Circulating extracellular vesicles expressing PD1 and PD-L1 predict response and mediate resistance to checkpoint inhibitors immunotherapy in metastatic melanoma

Background: The immunotherapy with immune checkpoints inhibitors (ICI) has changed the life expectancy in metastatic melanoma (MM) patients. Nevertheless, several patients do not respond hence, the identifcation and validation of novel biomarkers of response to ICI is of crucial importance. Circulating extracellular vesicles (EVs) such as PD-L1+ EV mediate resistance to anti-PD1, instead the role of PD1+ EV is not fully understood. Methods: We isolated the circulating EVs from the plasma of an observational cohort study of 71 metastatic melanoma patients and correlated the amount of PD-L1+ EVs and PD1+ EVs with the response to ICI. The analysis was performed according to the origin of EVs from the tumor and the immune cells. Subsequently, we analysed the data in a validation cohort of 22 MM patients to assess the reliability of identifed EV-based biomarkers. Additionally we assessed the involvement of PD1+ EVs in the seizure of nivolumab and in the perturbation of immune cells-mediated killing of melanoma spheroids. Results: The level of PD-L1+ EVs released from melanoma and CD8+ T cells and that of PD1+ EVs irrespective of the cellular origin were higher in non-responders. The Kaplan-Meier curves indicated that higher levels of PD1+ EVs were signifcantly correlated with poorer progression-free survival (PFS) and overall survival (OS). Signifcant correlations were found for PD-L1+ EVs only when released from melanoma and T cells. The multivariate analysis showed that high level of PD1+ EVs, from T cells and B cells, and high level of PD-L1+ EVs from melanoma cells, are independent biomarkers of response. The reliability of PD-L1+ EVs from melanoma and PD1+ EVs from T cells in predicting PFS was confrmed in the validation cohort through the univariate Cox-hazard regression analysis. Moreover we discovered that the circulating EVs captured nivolumab and reduced the T cells trafcking and tumor spheroids killing. Conclusion: Our study identifed circulating PD1+ EVs as driver of resistance to anti-PD1, and highlighted that the analysis of single EV population by liquid biopsy is a promising tool to stratify MM patients for immunotherapy

A model of anti-angiogenesis: differential transcriptosome profiling of microvascular endothelial cells from diffuse systemic sclerosis patients

The objective of this work was to identify genes involved in impaired angiogenesis by comparing the transcriptosomes of microvascular endothelial cells from normal subjects and patients affected by systemic sclerosis (SSc), as a unique human model disease characterized by insufficient angiogenesis. Total RNAs, prepared from skin endothelial cells of clinically healthy subjects and SSc patients affected by the diffuse form of the disease, were pooled, labeled with fluorochromes, and hybridized to 14,000 70 mer oligonucleotide microarrays. Genes were analyzed based on gene expression levels and categorized into different functional groups based on the description of the Gene Ontology (GO) consortium to identify statistically significant terms. Quantitative PCR was used to validate the array results. After data processing and application of the filtering criteria, the analyzable features numbered 6,724. About 3% of analyzable transcripts (199) were differentially expressed, 141 more abundantly and 58 less abundantly in SSc endothelial cells. Surprisingly, SSc endothelial cells over-express pro-angiogenic transcripts, but also show up-regulation of genes exerting a powerful negative control, and down-regulation of genes critical to cell migration and extracellular matrix-cytoskeleton coupling, all alterations that provide an impediment to correct angiogenesis. We also identified transcripts controlling haemostasis, inflammation, stimulus transduction, transcription, protein synthesis, and genome organization. An up-regulation of transcripts related to protein degradation and ubiquitination was observed in SSc endothelial cells. We have validated data on the main anti-angiogenesis-related genes by RT-PCR, western blotting, in vitro angiogenesis and immunohistochemistry. These observations indicate that microvascular endothelial cells of patients with SSc show abnormalities in a variety of genes that are able to account for defective angiogenesis

Cervical cancer benefits from trabectedin combination with the β-blocker propranolol: in vitro and ex vivo evaluations in patient-derived organoids

