Shedding of Syncytiotrophoblast-Derived Extracellular Vesicles Is Increased in Placenta Previa and Accreta Spectrum.

Placenta accreta spectrum (PAS) refers to excessive placental invasion into the maternal uterus and it is associated with high risk of obstetric haemorrhage and adverse maternal-neonatal outcomes. Currently, no specific circulating biomarkers of PAS have been identified. Given that in PAS disorders, the depth and the extension of placental invasion into the uterus are expected to be increased, in this study, we analysed plasma levels of syncytiotrophoblast-derived extracellular vesicles (STBEVs) in women with placenta previa (PP), at a high risk of PAS disorders, and pregnant women with normal placentation. Venous blood samples were collected from 35 women with ultrasonographic diagnosis of PP and 35 women with normal placentation, matched for gestational age. Plasma samples were ultracentrifuged at 120.000 g to collect extracellular vesicles (EVs). To identify and quantify plasma placenta-derived EVs (or STBEVs), EVs were analysed by flow cytometry using a monoclonal antibody against placental alkaline phosphatase (PLAP). Plasma levels of STBEVs were significantly higher in PP patients compared to controls. Plasma levels of STBEVs in women with PP and PAS showed a trend to a higher concentration compared to women with PP without PAS, although not reaching a statistical significance. Circulating STBEVs are potential candidates as biological markers to be integrated to ultrasonography in the antenatal screening programme for PAS. More studies are needed to confirm our observation in a larger cohort of patients and to analyse a possible association between high circulating levels of STBEVs and PAS.

Talniflumate abrogates mucin immune suppressive barrier improving efficacy of gemcitabine and nab-paclitaxel treatment in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. This is due to its aggressive course, late diagnosis and its intrinsic drugs resistance. The complexity of the tumor, in terms of cell components and heterogeneity, has led to the approval of few therapies with limited efficacy. The study of the early stages of carcinogenesis provides the opportunity for the identification of actionable pathways that underpin therapeutic resistance. METHODS: We analyzed 43 Intraductal papillary mucinous neoplasms (IPMN) (12 Low-grade and 31 High-grade) by Spatial Transcriptomics. Mouse and human pancreatic cancer organoids and T cells interaction platforms were established to test the role of mucins expression on T cells activity. Syngeneic mouse model of PDAC was used to explore the impact of mucins downregulation on standard therapy efficacy. RESULTS: Spatial transcriptomics showed that mucin O-glycosylation pathway is increased in the progression from low-grade to high-grade IPMN. We identified GCNT3, a master regulator of mucins expression, as an actionable target of this pathway by talniflumate. We showed that talniflumate impaired mucins expression increasing T cell activation and recognition using both mouse and human organoid interaction platforms. In vivo experiments showed that talniflumate was able to increase the efficacy of the chemotherapy by boosting immune infiltration. CONCLUSIONS: Finally, we demonstrated that combination of talniflumate, an anti-inflammatory drug, with chemotherapy effectively improves anti-tumor effect in PDAC.

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.

Smart nano-sized extracellular vesicles for cancer therapy: Potential theranostic applications in gastrointestinal tumors.

Extracellular vesicles (EVs) have gained tremendous interest in the search for next-generation therapeutics for the treatment of a range of pathologies, including cancer, especially due to their small size, biomolecular cargo, ability to mediate intercellular communication, high physicochemical stability, low immunogenicity and biocompatibility. The theranostic potential of EVs have been enhanced by adopting several strategies such as genetic or metabolic engineering, parental cell modification or direct functionalization to incorporate therapeutic compounds into these nanoplatforms. The smart nano-sized EVs indeed offer huge opportunities in the field of cancer, and current research is set at overcoming the existing pitfalls. Smart EVs are already being applied in the clinics despite the challenges faced. We provide, herein, an update on the technologies employed for EV functionalization in order to achieve optimal tumor cell targeting and EV tracking in vivo with bio-imaging modalities, as well as the preclinical and clinical studies making use of these modified EVs, in the context of gastrointestinal tumors.

Interferon signaling promotes tolerance to chromosomal instability during metastatic evolution in renal cancer.

