Viral Entry Inhibitors Protect against SARS-CoV-2-Induced Neurite Shortening in Differentiated SH-SY5Y Cells.

The utility of human neuroblastoma cell lines as in vitro model to study neuro-invasiveness and neuro-virulence of SARS-CoV-2 has been demonstrated by our laboratory and others. The aim of this report is to further characterize the associated cellular responses caused by a pre-alpha SARS-CoV-2 strain on differentiated SH-SY5Y and to prevent its cytopathic effect by using a set of entry inhibitors. The susceptibility of SH-SY5Y to SARS-CoV-2 was confirmed at high multiplicity-of-infection, without viral replication or release. Infection caused a reduction in the length of neuritic processes, occurrence of plasma membrane blebs, cell clustering, and changes in lipid droplets electron density. No changes in the expression of cytoskeletal proteins, such as tubulins or tau, could explain neurite shortening. To counteract the toxic effect on neurites, entry inhibitors targeting TMPRSS2, ACE2, NRP1 receptors, and Spike RBD were co-incubated with the viral inoculum. The neurite shortening could be prevented by the highest concentration of camostat mesylate, anti-RBD antibody, and NRP1 inhibitor, but not by soluble ACE2. According to the degree of entry inhibition, the average amount of intracellular viral RNA was negatively correlated to neurite length. This study demonstrated that targeting specific SARS-CoV-2 host receptors could reverse its neurocytopathic effect on SH-SY5Y.

Pharmacological targeting of netrin-1 inhibits EMT in cancer.

Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFß1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.

Netrin-1 blockade inhibits tumour growth and EMT features in endometrial cancer.

Netrin-1 is upregulated in cancers as a protumoural mechanism1. Here we describe netrin-1 upregulation in a majority of human endometrial carcinomas (ECs) and demonstrate that netrin-1 blockade, using an anti-netrin-1 antibody (NP137), is effective in reduction of tumour progression in an EC mouse model. We next examined the efficacy of NP137, as a first-in-class single agent, in a Phase I trial comprising 14 patients with advanced EC. As best response we observed 8 stable disease (8 out of 14, 57.1%) and 1 objective response as RECIST v.1.1 (partial response, 1 out of 14 (7.1%), 51.16% reduction in target lesions at 6 weeks and up to 54.65% reduction during the following 6 months). To evaluate the NP137 mechanism of action, mouse tumour gene profiling was performed, and we observed, in addition to cell death induction, that NP137 inhibited epithelial-to-mesenchymal transition (EMT). By performing bulk RNA sequencing (RNA-seq), spatial transcriptomics and single-cell RNA-seq on paired pre- and on-treatment biopsies from patients with EC from the NP137 trial, we noted a net reduction in tumour EMT. This was associated with changes in immune infiltrate and increased interactions between cancer cells and the tumour microenvironment. Given the importance of EMT in resistance to current standards of care2, we show in the EC mouse model that a combination of NP137 with carboplatin-paclitaxel outperformed carboplatin-paclitaxel alone. Our results identify netrin-1 blockade as a clinical strategy triggering both tumour debulking and EMT inhibition, thus potentially alleviating resistance to standard treatments.

A clinicopathological description of COVID-19-induced chilblains (COVID-toes) correlated with a published literature review.

BACKGROUND: The abundance of publications of COVID-19-induced chilblains has resulted in a confusing situation. METHODS: This is a prospective single-institution study from 15 March to 13 May 2020. Thirty-two patients received PCR nasopharyngeal swabs. Of these, 28 patients had a thoracic CT-scan, 31 patients had blood and urine examinations, 24 patients had skin biopsies including immunohistochemical and direct immunofluorescence studies, and four patients had electron microscopy. RESULTS: COVID-19-induced chilblains are clinically and histopathologically identical to chilblains from other causes. Although intravascular thrombi are sometimes observed, no patient had a systemic coagulopathy or severe clinical course. The exhaustive clinical, radiological, and laboratory work-up in this study ruled-out other primary and secondary causes. Electron microscopy revealed rare, probable viral particles whose core and spikes measured from 120 to 133 nm within endothelium and eccrine glands in two cases. CONCLUSION: This study provides further clinicopathologic evidence of COVID-19-related chilblains. Negative PCR and antibody tests do not rule-out infection. Chilblains represent a good prognosis, occurring later in the disease course. No systemic coagulopathy was identified in any patient. Patients presenting with acral lesions should be isolated, and chilblains should be distinguished from thrombotic lesions (livedo racemosa, retiform purpura, or ischemic acral necrosis).

