Basosquamous carcinoma (BSC) is an aggressive skin neoplasm with the features of both basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). While genetic drivers of BCC and SCC development ...have been extensively characterized, BSC has not been well studied, and it remains unclear whether these tumors originally derive from BCC or SCC. In addition, it is unknown which molecular pathways mediate the reprogramming of tumor keratinocytes toward basaloid or squamatized phenotypes. We sought to characterize the genomic alterations underlying sporadic BSC to elucidate the derivation of these mixed tumors. We identifed frequent Hedgehog (Hh) pathway mutations in BSCs, implicating Hh deregulation as the primary driving event in BSC. Principal component analysis of BCC and SCC driver genes further demonstrate the genetic similarity between BCC and BSC. In addition, 45% of the BSCs harbor recurrent mutations in the SWI/SNF complex gene, ARID1A, and evolutionary analysis revealed that ARID1A mutations occur after PTCH1 but before SCC driver mutations, indicating that ARID1A mutations may bestow plasticity enabling squamatization. Finally, we demonstrate mitogen-activated protein kinase pathway activation and the loss of Hh signaling associated with the squamatization of BSCs. Overall, these results support the genetic derivation of BSCs from BCCs and highlight potential factors involved in modulating tumor reprogramming between basaloid and squamatized phenotypes.
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas ...(SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.
A traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs).
...We examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing.
We found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC.
Contrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro.
In this issue, Albibas et al. investigate the mutational nature of p53-immunopositive patches, commonly observed in sun-damaged skin. p53-immunopositive patches have long been suspected to be lineal ...precursors to actinic keratoses and cutaneous squamous cell carcinomas. However, the mutations actually giving rise to p53-immunopositive patches, and their relationship to skin cancer, have never been defined. The considerable clinical and economic costs of monitoring and treating sun-damaged skin demand we better understand the evolution of these common premalignancies.
Single-cell RNA sequencing (scRNA-seq) allows for high-resolution analysis of transcriptionally dysregulated cell subpopulations in inflammatory diseases. However, it can be challenging to properly ...isolate viable immune cells from human skin for scRNA-seq due to its barrier properties. Here, we present a protocol to isolate high-viability human cutaneous immune cells. We describe steps for obtaining and enzymatically dissociating a skin biopsy specimen and isolating immune cells using flow cytometry. We then provide an overview of downstream computational techniques to analyze sequencing data.
For complete details on the use and execution of this protocol, please refer to Cook et al. (2022)1 and Liu et al. (2022).2
Display omitted
•Isolation of viable immune cells from human skin for single-cell RNA sequencing•Manual dissociation/enzymatic digestion without proprietary reagents•Basic workflow for computational analysis of immune cell subpopulations
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Single-cell RNA sequencing (scRNA-seq) allows for high-resolution analysis of transcriptionally dysregulated cell subpopulations in inflammatory diseases. However, it can be challenging to properly isolate viable immune cells from human skin for scRNA-seq due to its barrier properties. Here, we present a protocol to isolate high-viability human cutaneous immune cells. We describe steps for obtaining and enzymatically dissociating a skin biopsy specimen and isolating immune cells using flow cytometry. We then provide an overview of downstream computational techniques to analyze sequencing data.
Genetic variation in the NF-κB inhibitors, ABIN1 and A20, increase risk for psoriasis. While critical for hematopoietic immune cell function, these genes are believed to additionally inhibit ...psoriasis by dampening inflammatory signaling in keratinocytes. We dissected ABIN1 and A20’s regulatory role in human keratinocyte inflammation using an RNA sequencing–based comparative genomic approach. Here we show subsets of the IL-17 and tumor necrosis factor–α signaling pathways are robustly restricted by A20 overexpression. In contrast, ABIN1 overexpression inhibits these genes more modestly for IL-17, and weakly for tumor necrosis factor–α. Our genome-scale analysis also indicates that inflammatory program suppression appears to be the major transcriptional influence of A20/ABIN1 overexpression, without obvious influence on keratinocyte viability genes. Our findings thus enable dissection of the differing anti-inflammatory mechanisms of two distinct psoriasis modifiers, which may be directly exploited for therapeutic purposes. Importantly, we report that IL-17–induced targets of A20 show similar aberrant epidermal layer-specific transcriptional upregulation in keratinocytes from diseases as diverse as psoriasis, atopic dermatitis, and erythrokeratodermia variabilis, suggesting a contributory role for epidermal inflammation in a broad spectrum of rashes.
