Investigating the tumor-immune microenvironment through extracellular vesicles from frozen patient biopsies and 3D cultures

Melanomas are highly immunogenic tumors that have been shown to activate the immune response. Nonetheless, a significant portion of melanoma cases are either unresponsive to immunotherapy or relapsed due to acquired resistance. During melanomagenesis, melanoma and immune cells undergo immunomodulatory mechanisms that aid in immune resistance and evasion. The crosstalk within melanoma microenvironment is facilitated through the secretion of soluble factors, growth factors, cytokines, and chemokines. In addition, the release and uptake of secretory vesicles known as extracellular vesicles (EVs) play a key role in shaping the tumor microenvironment (TME). Melanoma-derived EVs have been implicated in immune suppression and escape, promoting tumor progression. In the context of cancer patients, EVs are usually isolated from biofluids such as serum, urine, and saliva. Nonetheless, this approach neglects the fact that biofluid-derived EVs reflect not only the tumor, but also include contributions from different organs and cell types. For that, isolating EVs from tissue samples allows for studying different cell populations resident at the tumor site, such as tumor-infiltrating lymphocytes and their secreted EVs, which play a central anti-tumor role. Herein, we outline the first instance of a method for EV isolation from frozen tissue samples at high purity and sensitivity that can be easily reproduced without the need for complicated isolation methods. Our method of processing the tissue not only circumvents the need for hard-to-acquire freshly isolated tissue samples, but also preserves EV surface proteins which allows for multiplex surface markers profiling. Tissue-derived EVs provide insight into the physiological role of EVs enrichment at tumor sites, which can be overlooked when studying circulating EVs coming from different sources. Tissue-derived EVs could be further characterized in terms of their genomics and proteomics to identify possible mechanisms for regulating the TME. Additionally, identified markers could be correlated to overall patient survival and disease progression for prognostic purposes

Perturbing resistance: a network perspective

The recent convergence of high-dimensional molecular datasets with small-molecule inhibitor pipelines allows for selective targeting of aberrantly regulated pathways in many cancer types. But intra-tumor heterogeneity, paradoxical activation, intrinsic or acquired therapeutic resistance, and non-linear pathway interactions confound most simple targeting strategies(Widmer et al., 2015). For instance, MAPK signaling is activated by hot-spot mutations in BRAF, which are found in about 40-50% of melanoma cases. This article is protected by copyright. All rights reserved

The Prognostic Value of a Single, Randomly Timed Circulating Tumor DNA Measurement in Patients with Metastatic Melanoma

Simple Summary In this study, we investigated the associations of circulating tumor DNA (ctDNA), measured at a random time point during the patient’s treatment, with tumor progression and routine blood markers (protein S100, lactate dehydrogenase (LDH), and C-reactive protein (CRP)) in a cohort of patients with metastatic melanoma. Detectable ctDNA was associated with the presence of extracerebral disease, tumor progression, and poorer overall survival (OS). Elevated S100 and CRP was correlated with detectable ctDNA, whereas LDH was not. Our results further support the use of ctDNA in the clinical management of patients with metastatic melanoma. Abstract Melanoma currently lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Circulating tumor DNA (ctDNA), originating from tumor cells and detectable in plasma, has emerged as a possible biomarker in patients with metastatic melanoma. In this retrospective, single-center study, we collected 129 plasma samples from 79 patients with stage IIIB–IV melanoma as determined by the American Joint Committee on Cancer (AJCC, 8th edition). For the determination of ctDNA levels, we used eight different assays of droplet digital polymerase chain reaction (ddPCR) to detect the most common hotspot mutations in the BRAF and NRAS genes. The aim of the study was to investigate the association of the detectability of ctDNA at a non-prespecified time point in a patient’s treatment with tumor progression, and to correlate ctDNA with commonly used biomarkers (protein S100, LDH, and CRP). Patients with detectable ctDNA progressed more frequently in PET-CT within 12 months than those without detectable ctDNA. Detectability of ctDNA was associated with shorter OS in univariate and multivariate analyses. ctDNA was detectable in a statistically significantly larger proportion of patients with distant metastases (79%) than in patients with no distant metastases or only intracranial metastases (32%). Elevated protein S100 and CRP correlated better with detectable ctDNA than LDH. This study supports the potential of ctDNA as a prognostic biomarker in patients with metastatic melanoma. However, additional prospective longitudinal studies with quantitative assessments of ctDNA are necessary to investigate the limitations and strengths of ctDNA as a biomarker. Keywords: ctDNA; melanoma; tumor progression; PET-CT; S100; biomarke

Combinational expression of tumor testis antigens NY-ESO-1, MAGE-A3, and MAGE-A4 predicts response to immunotherapy in mucosal melanoma patients

