The endothelin-1-driven tumor-stroma feed-forward loops in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HG-SOC) tumor microenvironment (TME) is constellated by cellular elements and a network of soluble constituents that contribute to tumor progression. In the multitude of the secreted molecules, the endothelin-1 (ET-1) has emerged to be implicated in the tumor/TME interplay; however, the molecular mechanisms induced by the ET-1-driven feed-forward loops (FFL) and associated with the HG-SOC metastatic potential need to be further investigated. The tracking of the patient-derived (PD) HG-SOC cell transcriptome by RNA-seq identified the vascular endothelial growth factor (VEGF) gene and its associated signature among those mostly up-regulated by ET-1 and down-modulated by the dual ET-1R antagonist macitentan. Within the ligand-receptor pairs concurrently expressed in PD-HG-SOC cells, endothelial cells and activated fibroblasts, we discovered two intertwined FFL, the ET-1/ET-1R and VEGF/VEGF receptors, concurrently activated by ET-1 and shutting-down by macitentan, or by the anti-VEGF antibody bevacizumab. In parallel, we observed that ET-1 fine-tuned the tumoral and stromal secretome toward a pro-invasive pattern. Into the fray of the HG-SOC/TME double and triple co-cultures, the secretion of ET-1 and VEGF, that share a common co-regulation, was inhibited upon the administration of macitentan. Functionally, macitentan, mimicking the effect of bevacizumab, interfered with the HG-SOC/TME FFL-driven communication that fuels the HG-SOC invasive behavior. The identification of ET-1 and VEGF FFL as tumor and TME actionable vulnerabilities, reveals how ET-1R blockade, targeting the HG-SOC cells and the TME simultaneously, may represent an effective therapeutic option for HG-SOC patients.

Scalable integration of multiomic single-cell data using generative adversarial networks.

MOTIVATION: Single-cell profiling has become a common practice to investigate the complexity of tissues, organs, and organisms. Recent technological advances are expanding our capabilities to profile various molecular layers beyond the transcriptome such as, but not limited to, the genome, the epigenome, and the proteome. Depending on the experimental procedure, these data can be obtained from separate assays or the very same cells. Yet, integration of more than two assays is currently not supported by the majority of the computational frameworks avaiable. RESULTS: We here propose a Multi-Omic data integration framework based on Wasserstein Generative Adversarial Networks suitable for the analysis of paired or unpaired data with a high number of modalities (>2). At the core of our strategy is a single network trained on all modalities together, limiting the computational burden when many molecular layers are evaluated. AVAILABILITY AND IMPLEMENTATION: Source code of our framework is available at https://github.com/vgiansanti/MOWGAN.

Unveiling the promising anticancer activity of palladium(II)-aryl complexes bearing diphosphine ligands: a structure-activity relationship analysis.

In continuation of our previous works on the cytotoxic properties of organopalladium compounds, in this contribution we describe the first systematic study of the anticancer activity of Pd(II)-aryl complexes. To this end, we have prepared and thoroughly characterized a wide range of palladium derivatives bearing different diphosphine, aryl and halide ligands, developing, when necessary, specific synthetic protocols. Most of the synthesized compounds showed remarkable cytotoxicity towards ovarian and breast cancer cell lines, with IC50 values often comparable to or lower than that of cisplatin. The most promising complexes ([PdI(Ph)(dppe)] and [PdI(p-CH3-Ph)(dppe)]), characterized by a diphosphine ligand with a low bite angle, exhibited, in addition to excellent cytotoxicity towards cancer cells, low activity on normal cells (MRC5 human lung fibroblasts). Specific immunofluorescence tests (cytochrome c and H2AX assays), performed to clarify the possible mechanism of action of this class of organopalladium derivatives, seemed to indicate DNA as the primary cellular target, whereas caspase 3/7 assays proved that the complex [PdI(Ph)(dppe)] was able to promote intrinsic apoptotic cell death. A detailed molecular docking analysis confirmed the importance of a diphosphine ligand with a reduced bite angle to ensure a strong DNA-complex interaction. Finally, one of the most promising complexes was tested towards patient-derived organoids, showing promising ex vivo cytotoxicity.

Extracellular vesicles secreted by human aneuploid embryos present a distinct transcriptomic profile and upregulate MUC1 transcription in decidualised endometrial stromal cells.

