applied to Raman spectra, as recently proposed. In this work the reliability of the EMSC procedure is tested by application to a well defined biological system: the 20 standard amino acids and their combination in peptides. The first step is the collection of a Raman database of these 20 amino acids, and subsequently EMSC processing is applied to retrieve quantitative information from amino acids mixtures and peptides. A critical review of the results is presented, showing that EMSC has to be carefully handled for complex biological systems.

Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition.

This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.

Microfluidics for 3D Cell and Tissue Cultures: Microfabricative and Ethical Aspects Updates.

The necessity to improve in vitro cell screening assays is becoming ever more important. Pharmaceutical companies, research laboratories and hospitals require technologies that help to speed up conventional screening and therapeutic procedures to produce more data in a short time in a realistic and reliable manner. The design of new solutions for test biomaterials and active molecules is one of the urgent problems of preclinical screening and the limited correlation between in vitro and in vivo data remains one of the major issues. The establishment of the most suitable in vitro model provides reduction in times, costs and, last but not least, in the number of animal experiments as recommended by the 3Rs (replace, reduce, refine) ethical guiding principles for testing involving animals. Although two-dimensional (2D) traditional cell screening assays are generally cheap and practical to manage, they have strong limitations, as cells, within the transition from the three-dimensional (3D) in vivo to the 2D in vitro growth conditions, do not properly mimic the real morphologies and physiology of their native tissues. In the study of human pathologies, especially, animal experiments provide data closer to what happens in the target organ or apparatus, but they imply slow and costly procedures and they generally do not fully accomplish the 3Rs recommendations, i.e., the amount of laboratory animals and the stress that they undergo must be minimized. Microfluidic devices seem to offer different advantages in relation to the mentioned issues. This review aims to describe the critical issues connected with the conventional cells culture and screening procedures, showing what happens in the in vivo physiological micro and nano environment also from a physical point of view. During the discussion, some microfluidic tools and their components are described to explain how these devices can circumvent the actual limitations described in the introduction.

Correlative Raman-Electron-Light (CREL) Microscopy Analysis of Lipid Droplets in Melanoma Cancer Stem Cells.

Among all neoplasms, melanoma is characterized by a very high percentage of cancer stem cells (CSCs). Several markers have been proposed for their identification, and lipid droplets (LDs) are among them. Different techniques are used for their characterization such as mass spectrometry, imaging techniques, and vibrational spectroscopies. Some emerging experimental approaches for the study of LDs are represented by correlative light-electron microscopy and by correlative Raman imaging-scanning electron microscopy (SEM). Based on these scientific approaches, we developed a novel methodology (CREL) by combining Raman micro-spectroscopy, confocal fluorescence microscopy, and SEM coupled with an energy-dispersive X-ray spectroscopy module. This procedure correlated cellular morphology, chemical properties, and spatial distribution from the same region of interest, and in this work, we presented the application of CREL for the analysis of LDs within patient-derived melanoma CSCs (MCSCs).

Direct Visualization and Identification of Membrane Voltage-Gated Sodium Channels from Human iPSC-Derived Neurons by Multiple Imaging and Light Enhanced Spectroscopy.

In this study, transmission electron microscopy atomic force microscopy, and surface enhanced Raman spectroscopy are combined through a direct imaging approach, to gather structural and chemical information of complex molecular systems such as ion channels in their original plasma membrane. Customized microfabricated sample holder allows to characterize Nav channels embedded in the original plasma membrane extracted from neuronal cells that are derived from healthy human induced pluripotent stem cells. The identification of the channels is accomplished by using two different approaches, one of them widely used in cryo-EM (the particle analysis method) and the other based on a novel Zernike Polynomial expansion of the images bitmap. This approach allows to carry out a whole series of investigations, one complementary to the other, on the same sample, preserving its state as close as possible to the original membrane configuration.

Nanoparticle microinjection and Raman spectroscopy as tools for nanotoxicology studies.

Microinjection techniques and Raman spectroscopy have been combined to provide a new methodology to investigate the cytotoxic effects due to the interaction of nanomaterials with cells. In the present work, this novel technique has been used to investigate the effects of Ag and Fe(3)O(4) nanoparticles on Hela cells. The nanoparticles are microinjected inside the cells and these latter ones are probed by means of Raman spectroscopy after a short incubation time, in order to highlight the first and impulsive mechanisms developed by the cells to counteract the presence of the nanoparticles. The results put in evidence a different behaviour of the cells treated with nanoparticles in comparison with the control cells; these differences are supposed to be generated by an emerging oxidative stress due to the nanoparticles. The achieved results demonstrate the suitability of the proposed method as a new tool for nanotoxicity studies.

