Bioengineered Surfaces for Real-Time Label-Free Detection of Cancer Cells

Biosensing technology is an advancing field that benefits from the properties of biological processes combined to functional materials. Recently, biosensors have emerged as essential tools in biomedical applications, offering advantages over conventional clinical techniques for diagnosis and therapy. Optical biosensors provide fast, selective, direct, and cost-effective analyses allowing label-free and real-time tests. They have also shown exceptional potential for integration in lab-on-a-chip (LOC) devices. The major challenge in the biosensor field is to achieve a fully operative LOC platform that can be used in any place at any time. The choice of an appropriate strategy to immobilize the biological element on the sensor surface becomes the key factor to obtain an applicable analytical tool. In this chapter, after a brief description of the main biofunctionalization procedures on silicon devices, two silicon-based chips that present an (i) IgG antibody or (ii) an Id-peptide as molecular probe, directed against the B-cell receptor of lymphoma cancer cells, will be presented. From a comparison in detecting cells, the Id-peptide device was able to detect lymphoma cells also at low cell concentrations (8.5 × 10−3 cells/μm2) and in the presence of a large amount of non-specific cells. This recognition strategy could represent a proof-of-concept for an innovative tool for the targeting of patient-specific neoplastic B cells during the minimal residual disease; in addition, it represents an encouraging starting point for the construction of a lab-on-a-chip system for the specific recognition of neoplastic cells in biological fluids enabling the follow-up of the changes of cancer cells number in patients, highly demanded for therapy monitoring applications

Cellular Interaction of Human Eukaryotic Elongation Factor 1A Isoforms

Besides its canonical role in protein synthesis, the eukaryotic translation elongation factor 1A (eEF1A) is also involved in many other cellular processes such as cell survival and apoptosis. We showed that eEF1A phosphorylation by C-Raf in vitro occurred only in the presence of eEF1A1 and eEF1A2, thus suggesting that both isoforms interacted in cancer cells (heterodimer formation). This hypothesis was recently investigated in COS-7 cells where fluorescent recombinant eEF1A isoforms colocalized at the level of cytoplasm with a FRET signal more intense at plasma membrane level. Here, we addressed our attention in highlighting and confirming this interaction in a different cell line, HEK 293, normally expressing eEF1A1 but lacking the eEF1A2 isoform. To this end, His-tagged eEF1A2 was expressed in HEK 293 cells and found to colocalize with endogenous eEF1A1 in the cytoplasm, also at the level of cellular membranes. Moreover, FRET analysis showed, in this case, the appearance of a stronger signal mainly at the level of the plasma membrane. These results confirmed what was previously observed in COS-7 cells and strongly reinforced the interaction among eEF1A isoforms. Moreover, the formation of eEF1A heterodimer in cancer cells could also be important for cytoskeleton rearrangements rather than for phosphorylation, most likely occurring during cell survival and apoptosis

Diffusional Kurtosis Imaging of White Matter Degeneration in Glaucoma

Glaucoma is an optic neuropathy characterized by death of retinal ganglion cells and loss of their axons, progressively leading to blindness. Recently, glaucoma has been conceptualized as a more diffuse neurodegenerative disorder involving the optic nerve and also the entire brain. Consistently, previous studies have used a variety of magnetic resonance imaging (MRI) techniques and described widespread changes in the grey and white matter of patients. Diffusion kurtosis imaging (DKI) provides additional information as compared with diffusion tensor imaging (DTI), and consistently provides higher sensitivity to early microstructural white matter modification. In this study, we employ DKI to evaluate differences among healthy controls and a mixed population of primary open angle glaucoma patients ranging from stage I to V according to Hodapp−Parrish−Anderson visual field impairment classification. To this end, a cohort of patients affected by primary open angle glaucoma (n = 23) and a group of healthy volunteers (n = 15) were prospectively enrolled and underwent an ophthalmological evaluation followed by magnetic resonance imaging (MRI) using a 3T MR scanner. After estimating both DTI indices, whole-brain, voxel-wise statistical comparisons were performed in white matter using Tract-Based Spatial Statistics (TBSS). We found widespread differences in several white matter tracts in patients with glaucoma relative to controls in several metrics (mean kurtosis, kurtosis anisotropy, radial kurtosis, and fractional anisotropy) which involved localization well beyond the visual pathways, and involved cognitive, motor, face recognition, and orientation functions amongst others. Our findings lend further support to a causal brain involvement in glaucoma and offer alternative explanations for a number of multidomain impairments often observed in glaucoma patients

