Single-cell phenotyping within transparent intact tissue through whole-body clearing.

Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.

Mechanisms of dermatological toxicities to immune checkpoint inhibitor cancer therapies.

The discovery of immune checkpoint inhibition (ICI) sparked a revolution in the era of targeted anticancer therapy. However, although monoclonal antibodies targeting the cytotoxic T-lymphocyte antigen-4 and programmed death-1 axes have improved survival in patients with advanced cancers, these immunotherapies are associated with a wide spectrum of dermatological immune-related adverse events (irAEs), ranging from mild to life-threatening. Several publications have addressed the clinical and histopathological classification of these skin-directed irAEs, their impact on anti-tumour immunity and survival, and the critical role of supportive oncological dermatology in their management. In this paper, we review the current understanding of the mechanistic drivers of immune-related skin toxicities with a focus on inflammatory, immunobullous and melanocyte/pigment-related reactions. We detail the specific immune-based mechanisms that may underlie different cutaneous reactions. We also discuss potential mechanisms as they relate to extracutaneous irAEs and the lessons learned from these, the potential overlap with cutaneous irAEs, techniques to study differences in immune-related vs. de novo skin reactions, and how treatment of these AEs impacts cancer treatment, patient quality of life and overall survival. An improved understanding of the mechanistic basis of cutaneous irAEs will allow clinicians to develop and use blood-based biomarkers that could help ultimately predict onset and/or severity of these irAEs, and to implement rational mechanistic-based treatment strategies that are targeted to the irAEs while potentially avoiding reducing the anti-tumour effect of ICIs.

Topological Arrangement of Cardiac Fibroblasts Regulates Cellular Plasticity.

RATIONALE: Cardiac fibroblasts do not form a syncytium but reside in the interstitium between myocytes. This topological relationship between fibroblasts and myocytes is maintained throughout postnatal life until an acute myocardial injury occurs, when fibroblasts are recruited to, proliferate and aggregate in the region of myocyte necrosis. The accumulation or aggregation of fibroblasts in the area of injury thus represents a unique event in the life cycle of the fibroblast, but little is known about how changes in the topological arrangement of fibroblasts after cardiac injury affect fibroblast function. OBJECTIVE: The objective of the study was to investigate how changes in topological states of cardiac fibroblasts (such as after cardiac injury) affect cellular phenotype. METHODS AND RESULTS: Using 2 and 3-dimensional (2D versus 3D) culture conditions, we show that simple aggregation of cardiac fibroblasts is sufficient by itself to induce genome-wide changes in gene expression and chromatin remodeling. Remarkably, gene expression changes are reversible after the transition from a 3D back to 2D state demonstrating a topological regulation of cellular plasticity. Genes induced by fibroblast aggregation are strongly associated and predictive of adverse cardiac outcomes and remodeling in mouse models of cardiac hypertrophy and failure. Using solvent-based tissue clearing techniques to create optically transparent cardiac scar tissue, we show that fibroblasts in the region of dense scar tissue express markers that are induced by fibroblasts in the 3D conformation. Finally, using live cell interferometry, a quantitative phase microscopy technique to detect absolute changes in single cell biomass, we demonstrate that conditioned medium collected from fibroblasts in 3D conformation compared with that from a 2D state significantly increases cardiomyocyte cell hypertrophy. CONCLUSIONS: Taken together, these findings demonstrate that simple topological changes in cardiac fibroblast organization are sufficient to induce chromatin remodeling and global changes in gene expression with potential functional consequences for the healing heart.

Effects of teriparatide on morphology of aortic calcification in aged hyperlipidemic mice.

Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit the onset of aortic calcification. Whether teriparatide affects the progression of preexisting aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over 1 yr to induce aortic calcification, treated for 4.5 wk with daily injections of control vehicle (PBS), 40 µg/kg teriparatide (PTH40), or 400 µg/kg teriparatide (PTH400), and assayed for aortic calcification by microcomputed tomography (microCT) before and after treatment. In a followup cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400 and assayed for aortic calcification by serial microCT and micropositron emission tomography. In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18F-NaF incorporation, detected by serial micropositron emission tomography, increased in the PBS-treated group (+14 ± 5%). In contrast, 18F-NaF incorporation decreased in the PTH400-treated group (-33 ± 20%, P = 0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400-treated group compared with the PBS-treated group ( P = 0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice that received PTH400 [2.1 ± 0.6% vs. control mice (0.5 ± 0.1%), P = 0.02]. In summary, although teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability. NEW & NOTEWORTHY Parathyroid hormone regulates bone mineralization and may also affect vascular calcification, which is an important issue, given that its active fragment, teriparatide, is widely used for the treatment of osteoporosis. To determine whether teriparatide alters vascular calcification, we imaged aortic calcification in mice treated with teriparatide and control mice. Although teriparatide did not affect the calcium content of cardiovascular deposits, it reduced their fluoride tracer uptake.

Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 µm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument.

Keratoacanthoma and squamous cell carcinoma are distinct from a molecular perspective.

Keratoacanthoma is a controversial entity. Some consider keratoacanthoma as a variant of squamous cell carcinoma, whereas others see it as a distinct self-resolving squamoproliferative lesion. Our objective is to examine the relationship of keratoacanthoma with squamous cell carcinoma and normal skin by using DNA microarrays. DNA microarray studies were performed on formalin-fixed and paraffin-embedded blocks from ten cases of actinic keratoacanthoma utilizing the U133plus2.0 array. These results were compared with our previously developed microarray database of ten squamous cell carcinoma and ten normal skin samples. Keratoacanthoma demonstrated 1449 differentially expressed genes in comparison with squamous cell carcinoma (>5-fold change: P<0.01) with 908 genes upregulated and 541 genes downregulated. Keratoacanthoma showed 2435 differentially expressed genes in comparison with normal skin (>5-fold change: P<0.01) with 1085 genes upregulated and 1350 genes downregulated. The most upregulated genes, comparing keratoacanthoma with normal skin included MALAT1, S100A8, CDR1, TPM4, and CALM1. The most downregulated genes included SCGB2A2, DCD, THRSP, ADIPOQ, adiponectin, and ADH1B. The molecular biological pathway analysis comparing keratoacanthoma with normal skin showed that cellular development, cellular growth and proliferation, cell death/apoptosis, and cell cycle pathways are prominently involved in the pathogenesis of keratoacanthoma. The most enriched canonical pathways were clathrin-mediated endocytosis signaling, molecular mechanisms of cancer and integrin signaling. The distinctive gene expression profile of keratoacanthoma reveals that it is molecularly distinct from squamous cell carcinoma. The molecular pathways and genes differentially expressed in comparing keratoacanthoma with normal skin suggest that keratoacanthoma is a neoplasm that can regress due to upregulation of the cell death/apoptosis pathway.

Comparison of Immunotherapy versus Targeted Therapy Effectiveness in BRAF-Mutant Melanoma Patients and Use of cGAS Expression and Aneuploidy as Potential Prognostic Biomarkers.

BRAF-mutant melanoma patients can be treated with targeted therapy or immunotherapies, and it is not clear which should be provided first. Targeted treatments do not work in up to one-third of cases, while immunotherapies may only be effective in up to 60% and come with a high risk of immune-related side effects. Determining which treatment to provide first is thus of critical importance. Recent studies suggest that chromosomal instability and aneuploidy and cyclic GMP-AMP synthase (cGAS) can act as biomarkers for cancer severity and patient outcome. Neither potential biomarker has been extensively studied in melanoma. We examined 20 BRAF-mutant melanomas treated with immunotherapy or targeted therapy and measured chromosomal aneuploidy and cGAS expression levels. Treatment type, aneuploidy, and cGAS expression were correlated with progression-free survival (PFS) in these patients. Those treated with immunotherapy first had significantly better outcomes than those treated with targeted therapy, suggesting immunotherapy should be strongly considered as the first-line therapy for patients bearing BRAF-mutant melanoma. We found that there was no correlation of aneuploidy with outcome while there was some positive correlation of cGAS levels with PFS. Further studies are needed to confirm these findings and to test other potential biomarkers.

