3D human foreskin model for testing topical formulations of sildenafil citrate.

Sildenafil citrate is an approved drug used for the treatment of erectile dysfunction and premature ejaculation. Despite a widespread application, sildenafil citrate shows numerous adverse cardiovascular effects in high-risk patients. Local transdermal drug delivery of this drug is therefore being explored as an interesting and noninvasive alternative administration method that avoids adverse effects arised from peak plasma drug concentrations. Although human and animal skin represents the most reliable models to perform penetration studies, they involve a series of ethical issues and restrictions. For these reasons new in vitro approaches based on artificially reconstructed human skin or "human skin equivalents" are being developed as possible alternatives for transdermal testing. There is little information, however, on the efficiency of such new in vitro methods on cutaneous penetration of active ingredients. The objective of the current study was to investigate the sildenafil citrate loaded in three commercial transdermal vehicles using 3D full-thickness skin equivalent and compare the results with the permeability experiments using porcine skin. Our results demonstrated that, while the formulation plays an imperative role in an appropriate dermal uptake of sildenafil citrate, the D coefficient results obtained by using the 3D skin equivalent are comparable to those obtained by using the porcine skin when a simple drug suspension is applied (1.17 × 10-10 ± 0.92 × 10-10 cm2/s vs 3.5 × 102 ± 3.3 × 102 cm2/s), suggesting that in such case, this 3D skin model can be a valid alternative for ex-vivo skin absorption experiments.

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.

Isolation of an "Early" Transit Amplifying Keratinocyte Population in Human Epidermis: A Role for the Low Affinity Neurotrophin Receptor CD271.

In the interfollicular epidermis (IFE), stem cells (KSC) generate transit amplifying (TA) cells that, after symmetric divisions, produce differentiating daughters. Here, we isolated and characterized the highly proliferative interfollicular epidermal basal cell population "early" TA (ETA) cells, based on their capacity to adhere to type IV collagen. Proliferation and colony-forming efficiency in ETA cells are lower than in KSC but higher than in "late" TA (LTA). Stemness, proliferation, and differentiation markers confirmed that ETA cells display a unique phenotype. Skin reconstructs derived from ETA cells present different features (epidermal thickness, Ki67, and Survivin expression), as compared to skin equivalents generated from either KSC or LTA cells. The low-affinity neurotrophin receptor CD271, which regulates the KSC to TA cell transition in the human epidermis through an on/off switch control mechanism, is predominantly expressed in ETA cells. Skin equivalents generated from siRNA CD271 ETA cells display a more proliferative and less differentiated phenotype, as compared to mock-derived reconstructs. Consistently, CD271 overexpression in LTA cells generates a more proliferative skin equivalent than mock LTA cells. Finally, the CD271 level declines with cellular senescence, while it induces a delay in p16INK4 expression. We conclude that ETA cells represent the first KSC progenitor with exclusive features. CD271 identifies and modulates ETA cells, thus participating in the early differentiation and regenerative capacity of the human epidermis.

Investigating Cutaneous Squamous Cell Carcinoma in vitro and in vivo: Novel 3D Tools and Animal Models.

Cutaneous Squamous Cell Carcinoma (cSCC) represents the second most common type of skin cancer, which incidence is continuously increasing worldwide. Given its high frequency, cSCC represents a major public health problem. Therefore, to provide the best patients' care, it is necessary having a detailed understanding of the molecular processes underlying cSCC development, progression, and invasion. Extensive efforts have been made in developing new models allowing to study the molecular pathogenesis of solid tumors, including cSCC tumors. Traditionally, in vitro studies were performed with cells grown in a two-dimensional context, which, however, does not represent the complexity of tumor in vivo. In the recent years, new in vitro models have been developed aiming to mimic the three-dimensionality (3D) of the tumor, allowing the evaluation of tumor cell-cell and tumor-microenvironment interaction in an in vivo-like setting. These models include spheroids, organotypic cultures, skin reconstructs and organoids. Although 3D models demonstrate high potential to enhance the overall knowledge in cancer research, they lack systemic components which may be solved only by using animal models. Zebrafish is emerging as an alternative xenotransplant model in cancer research, offering a high-throughput approach for drug screening and real-time in vivo imaging to study cell invasion. Moreover, several categories of mouse models were developed for pre-clinical purpose, including xeno- and syngeneic transplantation models, autochthonous models of chemically or UV-induced skin squamous carcinogenesis, and genetically engineered mouse models (GEMMs) of cSCC. These models have been instrumental in examining the molecular mechanisms of cSCC and drug response in an in vivo setting. The present review proposes an overview of in vitro, particularly 3D, and in vivo models and their application in cutaneous SCC research.

In Vivo Melanoma Cell Morphology Reflects Molecular Signature and Tumor Aggressiveness.