Molecular routes to metastatic dissemination are critical determinants of aggressive cancers. Through in vivo CRISPR-Cas9 genome editing, we generated somatic mosaic genetically engineered models that faithfully recapitulate metastatic renal tumors. Disruption of 9p21 locus is an evolutionary driver to systemic disease through the rapid acquisition of complex karyotypes in cancer cells. Cross-species analysis revealed that recurrent patterns of copy number variations, including 21q loss and dysregulation of the interferon pathway, are major drivers of metastatic potential. In vitro and in vivo genomic engineering, leveraging loss-of-function studies, along with a model of partial trisomy of chromosome 21q, demonstrated a dosage-dependent effect of the interferon receptor genes cluster as an adaptive mechanism to deleterious chromosomal instability in metastatic progression. This work provides critical knowledge on drivers of renal cell carcinoma progression and defines the primary role of interferon signaling in constraining the propagation of aneuploid clones in cancer evolution.

NTRK gene aberrations in triple-negative breast cancer: detection challenges using IHC, FISH, RT-PCR, and NGS.

Triple-negative breast cancer (TNBC) is usually an aggressive disease with a poor prognosis and limited treatment options. The neurotrophic tyrosine receptor kinase (NTRK) gene fusions are cancer type-agnostic emerging biomarkers approved by the Food and Drug Administration (FDA), USA, for the selection of patients for targeted therapy. The main aim of our study was to investigate the frequency of NTRK aberrations, i.e. fusions, gene copy number gain, and amplification, in a series of TNBC using different methods. A total of 83 TNBCs were analyzed using pan-TRK immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), and RNA-based next-generation sequencing (NGS). Of 83 cases, 16 showed pan-TRK positivity although no cases had NTRK-fusions. Indeed, FISH showed four cases carrying an atypical NTRK1 pattern consisting of one fusion signal and one/more single green signals, but all cases were negative for fusion by NGS and RT-PCR testing. In addition, FISH analysis showed six cases with NTRK1 amplification, one case with NTRK2 copy number gain, and five cases with NTRK3 copy number gain, all negative for pan-TRK IHC. Our data demonstrate that IHC has a high false-positive rate for the detection of fusions and molecular testing is mandatory; there is no need to perform additional molecular tests in cases negativity for NTRK by IHC. In conclusion, the NTRK genes are not involved in fusions in TNBC, but both copy number gain and amplification are frequent events, suggesting a possible predictive role for other NTRK aberrations.

Exploiting bioactive natural products of marine origin: Evaluation of the meroterpenoid metachromin V as a novel potential therapeutic drug for colorectal cancer.

Colorectal cancer (CRC) is the second most common cause of cancer death, leading to almost 1 million deaths per year. Despite constant progress in surgical and therapeutic protocols, the 5-year survival rate of advanced CRC patients remains extremely poor. Colorectal Cancer Stem Cells (CRC-CSCs) are endowed with unique stemness-related properties responsible for resistance, relapse and metastasis. The development of novel therapeutics able to tackle CSCs while avoiding undesired toxicity is a major need for cancer treatment. Natural products are a large reservoir of unexplored compounds with possible anticancer bioactivity, sustainability, and safety. The family of meroterpenoids derived from sponges share interesting bioactive properties. Bioassay-guided fractionation of a meroterpenoids extract led to the isolation of three compounds, all cytotoxic against several cancer cell lines: Metachromins U, V and W. In this study, we evaluated the anticancer potential of the most active one, Metachromins V (MV), on patient-derived CRC-CSCs. MV strongly impairs CSCs-viability regardless their mutational background and the cytotoxic effect is maintained on therapy-resistant metastatic CSCs. MV affects cell cycle progression, inducing a block in G2 phase in all the cell lines tested and more pronouncedly in CRC-CSCs. Moreover, MV triggers an important reorganization of the cytoskeleton and a strong reduction of Rho GTPases expression, impairing CRC-CSCs motility and invasion ability. By Proteomic analysis identified a potential molecular target of MV: CCAR1, that regulates apoptosis under chemotherapy treatments and affect ß-catenin pathway. Further studies will be needed to confirm and validate these data in in vivo experimental models.

Drug Repurposing in Pediatric Brain Tumors: Posterior Fossa Ependymoma and Diffuse Midline Glioma under the Looking Glass.

