SERS Multiplex Profiling of Melanoma Circulating Tumor Cells for Predicting the Response to Immune Checkpoint Blockade Therapy.

Immune checkpoint blockade (ICB) therapy has achieved remarkable success in many cancers including melanoma. However, ICB therapy benefits only a small proportion of patients and produces severe side effects for some patients. Thus, there is an urgent need to identify patients who are more likely to respond to ICB therapy to improve outcomes and minimize side effects. To predict ICB therapy responses, we design a surface-enhanced Raman scattering (SERS) assay for multiplex profiling of circulating tumor cells (CTCs) under basal and interferon-γ (IFN-γ) stimulation. Through simultaneous ensemble and single-cell measurements of CTCs, the SERS assay can reveal tumor heterogeneity and offer a comprehensive CTC phenotype for decision-making. Anisotropic gold-silver alloy nanoboxes are utilized as SERS plasmonic substrates for improved signal readouts of CTC surface biomarkers. By generating a unique CTC signature with four surface biomarkers, the developed assay enables the differentiation of CTCs from three different patient-derived melanoma cell lines. Significantly, in a cohort of 14 melanoma patients who received programmed cell death-1 blockade therapy, the changes of CTC signature induced by IFN-γ stimulation to CTCs show the potential to predict responders. We expect that the SERS assay can help select patients for receiving ICB therapy in other cancers.

Dynamic Monitoring of EMT in CTCs as an Indicator of Cancer Metastasis.

Epithelial to mesenchymal transition (EMT) results in the genesis of circulating tumor cells (CTCs) from tumor sites and promotes the metastatic capability of CTCs in circulation. In this study, we develop a multiplex surface-enhanced Raman scattering nanotechnology for comprehensive characterization of EMT-associated phenotypes in CTCs, to monitor cancer metastasis. We observe the downregulation of the CTC marker (EpCAM) and the epithelial marker (E-cadherin), as well as the upregulation of a mesenchymal marker (N-cadherin) and a stem cell marker (ABCB5) during the transforming growth factor-ß-induced EMT process in breast cancer cell line models. Additionally, we also find changes in the heterogeneity levels of these selected markers in cells. With this method, we successfully detect the presence of disease in samples from breast cancer patients and characterize EMT-associated phenotypes in their CTCs. Overall, this approach and findings provide a new means for monitoring the EMT process in cancer, insights into the detailed mechanistic progress of the diseases, and have potential for detecting the early occurrence of cancer metastasis.

Elongated microparticles tuned for targeting hyaluronic acid delivery to specific skin strata.

OBJECTIVE: Microneedle or fractional laser applications are the most common topical delivery enhancement platforms. However, these methods of drug delivery are not skin strata specific. Drug delivery approaches which could target specific stratum of the skin remains a challenge. Elongated microparticles (EMPs) have been used in enhancing drug delivery into the skin. The aim of this study was to evaluate, for the first time, elongated silica microparticles with two different length profiles to enhance delivery of hyaluronic acid into different strata of human skin. METHODS: Two types of EMPs-long (milled EMPs) or short (etched EMPs) length ranges were characterized. A prototypical liquid formulation (Fluorescent hyaluronic acid) with and without EMP enhancement were evaluated for hyaluronic acid delivery in ex-vivo human skin. High performance liquid chromatography, Typhoon fluorescence scanning system, laser scanning confocal microscopy and reflectance confocal microscopy (RCM) were used to validate F-HA stability, visualize fluorescein in the skin, image the depth of F-HA delivery in the skin and define EMP penetration in skin strata, respectively. Statistical analysis was conducted using GraphPad Prism 6 software (GraphPad Software Inc, USA). RESULTS: Fluorescein-hyaluronic acid was stable and EMP enhanced skin penetration. RCM revealed that 'etched EMP' penetrated the skin to the stratum spinosum level. The vast majority (97.8%; p < 0.001) of the etched EMP did not penetrate completely through the viable epidermis and no obvious penetration into the dermis. In contrast, milled EMP showed 41-fold increase in penetration compared to the etched EMP but penetrated beyond the dermoepidermal junction. CONCLUSION: EMPs can enhance delivery of hyaluronic acid. Using EMPs with defined length distributions, which can be tuned for a specific stratum of the skin, can achieve targeted hyaluronic acid delivery.

