The skin deploys multiple barriers to protect internal tissues and organs, as well as the skin itself, from the external environment. Each of the skin constituents-epidermis, dermis, sebaceous glands, sweat glands, hair, and adipose tissue-contributes to making these barriers competent to maintain proper biological functions. Among these constituents, the epidermis (the outermost layer of skin) is the first line of defense, so it is most responsible for skin barrier functions. A competent skin barrier is needed to protect against excess loss of water and other endogenous compounds from body, oxidative stress, sun and other sources of light irradiation, thermal stress, microbial invasion, substance permeation, and mechanical stress. In this chapter, we describe methods to assess both the epidermal permeability barrier and the light irradiation barrier using in vitro and ex vivo skin organ-cultured systems.

BACKGROUND AIMS:Human pluripotent stem cell (hPSC) manufacturing workflows frequently rely on suspension aggregation, yet inter-line and batch-to-batch variability in aggregate formation can compromise process consistency and downstream differentiation performance. We evaluated whether a short exposure to HA-100, a small-molecule inhibitor of protein kinase A and protein kinase C signaling, could be used as an upstream process intervention to improve aggregate uniformity without compromising hPSC identity or developmental competence. METHODS:Nine hPSC lines, including human embryonic stem cell and induced pluripotent stem cell lines, were examined in suspension culture. HA-100 was applied during the first 24 h of aggregation. Aggregate morphology and size distribution were assessed across lines. To investigate the cellular basis of this effect, we generated an mCherry-TJP1 reporter hESC line, which enabled live visualization of junction dynamics, including responses under calcium-depleted conditions and recovery of transepithelial electrical resistance. RESULTS:HA-100 treatment promoted more compact and spherical aggregates, increased aggregate size into a narrower range across lines, and reduced overall variability relative to medium alone. Across the nine-line panel, HA-100-treated aggregates fell within an empirically definesd size range of 25.37-33.95 x 10^-4 mm^3 after 24 h of suspension culture, providing a practical benchmark for process monitoring. In calcium-depleted conditions, HA-100 delayed disruption of intercellular contacts and accelerated recovery of transepithelial electrical resistance, consistent with improved junctional resilience. Transient exposure to HA-100 did not abolish pluripotency marker expression or tri-lineage differentiation capacity. CONCLUSIONS:These data support HA-100 as a practical upstream intervention to reduce aggregate heterogeneity in suspension hPSC cultures and improve reproducibility in manufacturing-oriented workflows requiring consistent aggregation.

Aging impairs epidermal differentiation and barrier function, making aged skin susceptible to dryness, dermatitis and infection. Here, we show that pharmacologically enhancing endoplasmic reticulum Ca2+ stores with CDN 1163, a small molecular SERCA2b allosteric activator, corrects age-related defects in Ca2+ storage in aged keratinocytes, partially corrects the abnormal Ca2+ gradient seen in aged epidermis, and enhances filaggrin expression in aged skin samples.

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer, caused by mutagenesis resulting from excess ultraviolet radiation or other types of oxidative stress. These stressors also upregulate production of a cutaneous innate immune element, cathelicidin antimicrobial peptide (CAMP), via endoplasmic reticulum (ER) stress-initiated, sphingosine-1-phosphate (S1P) signaling pathway. While CAMP has beneficial antimicrobial activities, it also can be pro-inflammatory and pro-carcinogenic. We addressed whether and how S1P-induced CAMP production leads to cSCC development. Our study demonstrated that: 1) CAMP expression is increased in cSCC cells and skin from cSCC patients; 2) S1P levels are elevated in cSCC cells, while inhibition of S1P production attenuates CAMP-stimulated cSCC growth; 3) exogenous CAMP stimulates cSCC, but not normal human keratinocyte growth; 4) blockade of formyl peptide receptor-like (FPRL) 1 protein, a CAMP receptor, attenuates cSCC growth as well as the growth and invasion of cSCC cells mediated by CAMP into an extracellular matrix-containing fibroblast substrate; 5) Foxp3+ regulatory T cell (which decreases anti-tumor immunity) levels increase in cSCC skin; and 6) CAMP induces ER stress in cSCC cells. Together, the ER stress-S1P-CAMP axis forms a vicious circle, creating a favorable environment for cSCC development, i.e., cSCC growth and invasion impedes anti-cancer immunity.