Deciphering functional tumor states at single-cell resolution.

Within a tumor, cancer cells exist in different states that are associated with distinct tumor functions, including proliferation, differentiation, invasion, metastasis, and resistance to anti-cancer therapy. The identification of the gene regulatory networks underpinning each state is essential for better understanding functional tumor heterogeneity and revealing tumor vulnerabilities. Here, we review the different studies identifying tumor states by single-cell sequencing approaches and the mechanisms that promote and sustain these functional states and regulate their transitions. We also describe how different tumor states are spatially distributed and interact with the specific stromal cells that compose the tumor microenvironment. Finally, we discuss how the understanding of tumor plasticity and transition states can be used to develop new strategies to improve cancer therapy.

NR2F2 controls malignant squamous cell carcinoma state by promoting stemness and invasion and repressing differentiation.

The nongenetic mechanisms required to sustain malignant tumor state are poorly understood. During the transition from benign tumors to malignant carcinoma, tumor cells need to repress differentiation and acquire invasive features. Using transcriptional profiling of cancer stem cells from benign tumors and malignant skin squamous cell carcinoma (SCC), we identified the nuclear receptor NR2F2 as uniquely expressed in malignant SCC. Using genetic gain of function and loss of function in vivo, we show that NR2F2 is essential for promoting the malignant tumor state by controlling tumor stemness and maintenance in mouse and human SCC. We demonstrate that NR2F2 promotes tumor cell proliferation, epithelial-mesenchymal transition and invasive features, while repressing tumor differentiation and immune cell infiltration by regulating a common transcriptional program in mouse and human SCCs. Altogether, we identify NR2F2 as a key regulator of malignant cancer stem cell functions that promotes tumor renewal and restricts differentiation to sustain a malignant tumor state.

Fat1 deletion promotes hybrid EMT state, tumour stemness and metastasis.

FAT1, which encodes a protocadherin, is one of the most frequently mutated genes in human cancers1-5. However, the role and the molecular mechanisms by which FAT1 mutations control tumour initiation and progression are poorly understood. Here, using mouse models of skin squamous cell carcinoma and lung tumours, we found that deletion of Fat1 accelerates tumour initiation and malignant progression and promotes a hybrid epithelial-to-mesenchymal transition (EMT) phenotype. We also found this hybrid EMT state in FAT1-mutated human squamous cell carcinomas. Skin squamous cell carcinomas in which Fat1 was deleted presented increased tumour stemness and spontaneous metastasis. We performed transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies, which revealed that loss of function of FAT1 activates a CAMK2-CD44-SRC axis that promotes YAP1 nuclear translocation and ZEB1 expression that stimulates the mesenchymal state. This loss of function also inactivates EZH2, promoting SOX2 expression, which sustains the epithelial state. Our comprehensive analysis identified drug resistance and vulnerabilities in FAT1-deficient tumours, which have important implications for cancer therapy. Our studies reveal that, in mouse and human squamous cell carcinoma, loss of function of FAT1 promotes tumour initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state.

Direct examination, histopathology and fungal culture for the diagnosis of onychomycosis: A retrospective, comparative study on 2245 specimens.