OBJECTIF: Les microaiguilles ou le laser fractionné sont couramment utilisés pour augmenter l'absorption d'actif appliqué par voie topique. Toutefois, ces approches ne permettent de cibler une strate spécifique de la peau. Ainsi les méthodes permettant de cibler spécifiquement l'épiderme reste un défi. Les microparticules allongées (EMP) ont déjà été utilisé pour augmenter l'absorption cutanée. L'objectif de l'étude est d'évaluer pour la première fois, la capacité de microparticules allongées de silice (de deux longueurs différentes) à délivrer l'acide hyaluronique dans les différentes couches de la peau. MÉTHODES: Deux types d'EMP, longues (EMP broyé) ou courtes (EMP gravé), ont été caractérisées. Une formulation liquide contenant de l'acide hyaluronique marquée avec une sonde fluorescente (F-HA) a été évaluée avec et sans EMP sur peau humaine ex vivo. La chromatographie liquide haute performance, le scanner à fluorescence Typhoon, la microscopie laser confocal à balayage et la microscopie confocale par réflectance (RCM) ont été utilisées respectivement pour contrôler la stabilité de la F-HA, visualiser le signal de la fluorescéine dans la peau, imager l'absorption du F-HA dans la peau en fonction de la profondeur et caractériser la pénétration des EMP. Les analyses statistiques ont été réalisées avec le logiciel GraphPad Prims 6 (GraphPad Software Inc, USA). RÉSULTATS: L'acide hyaluronique marquée avec la fluorescéine est stable et les EMP permettent d'augmenter son absorption cutanée. Le RCM a montré que les EMP gravées pénètrent dans la peau jusqu'au niveau du stratum spinosum. La grande majorité des EMP gravés (97.8% ; p < 0,001) ne pénètre pas complétement dans l'épiderme viable et aucune pénétration mesurable dans le derme. Au contraire, les EMP broyées ont montrées une pénétration 41 fois plus importantes que les EMP gravées et peuvent aller au-delà de la jonction derme-épiderme. CONCLUSION: Les EMP peuvent augmenter l'absorption cutanée de l'acide hyaluronique. En utilisant des EMP de longueur définie et en ajustant celle-ci, il est même possible de cibler spécifiquement une strate cutanée.

Tracking Drug-Induced Epithelial-Mesenchymal Transition in Breast Cancer by a Microfluidic Surface-Enhanced Raman Spectroscopy Immunoassay.

Epithelial-mesenchymal transition (EMT) is a primary mechanism for cancer metastasis. Detecting the activation of EMT can potentially convey signs of metastasis to guide treatment management and improve patient survival. One of the classic signatures of EMT is characterized by dynamic changes in cellular expression levels of E-cadherin and N-cadherin, whose soluble active fragments have recently been reported to be biomarkers for cancer diagnosis and prognosis. Herein, a microfluidic immunoassay (termed "SERS immunoassay") based on sensitive and simultaneous detection of soluble E-cadherin (sE-cadherin) and soluble N-cadherin (sN-cadherin) for EMT monitoring in patients' plasma is presented. The SERS immunoassay integrates in situ nanomixing and surface-enhanced Raman scattering readout to enable accurate detection of sE-cadherin and sN-cadherin from as low as 10 cells mL-1 . This assay enables tracking of a concurrent decrease in sE-cadherin and increase in sN-cadherin in breast cancer cells undergoing drug-induced mesenchymal transformation. The clinical potential of the SERS immunoassay is further demonstrated by successful detection of sE-cadherin and sN-cadherin in metastatic stage IV breast cancer patient plasma samples. The SERS immunoassay can potentially sense the activation of EMT to provide early indications of cancer invasions or metastasis.

A minimally invasive clinical model to test sunscreen toxicity based on oxidative stress levels using microbiopsy and confocal microscopy - a proof of concept study.