Methylglyoxal Adducts Levels in Blood Measured on Dried Spot by Portable Near-Infrared Spectroscopy.

The altered glucose metabolism characterising cancer cells determines an increased amount of methylglyoxal in their secretome. Previous studies have demonstrated that the methylglyoxal, in turn, modifies the protonation state (PS) of soluble proteins contained in the secretomes of cultivated circulating tumour cells (CTCs). In this study, we describe a method to assess the content of methylglyoxal adducts (MAs) in the secretome by near-infrared (NIR) portable handheld spectroscopy and the extreme learning machine (ELM) algorithm. By measuring the vibration absorption functional groups containing hydrogen, such as C-H, O-H and N-H, NIR generates specific spectra. These spectra reflect alterations of the energy frequency of a sample bringing information about its MAs concentration levels. The algorithm deciphers the information encoded in the spectra and yields a quantitative estimate of the concentration of MAs in the sample. This procedure was used for the comparative analysis of different biological fluids extracted from patients suspected of having cancer (secretome, plasma, serum, interstitial fluid and whole blood) measured directly on the solute left on a surface upon a sample-drop cast and evaporation, without any sample pretreatment. Qualitative and quantitative regression models were built and tested to characterise the different levels of MAs by ELM. The final model we selected was able to automatically segregate tumour from non-tumour patients. The method is simple, rapid and repeatable; moreover, it can be integrated in portable electronic devices for point-of-care and remote testing of patients.

Enhancing Antibodies' Binding Capacity through Oriented Functionalization of Plasmonic Surfaces.

Protein A has long been used in different research fields due to its ability to specifically recognize immunoglobulins (Ig). The protein derived from Staphylococcus aureus binds Ig through the Fc region of the antibody, showing its strongest binding in immunoglobulin G (IgG), making it the most used protein in its purification and detection. The research presented here integrates, for the first time, protein A to a silicon surface patterned with gold nanoparticles for the oriented binding of IgG. The signal detection is conveyed through a metal enhanced fluorescence (MEF) system. Orienting immunoglobulins allows the exposition of the fragment antigen-binding (Fab) region for the binding to its antigen, substantially increasing the binding capacity per antibody immobilized. Antibodies orientation is of crucial importance in many diagnostics devices, particularly when either component is in limited quantities.

Micro/Nanopatterned Superhydrophobic Surfaces Fabrication for Biomolecules and Biomaterials Manipulation and Analysis.

Superhydrophobic surfaces display an extraordinary repulsion to water and water-based solutions. This effect emerges from the interplay of intrinsic hydrophobicity of the surface and its morphology. These surfaces have been established for a long time and have been studied for decades. The increasing interest in recent years has been focused towards applications in many different fields and, in particular, biomedical applications. In this paper, we review the progress achieved in the last years in the fabrication of regularly patterned superhydrophobic surfaces in many different materials and their exploitation for the manipulation and characterization of biomaterial, with particular emphasis on the issues affecting the yields of the fabrication processes and the quality of the manufactured devices.

ssRNA Virus and Host Lipid Rearrangements: Is There a Role for Lipid Droplets in SARS-CoV-2 Infection?

Since its appearance, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has immediately alarmed the World Health Organization for its very high contagiousness and the complexity of patient clinical profiles. The worldwide scientific community is today gathered in a massive effort in order to develop safe vaccines and effective therapies in the shortest possible time. Every day, new pieces of SARS-CoV-2 infective puzzle are disclosed. Based on knowledge gained with other related coronaviruses and, more in general, on single-strand RNA viruses, we highlight underexplored molecular routes in which lipids and lipid droplets (LDs) might serve essential functions in viral infections. In fact, both lipid homeostasis and the pathways connected to lipids seem to be fundamental in all phases of the coronavirus infection. This review aims at describing potential roles for lipid and LDs in host-virus interactions and suggesting LDs as new and central cellular organelles to be investigated as potential targets against SARS-CoV-2 infection.

Emerging Designs of Electronic Devices in Biomedicine.

A long-standing goal of nanoelectronics is the development of integrated systems to be used in medicine as sensor, therapeutic, or theranostic devices. In this review, we examine the phenomena of transport and the interaction between electro-active charges and the material at the nanoscale. We then demonstrate how these mechanisms can be exploited to design and fabricate devices for applications in biomedicine and bioengineering. Specifically, we present and discuss electrochemical devices based on the interaction between ions and conductive polymers, such as organic electrochemical transistors (OFETs), electrolyte gated field-effect transistors (FETs), fin field-effect transistor (FinFETs), tunnelling field-effect transistors (TFETs), electrochemical lab-on-chips (LOCs). For these systems, we comment on their use in medicine.