STUDY QUESTION: Do extracellular vesicles (EVs) secreted by aneuploid human embryos possess a unique transcriptomic profile that elicits a relevant transcriptomic response in decidualized primary endometrial stromal cells (dESCs)? SUMMARY ANSWER: Aneuploid embryo-derived EVs contain transcripts of PPM1J, LINC00561, ANKRD34C, and TMED10 with differential abundance from euploid embryo-derived EVs and induce upregulation of MUC1 transcript in dESCs. WHAT IS KNOWN ALREADY: We have previously reported that IVF embryos secrete EVs that can be internalized by ESCs, conceptualizing that successful implantation to the endometrium is facilitated by EVs. Whether these EVs may additionally serve as biomarkers of ploidy status is unknown. STUDY DESIGN SIZE DURATION: Embryos destined for biopsy for preimplantation genetic testing for aneuploidy (PGT-A) were grown under standard conditions. Spent media (30 µl) were collected from euploid (n = 175) and aneuploid (n = 140) embryos at cleavage (Days 1-3) stage and from euploid (n = 187) and aneuploid (n = 142) embryos at blastocyst (Days 3-5) stage. Media samples from n = 35 cleavage-stage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocysts were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. PARTICIPANTS/MATERIALS SETTING METHODS: EVs were isolated from pools of media by differential centrifugation and EV-RNA sequencing was performed following a single-cell approach that circumvents RNA extraction. ESCs were decidualized (estradiol: 10 nM, progesterone: 1 µM, cAMP: 0.5 mM twice every 48 h) and incubated for 24 h with EVs (50 ng/ml). RNA sequencing was performed on ESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc = -5.13, P = 0.011), LINC00561 (log2fc = -7.87, P = 0.010), and ANKRD34C (log2fc = -7.30, P = 0.017) and more abundant in TMED10 (log2fc = 1.63, P = 0.025) compared to EVs of euploid embryos. Decidualization per se induced downregulation of MUC1 (log2fc = -0.54, P = 0.0028) in ESCs as a prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualized ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2fc = 0.85, P = 0.0201). LARGE SCALE DATA: Raw data have been uploaded to GEO (accession number GSE234338). LIMITATIONS REASONS FOR CAUTION: The findings of the study will require validation utilizing a second cohort of EV samples. WIDER IMPLICATIONS OF THE FINDINGS: The discovery that the transcriptomic profile of EVs secreted from aneuploid cleavage stage embryos differs from that of euploid embryos supports the possibility to develop a non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. STUDY FUNDING/COMPETING INTERESTS: The study was supported by a Marie Sklodowska-Curie Actions fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd. The authors have no conflicts of interest to declare.

Germline testing and genetic counseling in biliary tract cancer: an operative proposal to improve the state of art.

INTRODUCTION: A genetic predisposition seems to be involved in biliary tract cancer, but the prevalence of germline mutations in BTC remains unclear, and the therapeutic role of the germline pathologic variants is still unknown. AREA COVERED: The aim of the present work is to systematically review the data available on the hereditary predisposition of biliary tract cancer by a specific research on PubMed, in order to highlight the most important critical points and to define the current possible role of germinal testing and genetic counseling in this setting of patients. EXPERT OPINION: Basing on data already available, we decided to start in our institution a specific genetic protocol focused on biliary tract cancer patients, which includes genetic counseling and, if indicated, germline test. The inclusion criteria are: 1) Patient with personal history of oncologic disease other than BTC, 2) Patient with familiar history of oncologic disease (considering relatives of first and second grade), 3) Patient with ≤ 50 years old, 4) Patient presenting a somatic mutation in genes involved in DNA damage repair pathways and mismatch repair. The aim of the presented protocol is to identify germline pathogenic variants with prophylactic and therapeutic impact, and to collect and integrate a significant amount of clinical, familial, somatic, and genetic data.

Synergistic applications of cyclodextrin-based systems and metal-organic frameworks in transdermal drug delivery for skin cancer therapy.

This review article explores the innovative field of eco-friendly cyclodextrin-based coordination polymers and metal-organic frameworks (MOFs) for transdermal drug delivery in the case of skin cancer therapy. We critically examine the significant advancements in developing these nanocarriers, with a focus on their unique properties such as biocompatibility, targeted drug release, and enhanced skin permeability. These attributes are instrumental in addressing the limitations inherent in traditional skin cancer treatments and represent a paradigm shift towards more effective and patient-friendly therapeutic approaches. Furthermore, we discuss the challenges faced in optimizing the synthesis process for large-scale production while ensuring environmental sustainability. The review also emphasizes the immense potential for clinical applications of these nanocarriers in skin cancer therapy, highlighting their role in facilitating targeted, controlled drug release which minimizes systemic side effects. Future clinical applications could see these nanocarriers being customized to individual patient profiles, potentially revolutionizing personalized medicine in oncology. With further research and clinical trials, these nanocarriers hold the promise of transforming the landscape of skin cancer treatment. With this study, we aim to provide a comprehensive overview of the current state of research in this field and outline future directions for advancing the development and clinical application of these innovative nanocarriers.