OBJECTIVE: This proof-of-concept study demonstrated that using minimally invasive skin microsampling could enable significantly higher throughput of cosmetic testing in volunteers than conventional biopsy. Nanoparticle sunscreen was used as a model to test toxicity based on oxidative stress using microbiopsy and confocal imaging. METHODS: Six volunteers were recruited for this study (3 males and 3 females). Zinc oxide nanoparticle containing topical formulation was prepared at 10% w/v. Each volunteer had 3 areas of 4 cm2 each mapped on each inner forearm for a total of 6 treatment areas (intact/ tape-stripped and with/without treatment). The topical zinc-nanoparticle formulation was applied directly to volunteer skin (2mg/cm2 ) for 2 hrs. Microbiopsied tissue from each treatment group was stained for reactive oxygen and nitrogen species in addition to mitochondrial superoxide. The stained samples were then imaged using confocal microscopy prior to image analysis. RESULTS: Skin exposed to zinc oxide nanoparticles did not show any significant increases in oxidative stress. Zinc oxide nanoparticle tape-stripped skin resulted in signal significantly lower (P < 0.001) oxidative stress levels than t-butylated hydroxytoluene treated tape-stripped skin for oxidative stress markers. Topically applied zinc oxide nanoparticles had no detectable effect on the oxidative status in volunteer skin. No adverse reactions or effects were observed after all treatments including microbiopsy. CONCLUSION: The data support the hypothesis that microbiopsy is a viable approach to study cosmeceutical- skin interactions in volunteers with capacity for molecular assays and high throughput with very low risk to the volunteer.

OBJECTIFS: Cette étude de validation de concept a démontré que le microprélèvement cutané minimalement invasif pouvait augmenter considérablement la cadence des essais de produits cosmétiques sur des volontaires par rapport à une biopsie conventionnelle. Un écran solaire contenant des nanoparticules a été utilisé comme modèle pour tester la toxicité liée au stress oxydatif à l'aide de la microbiopsie et de l'imagerie confocale. MÉTHODES: Six volontaires ont été recrutés pour cette étude (3 hommes et 3 femmes). Une formulation topique contenant des nanoparticules d'oxyde de zinc a été préparée à 10 % p/v. Chaque volontaire disposait de 3 zones de 4 cm2 situées sur chaque pliure de coude pour un total de 6 zones de traitement (intactes / strippée et avec / sans traitement). La formulation topique contenant des nanoparticules d'oxyde de zinc a été appliquée directement sur la peau des volontaires (2 mg/cm2 ) pendant 2 heures. Les tissus microbiopsiés de chaque groupe de traitement ont été colorés pour détecter des espèces réactives de l'oxygène et de l'azote en plus de la superoxyde mitochondriale. Les échantillons colorés ont ensuite été examinés par microscopie confocale avant l'analyse des images. RÉSULTATS: La peau exposée aux nanoparticules d'oxyde de zinc n'a pas montré de hausse significative de stress oxydatif. La peau strippée traitée aux nanoparticules d'oxyde de zinc a entraîné des niveaux de stress oxydatif nettement inférieurs (p<0,001) comparés à ceux de la peau strippée traitée à l'hydroxytoluène t-butylé en ce que concerne les marqueurs de stress oxydatif. Les nanoparticules d'oxyde de zinc appliquées par voie topique n'ont eu aucun effet détectable sur l'état oxydatif de la peau des volontaires. Aucune réaction ou effet indésirable n'a été observé(e) après tous les traitements, y compris la microbiopsie. CONCLUSION: Les données obtenues étayent l'hypothèse selon laquelle la microbiopsie est une approche viable pour étudier les interactions des produits cosmétiques sur la peau des volontaires, avec la possibilité de réaliser des dosages moléculaires et à haut débit, avec un risque très faible pour les volontaires.

The fractional laser-induced coagulation zone characterized over time by laser scanning confocal microscopy-A proof of concept study.