MIF and CD74 as Emerging Biomarkers for Immune Checkpoint Blockade Therapy.

Immune checkpoint blockade (ICB) therapy is used to treat a wide range of cancers; however, some patients are at risk of developing treatment resistance and/or immune-related adverse events (irAEs). Thus, there is a great need for the identification of reliable predictive biomarkers for response and toxicity. The cytokine MIF (macrophage migration inhibitory factor) and its cognate receptor CD74 are intimately connected with cancer progression and have previously been proposed as prognostic biomarkers for patient outcome in various cancers, including solid tumors such as malignant melanoma. Here, we assess their potential as predictive biomarkers for response to ICB therapy and irAE development. We provide a brief overview of their function and roles in the context of cancer and autoimmune disease. We also review the evidence showing that MIF and CD74 may be of use as predictive biomarkers of patient response to ICB therapy and irAE development. We also highlight that careful consideration is required when assessing the potential of serum MIF levels as a biomarker due to its reported circadian expression in human plasma. Finally, we suggest future directions for the establishment of MIF and CD74 as predictive biomarkers for ICB therapy and irAE development to guide further research in this field.

Optimization of Tissue Digestion Methods for Characterization of Photoaged Skin by Single Cell RNA Sequencing Reveals Preferential Enrichment of T Cell Subsets.

Healthy human skin tissue is often used as a control for comparison to diseased skin in patients with skin pathologies, including skin cancers or other inflammatory conditions such as atopic dermatitis or psoriasis. Although non-affected skin from these patients is a more appropriate choice for comparison, there is a paucity of studies examining such tissue. This lack is exacerbated by the difficulty of processing skin tissue for experimental analysis. In addition, choosing a processing protocol for skin tissue which preserves cell viability and identity while sufficiently dissociating cells for single-cell analysis is not a trivial task. Here, we compare three digestion methods for human skin tissue, evaluating the cell yield and viability for each protocol. We find that the use of a sequential dissociation method with multiple enzymatic digestion steps produces the highest cell viability. Using single-cell sequencing, we show this method results in a relative increase in the proportion of non-antigen-presenting mast cells and CD8 T cells as well as a relative decrease in the proportion of antigen-presenting mast cells and KYNU+ CD4 T cells. Overall, our findings support the use of this sequential digestion method on freshly processed human skin samples for optimal cell yield and viability.

DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells.

How the biochemical reactions that lead to the repair of DNA damage are controlled by the diffusion and availability of protein reactants within the nucleoplasm is poorly understood. Here, we use gene targeting to replace Brca2 (a cancer suppressor protein essential for DNA repair) with a functional enhanced green fluorescent protein (EGFP)-tagged form, followed by fluorescence correlation spectroscopy to measure Brca2-EGFP diffusion in the nucleoplasm of living cells exposed to DNA breakage. Before damage, nucleoplasmic Brca2 molecules exhibit complex states of mobility, with long dwell times within a sub-fL observation volume, indicative of restricted motion. DNA damage significantly enhances the mobility of Brca2 molecules in the S/G2 phases of the cell cycle, via signaling through damage-activated protein kinases. Brca2 mobilization is accompanied by increased binding within the nucleoplasm to its cargo, the Rad51 recombinase, measured by fluorescence cross-correlation spectroscopy. Together, these results suggest that DNA breakage triggers the redistribution of soluble nucleoplasmic Brca2 molecules from a state of restricted diffusion, into a mobile fraction available for Rad51 binding. Our findings identify signal-regulated changes in nucleoplasmic protein diffusion as a means to control biochemical reactions in the cell nucleus.

Survival Among Veterans Receiving Steroids for Immune-Related Adverse Events After Immune Checkpoint Inhibitor Therapy.

