Lysis-on-chip of single target cells following forced interaction with CTLs or NK cells on a dielectrophoresis-based array.

Guiding the interaction of single cells acting as partners in heterotypic interactions (e.g., effectors and targets of immune lysis) and monitoring the outcome of these interactions are regarded as crucial biomedical achievements. In this study, taking advantage of a dielectrophoresis (DEP)-based Laboratory-on-a-chip platform (the DEPArray), we show that it is possible to generate closed DEP cages entrapping CTLs and NK cells as either single cells or clusters; reversibly immobilize a single virus-presenting or tumor cell within the chip at a selected position; move cages and their content to predetermined spatial coordinates by software-guided routing; force a cytotoxic effector to physically interact with a putative target within a secluded area by merging their respective cages; generate cages containing effector and target cells at predetermined E:T ratios; accurately assess cytotoxicity by real-time quantitation of the release kinetics of the fluorescent dye calcein from target cells (>50 lytic events may be tested simultaneously); estimate end points of calcein release within 16 min of initial E:T cell contact; simultaneously deliver Ab-based phenotyping and on-chip lysis assessment; and identify lytic and nonlytic E:T combinations and discriminate nonlytic effector phenotypes from target refractoriness to immune lysis. The proof of principle is provided that DEPArray technology, previously used to levitate and move single cells, can be used to identify highly lytic antiviral CTLs and tumor cells that are particularly refractory to NK cell lysis. These findings are of primary interest in targeted immunotherapy.

Antibody dependent cellular cytotoxicity-inducing anti-EGFR antibodies as effective therapeutic option for cutaneous melanoma resistant to BRAF inhibitors.

Introduction: About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots. Methods and results: By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors' and patients' peripheral blood cells. Conclusion: Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

High yield patterning of single cells from extremely small populations.

Many biological assays require the ability to isolate and process single cells. Some research fields, such as the characterization of rare cells, the in vitro processing of stem cells, and the study of early stage cell differentiation, call for the additional and typically unmet ability to work with extremely low-count cell populations. In all these cases, efficient single-cell handling must be matched with the ability to work on a limited number of cells with a low cell loss rate. In this paper, we present a platform combining flow-through processing with deterministic (nonstatistical) patterning of cells coming from extremely small cell populations. We describe here modules using dielectrophoresis to control the position of cells flowing in microchannels and to pattern them in open microwells where cells were further analyzed. K562 cells continuously flowing at a speed of up to 100 µm/s were tridimensionally focused, aligned, and patterned inside microwells. A high-patterning yield and low cell loss rate were demonstrated experimentally: 15uL drops, containing an average of 15 cells, were transferred to the microchannel with an 83% yield, and cells were then patterned into microwells with a 100% yield. The deterministic patterning of cells was demonstrated both by isolating single cells in microwells and by creating clusters composed of a predetermined number of cells. Cell proliferation was assessed by easily recovering cells from open microwells, and a growth rate comparable to the control was obtained.

Programmable interactions of functionalized single bioparticles in a dielectrophoresis-based microarray chip.

Manipulating single biological objects is a major unmet challenge of biomedicine. Herein, we describe a lab-on-a-chip platform based on dielectrophoresis (DEP). The DEParray is a prototypal version consisting of 320 × 320 arrayed electrodes generating >10,000 spherical DEP cages. It allows the capture and software-guided movement to predetermined spatial coordinates of single biological objects. With the DEParray we demonstrate (a) forced interaction between a single, preselected target cell and a programmable number of either microspheres or natural killer (NK) cells, (b) on-chip immunophenotypic discrimination of individual cells based on differential rosetting with microspheres functionalized with monoclonal antibodies to an inhibitory NK cell ligand (HLA-G), (c) on-chip, real-time (few minutes) assessment of immune lysis by either visual inspection or semiautomated, time-lapse reading of a fluorescent dye released from NK cell-sensitive targets, and (d) manipulation and immunophenotyping with limiting amounts (about 500) cells. To our knowledge, this is the first report describing a DEP-based lab-on-a-chip platform for the quick, arrayed, software-guided binding of individually moved biological objects, the targeting of single cells with microspheres, and the real-time characterization of immunophenotypes. The DEParray candidates as a discovery tool for novel cell:cell interactions with no prior (immuno)phenotypic knowledge.