Melanoma is the deadliest type of skin cancer characterized by high cellular heterogeneity, which contributes to therapy resistance and unpredictable disease outcome. Recently, by correlating reflectance confocal microscopy morphology with histopathological type, we identified four distinct melanoma subtypes: dendritic cell, round cell, dermal nest, and combined-type melanomas. In this study, each reflectance confocal microscopy melanoma subtype expressed a specific biomolecular profile and biological behavior in vitro. Markers of tumor aggressiveness, including Ki-67, MERTK, nestin, and stemness markers were highest in the most invasive combined-type and dermal nest melanomas than in dendritic cell and round cell melanomas. This was also confirmed in multicellular tumor spheroids. Transcriptomic analysis showed modulation of cancer progression-associated genes from dendritic cell to combined-type melanomas. The switch from E- to N-cadherin expression proved the epithelial-to-mesenchymal transition from dendritic cell to combined-type subtypes. The dermal nest melanoma was predominantly located in the dermis, as also shown in skin reconstructs. It displayed a unique behavior and a molecular profile associated with a high degree of aggressiveness. Altogether, our results show that each reflectance confocal microscopy melanoma subtype has a distinct biological and gene expression profile related to tumor aggressiveness, confirming that reflectance confocal microscopy can be a dependable tool for in vivo detection of different types of melanoma and for early diagnostic screening.

Activation of cGMP-Dependent Protein Kinase Restricts Melanoma Growth and Invasion by Interfering with the EGF/EGFR Pathway.

Drug resistance mechanisms still characterize metastatic melanoma, despite the new treatments that have been recently developed. Targeting of the cGMP/protein kinase G pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric-linked dimeric cGMP analogs able to bind and activate protein kinase G, called protein kinase G activators (PAs) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor-specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analog. We defined that by triggering protein kinase G, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of protein kinase B and extracellular signal‒regulated kinase 1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analog PA5 as a potential therapeutic strategy for melanoma.

Specific Activation of the CD271 Intracellular Domain in Combination with Chemotherapy or Targeted Therapy Inhibits Melanoma Progression.

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short ß-amyloid-derived peptide (Aß(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aß(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aß(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aß(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy. SIGNIFICANCE: The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.

A Novel Multi-Action Emollient Plus Cream Improves Skin Barrier Function in Patients with Atopic Dermatitis: In vitro and Clinical Evidence.

BACKGROUND: Emollients capable of restoring the skin barrier function would extend their role beyond basic maintenance therapy in atopic dermatitis (AD). OBJECTIVES: Investigate the effect of a novel emollient plus cream (EC; Dermoflan®) on the skin barrier in vitro and in patients with mild-to-moderate AD. METHODS: The effect of EC on the skin barrier recovery was evaluated using a tape-stripping (TS) model. After TS, organ cultures were treated with EC (undiluted or diluted 1:1 with water) and analyzed at 18-120 h using hematoxylin and eosin, Oil Red O, immunohistochemical, and immunofluorescent techniques. In a double-blind, randomized study, EC or placebo was applied once daily for 2 months to antecubital folds of the upper and lower limbs of patients with mild-to-moderate AD in clinical remission. Epidermal thickness, vascularization, and epidermal hydration were assessed by optical coherence tomography and corneometry, respectively, at baseline, and 1 and 2 months following treatment initiation. RESULTS: Following TS, EC treatment significantly increased epidermal thickness and lipid content versus diluent in the skin organ culture, as well as claudin-1, involucrin, and caspase-14 expression, suggesting skin barrier repair. EC treatment also decreased keratin-16 expression and increased levels of Toll-like receptors 1 and 2 versus diluent, suggesting involvement in regulating the epidermal immune response. In 20 patients randomized 1:1 to EC or placebo, EC treatment at the elbow fold/popliteal fossa significantly decreased epidermal thickness after 2 months, and the number of blood vessels at the elbow fold after 1 and 2 months, versus placebo. EC significantly improved the skin hydration after 2 months versus baseline. CONCLUSIONS: This novel multi-action EC may help to restore epidermal homeostasis and improve the skin of patients with AD. Results indicate that this novel multi-action EC could be a valid adjuvant therapy in patients with AD. Key Message: Novel multi-action emollient cream helps to restore epidermal homeostasis and improves the skin affected by AD.

Progress in melanoma modelling in vitro.

Melanoma is one of the most studied neoplasia, although laboratory techniques used for investigating this tumor are not fully reliable. Animal models may not predict the human response due to differences in skin physiology and immunity. In addition, international guidelines recommend to develop processes that contribute to the reduction, refinement and replacement of animals for experiments (3Rs). Adherent cell culture has been widely used for the study of melanoma to obtain important information regarding melanoma biology. Nonetheless, these cells grow in adhesion on the culture substrate which differs considerably from the situation in vivo. Melanoma grows in a 3D spatial conformation where cells are subjected to a heterogeneous exposure to oxygen and nutrient. In addition, cell-cell and cell-matrix interaction play a crucial role in the pathobiology of the tumor as well as in the response to therapeutic agents. To better study, melanoma new techniques, including spherical models, tumorospheres and melanoma skin equivalents, have been developed. These 3D models allow to study tumors in a microenvironment that is more close to the in vivo situation and are less expensive and time-consuming than animal studies. This review will also describe the new technologies applied to skin reconstructs such as organ-on-a-chip that allows skin perfusion through microfluidic platforms. 3D in vitro models, based on the new technologies, are becoming more sophisticated, representing at a great extent the in vivo situation, the "perfect" model that will allow less involvement of animals up to their complete replacement, is still far from being achieved.