Importance: Systemic steroids are commonly used to manage immune-related adverse events (irAEs), but it remains unclear whether they may undermine immune checkpoint inhibitor (ICI) therapy outcomes. Few studies have assessed the impact of steroid timing and its association with continuation or cessation of ICI therapy. Objective: To characterize how systemic steroids and steroid timing for irAEs are associated with survival in patients receiving ICI therapy. Design, Setting, and Participants: This multicenter retrospective cohort study encompassed veterans receiving ICI for cancer between January 1, 2010, and December 31, 2021. Data analysis was conducted September 8, 2023. Exposures: Identifiable primary diagnosis of cancer. Patients were categorized into 3 cohorts: those receiving no steroids, systemic steroids for irAEs, and steroids for non-irAE-associated reasons. All eligible patients received 1 or more doses of an ICI (atezolizumab, avelumab, cemiplimab, durvalumab, ipilimumab, nivolumab, or pembrolizumab). Eligible patients in the steroid group received at least 1 dose (intravenous, intramuscular, or oral) of dexamethasone, hydrocortisone, methylprednisolone, prednisone, or prednisolone. Steroid use at baseline for palliation or infusion prophylaxis or delivered as a single dose was deemed to be non-irAE associated. All other patterns of steroid use were assumed to be for irAEs. Main Outcomes and Measures: The primary outcome was overall survival, with a 5-year follow-up after ICI initiation. Kaplan-Meier survival analyses were performed with pairwise log-rank tests to determine significance. Risk was modeled with Cox proportional hazard regression. Results: The cohort consisted of 20 163 veterans receiving ICI therapy including 12 221 patients (mean [SD] age, 69.5 [8.0] years; 11 830 male patients [96.8%]; 9394 White patients [76.9%]) who received systemic steroids during ICI treatment and 7942 patients (mean [SD] age, 70.3 [8.5] years; 7747 male patients [97.5%]; 6085 White patients [76.6%]) who did not. Patients with an irAE diagnosis had significantly improved overall survival (OS) compared with those without (median [IQR] OS, 17.4 [6.6 to 48.5] months vs 10.5 [3.5 to 36.8] months; adjusted hazard ratio, 0.84; 95% CI, 0.81-0.84; P < .001). For patients with irAEs, systemic steroids for irAEs were associated with significantly improved survival compared with those who received steroids for non-irAE-related reasons or no steroid treatment (median [IQR] OS, 21.3 [9.3 to 58.2] months vs 13.6 [5.5 to 33.7] months vs 15.8 [4.9 to not reached] months; P <.001). However, among those who received steroids for irAEs, early steroid use (<2 months after ICI initiation) was associated with reduced relative survival benefit vs later steroid use, regardless of ICI continuation or cessation following steroid initiation (median [IQR] OS after ICI cessation 4.4 [1.9 to 19.5] months vs 16.0 [8.0 to 42.2] months; median [IQR] OS after ICI continuation, 16.0 [7.1 to not reached] months vs 29.2 [16.5 to 53.5] months; P <.001). Conclusions and Relevance: This study suggests that steroids for irAE management may not abrogate irAE-associated survival benefits. However, early steroid administration within 2 months of ICI initiation is associated with shorter survival despite continuation of ICI therapy.

Advances in Early Detection of Melanoma and the Future of At-Home Testing.

The past decade has seen numerous advancements in approaches to melanoma detection, each with the common goal to stem the growing incidence of melanoma and its mortality rate. These advancements, while well documented to increase early melanoma detection, have also garnered considerable criticism of their efficacy for improving survival rates. In this review, we discuss the current state of such early detection approaches that do not require direct dermatologist intervention. Our findings suggest that a number of at-home and non-specialist methods exist with high accuracy for detecting melanoma, albeit with a few notable concerns worth further investigation. Additionally, research continues to find new approaches using artificial intelligence which have promise for the future.

Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of melanoma, version 3.0.