Background: Cervical cancer (CC) is characterized by genomic alterations in DNA repair genes, which could favor treatment with agents causing DNA double-strand breaks (DSBs), such as trabectedin. Hence, we evaluated the capability of trabectedin to inhibit CC viability and used ovarian cancer (OC) models as a reference. Since chronic stress may promote gynecological cancer and may hinder the efficacy of therapy, we investigated the potential of targeting β-adrenergic receptors with propranolol to enhance trabectedin efficacy and change tumor immunogenicity.Methods: OC cell lines, Caov-3 and SK-OV-3, CC cell lines, HeLa and OV2008, and patient-derived organoids were used as study models. MTT and 3D cell viability assays were used for drug(s) IC50 determination. The analysis of apoptosis, JC-1 mitochondrial membrane depolarization, cell cycle, and protein expression was performed by flow cytometry. Cell target modulation analyses were carried out by gene expression, Western blotting, immunofluorescence, and immunocytochemistry.Results: Trabectedin reduced the proliferation of both CC and OC cell lines and notably of CC patient-derived organoids. Mechanistically, trabectedin caused DNA DSBs and S-phase cell cycle arrest. Despite DNA DSBs, cells failed the formation of nuclear RAD51 foci and underwent apoptosis. Under norepinephrine stimulation, propranolol enhanced trabectedin efficacy, further inducing apoptosis through the involvement of mitochondria, Erk1/2 activation, and the increase of inducible COX-2. Notably, trabectedin and propranolol affected the expression of PD1 in both CC and OC cell lines.Conclusion: Overall, our results show that CC is responsive to trabectedin and provide translational evidence that could benefit CC treatment options. Our study pointed out that combined treatment offset trabectedin resistance caused by β-adrenergic receptor activation in both ovarian and cervical cancer models

uPAR+ extracellular vesicles: a robust biomarker of resistance to checkpoint inhibitor immunotherapy in metastatic melanoma patients

Background Emerging evidence has highlighted the importance of extracellular vesicle (EV)-based biomarkers of resistance to immunotherapy with checkpoint inhibitors in metastatic melanoma. Considering the tumor-promoting implications of urokinase-type plasminogen activator receptor (uPAR) signaling, this study aimed to assess uPAR expression in the plasma-derived EVs of patients with metastatic melanoma to determine its potential correlation with clinical outcomes.Methods Blood samples from 71 patients with metastatic melanoma were collected before initiating immunotherapy. Tumor-derived and immune cell-derived EVs were isolated and analyzed to assess the relative percentage of uPAR+ EVs. The associations between uPAR and clinical outcomes, sex, BRAF status, baseline lactate dehydrogenase levels and number of metastatic sites were assessed.Results Responders had a significantly lower percentage of tumor-derived, dendritic cell (DC)-derived and CD8+ T cell-derived uPAR +EVs at baseline than non-responders. The Kaplan-Meier survival curves for the uPAR+EV quartiles indicated that higher levels of melanoma-derived uPAR+ EVs were strongly correlated with poorer progression-free survival (p<0.0001) and overall survival (p<0.0001). We also found a statistically significant correlation between lower levels of uPAR+ EVs from both CD8+ T cells and DCs and better survival.Conclusions Our results indicate that higher levels of tumor-derived, DC-derived and CD8+ T cell-derived uPAR+ EVs in non-responders may represent a new biomarker of innate resistance to immunotherapy with checkpoint inhibitors. Moreover, uPAR+ EVs represent a new potential target for future therapeutic approaches

Salting-Out Approach Is Worthy of Comparison with Ultracentrifugation for Extracellular Vesicle Isolation from Tumor and Healthy Models

The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation

EGFR/uPAR interaction as druggable target to overcome vemurafenib acquired resistance in melanoma cellsResearch in context

Background: BRAF inhibitor (BRAF-I) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms behind BRAF-I responsiveness and acquired resistance is therefore an important issue. Here we assessed the role of urokinase type plasminogen activator receptor (uPAR) as a potentially valuable biomarker in the acquisition of BRAF-I resistance in V600E mutant melanoma cells. Methods: We examined uPAR and EGFR levels by real time PCR and western blot analysis. uPAR loss of function was realized by knocking down uPAR by RNAi or using M25, a peptide that uncouples uPAR-integrin interaction. We investigated uPAR-β1integrin-EGFR association by co-immunoprecipitation and confocal immuno-fluorescence analysis. Acquired resistance to BRAF-I was generated by chronic exposure of cells to vemurafenib. Findings: We proved that uPAR knockdown in combination with vemurafenib inhibits melanoma cell proliferation to greater extent than either treatment alone causing a decrease in AKT and ERK1/2 phosphorylation. Conversely, we demonstrated that uPAR enforced over-expression results in reduced sensitivity to BRAF inhibition. Moreover, by targeting uPAR and EGFR interaction with an integrin antagonist peptide we restored vemurafenib responsiveness in melanoma resistant cells. Furthermore, we found significant detectable uPAR and EGFR levels in tumor biopsies of 4 relapsed patients. Interpretation: We disclosed an unpredicted mechanism of reduced sensitiveness to BRAF inhibition, driven by elevated levels of uPAR and identified a potential therapeutic strategy to overcome acquired resistance. Funds: Associazione Italiana Ricerca sul Cancro (AIRC); Ente Cassa di Risparmio di Firenze. Keywords: Vemurafenib, Acquired resistance, Melanoma, uPAR, EGF