KDM6A Regulates Immune Response Genes in Multiple Myeloma.

The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration. Statement of significance: We show that KDM6A participates in immune recognition of myeloma tumor cells by directly regulating the expression of the master regulators of MHC-I and II, NLRC5 and CIITA. The expression of these regulators can by rescued by the HDAC3 inhibitors in KDM6A-null cell lines.

UNCAN.eu: Toward a European Federated Cancer Research Data Hub.

To enable a collective effort that generates a new level of UNderstanding CANcer (UNCAN.eu) [Cancer Discov (2022) 12 (11): OF1], the European Union supports the creation of a sustainable platform that connects cancer research across Member States. A workshop hosted in Heidelberg gathered European cancer experts to identify ongoing initiatives that may contribute to building this platform and discuss the governance and long-term evolution of a European Federated Cancer Data Hub.

Cancer Evolution: A Multifaceted Affair.

Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery. SIGNIFICANCE: Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution.

Myeloma and DNA damage.

ABSTRACT: DNA-damaging agents have represented the first effective treatment for the blood cancer multiple myeloma, and after 65 years since their introduction to the clinic, they remain one of the mainstay therapies for this disease. Myeloma is a cancer of plasma cells. Despite exceedingly slow proliferation, myeloma cells present extended genomic rearrangements and intense genomic instability, starting at the premalignant stage of the disease. Where does such DNA damage stem from? A reliable model argues that the powerful oncogenes activated in myeloma as well the phenotypic peculiarities of cancer plasma cells, including the dependency on the proteasome for survival and the constant presence of oxidative stress, all converge on modulating DNA damage and repair. Beleaguered by these contraposing forces, myeloma cells survive in a precarious balance, in which the robust engagement of DNA repair mechanisms to guarantee cell survival is continuously challenged by rampant genomic instability, essential for cancer cells to withstand hostile selective pressures. Shattering this delicate equilibrium has been the goal of the extensive use of DNA-damaging agents since their introduction in the clinic, now enriched by novel approaches that leverage upon synthetic lethality paradigms. Exploiting the impairment of homologous recombination caused by myeloma genetic lesions or treatments, it is now possible to design therapeutic combinations that could target myeloma cells more effectively. Furthermore, DNA-damaging agents, as demonstrated in solid tumors, may sensitize cells to immune therapies. In all, targeting DNA damage and repair remains as central as ever in myeloma, even for the foreseeable future.

In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases.

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

Evolutionary fingerprints of EMT in pancreatic cancers.

Mesenchymal plasticity has been extensively described in advanced and metastatic epithelial cancers; however, its functional role in malignant progression, metastatic dissemination and therapy response is controversial. More importantly, the role of epithelial mesenchymal transition (EMT) and cell plasticity in tumor heterogeneity, clonal selection and clonal evolution is poorly understood. Functionally, our work clarifies the contribution of EMT to malignant progression and metastasis in pancreatic cancer. We leveraged ad hoc somatic mosaic genome engineering, lineage tracing and ablation technologies and dynamic genetic reporters to trace and ablate tumor-specific lineages along the phenotypic spectrum of epithelial to mesenchymal plasticity. The experimental evidences clarify the essential contribution of mesenchymal lineages to pancreatic cancer evolution and metastatic dissemination. Spatial genomic analysis combined with single cell transcriptomic and epigenomic profiling of epithelial and mesenchymal lineages reveals that EMT promotes with the emergence of chromosomal instability (CIN). Specifically tumor lineages with mesenchymal features display highly conserved patterns of genomic evolution including complex structural genomic rearrangements and chromotriptic events. Genetic ablation of mesenchymal lineages robustly abolished these mutational processes and evolutionary patterns, as confirmed by cross species analysis of pancreatic and other human epithelial cancers. Mechanistically, we discovered that malignant cells with mesenchymal features display increased chromatin accessibility, particularly in the pericentromeric and centromeric regions, which in turn results in delayed mitosis and catastrophic cell division. Therefore, EMT favors the emergence of high-fitness tumor cells, strongly supporting the concept of a cell-state, lineage-restricted patterns of evolution, where cancer cell sub-clonal speciation is propagated to progenies only through restricted functional compartments. Restraining those evolutionary routes through genetic ablation of clones capable of mesenchymal plasticity and extinction of the derived lineages completely abrogates the malignant potential of one of the most aggressive form of human cancer.