Cutaneous squamous cell carcinoma (cSCC) is a common cancer, caused by mutagenesis from excess ultraviolet radiation (UVR) or other types of oxidative stress. These stressors also upregulate production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), via endoplasmic reticulum (ER) stress-initiated, sphingosine-1-phosphate (S1P) signaling. While CAMP has beneficial antimicrobial activities, it also can be pro-inflammatory and pro-carcinogenic. We addressed whether/how S1P-induced CAMP production leads to cSCC development. We found that: 1) CAMP expression was markedly increased in cSCC cells and in cSCC patients' skin; 2) inhibition of S1P production attenuated CAMP-stimulated cSCC growth; 3) exogenous CAMP stimulates cSCC growth, but not growth of normal human keratinocytes; 4) blockade of formyl peptide receptor-like (FPRL) 1 protein (a receptor of CAMP) activation attenuated cSCC growth as well as the growth-promoting effects of exogenously applied CAMP on cSCC cells, and also slowed invasion of cSCC cells into an extracellular matrix-containing fibroblast substrate; and 5) Foxp3+ regulatory T (Treg) cells (which diminish anti-tumor immunity) levels were increased in cSCC skin. These results suggest that the relationship of S1P signaling to CAMP overproduction is crucial for cSCC development. Since Treg cells in a cancer microenvironment weaken anti-cancer immunity, we will also address roles of CAMP in Treg function in cSCC.

Systemic inflammation, associated with impaired epidermal barrier function, increases starting in middle age. However, transepidermal water loss (TEWL), the traditional measure of "inside-out" barrier function, remains intact in middle-aged and aged skin. We hypothesized that an additional source of age-related inflammation is a defective barrier to outside agents ("outside-in"). We first compared epidermal permeability to outside agents in untreated newborn vs. aged human epidermis, using a previously validated fluorescent tracer, Ca2+ Green. Permeability, as quantified by penetration volume, was 2-3x higher in aged skin. Since occlusion does not increase inflammation in young skin but increases penetration of agents on the surface of the stratum corneum (SC), we hypothesized that using occlusion to increase penetration through a defective barrier in aged skin would induce inflammation in middle-aged (12 mos) but not young (8-12 weeks) mice. Occlusion decreased SC hydration but did not change TEWL or the immune cell profiles in both young and aged mice. Occlusion increased serum IL-1 beta and IL-6, both known to be derived from epidermis, to a much greater extent in middle-aged mice and significantly enhanced Ca2+ Green permeability in middle-aged but not young mice. Occlusion did not increase serum TNF-alpha, consistent with the minimal changes seen in immune parameters. Finally, we demonstrated that larger but more widely spaced, experimentally induced barrier defects that mimic those seen with decreased hydration lead to increased outside-in penetration without increasing TEWL. These findings suggest that an "outside-in" mechanism, linked to hydration defects, might induce systemic inflammation derived from aging epidermis.

FLG variants underlie ichthyosis vulgaris and increased risk of atopic dermatitis, conditions typified by disruption of the skin microbiome and cutaneous immune response. Yet, it remains unclear whether neonatal skin barrier compromise because of FLG deficiency alters the quality of commensal-specific T cells and the functional impact of such responses. To address these questions, we profiled changes in the skin barrier and early cutaneous immune response of neonatal C57BL/6 Flg-/- and wild-type mice using single-cell RNA sequencing, flow cytometry, and other modalities. Flg-/- neonates showed little alteration in transepidermal water loss or lipid- or corneocyte-related gene expression. However, they showed increases in barrier disruption genes, epidermal dye penetration, and numbers of skin CD4+ T cells. Using an engineered strain of Staphylococcus epidermidis (S. epidermidis 2W) to study the response to neonatal skin colonization, we found that commensal-specific CD4+ T cells were skewed in Flg-/- pups toward effector rather than regulatory T cells. This altered response persisted into adulthood, where it was typified by T helper 17 (Th17) cells and associated with increased susceptibility to imiquimod-induced skin inflammation. Thus, subtle but impactful differences in neonatal barrier function in Flg-/- mice are accompanied by a skewed commensal-specific CD4+ response, with enduring consequences for skin immune homeostasis.

Mutations in Ras family proteins are implicated in 33% of human cancers, but direct pharmacological inhibition of Ras mutants remains challenging. As an alternative to direct inhibition, we screened for sensitivities in Ras-mutant cells and discovered 249C as a Ras-mutant selective cytotoxic agent with nanomolar potency against a spectrum of Ras-mutant cancers. 249C binds to vacuolar (V)-ATPase with nanomolar affinity and inhibits its activity, preventing lysosomal acidification and inhibiting autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. Unexpectedly, potency of 249C varies with the identity of the Ras driver mutation, with the highest potency for KRASG13D and G12V both in vitro and in vivo, highlighting a mutant-specific dependence on macropinocytosis and lysosomal pH. Indeed, 249C potently inhibits tumor growth without adverse side effects in mouse xenografts of KRAS-driven lung and colon cancers. A comparison of isogenic SW48 xenografts with different KRAS mutations confirmed that KRASG13D/+ (followed by G12V/+) mutations are especially sensitive to 249C treatment. These data establish proof-of-concept for targeting V-ATPase in cancers driven by specific KRAS mutations such as KRASG13D and G12V.