Autophagy in BRAF-mutant cutaneous melanoma: recent advances and therapeutic perspective.

Macroautophagy, hereafter referred to as autophagy, represents a highly conserved catabolic process that maintains cellular homeostasis. At present, the role of autophagy in cutaneous melanoma (CM) is still controversial, since it appears to be tumor-suppressive at early stages of malignant transformation and cancer-promoting during disease progression. Interestingly, autophagy has been found to be often increased in CM harboring BRAF mutation and to impair the response to targeted therapy. In addition to autophagy, numerous studies have recently conducted in cancer to elucidate the molecular mechanisms of mitophagy, a selective form of mitochondria autophagy, and secretory autophagy, a process that facilitates unconventional cellular secretion. Although several aspects of mitophagy and secretory autophagy have been investigated in depth, their involvement in BRAF-mutant CM biology has only recently emerged. In this review, we aim to overview autophagy dysregulation in BRAF-mutant CM, along with the therapeutic advantages that may arise from combining autophagy inhibitors with targeted therapy. In addition, the recent advances on mitophagy and secretory autophagy involvement in BRAF-mutant CM will be also discussed. Finally, since a number of autophagy-related non-coding RNAs (ncRNAs) have been identified so far, we will briefly discussed recent advances linking ncRNAs to autophagy regulation in BRAF-mutant CM.

LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer.

BACKGROUND AND PURPOSE: Currently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation. RESULTS: In this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival-OS: 28.1% for LINE-1 methylation < 35% vs. 69.1% for ≥ 55%; p < 0.0001). When LINE-1 methylation was dichotomized as < 55% versus ≥ 55%, interaction with HPV16 emerged: compared with hypermethylated HPV16-positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR 4.83, 95% CI 2.24-10.38) and progression (HR 4.54, 95% CI 2.18-9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53 ≥ 50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53 ≥ 50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine-phosphate-guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences. CONCLUSIONS: Evaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations and lead to altered gene expression in OPSCC.

Long Noncoding RNAs as Innovative Urinary Diagnostic Biomarkers.

The characterization of circulating tumor cells (CTCs) is now widely studied as a promising source of cancer-derived biomarkers because of their role in tumor formation and progression. However, CTCs analysis presents some limitations and no standardized method for CTCs isolation from urine has been defined so far. In fact, besides blood, urine represents an ideal source of noninvasive biomarkers, especially for the early detection of genitourinary tumors. Besides CTCs, long noncoding RNAs (lncRNAs) have also been proposed as potential noninvasive biomarkers, and the evaluation of the diagnostic accuracy of urinary lncRNAs has dramatically increased over the last years, with many studies being published. Therefore, this review provides an update on the clinical utility of urinary lncRNAs as novel biomarkers for the diagnosis of bladder and prostate cancers.

Prognostic Significance of PD-L1 Expression In Patients With Primary Oropharyngeal Squamous Cell Carcinoma: A Meta-Analysis.

BACKGROUND: At present, the prognostic significance of programmed cell death receptor ligand 1 (PD-L1) expression in oropharyngeal squamous cell carcinoma (OPSCC) patients is still controversial. In this study, we aim to synthesize relevant studies that have assessed the prognostic value of PD-L1 in patients with primary OPSCC treated according to the current standard-of-care. METHODS: A systematic search of Medline/PubMed, Cochrane, Embase, Web of Science, and Scopus was conducted to define the prognostic role of PD-L1 expression in OPSCC. All studies published before July 31, 2021 were screened. Summary hazard ratios (sHR) with 95% confidence intervals (CIs) were calculated using a random-effects model. RESULTS: A total of 1522 OPSCC patients from 12 studies were included. PD-L1 expression in OPSCC tumor cells (TCs) was significantly associated with longer overall survival (sHR=0.63, 95% CI 0.50-0.79), and progression-free survival (sHR=0.62, 95% CI 0.49-0.79). A benefit in survival was also observed in PD-L1-positive OPSCC patients who underwent surgery (sHR=0.34, 95% CI 0.18-0.65). Finally, although PD-L1-positive expression was related to better outcomes both in HPV-negative and HPV-positive OPSCC, the difference reached the statistical significance only in the HPV-positive subgroup (sHR=0.37, 95% CI 0.19-0.73). No heterogeneity emerged between studies for all considered outcomes, with I 2 ranging from 0% for progression-free survival to 11% for overall survival. CONCLUSIONS: PD-L1 expression on TCs associated with improved survival in OPSCC. In particular, HPV-positive OPSCC most benefited from PD-L1 expression when compared to the PD-L1 negative counterpart. Thus, PD-L1 might represent a useful biomarker to stratify prognosis in OPSCC in addition to HPV status.