Since the first approval for immune checkpoint inhibitors (ICIs) for the treatment of cutaneous melanoma more than a decade ago, immunotherapy has completely transformed the treatment landscape of this chemotherapy-resistant disease. Combination regimens including ICIs directed against programmed cell death protein 1 (PD-1) with anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) agents or, more recently, anti-lymphocyte-activation gene 3 (LAG-3) agents, have gained regulatory approvals for the treatment of metastatic cutaneous melanoma, with long-term follow-up data suggesting the possibility of cure for some patients with advanced disease. In the resectable setting, adjuvant ICIs prolong recurrence-free survival, and neoadjuvant strategies are an active area of investigation. Other immunotherapy strategies, such as oncolytic virotherapy for injectable cutaneous melanoma and bispecific T-cell engager therapy for HLA-A*02:01 genotype-positive uveal melanoma, are also available to patients. Despite the remarkable efficacy of these regimens for many patients with cutaneous melanoma, traditional immunotherapy biomarkers (ie, programmed death-ligand 1 expression, tumor mutational burden, T-cell infiltrate and/or microsatellite stability) have failed to reliably predict response. Furthermore, ICIs are associated with unique toxicity profiles, particularly for the highly active combination of anti-PD-1 plus anti-CTLA-4 agents. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to develop this clinical practice guideline on immunotherapy for the treatment of melanoma, including rare subtypes of the disease (eg, uveal, mucosal), with the goal of improving patient care by providing guidance to the oncology community. Drawing from published data and clinical experience, the Expert Panel developed evidence- and consensus-based recommendations for healthcare professionals using immunotherapy to treat melanoma, with topics including therapy selection in the advanced and perioperative settings, intratumoral immunotherapy, when to use immunotherapy for patients with BRAFV600-mutated disease, management of patients with brain metastases, evaluation of treatment response, special patient populations, patient education, quality of life, and survivorship, among others.

Early Detection and Prognostic Assessment of Cutaneous Melanoma: Consensus on Optimal Practice and the Role of Gene Expression Profile Testing.

Importance: Therapy for advanced melanoma has transformed during the past decade, but early detection and prognostic assessment of cutaneous melanoma (CM) remain paramount goals. Best practices for screening and use of pigmented lesion evaluation tools and gene expression profile (GEP) testing in CM remain to be defined. Objective: To provide consensus recommendations on optimal screening practices and prebiopsy diagnostic, postbiopsy diagnostic, and prognostic assessment of CM. Evidence Review: Case scenarios were interrogated using a modified Delphi consensus method. Melanoma panelists (n = 60) were invited to vote on hypothetical scenarios via an emailed survey (n = 42), which was followed by a consensus conference (n = 51) that reviewed the literature and the rationale for survey answers. Panelists participated in a follow-up survey for final recommendations on the scenarios (n = 45). Findings: The panelists reached consensus (≥70% agreement) in supporting a risk-stratified approach to melanoma screening in clinical settings and public screening events, screening personnel recommendations (self/partner, primary care provider, general dermatologist, and pigmented lesion expert), screening intervals, and acceptable appointment wait times. Participants also reached consensus that visual and dermoscopic examination are sufficient for evaluation and follow-up of melanocytic skin lesions deemed innocuous. The panelists reached consensus on interpreting reflectance confocal microscopy and some but not all results from epidermal tape stripping, but they did not reach consensus on use of certain pigmented lesion evaluation tools, such as electrical impedance spectroscopy. Regarding GEP scores, the panelists reached consensus that a low-risk prognostic GEP score should not outweigh concerning histologic features when selecting patients to undergo sentinel lymph node biopsy but did not reach consensus on imaging recommendations in the setting of a high-risk prognostic GEP score and low-risk histology and/or negative nodal status. Conclusions and Relevance: For this consensus statement, panelists reached consensus on aspects of a risk-stratified approach to melanoma screening and follow-up as well as use of visual examination and dermoscopy. These findings support a practical approach to diagnosing and evaluating CM. Panelists did not reach consensus on a clearly defined role for GEP testing in clinical decision-making, citing the need for additional studies to establish the clinical use of existing GEP assays.

Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

Circulating biomarkers of response to immunotherapy and immune-related adverse events.