BACKGROUND: Ablative fractional laser (AFXL) is an acknowledged technique to increase uptake of topical agents in skin. Micro thermal ablation zones (MAZs) consist of ablated vertical channels surrounded by a coagulation zone (CZ). Laser scanning confocal microscopy (LSCM) images individual MAZs at 733 nm (reflectance confocal microscopy (RCM)). Further, LSCM can image sodium fluorescein (NaF) fluorescence with 488 nm excitation (fluorescence confocal microcopy (FCM)), a small hydrophilic test molecule (370 MW, log P -1.52), which may simulate uptake, bio-distribution and kinetics of small hydrophilic drugs. OBJECTIVES: To explore LSCM for combined investigations of CZ thickness and uptake, bio-distribution and kinetics of NaF in AFXL-exposed skin. STUDY DESIGNS/METHODS AND MATERIALS: Excised human abdominal skin samples were exposed to AFXL (15 mJ/microbeam, 2% density) and NaF gel (1000 µg/ml, 10 µl/cm2) in six repetitions, including untreated control samples. CZ thickness and spatiotemporal fluorescence intensities (FI) were quantified up to four hours after NaF application by RCM and FCM. Test sites were scanned to a depth of 200 µm, quantifying thickness of skin compartments (stratum corneum, epidermis, upper dermis), individual CZ thicknesses and FI in CZ and surrounding skin. RESULTS: RCM images established skin morphology to a depth of 200 µm. The CZ thickness measurements were feasible to a depth of 50 µm, and remained unchanged over time at 50 µm (P > 0.5). FI were detected to a depth of 160 µm and remained constant in CZ up to four hours after NaF application (15 minutes: 79 AU (73-92 AU), 60 minutes: 72 AU (58-82 AU), four hours: 78 AU (71-90 AU), P > 0.1). In surrounding skin, FI increased significantly over time, but remained lower than FI in CZ (15 minutes: 21 AU (17-22 AU), 60 minutes: 21 AU (19-26 AU), four hours: 42 (31- 48 AU), P = 0.03). AFXL-processed skin generated higher FI compared to non-laser processed skin in epidermis and upper dermis at 60 minutes and four hours (P = 0.03). CONCLUSIONS: By LSCM, assessment of the AFXL-induced CZ thickness was feasible to a depth of 50 µm, and assessment of FI from a small hydrophilic test molecule, NaF in CZ and surrounding skin feasible to a depth of 160 µm. Lasers Surg. Med. 50:70-77, 2018. © 2017 Wiley Periodicals, Inc.

Noninvasive methods for the assessment of photoageing.

Although histopathological dermal elastosis is the current gold standard for the diagnosis of photoageing, noninvasive methods for quantifying the amount of photodamage to skin are clearly preferable. This study is the first to survey five noninvasive methods of assessing photoageing (clinical examination, spectrophotometry, skin surface topography, reflectance confocal microscopy and fluorescence lifetime imaging microscopy) in the same individual. Measurements for each noninvasive method were compared across nine individuals from three participant groups ('younger', 'older' and 'photodamaged') in UV-protected volar and UV-exposed dorsal forearm skin. Overall, participants in the younger group had the lowest measures of photodamage, while those in the photodamaged group had the highest, as indicated by each modality. The five noninvasive strategies surveyed in this study may demonstrate potential as a suitable methodology for the quantification of photoageing. The advantage of such noninvasive methods is that they allow for skin visualisation in vivo and repeated assessments of the same site. The main limitation of this study was its small sample size, which may have precluded many findings of statistical significance.

A blueprint for staging of murine melanocytic lesions based on the Cdk4 ( R24C/R24C ) ::Tyr- NRAS ( Q ) ( 61K ) model.

It has been shown that gene mutations which drive the development of malignant melanoma (MM) in humans also lead to emergence of MM when engineered mice. However, little attention has been paid to the clinical and histopathological features of melanocytic lesions and their natural history in a given mouse model. This knowledge is crucial to enable us to understand how engineered mutations influence the initiation and evolution of melanocytic lesions, and/or for the use of mice as a preclinical model to test specific treatments. We recently reported the development of melanocytic proliferations along the spectrum of naevi to MM in a Cdk4 ( R24C/R24C ) ::Tyr- NRAS ( Q ) ( 61K ) mouse model. In this study, we followed the development of lesions over time using digital photography and dermoscopy with the aim to correlate the clinical and histopathological features of lesions developing in this model. We identified two types of lesions. The first are slow-growing dermal MMs that emanate from dermal naevi. The second did not emanate from naevi, grew rapidly, and appeared to be solely confined to the subcutaneous fat. We present a simple staging system for the MMs that progress from naevi, based on depth of extension into the dermis and subcutis. This represents a blueprint for documentation and follow-up of MMs in the live animal, which is critical for the proper use of murine melanoma models.