Chapter 9: Silica-based Nanovectors: From Mother Nature to Biomedical Applications (Book chapter)

Diatomite is a natural porous silica material of sedimentary origin, formed by remains of diatom skeletons called “frustules.” The abundance in many areas of the world and the peculiar physico-chemical properties made diatomite an intriguing material for several applications ranging from food production to pharmaceutics. However, diatomite is a material still rarely used in biomedical applications. In this chapter, the properties of diatom frustules reduced to nanoparticles, with an average diameter less than 350 nm, as potential drug vectors are described. Their biocompatibility, cellular uptake, and capability to transport molecules inside cancer cells are discussed. Preliminary studies of in vivo toxicity are also presented.Peer reviewe

Intratumor Heterogeneity of ALK-Rearrangements and Homogeneity of EGFR-Mutations in Mixed Lung Adenocarcinoma

BACKGROUND: Non Small Cell Lung Cancer is a highly heterogeneous tumor. Histologic intratumor heterogeneity could be 'major', characterized by a single tumor showing two different histologic types, and 'minor', due to at least 2 different growth patterns in the same tumor. Therefore, a morphological heterogeneity could reflect an intratumor molecular heterogeneity. To date, few data are reported in literature about molecular features of the mixed adenocarcinoma. The aim of our study was to assess EGFR-mutations and ALK-rearrangements in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas and adenosquamous carcinomas. METHODS: 590 Non Small Cell Lung Carcinomas tumor samples were revised in order to select mixed adenocarcinomas with available tumor components. Finally, only 105 mixed adenocarcinomas and 17 adenosquamous carcinomas were included in the study for further analyses. Two TMAs were built selecting the different intratumor histotypes. ALK-rearrangements were detected through FISH and IHC, and EGFR-mutations were detected through IHC and confirmed by RT-PCR. RESULTS: 10/122 cases were ALK-rearranged and 7 from those 10 showing an intratumor heterogeneity of the rearrangements. 12/122 cases were EGFR-mutated, uniformly expressing the EGFR-mutated protein in all histologic components. CONCLUSION: Our data suggests that EGFR-mutations is generally homogeneously expressed. On the contrary, ALK-rearrangement showed an intratumor heterogeneity in both mixed adenocarcinomas and adenosquamous carcinomas. The intratumor heterogeneity of ALK-rearrangements could lead to a possible impact on the therapeutic responses and the disease outcomes

Epithelial to Mesenchymal Transition by TGFβ-1 Induction Increases Stemness Characteristics in Primary Non Small Cell Lung Cancer Cell Line

Cancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line.A549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed.TGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable.The induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers

ECMO for COVID-19 patients in Europe and Israel

Since March 15th, 2020, 177 centres from Europe and Israel have joined the study, routinely reporting on the ECMO support they provide to COVID-19 patients. The mean annual number of cases treated with ECMO in the participating centres before the pandemic (2019) was 55. The number of COVID-19 patients has increased rapidly each week reaching 1531 treated patients as of September 14th. The greatest number of cases has been reported from France (n = 385), UK (n = 193), Germany (n = 176), Spain (n = 166), and Italy (n = 136) .The mean age of treated patients was 52.6 years (range 16–80), 79% were male. The ECMO configuration used was VV in 91% of cases, VA in 5% and other in 4%. The mean PaO2 before ECMO implantation was 65 mmHg. The mean duration of ECMO support thus far has been 18 days and the mean ICU length of stay of these patients was 33 days. As of the 14th September, overall 841 patients have been weaned from ECMO support, 601 died during ECMO support, 71 died after withdrawal of ECMO, 79 are still receiving ECMO support and for 10 patients status n.a. . Our preliminary data suggest that patients placed on ECMO with severe refractory respiratory or cardiac failure secondary to COVID-19 have a reasonable (55%) chance of survival. Further extensive data analysis is expected to provide invaluable information on the demographics, severity of illness, indications and different ECMO management strategies in these patients