BACKGROUND: Onychomycosis affects 5.5% of the general population and represents up to 50% of all nail diseases. Diagnosis and pathogen identification are essential in order to plan an adequate treatment. Many diagnostic techniques are available, and however, no solid data regarding comparison between different techniques over a large number of specimens are available to date. OBJECTIVES: To compare sensitivity and specificity of direct examination, histopathology and fungal culture in our referral mycology laboratory. METHODS: Nail specimens received at the cutaneous pathology and mycology laboratory of the University Hospital Saint-Pierre (Brussels, Belgium) between 1 January and 15 May 2018 were retrospectively analysed. All specimens were submitted to direct examination and culture. In cases of adequate specimen size, histopathology was performed. Fungal culture was considered the gold standard for diagnosis. RESULTS: A total of 2245 nail samples were included in the study. Onychomycosis was diagnosed in 1266 specimens. Sensitivity and positive predictive value were found to be higher for direct examination compared to histopathology, while sensitivity of direct examination was found to be lower. Combined approach with all the three techniques showed the highest rate of positivity, followed by the association of direct examination and histopathology. CONCLUSIONS: To our knowledge, this study included the largest number of nail specimens to date, allowing a comparison between direct examination, culture and histopathology. Direct examination showed to be the most performing technique in routine practice. Histopathology represents the most effective option in cases where both specimen size and laboratory resources are adequate. Our paper adds to the literature the 'real-life' experience of the mycology laboratory of a referral centre for nail diseases.

A Novel Approach for Quantifying Cancer Cells Showing Hybrid Epithelial/Mesenchymal States in Large Series of Tissue Samples: Towards a New Prognostic Marker.

In cancer biology, epithelial-to-mesenchymal transition (EMT) is associated with tumorigenesis, stemness, invasion, metastasis, and resistance to therapy. Evidence of co-expression of epithelial and mesenchymal markers suggests that EMT should be a stepwise process with distinct intermediate states rather than a binary switch. In the present study, we propose a morphological approach that enables the detection and quantification of cancer cells with hybrid E/M states, i.e., which combine partially epithelial (E) and partially mesenchymal (M) states. This approach is based on a sequential immunohistochemistry technique performed on the same tissue section, the digitization of whole slides, and image processing. The aim is to extract quantitative indicators able to quantify the presence of hybrid E/M states in large series of human cancer samples and to analyze their relationship with cancer aggressiveness. As a proof of concept, we applied our methodology to a series of about a hundred urothelial carcinomas and demonstrated that the presence of cancer cells with hybrid E/M phenotypes at the time of diagnosis is strongly associated with a poor prognostic value, independently of standard clinicopathological features. Although validation on a larger case series and other cancer types is required, our data support the hybrid E/M score as a promising prognostic biomarker for carcinoma patients.

Perineural Invasion as a Clue to Malignant Behavior in a Dermatofibroma.

Dermatofibroma (DF) represents one of the most common mesenchymal proliferations of the skin. Their recurrence rate, even when incompletely excised, is very low, whereas the atypical, aneurysmal, and cellular variants have recurrence rates of up to 20% each. Extraordinary rare malignant lesions with metastases to lymph nodes and/or lung have been described. We report a 64-year-old woman with a long history (years) of a skin lesion on her right arm that became painful during the last months. Histologically, it consisted of a conventional cellular DF in which perineural invasion was present. Subsequently, the lesion showed a clinically aggressive course with recurrences, sarcomatous transformation, and pulmonary metastases. Given that no predictive morphological features have been identified to separate classical benign DF from rare metastasizing forms, perineural invasion in an otherwise conventional DF could be a histopathologic clue for an adverse prognosis and should provoke a closer clinical follow-up.

Context Dependency of Epithelial-to-Mesenchymal Transition for Metastasis.

Epithelial-to-mesenchymal transition (EMT) has been proposed to be important for metastatic dissemination. However, recent studies have challenged the requirement of EMT for metastasis. Here, we assessed in different models of primary skin squamous cell carcinomas (SCCs) whether EMT is associated with metastasis. The incidence of metastasis was much higher in SCCs presenting EMT compared to SCCs without EMT, supporting the notion that a certain degree of EMT is required to initiate the metastatic cascade in primary skin SCCs. Most circulating tumor cells presented EMT, whereas most lung metastasis did not present EMT, showing that mesenchymal-to-epithelial transition is important for metastatic colonization. In contrast, immunodeficient mice transplanted with SCCs, whether displaying EMT or not, presented metastasis. Altogether, our data demonstrate that the association of EMT and metastasis is model dependent, and metastasis of primary skin SCCs is associated with EMT.