Epidermal basement membrane, a tightly packed network of extracellular matrix (ECM) components, is a source of physical, chemical, and biological factors required for the structural and functional homeostasis of the epidermis. Variations within the ECM create distinct environments, which can affect the property of cells in the basal layer of the epidermis and subsequently affect keratinocyte differentiation and stratification. Very little attention has been paid to mimicking basement membrane in organotypic cultures. In this study, using parameters outlined in a consensus on the quality standard of organotypic models suitable for dermatological research, we have evaluated three basement membrane substitutes. We compared fibronectin with three complex three-dimensional matrices: Matrigel, decellularized dermal fibroblast‒produced and ‒assembled ECM, and a dry human amniotic membrane. Our results suggest that Matrigel is not a suitable substrate for human epidermal equivalent culture, whereas the two other complex three-dimensional substitutes, decellularized dermal fibroblast‒produced and ‒assembled ECM and dry human amniotic membrane, were superior to single layer fibronectin coating. Human epidermal equivalents cultured on either decellularized dermal fibroblast‒produced and ‒assembled ECM or on dry human amniotic membrane generated hemidesmosomes, whereas those on fibronectin did not. In addition, human epidermal equivalent cultured on decellularized dermal fibroblast‒produced and ‒assembled ECM and on dry human amniotic membrane can be maintained in culture 4 days longer than human epidermal equivalent cultured on fibronectin without compromising the barrier function.

The calcium-sensing receptor (CaSR) drives essential calcium ion (Ca2+) and E-cadherin-mediated processes in the epidermis, including differentiation, cell-to-cell adhesion, and epidermal barrier homeostasis in cells and in young adult mice. We now report that decreased CaSR expression leads to impaired Ca2+ signal propagation in aged mouse (aged >22 months) epidermis and human (aged >79 years, donor age) keratinocytes. Baseline cytosolic Ca2+ concentrations were higher, and capacitive Ca2+ entry was lower in aged than in young keratinocytes. As in Casr-knockout mice ((CaSR-/-)-Ca-Epid), decreased CaSR expression led to decreased E-cadherin and phospholipase C-gamma expression and to a compensatory upregulation of STIM1. Pretreatment with the CaSR agonist N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine normalized Ca2+ propagation and E-cadherin organization after experimental wounding. These results suggest that age-related defects in CaSR expression dysregulate normal keratinocyte and epidermal Ca2+ signaling, leading to impaired E-cadherin expression, organization, and function. These findings show an innovative mechanism whereby Ca2+- and E-cadherin-dependent functions are impaired in aging epidermis and suggest a new therapeutic approach by restoring CaSR function.

Background Ceramide kinase-like protein (CERKL) was originally described in retinal tissue. CERKL has been shown to protect cells from oxidative stress, and mutations in CERKL underlie the inherited disease retinitis pigmentosa. CERKL expression maintains cellular sphingolipids via an unknown mechanism. Objectives To determine whether CERKL is expressed in epidermis and cutaneous squamous cell carcinoma (cSCC) and whether CERKL expression affects cSCC sphingolipid metabolism and susceptibility to oxidative stress. Methods CERKL expression was determined by RNA-Seq, quantitative polymerase chain reaction and immunohistochemistry. CERKL was knocked down in cSCC cells using small interfering RNA. Sphingolipid content was analysed by liquid chromatography-mass spectrometry. Oxidative stress was induced by treatment with H2O2, and apoptosis was measured using flow cytometry to determine annexin V binding. Results CERKL mRNA and protein are highly expressed in actinic keratosis and cSCC in comparison with normal epidermis. CERKL is also expressed in metabolically active epithelial cells in normal hair bulbs and sebaceous glands. CERKL knockdown in cultured cSCC cells reduces cellular sphingolipid content and enhances susceptibility to oxidative stress. Conclusions These findings suggest that CERKL may be important in cSCC progression and could lead to novel strategies for prevention and treatment of cSCC.