Prognostic Nutritional Index Predicts Toxicity in Head and Neck Cancer Patients Treated with Definitive Radiotherapy in Association with Chemotherapy.

BACKGROUND: The Prognostic Nutritional Index (PNI) is a parameter of nutritional and inflammation status related to toxicity in cancer treatment. Since data for head and neck cancer are scanty, this study aims to investigate the association between PNI and acute and late toxicity for this malignancy. METHODS: A retrospective cohort of 179 head and neck cancer patients treated with definitive radiotherapy with induction/concurrent chemotherapy was followed-up (median follow-up: 38 months) for toxicity and vital status between 2010 and 2017. PNI was calculated according to Onodera formula and low/high PNI levels were defined according to median value. Odds ratio (OR) for acute toxicity were calculated through logistic regression model; hazard ratios (HR) for late toxicity and survival were calculated through the Cox proportional hazards model. RESULTS: median PNI was 50.0 (interquartile range: 45.5-53.5). Low PNI was associated with higher risk of weight loss > 10% during treatment (OR = 4.84, 95% CI: 1.73-13.53 for PNI < 50 versus PNI ≥ 50), which was in turn significantly associated with worse overall survival, and higher risk of late mucositis (HR = 1.84; 95% CI:1.09-3.12). PNI predicts acute weight loss >10% and late mucositis. CONCLUSIONS: PNI could help clinicians to identify patients undergoing radiotherapy who are at high risk of acute and late toxicity.

Horizontal nDEP cages within open microwell arrays for precise positioning of cells and particles.

We present the structure of an open microwell, i.e. a microwell open at both the top and bottom ends, which enables single-cells to be handled, processed and recovered after the experiment. The microwell has a novel architecture which allows particles to be trapped and forced to interact by means of a cylindrical negative dielectrophoretic cage. Particles are aligned along a horizontal axis where the electric field minimum is placed. Arrays of open microwells are fabricated using flexible printed circuit board (PCB) technology providing cheap and disposable devices. Levitation and precise positioning of both polystyrene beads and K562 cells were experimented, confirming the results of physical simulations. Assessment of cell viability after 20 min exposure to the electric field was performed through a standard calcein-release assay.

Effects of biomaterials for Lab-on-a-chip production on cell growth and expression of differentiated functions of leukemic cell lines.

The rapid increase of the applications for Lab-on-a-chip devices has attracted the interest of researchers and engineers on standard process of the electronics industry for low production costs and large scale development, necessary for disposable applications. The printed circuit board technology could be used for this purpose, in particular for the wide range of materials available. In this paper, assays on biocompatibility of materials used for Lab-on-a-chip fabrication has been carried out using two tumor cell lines growing in suspension, the human chronic myelogenous leukemia K562 cell line, able to undergo erythroid differentiation when cultured with chemical inducers, and the lymphoblastoid cell line (LCL), extensively used for screening of cytotoxic T-lymphocytes (CTLs). We have demonstrated that some materials strongly inhibit cell proliferation of both the two cell lines to an extent higher that 70-75%, but only after a prolonged exposure of 3-6 days (Copper, Gold over Nickel, Aramid fiber filled epoxy uncured, b-stage epoxy die attach film, Tesa 4985 adhesive tape, Pyralux uncured, Copper + 1-octodecanethiol). However, when experiments were performed with short incubation time (1 h), only Aramid fiber filled epoxy uncured was cytotoxic. Variation of the results concerning the other materials was appreciable when the experiments performed on two cell lines were compared together. Furthermore, the effects of the materials on erythroid differentiation and CTL-mediated LCL lysis confirmed, in most of the cases, the data obtained in cytotoxic and antiproliferative tests.