Soluble Fas Ligand Is Essential for Blister Formation in Pemphigus.

Pemphigus is a blistering disease characterized by pemphigus autoantibodies (PVIgG) directed mostly against desmogleins (Dsgs), resulting in the loss of keratinocyte adhesion (acantholysis). Yet, the mechanisms underlying blister formation remain to be clarified. We have shown previously that anti-Fas ligand (FasL) antibody (Ab) prevents PVIgG-induced caspase-8 activation and Dsg cleavage in human keratinocytes, and that sera from pemphigus patients contain abnormally increased levels of FasL. Here, we demonstrate that recombinant FasL induces the activation of caspases prior to Dsg degradation, and anti-FasL Ab prevents acantholysis in cultured keratinocytes. Moreover, the silencing of FasL reduces PVIgG-induced caspase-8 activation and Dsg3 cleavage. Following injection of PVIgG into mice, FasL is upregulated at 1-3 h and is followed by caspase-8-mediated keratinocyte apoptosis, before blister formation. The administration of anti-FasL Ab after PVIgG injection blocks blister formation in mice. Furthermore, we injected PVIgG into two different gene-targeted mutant mice that selectively lack either secreted soluble FasL (sFasL), FasLΔs/Δs mice, or the membrane-bound form of FasL (mFasL), FasLΔm/Δm mice. After PVIgG treatment, blisters are only visible in FasLΔm/Δm animals, lacking mFasL, but still producing sFasL, similar to wild-type (C57BL/6) animals. By contrast, a significant decrease in the relative acantholytic area is observed in the FasLΔs/Δs animals. These results demonstrate that soluble FasL plays a crucial role in the mechanisms of blister formation, and blockade of FasL could be an effective therapeutic approach for pemphigus.

CD271 Down-Regulation Promotes Melanoma Progression and Invasion in Three-Dimensional Models and in Zebrafish.

CD271 is a neurotrophin receptor variably expressed in melanoma. Although contradictory data are reported on its role as a marker of tumor-initiating cells, little is known about its function in tumor progression. CD271 expression was higher in spheroids derived from freshly isolated cells of primary melanomas and in primary WM115 and WM793-B cell lines, and it decreased during progression to advanced stages in cells isolated from metastatic melanomas and in metastatic WM266-4 and 1205Lu cell lines. Moreover, CD271 was scarcely detected in the highly invasive spheroids (SKMEL28 and 1205Lu). CD271, originally expressed in the epidermis of skin reconstructs, disappeared when melanoma started to invade the dermis. SKMEL8 CD271(-) cells showed greater proliferation and invasiveness in vitro and were associated with a higher number of metastases in zebrafish compared with CD271(+) cells. CD271 silencing in WM115 induced a more aggressive phenotype in vitro and in vivo. On the contrary, CD271 overexpression in SKMEL28 cells reduced invasion in vitro, and CD271 overexpressing 1205Lu cells was associated with a lower percentage of metastases in zebrafish. A reduced cell-cell adhesion was also observed in the absence of CD271. Taken together, these results indicate that CD271 loss is critical for melanoma progression and metastasis.

Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo.

Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, ß1-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in Ras(G12V)-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development.

Phosphodiesterase 4 in inflammatory diseases: Effects of apremilast in psoriatic blood and in dermal myofibroblasts through the PDE4/CD271 complex.

Phosphodiesterases 4 (PDE4) act as proinflammatory enzymes via degradation of cAMP, whereas PDE4 inhibitors play an anti-inflammatory role in vitro and in vivo. In particular, apremilast has been recently approved for the treatment of psoriasis and psoriatic arthritis. However, little is known on the expression pattern of PDE4 in psoriasis. We report that PDE4B and PDE4D mRNA are overexpressed in peripheral blood mononuclear cells (PBMC) from psoriasis, as compared with normal controls, while apremilast reduces PBMC production of a number of pro-inflammatory cytokines and increases the levels of anti-inflammatory mediators. PDE4 expression is up-regulated in psoriatic dermis as compared with normal skin, with particular regard to fibroblasts. This is confirmed in vitro, where both dermal fibroblasts (DF) and, to a greater extent, myofibroblasts (DM) express all PDE4 isoforms at the mRNA and protein level. Because PDE4 interacts with the nerve growth factor (NGF) receptor CD271 in lung fibroblasts, we evaluated the relationship and function of PDE4 and CD271 in normal human skin fibroblasts. All PDE4 isoforms co-immunoprecipitate with CD271 in DM, while apremilast inhibits apoptosis induced by ß-amyloid, a CD271 ligand, in DM. Furthermore, apremilast significantly reduces NGF- and transforming growth factor-ß1 (TGF-ß1)-induced fibroblast migration, and inhibits DF differentiation into DM mediated by NGF or TGF-ß1. Finally, in DM, apremilast significantly reduces cAMP degradation induced by treatment with ß-amyloid. Taken together, these results indicate that PDE4 play an important role in psoriasis. In addition, the study reveals that the PDE4/CD271 complex could be important in modulating fibroblast functions.