Treatment of solid cancers with chimeric antigen receptor (CAR) T cells is plagued by the lack of ideal target antigens that are both absolutely tumor specific and homogeneously expressed. We show that multi-antigen prime-and-kill recognition circuits provide flexibility and precision to overcome these challenges in the context of glioblastoma. A synNotch receptor that recognizes a specific priming antigen, such as the heterogeneous but tumor-specific glioblastoma neoantigen epidermal growth factor receptor splice variant III (EGFRvIII) or the central nervous system (CNS) tissue-specific antigen myelin oligodendrocyte glycoprotein (MOG), can be used to locally induce expression of a CAR. This enables thorough but controlled tumor cell killing by targeting antigens that are homogeneous but not absolutely tumor specific. Moreover, synNotch-regulated CAR expression averts tonic signaling and exhaustion, maintaining a higher fraction of the T cells in a naïve/stem cell memory state. In immunodeficient mice bearing intracerebral patient-derived xenografts (PDXs) with heterogeneous expression of EGFRvIII, a single intravenous infusion of EGFRvIII synNotch-CAR T cells demonstrated higher antitumor efficacy and T cell durability than conventional constitutively expressed CAR T cells, without off-tumor killing. T cells transduced with a synNotch-CAR circuit primed by the CNS-specific antigen MOG also exhibited precise and potent control of intracerebral PDX without evidence of priming outside of the brain. In summary, by using circuits that integrate recognition of multiple imperfect but complementary antigens, we improve the specificity, completeness, and persistence of T cells directed against glioblastoma, providing a general recognition strategy applicable to other solid tumors.

Organotypic cultures for human skin and its compartments (also called three-dimensional skin models, human skin equivalents, or human epidermal equivalents), generated from keratinocytes alone or keratinocytes plus fibroblasts derived from primary cell sources or immortalized cell lines, have become an important experimental approach to assess how changes in specific processes affect epidermal growth, differentiation, permeation, and barrier function. Innovations in this field have led to a wide variety in models based on different cell sources, dermal substrates, and cell culture media.

ABSTRACTMutations in the Ras family of oncogenes are implicated in 33% of human cancers, making Ras an intensely pursued target in drug discovery. As an alternative to direct pharmacological inhibition of Ras, we looked for sensitivities in RAS mutant cells. Using a small molecule screen in cell lines with mutations in Ras and its effector Raf, we discovered 249C as a Ras-mutant selective cytotoxic agent against a spectrum of RAS-mutant cancers. By combining CRISPR chemical-genetic screening, comparative profiling and chemoproteomics, we identified that 249C binds to a unique subunit on vacuolar (V)-ATPase with nanomolar affinity, inhibiting its biochemical activity and, unexpectedly, altering V-ATPase translocation in Ras-induced macropinocytosis. Via binding to V-ATPase, 249C prevents lysosomal acidification and inhibits autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. In characterizing 249C’s mechanism, we show that potency varies with the identity of the RAS driver mutation highlighting a mutant-specific dependence on autophagy and macropinocytosis. Indeed, 249C potently inhibits tumor growth without adverse side effects in a mouse xenograft model ofKRAS-driven non-small cell lung cancer. These data establish proof-of-concept for targeting V-ATPase as a way to indirectly target specific Ras mutants, and provide a fundamental link between V-ATPase localization and specific Ras mutant tumor-related activity.

The epidermal permeability barrier serves as a multifunctional partition to protect its host from the external environment. Most epidermal permeability barrier studies have been conducted using in vivo human and experimental animals, although some studies have used in vitro cultured cells. There currently is an increased demand for these cultured models, thus avoiding the use of laboratory animals. Here, we first summarize required features that need to be recaptured in cultured keratinocytes for an epidermal permeability barrier study and second, we describe a method for culturing these cells. We also introduce methods to analyze epidermal permeability barrier function using cultured keratinocytes.

The epithelial tight junction regulates barrier function and is responsive to extracellular stimuli. Here we demonstrated that contact of synthetic surfaces with defined nanotopography at the apical surface of epithelial monolayers increased paracellular permeability of macromolecules. To monitor changes in tight junction morphology in live cells, we fluorescently tagged the scaffold protein zonula occludens-1 (ZO-1) through CRISPR/Cas9-based gene editing. Contact between cells and nanostructured surfaces destabilized junction-associated ZO-1 and promoted its arrangement into highly dynamic non-junctional cytosolic complexes that averaged ∼2 μm in diameter. Junction-associated ZO-1 rapidly remodeled, and we also observed the direct transformation of cytosolic complexes into junction-like structures. Claudin-family tight junction transmembrane proteins and F-actin also were associated with these ZO-1 containing cytosolic complexes. These data suggest that the cytosolic structures are novel intermediates formed in response to nanotopographic cues that facilitate rapid tight junction remodeling in order to regulate paracellular permeability.