Circulating extracellular vesicles are monitoring biomarkers of anti-PD1 response and enhancer of tumor progression and immunosuppression in metastatic melanoma

Abstract Background Clinical drawback in checkpoint inhibitors immunotherapy (ICI) of metastatic melanoma (MM) is monitoring clinical benefit. Soluble forms of PD1(sPD1) and PD-L1(sPD-L1) and extracellular vesicles (EVs) expressing PD1 and PD-L1 have recently emerged as predictive biomarkers of response. As factors released in the blood, EVs and soluble forms could be relevant in monitoring treatment efficacy and adaptive resistance to ICI. Methods We used pre-therapy plasma samples of 110 MM patients and longitudinal samples of 46 patients. Elisa assay and flow cytometry (FCM) were used to measure sPD-L1 and sPD1 concentrations and the percentage of PD1+ EVs and PD-L1+ EVs, released from tumor and immune cells in patients subsets. Transwell assays were conducted to investigate the impact of EVs of each patient subset on MM cells invasion and interaction between tumor cells and macrophages or dendritic cells. Viability assays were performed to assess EVs effect on MM cells and organoids sensitivity to anti-PD1. FCM was used to investigate immunosuppressive markers in EVs and immune cells. Results The concentrations of sPD1 and sPD-L1 in pre-treatment and longitudinal samples did not correlate with anti-PD1 response, instead only tumor-derived PD1+ EVs decreased in long responders while increased during disease progression in responders. Notably, we observed reduction of T cell derived EVs expressing LAG3+ and PD1+ in long responders and their increase in responders experiencing progression. By investigating the impact of EVs on disease progression, we found that those isolated from non-responders and from patients with progression disease accelerated tumor cells invasiveness and migration towards macrophages, while EVs of long responders reduced the metastatic potential of MM cells and neo-angiogenesis. Additionally, the EVs of non-responders and of progression disease patients subset reduced the sensitivity of MM cells and organoids of responder to anti-PD1 and the recruitment of dendritic cells, while the EVs of progression disease subset skewed macrophages to express higher level of PDL-1. Conclusion Collectively, we suggest that the detection of tumor-derived PD1 + EVs may represent a useful tool for monitoring the response to anti-PD1 and a role for EVs shed by tumor and immune cells in promoting tumor progression and immune dysfunction. Graphical Abstrac

CAFs and TGF-β Signaling Activation by Mast Cells Contribute to Resistance to Gemcitabine/Nabpaclitaxel in Pancreatic Cancer

Tumor–stroma interactions are of key importance for pancreatic ductal adenocarcinoma (PDAC) progression. Our aim was to investigate whether cancer associated fibroblasts (CAFs) and mast cells (MC) affected the sensitivity of PDAC cells to gemcitabine/nabpaclitaxel (GEM/NAB). For this purpose, the combination cytotoxicity and the effect on tumor invasion and angiogenesis were evaluated with or without a conditioned medium from the mast cell line HMC-1 (human mast cell line-1 cells) and CAFs. Beside the clinical outcome of a homogenous population of PDAC patients, receiving GEM/NAB, was correlated to the circulating levels of mast cell tryptase and to a panel of inflammatory and immunosuppressive cytokines. CAFs neither affected drugs’ cytotoxicity nor the inhibition of angiogenesis, but promoted tumor cell invasion. The MC instead, caused resistance to drugs by reducing apoptosis, by activating the TGF-β signalling and by promoting tumor invasion. Indeed, the inhibition of TβRI serine/threonine kinase activity by galunisertib restored drugs cytotoxicity. Moreover, MC induced the release of TGF-β1, and increased expression of PAR-2, ERK1/2 and Akt activation. Accordingly, TGF-β1, tryptase and other pro-inflammatory and immunosuppressive cytokines increased in the unresponsive patients. In conclusion, MC play a pivotal role in the resistance to GEM/NAB. A correlation between high level of circulating pro-inflammatory/ immunosuppressive cytokines and unresponsiveness was found in PDAC patients