Changes in the skin microbiome associated with squamous cell carcinoma in transplant recipients.

Actinic keratoses (AK) arise in severely photo-damaged skin and can progress to squamous cell carcinomas (SCC). AK and SCC are common in Caucasian populations, and immunosuppressed individuals have a markedly higher risk of developing SCC. An overabundance of Staphylococcus aureus has been reported in AK and SCC lesions of immunocompetent individuals, however, the AK/SCC microbiome in immunosuppressed cohorts has not been investigated. Here, the microbial profile and bacterial load of AK, SCC and control skin swabs from 32 immunosuppressed organ transplant recipients were characterised via SSU rRNA gene sequencing and qPCR, and compared to a previously described immunocompetent cohort. Although the taxonomic composition of skin swab samples was mostly subject-specific, significant differences were observed between control skin, AK, and SCC in both cohorts. Surface bacterial load was increased and alpha diversity decreased in AK and SCC compared to control skin due to an increased abundance of Staphylococcus species and relative decrease of skin commensals. Staphylococcus epidermidis predominated on SCC from transplant recipients in contrast to SCC of immunocompetent subjects dominated by S. aureus. In conclusion, AK and SCC of immunosuppressed and immunocompetent subjects present with distinctive microbial dysbioses, which may be relevant to SCC pathogenesis and progression.

Cytokine/Chemokine assessment as a complementary diagnostic tool for inflammatory skin diseases.

Inflammatory skin conditions are the 4th leading cause of non-fatal health burden in the general population worldwide. The diagnosis of skin lesions due to systemic drug reactions, viral or bacterial exanthems, or in patients with psoriasis, atopic dermatitis or contact dermatitis is often difficult and relies heavily upon conventional histopathologic examination. Conversely, it is widely accepted that the cutaneous profile of inflammatory markers, or 'inflammatory signature', is differentially expressed in various skin conditions. In this pilot study, we investigated the possibility of inflammatory skin disease diagnosis from an immunological perspective in small punch biopsies. We collected lesional and perilesional punch biopsies from 139 patients suffering from a variety of inflammatory skin conditions and attending the Dermatology Department at the Princess Alexandra Hospital in Brisbane, Australia. Using bead-based immunoassays we were able to measure 13 out of 17 inflammatory markers from a pre-selected multi-analyte panel and to detect significant differences between lesional and perilesional biopsies from each individual patient. Hierarchical and unbiased clustering methods based on inflammatory signatures grouped psoriasis and atopic dermatitis lesions into individual clusters in contrast to other skin conditions, highlighting the potential of inflammatory signatures to be used as diagnostic differentiators and to inform alternative targets in anti-inflammatory treatment strategies.

Time-correlated single photon counting for simultaneous monitoring of zinc oxide nanoparticles and NAD(P)H in intact and barrier-disrupted volunteer skin.

PURPOSE: There is a lack of relevant, non-animal alternatives for assessing exposure and toxicity of nanoparticle-containing cosmetics, e.g. sunscreens. Our goal was to evaluate timecorrelated single photon counting (TCSPC) for simultaneous monitoring of zinc oxide nanoparticles (ZnO-NP) and the metabolic state of volunteer skin. METHODS: We separated the fluorescence lifetime signatures of endogenous fluorophore signals (i.e. nicotinamide adenine dinucleotide phosphate, NAD(P)H and keratin) and the ZnO-NP signal using advanced TCSPC to simultaneously determine ZnO-NP penetration profiles and NAD(P)H changes in subjects with altered barrier function, including tape-stripped skin and in psoriasis or atopic dermatitis lesions. RESULTS: We detected no ZnO-NP penetration into viable human skin in any group. ZnO-NP signal was significantly increased (p < 0.01) on the surface of tape-stripped and lesional skin after 4 and 2 h of treatment, respectively. Free NAD(P)H signal significantly increased in tape-stripped viable epidermis treated for 4 h of ZnO-NP compared to vehicle control. No significant NAD(P)H changes were noted in the lesional study. CONCLUSION: TCSPC techniques enabled simultaneous, real-time quantification of ZnO-NP concentration and NAD(P)H via non-invasive imaging in the stratum corneum and viable epidermis of volunteers.

