Do DLX3 and CD271 Protect Human Keratinocytes from Squamous Tumor Development?

Well-regulated epidermal homeostasis depends on the function of different classes of factors, such as transcription regulators and receptors. Alterations in this homeostatic balance may lead to the development of cutaneous squamous tumorigenesis. The homeobox transcription factor DLX3 is determinant for a p53-dependent regulation of epidermal differentiation and modulates skin carcinogenesis. The maintenance of skin homeostasis also involves the action of neurotrophins (NTs) and their receptors, Trk and CD271. While Trk receptor overexpression is a hallmark of cancer, there are conflicting data on CD271 expression and function in cutaneous SCC (cSCC). Previous studies have reported NT receptors expression in head and neck SSC (HNSCC). We show that CD271 is expressed at low levels in primary cSCC cells and the number of CD271+ cells correlates with cell cohesion in SCC spheroids. In normal epidermis, CD271 is expressed in proliferative progenitor cells and DLX3 in terminally differentiated keratinocytes. Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) increase DLX3 expression. In the absence of a functional BDNF receptor TrkB in keratinocytes, we hypothesize that the BDNF-dependent DLX3 response could be mediated via CD271. Altogether, our results support a putative CD271-DLX3 connection in keratinocytes, which might be crucial to preventing squamous skin cancer

Towards a Universal Method for the Stable and Clean Functionalization of Inert Perfluoropolymer Nanoparticles : Exploiting Photopolymerizable Amphiphilic Diacetylenes

Highly fluorinated materials are being widely investigated due to a number of peculiar properties, which are potentially useful for various applications, including use as lubricants, anti-adhesive films, and substitutes for biological fluids for biomedical utilization. However, at present such potential is still poorly exploited. One of the major drawbacks that hampers the rapid development of nanoscale fluoro-hybrid devices is the remarkable inertness of perfluoropolymeric materials that lack reactive functionalities, as they do not offer any functional groups that can be employed to covalently anchor organic molecules on their surface. In this paper, a convenient method for the stable biofunctionalization of strongly unreactive perfluoropolymer nanoparticles (PnPs) is reported. PnPs are easily coated with newly synthesized asymmetric diacetylenic monomer compounds (ADMs), thanks to PnP's high propensity to interact with hydrophobic moieties. Once monomerically adsorbed onto PnPs, such suitably designed ADMs enable the formation of a robust polymeric shell around the perfluoroelastomer core via a clean UV-promoted localized photopolymerization. Given the peculiar optical characteristics of PnPs, the coating of the particles can be monitored step by step using light scattering, which also allows estimation of the fraction of reacted monomers by competitive adsorption with smaller particles. The potential of this method for the biofunctionalization of PnPs is demonstrated with representative proteins and carbohydrates. Among them, the extension to avidin-biotin technology may broaden the scope and applicability of this strategy to potentially a large number of molecules of biomedical interest. Making the unreactive reactive: A smart method for the biofunctionalization of strongly inert perfluoropolymer nanoparticles (PnPs) is presented, using a stable coating with novel diacetylenic compounds followed by clean UV photopolymerization to generate reactive functionalities on the PnP surface. This method further allows fine tuning of the amount of conjugated biomolecules, which can be sensitively and straightforwardly quantified

CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes

Background: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. Methods: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. Results: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. Conclusion: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development

Impact of doxorubicin-loaded ferritin nanocages (FerOX) vs. free doxorubicin on T lymphocytes: a translational clinical study on breast cancer patients undergoing neoadjuvant chemotherapy

Despite the advent of numerous targeted therapies in clinical practice, anthracyclines, including doxorubicin (DOX), continue to play a pivotal role in breast cancer (BC) treatment. DOX directly disrupts DNA replication, demonstrating remarkable efficacy against BC cells. However, its non-specificity toward cancer cells leads to significant side effects, limiting its clinical utility. Interestingly, DOX can also enhance the antitumor immune response by promoting immunogenic cell death in BC cells, thereby facilitating the presentation of tumor antigens to the adaptive immune system. However, the generation of an adaptive immune response involves highly proliferative processes, which may be adversely affected by DOX-induced cytotoxicity. Therefore, understanding the impact of DOX on dividing T cells becomes crucial, to deepen our understanding and potentially devise strategies to shield anti-tumor immunity from DOX-induced toxicity. Our investigation focused on studying DOX uptake and its effects on human lymphocytes. We collected lymphocytes from healthy donors and BC patients undergoing neoadjuvant chemotherapy (NAC). Notably, patient-derived peripheral blood mononuclear cells (PBMC) promptly internalized DOX when incubated in vitro or isolated immediately after NAC. These DOX-treated PBMCs exhibited significant proliferative impairment compared to untreated cells or those isolated before treatment initiation. Intriguingly, among diverse lymphocyte sub-populations, CD8 + T cells exhibited the highest uptake of DOX. To address this concern, we explored a novel DOX formulation encapsulated in ferritin nanocages (FerOX). FerOX specifically targets tumors and effectively eradicates BC both in vitro and in vivo. Remarkably, only T cells treated with FerOX exhibited reduced DOX internalization, potentially minimizing cytotoxic effects on adaptive immunity

High-Resolution Raman Imaging of >300 Patient-Derived Cells from Nine Different Leukemia Subtypes:A Global Clustering Approach