Beyond MicroRNAs: Emerging Role of Other Non-Coding RNAs in HPV-Driven Cancers.

Persistent infection with high-risk Human Papilloma Virus (HPV) leads to the development of several tumors, including cervical, oropharyngeal, and anogenital squamous cell carcinoma. In the last years, the use of high-throughput sequencing technologies has revealed a number of non-coding RNA (ncRNAs), distinct from micro RNAs (miRNAs), that are deregulated in HPV-driven cancers, thus suggesting that HPV infection may affect their expression. However, since the knowledge of ncRNAs is still limited, a better understanding of ncRNAs biology, biogenesis, and function may be challenging for improving the diagnosis of HPV infection or progression, and for monitoring the response to therapy of patients affected by HPV-driven tumors. In addition, to establish a ncRNAs expression profile may be instrumental for developing more effective therapeutic strategies for the treatment of HPV-associated lesions and cancers. Therefore, this review will address novel classes of ncRNAs that have recently started to draw increasing attention in HPV-driven tumors, with a particular focus on ncRNAs that have been identified as a direct target of HPV oncoproteins.

Creamino: A Cost-Effective, Open-Source EEG-Based BCI System.

This paper presents an open source framework called Creamino. It consists of an Arduino-based cost-effective quick-setup EEG platform built with off-the-shelf components and a set of software modules that easily allow users to connect this system to Simulink or BCI-oriented tools (such as BCI2000 or OpenViBE) and set up a wide number of neuroscientific experiments. Creamino is capable of processing multiple EEG channels in real-time and operates under Windows, Linux, and Mac OS X in real-time on a standard PC. Its objective is to provide a system that can be readily fabricated and used for neurophysiological experiments and, at the same time, can serve as the basis for development of novel BCI platforms by accessing and modifying its open source hardware and software libraries. Schematics, gerber files, bill of materials, source code, software modules, demonstration videos, and instructions on how to use these modules are available free of charge for research and educational purposes online at https://github.com/ArcesUnibo/creamino. Application cases show how the system can be used for neuroscientific or BCI experiments. Thanks to its low production cost and its compatibility with open-source BCI tools, the system presented is particularly suitable for use in BCI research and educational applications.

Publisher Correction: Innovative mouse model mimicking human-like features of spinal cord injury: efficacy of Docosahexaenoic acid on acute and chronic phases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Innovative mouse model mimicking human-like features of spinal cord injury: efficacy of Docosahexaenoic acid on acute and chronic phases.

Traumatic spinal cord injury has dramatic consequences and a huge social impact. We propose a new mouse model of spinal trauma that induces a complete paralysis of hindlimbs, still observable 30 days after injury. The contusion, performed without laminectomy and deriving from the pressure exerted directly on the bone, mimics more closely many features of spinal injury in humans. Spinal cord was injured at thoracic level 10 (T10) in adult anesthetized female CD1 mice, mounted on stereotaxic apparatus and connected to a precision impactor device. Following severe injury, we evaluated motor and sensory functions, and histological/morphological features of spinal tissue at different time points. Moreover, we studied the effects of early and subchronic administration of Docosahexaenoic acid, investigating functional responses, structural changes proximal and distal to the lesion in primary and secondary injury phases, proteome modulation in injured spinal cord. Docosahexaenoic acid was able i) to restore behavioural responses and ii) to induce pro-regenerative effects and neuroprotective action against demyelination, apoptosis and neuroinflammation. Considering the urgent health challenge represented by spinal injury, this new and reliable mouse model together with the positive effects of docosahexaenoic acid provide important translational implications for promising therapeutic approaches for spinal cord injuries.

Electronic sorting and recovery of single live cells from microlitre sized samples.

Sorting and recovering specific live cells from samples containing less than a few thousand cells have become major hurdles in rare cell exploration such as stem cell research, cell therapy and cell based diagnostics. We describe here a new technology based on a microelectronic chip integrating an array of over 100,000 independent electrodes and sensors which allow individual and parallel single cell manipulation of up to 10,000 cells while maintaining viability and proliferation capabilities. Manipulation is carried out using dynamic dielectrophoretic traps controlled by an electronic interface. We also demonstrate the capabilities of the chip by sorting and recovering individual live fluorescent cells from an unlabeled population.