Gold nanoparticle penetration and reduced metabolism in human skin by toluene.

PURPOSE: To measure penetration and metabolic effects of ion-stabilized, polar, 15 nm gold nanoparticles in aqueous solution (AuNP-Aq) and sterically stabilized, non-polar, 6 nm gold nanoparticles in toluene (AuNP-TOL) on excised human skin. METHODS: Gold nanoparticles were characterized with dynamic light scattering and transmission electron microscopy (TEM). Skin penetration studies were done on frozen or fresh excised skin using static Franz diffusion cells. Viable treated skin was assessed by dermoscopy, reflectance confocal microscopy (RCM), multiphoton tomography (MPT) with fluorescence lifetime imaging microscopy (FLIM), and TEM. RESULTS: Dermoscopy and RCM showed large aggregates in the furrows of AuNP-Aq-treated skin. Treatment of thawed and viable skin only showed enhanced permeability to nanoparticles in the AuNP-TOL group with MPT and FLIM imaging to stratum spinosum of epidermis. TEM analysis revealed gold nanoparticles within AuNP-treated stratum corneum. FLIM analysis of NAD(P)H showed a significant decrease in total NAD(P)H in all toluene-treated groups. CONCLUSIONS: Gold nanoparticles, 15 nm, in aqueous solution aggregated on the skin surface. Toluene treatment eliminated skin metabolism; skin treated with toluene/gold nanoparticles (6 nm) for 24 h, but not at 4 h, showed increased nanoparticle permeability. These results are of value to nanotoxicology.

Tracking extracellular vesicle phenotypic changes enables treatment monitoring in melanoma.

Monitoring targeted therapy in real time for cancer patients could provide vital information about the development of drug resistance and improve therapeutic outcomes. Extracellular vesicles (EVs) have recently emerged as a promising cancer biomarker, and EV phenotyping shows high potential for monitoring treatment responses. Here, we demonstrate the feasibility of monitoring patient treatment responses based on the plasma EV phenotypic evolution using a multiplex EV phenotype analyzer chip (EPAC). EPAC incorporates the nanomixing-enhanced microchip and the multiplex surface-enhanced Raman scattering (SERS) nanotag system for direct EV phenotyping without EV enrichment. In a preclinical model, we observe the EV phenotypic heterogeneity and different phenotypic responses to the treatment. Furthermore, we successfully detect cancer-specific EV phenotypes from melanoma patient plasma. We longitudinally monitor the EV phenotypic evolution of eight melanoma patients receiving targeted therapy and find specific EV profiles involved in the development of drug resistance, reflecting the potential of EV phenotyping for monitoring treatment responses.

A high-resolution study of in situ surface-enhanced Raman scattering nanotag behavior in biological systems.

The colloidal stability of surface-enhanced Raman scattering (SERS) nanotags (Raman reporter-conjugated plasmonic nanoparticles) significantly affects the accuracy and reproducibility of SERS measurements, particularly in biological systems. Limited understanding of SERS nanotag stability may partly hamper the translation of SERS nanotags from the laboratory to their use in the clinic. In this contribution, we utilized differential centrifugal sedimentation (DCS), a reliable and straightforward technique to comprehensively analyze the colloidal stability of SERS nanotags in biological systems. Compared with other particle characterization techniques, DCS has been shown to have a unique advantage for high-resolution and high-throughput polydisperse particle characterization. DCS data revealed that the universal aggregation prevention practice of coating SERS nanotags with silica or bovine serum albumin layers did not sufficiently stabilize them in common measurement environments (e.g., 1 × PBS). Combined DCS and SERS measurements established a strong correlation between the degrees of nanotag aggregation and signal intensities, further reinforcing the necessity of characterizing SERS nanotag stability for every condition in which they are used. We also found that increasing the protein thickness by the inclusion of extra protein components in the detection environments and antibody functionalization can improve the stability of SERS nanotags. We believe that this study can provide guidelines on appropriate measurement techniques and particle design considerations to assess and improve SERS nanotag stability in complex biological systems.

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis.

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

Phase 1 Safety, Pharmacokinetics, and Fluorescence Imaging Study of Tozuleristide (BLZ-100) in Adults With Newly Diagnosed or Recurrent Gliomas.