PURPOSE: Immunotherapy using immune checkpoint inhibitors (ICI) has revolutionized cancer treatment in recent years, particularly in melanoma. While response to immunotherapy is associated with high tumor mutational burden (TMB), PD-L1 expression, and microsatellite instability in several cancers, tumors lacking these biomarkers can still respond to this treatment. Especially, mucosal melanoma, commonly exhibiting low TMB compared to cutaneous melanoma, may respond to immunotherapy with immune checkpoint inhibitors. Therefore, the aim of our study was to investigate novel biomarkers in mucosal melanoma that predict response to combined ipilimumab and nivolumab. METHODS: We investigated 10 tumor samples from 10 patients (three responders, seven non-responders) before treatment and six tumor samples from five patients after progression using a targeted Next Generation Sequencing (NGS) gene expression panel. The findings were corroborated with an independent method (i.e., immunohistochemical staining) on the same 10 tumor samples before treatment and, to increase the cohort, in addition on three tumor samples before treatment of more recent patients (one responder, two non-responders). RESULTS: With the targeted gene expression panel, we found the three tumor testis antigens CTAG1B (NY-ESO-1), MAGE-A3, and MAGE-A4 to be predominantly expressed in responding tumors. This marker panel was either not or not completely expressed in non-responders (p < 0.01). Using immunohistochemistry for all three markers, we could confirm the elevated expression in tumors responding to the ipilimumab/nivolumab combination therapy. CONCLUSION: In conclusion, these three biomarkers await validation in a larger patient cohort and could be easily used in future routine diagnostics to predict the outcome of ipilimumab/nivolumab combination therapy in mucosal melanoma patients

Phase-specific signatures of wound fibroblasts and matrix patterns define cancer-associated fibroblast subtypes

Healing wounds and cancers present remarkable cellular and molecular parallels, but the specific roles of the healing phases are largely unknown. We developed a bioinformatics pipeline to identify genes and pathways that define distinct phases across the time-course of healing. Their comparison to cancer transcriptomes revealed that a resolution phase wound signature is associated with increased severity in skin cancer and enriches for extracellular matrix-related pathways. Comparisons of transcriptomes of early- and late-phase wound fibroblasts vs skin cancer-associated fibroblasts (CAFs) identified an "early wound" CAF subtype, which localizes to the inner tumor stroma and expresses collagen-related genes that are controlled by the RUNX2 transcription factor. A "late wound" CAF subtype localizes to the outer tumor stroma and expresses elastin-related genes. Matrix imaging of primary melanoma tissue microarrays validated these matrix signatures and identified collagen- vs elastin-rich niches within the tumor microenvironment, whose spatial organization predicts survival and recurrence. These results identify wound-regulated genes and matrix patterns with prognostic potential in skin cancer

Focused Local Search for Random 3-Satisfiability

A local search algorithm solving an NP-complete optimisation problem can be viewed as a stochastic process moving in an 'energy landscape' towards eventually finding an optimal solution. For the random 3-satisfiability problem, the heuristic of focusing the local moves on the presently unsatisfiedclauses is known to be very effective: the time to solution has been observed to grow only linearly in the number of variables, for a given clauses-to-variables ratio $\alpha$ sufficiently far below the critical satisfiability threshold $\alpha_c \approx 4.27$. We present numerical results on the behaviour of three focused local search algorithms for this problem, considering in particular the characteristics of a focused variant of the simple Metropolis dynamics. We estimate the optimal value for the ``temperature'' parameter $\eta$ for this algorithm, such that its linear-time regime extends as close to $\alpha_c$ as possible. Similar parameter optimisation is performed also for the well-known WalkSAT algorithm and for the less studied, but very well performing Focused Record-to-Record Travel method. We observe that with an appropriate choice of parameters, the linear time regime for each of these algorithms seems to extend well into ratios $\alpha > 4.2$ -- much further than has so far been generally assumed. We discuss the statistics of solution times for the algorithms, relate their performance to the process of ``whitening'', and present some conjectures on the shape of their computational phase diagrams.Comment: 20 pages, lots of figure

Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling

Ample evidence pinpoints the phenotypic diversity of blood vessels (BVs) and site-specific functions of their lining endothelial cells (ECs). We harnessed single-cell RNA sequencing (scRNA-seq) to dissect the molecular heterogeneity of blood vascular endothelial cells (BECs) in healthy adult human skin and identified six different subpopulations, signifying arterioles, post-arterial capillaries, pre-venular capillaries, post-capillary venules, venules and collecting venules. Individual BEC subtypes exhibited distinctive transcriptomic landscapes associated with diverse biological pathways. These functionally distinct dermal BV segments were characterized by their unique compositions of conventional and novel markers (e.g., arteriole marker GJA5; arteriole capillary markers ASS1 and S100A4; pre-venular capillary markers SOX17 and PLAUR; venular markers EGR2 and LRG1), many of which have been implicated in vascular remodeling upon inflammatory responses. Immunofluorescence staining of human skin sections and whole-mount skin blocks confirmed the discrete expression of these markers along the blood vascular tree in situ, further corroborating BEC heterogeneity in human skin. Overall, our study molecularly refines individual BV compartments, whilst the identification of novel subtype-specific signatures provides more insights for future studies dissecting the responses of distinct vessel segments under pathological conditions