A Driving Right Leg Circuit (DgRL) for Improved Common Mode Rejection in Bio-Potential Acquisition Systems.

The paper presents a novel Driving Right Leg (DgRL) circuit designed to mitigate the effect of common mode signals deriving, say, from power line interferences. The DgRL drives the isolated ground of the instrumentation towards a voltage which is fixed with respect to the common mode potential on the subject, therefore minimizing common mode voltage at the input of the front-end. The paper provides an analytical derivation of the common mode rejection performances of DgRL as compared to the usual grounding circuit or Driven Right Leg (DRL) loop. DgRL is integrated in a bio-potential acquisition system to show how it can reduce the common mode signal of more than 70 dB with respect to standard patient grounding. This value is at least 30 dB higher than the reduction achievable with DRL, making DgRL suitable for single-ended front-ends, like those based on active electrodes. EEG signal acquisition is performed to show how the system can successfully cancel power line interference without any need for differential acquisition, signal post-processing or filtering.

Parallel Solver for Diffuse Optical Tomography on Realistic Head Models With Scattering and Clear Regions.

Diffuse optical tomography is an imaging technique, based on evaluation of how light propagates within the human head to obtain the functional information about the brain. Precision in reconstructing such an optical properties map is highly affected by the accuracy of the light propagation model implemented, which needs to take into account the presence of clear and scattering tissues. We present a numerical solver based on the radiosity-diffusion model, integrating the anatomical information provided by a structural MRI. The solver is designed to run on parallel heterogeneous platforms based on multiple GPUs and CPUs. We demonstrate how the solver provides a 7 times speed-up over an isotropic-scattered parallel Monte Carlo engine based on a radiative transport equation for a domain composed of 2 million voxels, along with a significant improvement in accuracy. The speed-up greatly increases for larger domains, allowing us to compute the light distribution of a full human head ( ≈ 3 million voxels) in 116 s for the platform used.

Dielectrophoresis-based 'Lab-on-a-chip' devices for programmable binding of microspheres to target cells.

There is a general agreement on the fact that the Laboratory on chip (Lab-on-a-chip) technology will enable laboratory testing to move from laboratories employing complex equipments into non-laboratory settings. In this respect, dielectrophoresis (DEP) is a very valuable approach to design and produce Lab-on-a-chip devices able to manipulate microparticles and cells. In this study, we report the application of DEP-based devices for facilitating programmable interactions between microspheres and target tumor cells. We used two Lab-on-a-chip devices, one (the SmartSlide) carrying 193 parallel electrodes and generating up to 50 cylinder-shaped DEP cages, the other (the DEP array) carrying 102,400 arrayed electrodes and generating more than 10,000 spherical DEP cages. We determined whether these devices can be used to levitate and move microspheres and cells in order to obtain a forced interaction between microspheres and target cells. The first major point of this manuscript is that the DEP-based SmartSlide can be used for transfection experiments in which microspheres and target cells are forced to share the same DEP cage, leading to efficient binding of the microspheres to target cells. The data obtained using the DEP array show that this system allows the sequential, software-controlled binding of individually and independently moved single microspheres to a single target tumor cell. To our knowledge, this is the first report on the possible use of a DEP-based Lab-on-a-chip device for guided multiple binding of singularly moved microspheres to a single tumor cell. This approach can be of interest in the field of drug discovery, delivery and diagnosis.

Separation of white blood cells from erythrocytes on a dielectrophoresis (DEP) based 'Lab-on-a-chip' device.

The 'Lab-on-a-chip technology' involves miniaturization of complex analytical procedures and is expected to enable laboratory testing to move from the central laboratory employing complex equipment into non-laboratory settings. We report the application of a printed circuit board (PCB)-based chip, generating dielectrophoretic (DEP)-based cylinder-shaped cages for separation and recovery of white blood cells from erythrocytes. This possibility is of interest to develop low-cost Lab-on-a-chip devices for diagnostic purposes. Accordingly, we demonstrate that white blood cells recovered from this Lab-on-a-chip device are suitable for PCR-based molecular diagnosis procedures employing DNA sequencing or biospecific interaction analysis using surface plasmon resonance and biosensor technology.