BACKGROUND: Fluorescence-guided surgery (FGS) can improve extent of resection in gliomas. Tozuleristide (BLZ-100), a near-infrared imaging agent composed of the peptide chlorotoxin and a near-infrared fluorophore indocyanine green, is a candidate molecule for FGS of glioma and other tumor types. OBJECTIVE: To perform a phase 1 dose-escalation study to characterize the safety, pharmacokinetics, and fluorescence imaging of tozuleristide in adults with suspected glioma. METHODS: Patients received a single intravenous dose of tozuleristide 3 to 29 h before surgery. Fluorescence images of tumor and cavity in Situ before and after resection and of excised tissue ex Vivo were acquired, along with safety and pharmacokinetic measures. RESULTS: A total of 17 subjects received doses between 3 and 30 mg. No dose-limiting toxicity was observed, and no reported adverse events were considered related to tozuleristide. At doses of 9 mg and above, the terminal serum half-life for tozuleristide was approximately 30 min. Fluorescence signal was detected in both high- and low-grade glial tumors, with high-grade tumors generally showing greater fluorescence intensity compared to lower grade tumors. In high-grade tumors, signal intensity increased with increased dose levels of tozuleristide, regardless of the time of dosing relative to surgery. CONCLUSION: These results support the safety of tozuleristide at doses up to 30 mg and suggest that tozuleristide imaging may be useful for FGS of gliomas.

RNA-seq reveals more consistent reference genes for gene expression studies in human non-melanoma skin cancers.

Identification of appropriate reference genes (RGs) is critical to accurate data interpretation in quantitative real-time PCR (qPCR) experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq) to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer.

BRAF wild-type melanoma in situ arising in a BRAF V600E mutant dysplastic nevus.

IMPORTANCE: The BRAF V600E mutation accounts for the majority of BRAF mutations found in cutaneous melanoma and is also commonly found in nevi. We used dermoscopy-targeted sampling and a microbiopsy device coupled with DNA sequence analysis to highlight BRAF V600E heterogeneity within a multicomponent melanocytic proliferation. This sampling technique demonstrates the prospect of in vivo application in a clinical setting. OBSERVATIONS: A man in his 50s with Fitzpatrick skin type II presented with an irregularly pigmented melanocytic lesion on his back that met melanoma-specific dermoscopic criteria, and diagnostic shave excision of the lesion was performed. Histopathologic analysis revealed a melanoma in situ arising in a dysplastic nevus. Dermoscopy-targeted microbiopsy specimens were taken across the lesion, and genotyping was carried out on extracted DNA samples for BRAF and NRAS mutations. The melanoma in situ showed only BRAF wild-type results, while the dysplastic nevus showed both BRAF wild-type and BRAF V600E mutations. Sequencing in all DNA samples revealed NRAS wild-type genotype. CONCLUSIONS AND RELEVANCE: Dermoscopy-targeted sampling and genotyping of a melanoma in situ arising in a dysplastic nevus revealed a phenotype-genotype paradox that confounds the exclusive significance of BRAF and NRAS mutations in melanoma pathogenesis. Further studies are required to investigate the importance of other candidate genes linked to melanomagenesis.

Microneedle enhanced delivery of cosmeceutically relevant peptides in human skin.

Peptides and proteins play an important role in skin health and well-being. They are also found to contribute to skin aging and melanogenesis. Microneedles have been shown to substantially enhance skin penetration and may offer an effective means of peptide delivery enhancement. The aim of this investigation was to assess the influence of microneedles on the skin penetration of peptides using fluorescence imaging to determine skin distribution. In particular the effect of peptide chain length (3, 4, 5 amino acid chain length) on passive and MN facilitated skin penetration was investigated. Confocal laser scanning microscopy was used to image fluorescence intensity and the area of penetration of fluorescently tagged peptides. Penetration studies were conducted on excised full thickness human skin in Franz type diffusion cells for 1 and 24 hours. A 2 to 22 fold signal improvement in microneedle enhanced delivery of melanostatin, rigin and pal-KTTKS was observed. To our knowledge this is the first description of microneedle enhanced skin permeation studies on these peptides.