Ivermectin is the only drug currently recommended for the treatment of onchocerciasis, the second leading infectious cause of blindness in the world. This drug kills only the first stage ...larvae-microfilariae (mf) of Onchocerca volvulus and is to be used cautiously in areas where Loa loa is prevalent because of severe adverse events observed with coinfected patients.
This study investigated the anti-filarial activities of two Cameroonian medicinal plants, Lantana camara and Tamarindus indica locally used to treat onchocerciasis. Twelve (12) extracts were prepared and tested in vitro on the bovine model parasite, O. ochengi as well as L. loa mf. Both mf and adult male worm viabilities were assessed by motility scoring, while adult female worm viability was determined biochemically by standard MTT/formazan colorimetry. Cytotoxicity and acute toxicity were determined respectively, in monkey kidney epithelial cells and in BALB/c mice. Pure compounds were isolated by LC/MS using a bio-assay guided strategy. All the extracts showed 100% activity at 500 μg/mL against O. ochengi adult worms and mf. The highest activity against O. ochengi was observed with the hexane extract of L. camara leaves (LCLhex), with IC50 of 35.1 μg/mL for adult females and 3.8 μg/mL for the mf. Interestingly, this extract was more active against O. ochengi mf than L. loa mf. Further studies on the extracts led to the isolation of lantadene A from the methylene chloride extract of L. camara leaves, with IC50s of 7.85 μg/mL for adult males, 10.38 μg/mL for adult females, 10.84 μg/mL for O. ochengi mf and 20.13 μg/mL for L. loa mf.
We report for the first time the anti-onchocercal activities of these locally consumed medicinal plants and lantadene A, a potential lead for further development as an onchocerciasis cure.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Inflammatory conditions represent the largest class of chronic skin disease, but the molecular dysregulation underlying many individual cases remains unclear. Single-cell RNA sequencing (scRNA-seq) ...has increased precision in dissecting the complex mixture of immune and stromal cell perturbations in inflammatory skin disease states. We single-cell-profiled CD45
immune cell transcriptomes from skin samples of 31 patients (7 atopic dermatitis, 8 psoriasis vulgaris, 2 lichen planus (LP), 1 bullous pemphigoid (BP), 6 clinical/histopathologically indeterminate rashes, and 7 healthy controls). Our data revealed active proliferative expansion of the T
and Trm components and universal T cell exhaustion in human rashes, with a relative attenuation of antigen-presenting cells. Skin-resident memory T cells showed the greatest transcriptional dysregulation in both atopic dermatitis and psoriasis, whereas atopic dermatitis also demonstrated recurrent abnormalities in ILC and CD8
cytotoxic lymphocytes. Transcript signatures differentiating these rash types included genes previously implicated in T helper cell (T
2)/T
17 diatheses, segregated in unbiased functional networks, and accurately identified disease class in untrained validation data sets. These gene signatures were able to classify clinicopathologically ambiguous rashes with diagnoses consistent with therapeutic response. Thus, we have defined major classes of human inflammatory skin disease at the molecular level and described a quantitative method to classify indeterminate instances of pathologic inflammation. To make this approach accessible to the scientific community, we created a proof-of-principle web interface (RashX), where scientists and clinicians can visualize their patient-level rash scRNA-seq-derived data in the context of our T
2/T
17 transcriptional framework.
Massively parallel approaches to nucleic acid sequencing have matured from proof-of-concept to commercial products during the past 5 years. These technologies are now widely accessible, increasingly ...affordable, and have already exerted a transformative influence on the study of human cancer. Here, we review new features of cancer genomes that are being revealed by large-scale applications of these technologies. We focus on those insights most likely to affect future clinical practice. Foremost among these lessons, we summarize the formidable genetic heterogeneity within given cancer types that is appreciable with higher resolution profiling and larger sample sets. We discuss the inherent challenges of defining driving genomic events in a given cancer genome amidst thousands of other somatic events. Finally, we explore the organizational, regulatory and societal challenges impeding precision cancer medicine based on genomic profiling from assuming its place as standard-of-care.
Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE ...samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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