Phenotypic Plasticity: Driver of Cancer Initiation, Progression, and Therapy Resistance.

Our traditional understanding of phenotypic plasticity in adult somatic cells comprises dedifferentiation and transdifferentiation in the context of tissue regeneration or wound healing. Although dedifferentiation is central to tissue repair and stemness, this process inherently carries the risk of cancer initiation. Consequently, recent research suggests phenotypic plasticity as a new paradigm for understanding cancer initiation, progression, and resistance to therapy. Here, we discuss how cells acquire plasticity and the role of plasticity in initiating cancer, cancer progression, and metastasis and in developing therapy resistance. We also highlight the epithelial-to-mesenchymal transition (EMT) and known molecular mechanisms underlying plasticity and we consider potential therapeutic avenues.

EMT Transition States during Tumor Progression and Metastasis.

Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells acquire mesenchymal features. In cancer, EMT is associated with tumor initiation, invasion, metastasis, and resistance to therapy. Recently, it has been demonstrated that EMT is not a binary process, but occurs through distinct cellular states. Here, we review the recent studies that demonstrate the existence of these different EMT states in cancer and the mechanisms regulating their functions. We discuss the different functional characteristics, such as proliferation, propagation, plasticity, invasion, and metastasis associated with the distinct EMT states. We summarize the role of the transcriptional and epigenetic landscapes, gene regulatory network and their surrounding niche in controlling the transition through the different EMT states.

Identification of the tumour transition states occurring during EMT.

In cancer, the epithelial-to-mesenchymal transition (EMT) is associated with tumour stemness, metastasis and resistance to therapy. It has recently been proposed that, rather than being a binary process, EMT occurs through distinct intermediate states. However, there is no direct in vivo evidence for this idea. Here we screen a large panel of cell surface markers in skin and mammary primary tumours, and identify the existence of multiple tumour subpopulations associated with different EMT stages: from epithelial to completely mesenchymal states, passing through intermediate hybrid states. Although all EMT subpopulations presented similar tumour-propagating cell capacity, they displayed differences in cellular plasticity, invasiveness and metastatic potential. Their transcriptional and epigenetic landscapes identify the underlying gene regulatory networks, transcription factors and signalling pathways that control these different EMT transition states. Finally, these tumour subpopulations are localized in different niches that differentially regulate EMT transition states.

Heteronanoparticles by self-Assembly of Doxorubicin and Cyclopamine Conjugates.

The preparation of heteronanoparticles (NPs) with doxorubicin (DOXO) and cyclopamine (CYP) conjugates is presented. Biological evaluation on A431 cell lines confirms the maintenance of the activity of the parental drugs. The in vivo study shows that self-assembled NPs reduce tumor growth and toxicity of chemotherapy.

[Apocrine hidrocystoma associated with gouty tophi of the pinna]. / Hidrocistoma apocrino asociado con tofo gotoso en pabellón auricular.

BACKGROUND: Apocrine hidrocystomas, also known as apocrine cystadenomas, are bening cystic tumours derived from the secretory portion of apocrine sweat glands. CLINICAL CASE: A 78-year old female was referred to our division for assesment an asymptomatic translucent, well-defined cystic lesion located on the upper helix. The histological features were consistent with apocrine hidrocystoma coexisting with gouty tophi. CONCLUSIONS: We report the second case of apocrine hydrocystoma located in the pinna, outside of ear canal and the first case of its association with gouty tophi described. It is difficult to know which lesion was first established and if the tophi may lead to ductal obstruction and subsequent cystic retention.

Introducción: los hidrocistomas apocrinos, también conocidos como cistoadenomas apocrinos, son lesiones quísticas benignas derivadas de la porción secretora de las glándulas apocrinas. Caso clínico: remiten al servicio de Dermatología a una mujer de 78 años para valorar una lesión asintomática translúcida, bien definida, localizada en la porción superior del hélix. Los hallazgos histológicos fueron compatibles con un hidrocistoma apocrino asociado a tofo gotoso. Conclusiones: reportamos el segundo caso de hidrocistoma apocrino localizado en el pabellón auricular externo y el primer caso asociado a tofo gotoso. Es difícil de conocer qué lesión se estableció primero, y si el tofo gotoso pudo provocar una obstrucción ductal con la subsiguiente retención quística.