Leukemia comprises a diverse group of bone marrow tumors marked by cell proliferation. Current diagnosis involves identifying leukemia subtypes through visual assessment of blood and bone marrow smears, a subjective and time-consuming method. Our study introduces the characterization of different leukemia subtypes using a global clustering approach of Raman hyperspectral maps of cells. We analyzed bone marrow samples from 19 patients, each presenting one of nine distinct leukemia subtypes, by conducting high spatial resolution Raman imaging on 319 cells, generating over 1.3 million spectra in total. An automated preprocessing pipeline followed by a single-step global clustering approach performed over the entire data set identified relevant cellular components (cytoplasm, nucleus, carotenoids, myeloperoxidase (MPO), and hemoglobin (HB)) enabling the unsupervised creation of high-quality pseudostained images at the single-cell level. Furthermore, this approach provided a semiquantitative analysis of cellular component distribution, and multivariate analysis of clustering results revealed the potential of Raman imaging in leukemia research, highlighting both advantages and challenges associated with global clustering.</p

A Bioorthogonal Probe for Multiscale Imaging by 19F-MRI and Raman Microscopy: From Whole Body to Single Cells

Molecular imaging techniques are essential tools for better investigating biological processes and detecting disease biomarkers with improvement of both diagnosis and therapy monitoring. Often, a single imaging technique is not sufficient to obtain comprehensive information at different levels. Multimodal diagnostic probes are key tools to enable imaging across multiple scales. The direct registration of in vivo imaging markers with ex vivo imaging at the cellular level with a single probe is still challenging. Fluorinated (19F) probes have been increasingly showing promising potentialities for in vivo cell tracking by 19F-MRI. Here we present the unique features of a bioorthogonal 19F-probe that enables direct signal correlation of MRI with Raman imaging. In particular, we reveal the ability of PERFECTA, a superfluorinated molecule, to exhibit a remarkable intense Raman signal distinct from cell and tissue fingerprints. Therefore, PERFECTA combines in a single molecule excellent characteristics for both macroscopic in vivo19F-MRI, across the whole body, and microscopic imaging at tissue and cellular levels by Raman imaging

Determination of the quality of lipoproteins by Raman spectroscopy in obese and healthy subjects

Lipoproteins (LPs) are multimolecular complexes of lipids and proteins responsible for transporting fatty acids, cholesterol, and micronutrients (carotenoids) through the body. The quantification of triglycerides and cholesterol carried by lipoproteins is a leading clinical parameter to assess the increased risk of cardiovascular events. However, in recent times, the study of the overall "quality" of lipoproteins, defined by their biochemical composition and oxidation state, has emerged as necessary to improve the definition of the cardiovascular risk. In this work, we present Raman spectroscopy (RS) as an effective method to immediately detect the functional groups relative to the principal biochemical components and the level of unsaturated lipids present in LPs. Furthermore, we show how RS can reveal the differences in the biochemical composition and oxidation state of LPs extracted from a cohort of obese patients (Ob) and a control group of healthy subjects (HC). In particular, RS revealed how low-density lipoproteins (LDLs) from obese patients are enriched in triglycerides and more oxidized than those from the control group, while high-density lipoproteins (HDLs) from Ob patients were depleted in cholesterol and phospholipids. RS analysis also allowed the study of the relationship between the levels of carotenoids present in the different classes of LPs highlighting how this parameter depends on the disease severity. Overall, these results demonstrated that RS is a viable approach for quickly and effectively gaining information on LPs' biochemical composition and oxidation state, providing an immediate measure of their quality. Besides, RS further proved the role of LPs in obesity and metabolic dysfunctions

Metabolic Profiling of Type 2 Diabetes Patients after Bariatric Surgery by Raman Spectroscopy

Background: Bariatric surgery (BS) is an important procedure used for the treatment of morbid obesity and has been proven to improve, or even cure, type 2 diabetes mellitus (T2DM). However, of the patients with T2DM who initially go into remission, a proportion experience a relapse during the follow-up. In this context, Raman spectroscopy (RS) could be a promising technique for monitoring the metabolic profile of patients after surgical treatment with the aim of improving their postsurgical management. Methods: Fourteen obese patients with T2DM were recruited. Clinical parameters, adipokines, ghrelin, Fibroblast growth factor 19 (FGF-19) values, and Raman spectra were collected and analyzed before and after surgery. RS results were compared with profiles obtained from 23 healthy subjects (HC), to observe whether the metabolic fingerprint of bariatric patients normalized during the surgical follow-up. Results: The reduction in anthropometric measures and improved glycemic control and lipid profile after surgical treatment highlighted the benefits of BS. Consequently, adipokines, ghrelin and FGF-19 concentration returned to normal values after surgery. However, RS data highlighted an altered metabolic profile even after BS. Conclusion: RS suggests that BS does not fully restore the metabolic profile of patients in the immediate follow-up after the surgery

Stable and scalable SERS tags conjugated with neutravidin for the detection of fibroblast activation protein (FAP) in primary fibroblasts

SERS tags are a class of nanoparticles with great potential in advanced imaging experiments. The preparation of SERS tags however is complex, as they suffer from the high variability of the SERS signals observed even at the slightest sign of aggregation. Here, we developed a method for the preparation of SERS tags based on the use of gold nanostars conjugated with neutravidin. The SERS tags here obtained are extremely stable in all biological buffers commonly employed and can be prepared at a relatively large scale in very mild conditions. The obtained SERS tags have been used to monitor the expression of fibroblast activation protein alpha (FAP) on the membrane of primary fibroblasts obtained from patients affected by Crohn’s disease. The SERS tags allowed the unambiguous identification of FAP on the surface of cells thus suggesting the feasibility of semi-quantitative analysis of the target protein. Moreover, the use of the neutravidin–biotin system allows to apply the SERS tags for any other marker detection, for example, different cancer cell types, simply by changing the biotinylated antibody chosen in the analysis