Clinical Presentation and Prognostic Features in Patients with Immunotherapy-Induced Vitiligo-like Depigmentation: A Monocentric Prospective Observational Study

Vitiligo-like depigmentation (VLD) is an immune-related adverse event (irAE) of checkpoint-inhibitor (CPI) treatment, which has previously been associated with a favourable outcome. The aim of this study was to explore clinical, biological and prognostic features of melanoma patients with VLD under CPI-treatment and to explore whether they exhibit a characteristic immune response profile in peripheral blood. Melanoma patients developing VLD under CPI were included in a prospective observational single-center cohort study. We collected and analysed clinical parameters, photographs and serum from 28 VLD patients. They received pembrolizumab (36%), nivolumab (11%), ipilimumab/nivolumab (32%) or clinical trial medications (21%). We performed a high-throughput proteomics assay (Olink), in which we identified a distinct proteomic signature in VLD patients in comparison to non-VLD CPI patients. Our clinical assessments revealed that VLD lesions had a predominantly symmetrical distribution pattern, with mostly smaller “freckle-like” macules and a preferential distribution in UV-exposed areas. Patients with previous targeted therapy showed a significantly longer time lapse between CPI initiation and VLD onset compared to non-pre-treated patients (12.5 vs. 6.25 months). Therapy responders exhibited a distinct proteomic profile when compared with non-responders in VLD such as upregulation of EDAR and downregulation of LAG3. ITGA11 was elevated in the VLD-group when compared to non-VLD-CPI-treated melanoma patients. Our findings demonstrate that on a proteomic level, VLD is characterized by a distinct immune signature when compared to CPI-treated patients without VLD and that therapy responsiveness is reflected by a characteristic immune profile. The pathomechanisms underlying these findings and how they could relate to the antitumoral response in melanoma remain to be elucidated

NRASQ61K melanoma tumor formation is reduced by p38-MAPK14 activation in zebrafish models and NRAS-mutated human melanoma cells.

Oncogenic BRAF and NRAS mutations drive human melanoma initiation. We used transgenic zebrafish to model NRAS mutant melanoma and the rapid tumor onset allowed us to study candidate tumor suppressors. We identified P38α-MAPK14 as a potential tumor suppressor in The Cancer Genome Atlas melanoma cohort of NRAS mutant melanomas, and overexpression significantly increased the time to tumor onset in transgenic zebrafish with NRAS-driven melanoma. Pharmacological activation of P38α-MAPK14 using anisomycin reduced in vitro viability of melanoma cultures, which we confirmed by stable overexpression of p38α. We observed that the viability of MEK-inhibitor resistant melanoma cells could be reduced by combined treatment of anisomycin and MEK-inhibition. Our study demonstrates that activating the p38α-MAPK14 pathway in the presence of oncogenic NRAS abrogates melanoma in vitro and in vivo.This project has received funding from the European Union’s Horizon 2020 432 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641458. The 433 work carried out at the University of Edinburgh was partly funded by EEP, MRC HGU Programme 434 (MC_UU_00007/9), European Research Council (ZF-MEL-CHEMBIO-648489), and L'Oreal-Melanoma 435 Research Alliance (401181)

Frequency, Treatment and Outcome of Immune-Related Toxicities in Patients with Immune-Checkpoint Inhibitors for Advanced Melanoma: Results from an Institutional Database Analysis

Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events (irAEs), which may result in treatment discontinuation. We sought to describe the onset, frequency, and kinetics of irAEs in melanoma patients in a real-life setting and to further investigate the prognostic role of irAEs in treatment outcomes. In this retrospective single-center cohort study, we included 249 melanoma patients. Onset, grade, and resolution of irAEs and their treatment were analyzed. A total of 191 (74.6%) patients in the non-adjuvant and 65 (25.3%) in the adjuvant treatment setting were identified. In the non-adjuvant setting, 29 patients (59.2%) with anti-CTLA4, 43 (58.1%) with anti-PD1, and 54 (79.4%) with anti-PD1/anti-CTLA4 experienced some grade of irAE and these had an improved outcome. In the adjuvant setting, the frequency of irAEs was 84.6% in anti-CTLA4 and 63.5% in anti-PD1, but no correlation with disease relapse was observed. Patients with underlying autoimmune conditions have a risk of disease exacerbation. Immunomodulatory agents had no impact on treatment efficacy. IrAEs are correlated with increased treatment efficacy in the non-adjuvant setting. Application of steroids and immunomodulatory agents, such as anti-TNF-alpha or anti-IL6, did not affect ICI efficacy. These data support irAEs as possible prognostic markers for ICI treatment