Increased Angiogenesis and Lymphangiogenesis in Metastatic Sentinel Lymph Nodes Is Associated With Nonsentinel Lymph Node Involvement and Distant Metastasis in Patients With Melanoma.

Lymph node angio- and lymphangio-genesis have been shown to play an important role in the premetastatic niche of sentinel lymph nodes. In the current study we have investigated the association of angio- and lympangio-genesis related parameters in metastatic sentinel lymph nodes of patients with melanoma with the presence of nonsentinel and distant organ metastasis. Peritumoral and intratumoral relative blood and lymphatic vessel areas (evaluated by Chalkley method), blood and lymphatic microvessel densities, and the rates of blood and lymphatic vessel proliferation were assessed in primary tumors and sentinel lymph node metastasis of 44 patients with melanoma using CD34/Ki-67 and D240/Ki-67 immunohistochemical double staining. Primary melanoma exhibited significantly higher rate of lymphatic proliferation compared with its lymph node metastasis (P < 0.05), while lymph node metastasis showed significantly higher rate of blood vessel proliferation (P < 0.05). Using multivariate logistic regression model, the rate of peritumoral lymphatic proliferation was inversely associated with positive nonsentinel lymph node status (P < 0.05), whereas the rate of intratumoral blood vessel proliferation was associated with distant organ metastasis (P < 0.05). Using multivariate Cox regression analysis, the rate of intratumoral blood vessel proliferation was also inversely associated with overall survival of patients with melanoma (P < 0.05).

Peritumoral D2-40 Chalkley score independently predicts metastases and survival in patients with cutaneous malignant melanoma.

BACKGROUND: Many observational studies investigated the prognostic significance of angiogenesis and lymphangiogenesis in patients with melanoma. However, the obtained results are rather contradictory, probably due to the lack of the consensus methodology. METHODS: To investigate the prognostic significance of angiogenesis and lymphangiogenesis-related parameters in patients with melanoma, we performed a retrospective investigation following the consensus recommendations for angiogenesis and lymphangiogenesis quantification in solid tumors and reporting recommendations for tumor marker (REMARK) criteria for reporting the results. Blood and lymphatic vessel Chalkley scores, endothelial cell proliferation fractions and microvessel densities were quantified using a double immunostaining for endothelial marker CD34 or lymphendothelial marker D240 and the proliferation marker Ki-67 in 196 patients with melanoma. These parameters were evaluated separately for peritumoral (PT) and intratumoral areas and were correlated with outcome. RESULTS: In multivariate analysis PT D240 Chalkley score was identified as a strongest predictor for sentinel lymph node metastases, non-sentinel lymph node metastases, distant metastases, disease free survival and overall survival in patients with melanoma. CONCLUSIONS: If additional studies corroborate our findings, we believe that the inclusion of PT D240 Chalkley counts to the routine pathology examination of melanoma samples would provide additional information for identifying high-risk patients.

Melanomas arising on skin with chronic sun-induced damage exhibit low degree of angiogenesis and lymphangiogenesis.

Differences in gene expression between melanomas arising on skin intermittently and chronically sun-exposed areas were described. Additionally, several studies have shown differences in clinical characteristics and prognosis, suggesting distinct biological pathways in the development of these tumors. We performed a retrospective investigation aimed on evaluation of the differences in angiogenesis and lymphangiogenesis between melanomas arising on skin with and without signs of chronic sun-induced damage. For that purpose, we evaluated relative blood and lymphatic vessel areas, blood and lymphatic endothelial cell proliferation fractions, separately for peritumoral and intratumoral areas. We have shown that melanomas arising on sun-exposed skin exhibit lower angiogenic and lymphangiogenic potentials and better prognosis than those arising on skin without signs of chronic sun-induced damage.