Gold(I)-N-Heterocyclic Carbene Synthons in Organometallic Synthesis.

The prominent role of gold-N-heterocyclic carbene (NHC) complexes in numerous research areas such as homogeneous (photo)catalysis, medicinal chemistry and materials science has prompted organometallic chemists to design gold-based synthons that permit access to target complexes through simple synthetic steps under mild conditions. In this review, the main gold-NHC synthons employed in organometallic synthesis are discussed. Mechanistic aspects involved in their synthesis and reactivity as well as applications of gold-NHC synthons as efficient pre-catalysts, antitumor agents and/or photo-emissive materials are presented.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Preexisting Immunity Drives the Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma.

Current treatment for patients with locally advanced esophageal adenocarcinoma (EAC) is neoadjuvant chemotherapy (nCT), alone or combined with radiotherapy, before surgery. However, fewer than 30% of treated patients show a pathologic complete response to nCT, which correlates with increased 5-year survival compared with nonresponders. Understanding the mechanisms of response to nCT is pivotal to better stratify patients and inform more efficacious therapies. Here, we investigated the immune mechanisms involved in nCT response by multidimensional profiling of pretreatment tumor biopsies and blood from 68 patients with EAC (34 prospectively and 34 retrospectively collected), comparing complete responders versus nonresponders to nCT. At the tumor level, complete response to nCT was associated with molecular signatures of immune response and proliferation, increased putative antitumor tissue-resident memory CD39+ CD103+ CD8+ T cells, and reduced immunosuppressive T regulatory cells (Treg) and M2-like macrophages. Systemically, complete responders showed higher frequencies of immunostimulatory CD14+ CD11c+ HLA-DRhigh cells, and reduced programmed cell death ligand 1-positive (PD-L1+) monocytic myeloid-derived suppressor cells, along with high plasma GM-CSF (proinflammatory) and low IL4, CXCL10, C3a, and C5a (suppressive). Plasma proinflammatory and suppressive cytokines correlated directly and inversely, respectively, with the frequency of tumor-infiltrating CD39+ CD103+ CD8+ T cells. These results suggest that preexisting immunity in baseline tumor drives the clinical activity of nCT in locally advanced EAC. Furthermore, it may be possible to stratify patients based on predictive immune signatures, enabling tailored neoadjuvant and/or adjuvant regimens. SIGNIFICANCE: Multidimensional profiling of pretreatment esophageal adenocarcinoma shows patient response to nCT is correlated with active preexisting immunity and indicates molecular pathways of resistance that may be targeted to improve clinical outcomes.

Analyzing genomic and epigenetic profiles in single cells by hybrid transposase (scGET-seq).

scGET-seq simultaneously profiles euchromatin and heterochromatin. scGET-seq exploits the concurrent action of transposase Tn5 and its hybrid form TnH, which targets H3K9me3 domains. Here we present a step-by-step protocol to profile single cells by scGET-seq using a 10× Chromium Controller. We describe steps for transposomes preparation and validation. We detail nuclei preparation and transposition, followed by encapsulation, library preparation, sequencing, and data analysis. For complete details on the use and execution of this protocol, please refer to Tedesco et al. (2022)1 and de Pretis and Cittaro (2022).2.

RNA sequencing of blood from sex- and age-matched discordant siblings supports immune and transcriptional dysregulation in autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with onset in early childhood, still diagnosed only through clinical observation due to the lack of laboratory biomarkers. Early detection strategies would be especially useful in screening high-risk newborn siblings of children already diagnosed with ASD. We performed RNA sequencing on peripheral blood, comparing 27 pairs of ASD children vs their sex- and age-matched unaffected siblings. Differential gene expression profiling, performed applying an unpaired model found two immune genes, EGR1 and IGKV3D-15, significantly upregulated in ASD patients (both p adj = 0.037). Weighted gene correlation network analysis identified 18 co-expressed modules. One of these modules was downregulated among autistic individuals (p = 0.035) and a ROC curve using its eigengene values yielded an AUC of 0.62. Genes in this module are primarily involved in transcriptional control and its hub gene, RACK1, encodes for a signaling protein critical for neurodevelopment and innate immunity, whose expression is influenced by various hormones and known "endocrine disruptors". These results indicate that transcriptomic biomarkers can contribute to the sensitivity of an intra-familial multimarker panel for ASD and provide further evidence that neurodevelopment, innate immunity and transcriptional regulation are key to ASD pathogenesis.