INTRODUCTION: Immune checkpoint blockade has revolutionized cancer treatment. However, response rates vary, and these treatments have a high rate of immune-related side effects, which can be limiting. Thus, tests to predict who will respond and who may experience side effects are of critical importance toward realizing the ultimate goal of precision oncology. AREAS COVERED: We review several of the most recent advances in circulating biomarkers that have been reported to be useful in predicting response and immune-related adverse events (irAE) to checkpoint blockade immunotherapies (CBI). We focus on high-quality studies published within the last few years. We highlight significant findings, identify areas for improvement, and provide recommendations on how these biomarkers may be translated into clinical utility. EXPERT OPINION: As newer immunotherapies are developed, there is a pressing need to identify circulating biomarkers that can help predict responses and side effects. Current studies are mostly small-scale and retrospective; there is a need for larger-scale and prospective studies to help validate several of the biomarkers detailed here. As oncology focuses more on precision-based approaches, it is likely that a combination of biomarkers, including circulating ones as detailed here, will have critical utility in guiding clinical decisions.

A Novel Potential Role for Monocytes Revealed by Single Cell Analysis of Immunotherapy Induced Immune Related Adverse Events.

Immune related adverse events (irAEs) are one of the leading causes of discontinuation of cancer immunotherapy treatment. Despite extensive research into the frequency and types of irAEs, little is known about the cell types and pathways through which these drugs cause the observed side effects. To identify cell types and pathways of interest, we have analyzed single cell sequencing data of PBMCs from patients who developed skin irAEs as a result of their immunotherapy treatment. Using Azimuth's cell type identification software for PBMCs and GSEA pathway analysis, we found macrophage cell populations and reactive oxygen species related pathways to be upregulated. These results provide important groundwork to build a complete picture of the mechanisms which cause irAEs and finding ways to more effectively treat them.

Single-Cell Identification of Melanoma Biomarkers in Circulating Tumor Cells.

The current standard for investigating tumors is surgical biopsy, which is costly, invasive, and difficult to perform serially. As an adjunct, circulating tumor cells (CTCs)-cells that have broken away from the primary tumor or metastatic sites-can be obtained from a blood draw and offer the potential for obtaining serial genetic information and serving as biomarkers. Here, we detail the potential for melanoma CTCs to serve as biomarkers and discuss a clinically viable methodology for single-cell CTC isolation and analysis that overcomes previous limitations. We explore the use of melanoma CTC biomarkers by isolating and performing single-cell RNA sequencing on CTCs from melanoma patients. We then compared transcriptional profiles of single melanoma CTCs against A375 cells and peripheral blood mononuclear cells to identify unique genes differentially regulated in circulating melanoma tumor cells. The information that can be obtained via analysis of these CTCs has significant potential in disease tracking.

Slip versus Slop: A Head-to-Head Comparison of UV-Protective Clothing to Sunscreen.

Ultraviolet radiation (UVR) exposure is the most important modifiable risk factor for skin cancer development. Although sunscreen and sun-protective clothing are essential tools to minimize UVR exposure, few studies have compared the two modalities head-to-head. This study evaluates the UV-protective capacity of four modern, sun-protective textiles and two broad-spectrum, organic sunscreens (SPF 30 and 50). Sun Protection Factor (SPF), Ultraviolet Protection Factor (UPF), Critical Wavelength (CW), and % UVA- and % UVB-blocking were measured for each fabric. UPF, CW, % UVA- and % UVB-blocking were measured for each sunscreen at 2 mg/cm2 (recommended areal density) and 1 mg/cm2 (simulating real-world consumer application). The four textiles provided superior UVR protection when compared to the two sunscreens tested. All fabrics blocked erythemogenic UVR better than the sunscreens, as measured by SPF, UPF, and % UVB-blocking. Each fabric was superior to the sunscreens in blocking full-spectrum UVR, as measured by CW and % UVA-blocking. Our data demonstrate the limitations of sunscreen and UV-protective clothing labeling and suggest the combination of SPF or UPF with % UVA-blocking may provide more suitable measures for broad-spectrum protection. While sunscreen remains an important photoprotective modality (especially for sites where clothing is impractical), these data suggest that clothing should be considered the cornerstone of UV protection.

Overstretched and overlooked: solving challenges faced by early-career investigators after the pandemic.

The coronavirus disease 2019 (COVID-19) pandemic has had a detrimental effect on research. However, little has been done to identify and solve the unique challenges faced by early career investigators (ECIs). As a group of American Cancer Society-funded ECIs, we provide recommendations for solving these challenges in the aftermath of the pandemic.