Tumors of the Central Nervous System (CNS) represent the leading cause of cancer-related deaths in children. Current treatment options are not curative for most malignant histologies, and intense preclinical and clinical research is needed to develop more effective therapeutic interventions against these tumors, most of which meet the FDA definition for orphan diseases. Increased attention is being paid to the repositioning of already-approved drugs for new anticancer indications as a fast-tracking strategy for identifying new and more effective therapies. Two pediatric CNS tumors, posterior fossa ependymoma (EPN-PF) type A and diffuse midline glioma (DMG) H3K27-altered, share loss of H3K27 trimethylation as a common epigenetic hallmark and display early onset and poor prognosis. These features suggest a potentially common druggable vulnerability. Successful treatment of these CNS tumors raises several challenges due to the location of tumors, chemoresistance, drug blood-brain barrier penetration, and the likelihood of adverse side effects. Recently, increasing evidence demonstrates intense interactions between tumor cell subpopulations and supportive tumor microenvironments (TMEs) including nerve, metabolic, and inflammatory TMEs. These findings suggest the use of drugs, and/or multi-drug combinations, that attack both tumor cells and the TME simultaneously. In this work, we present an overview of the existing evidence concerning the most preclinically validated noncancer drugs with antineoplastic activity. These drugs belong to four pharmacotherapeutic classes: antiparasitic, neuroactive, metabolic, and anti-inflammatory. Preclinical evidence and undergoing clinical trials in patients with brain tumors, with special emphasis on pediatric EPN-PF and DMG, are summarized and critically discussed.

DNA methylation of hematopoietic stem/progenitor cells from donor peripheral blood to patient bone marrow: implications for allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) is a life-saving treatment for selected hematological malignancies. So far, it remains unclear whether transplanted hematopoietic stem/progenitor cells (HSPCs) undergo epigenetic changes upon engraftment in recipient bone marrow (BM) after AHSCT and whether these changes might be useful in the transplant diagnostics. The purpose of this study was to characterize the whole genome methylation profile of HSPCs following AHSCT. Moreover, the relationship between the observed methylation signature and patient outcome was analyzed. Mobilized peripheral blood (mPB)-HSPCs from seven donors and BM-HSPCs longitudinally collected from transplanted patients with hematological malignancies up to one year from AHSCT (a total of twenty-eight samples) were analyzed using DNA methylation based-arrays. The obtained data showed that DNA methylation of mPB-HSPCs differs between young and adult donors and changes following HSPC engraftment in the BM of recipient patients. Looking at methylation in promoter regions, at 30 days post-AHSCT, BM-HSPCs showed a higher number of differentially methylated genes (DMGs) compared to those of mPB-HSPCs, with a prevalent hyper-methylation. These changes were maintained during all the analyzed time points, and methylation became like the donors after one year from transplant. Functional analysis of these DMGs showed an enrichment in cell adhesion, differentiation and cytokine (interleukin-2, -5 and -7) production and signaling pathways. Of note, DNA methylation analysis allowed to identify a potential "cancer/graft methylation signature" of transplant failure. It was evident in the latest available post-transplant BM-HSPC sample (at 160 days) and surprisingly already in early phase (at 30 days) in patients whose transplant was doomed to fail. Overall, the analysis of HSPC methylation profile could offer useful prognostic information to potentially assess engraftment success and predict graft failure in AHSCT.

Gene Regulatory Network Characterization of Gastric Cancer's Histological Subtypes: Distinctive Biological and Clinically Relevant Master Regulators.

Gastric cancer (GC) molecular heterogeneity represents a major determinant for clinical outcomes, and although new molecular classifications have been introduced, they are not easy to translate from bench to bedside. We explored the data from GC public databases by performing differential gene expression analysis (DEGs) and gene network reconstruction to identify master regulators (MRs), as well as a gene set analysis (GSA) to reveal their biological features. Moreover, we evaluated the association of MRs with clinicopathological parameters. According to the GSA, the Diffuse group was characterized by an epithelial-mesenchymal transition (EMT) and inflammatory response, while the Intestinal group was associated with a cell cycle and drug resistance pathways. In particular, the regulons of Diffuse MRs, such as Vgll3 and Ciita, overlapped with the EMT and interferon-gamma response, while the regulons Top2a and Foxm1 were shared with the cell cycle pathways in the Intestinal group. We also found a strict association between MR activity and several clinicopathological features, such as survival. Our approach led to the identification of genes and pathways differentially regulated in the Intestinal and Diffuse GC histotypes, highlighting biologically interesting MRs and subnetworks associated with clinical features and prognosis, suggesting putative actionable candidates.

Radiation-induced sarcomas: A single referral cancer center experience and literature review.

Background and objective: The oncogenic effect of ionizing radiation is widely known. Sarcomas developing after radiation therapy (RT), termed "iatrogenic disease of success", represent a growing problem, since the advancements in cancer management and screening programs have increased the number of long-term cancer survivors. Although many patients have been treated with radiation therapy, only few data are available on radiation-induced sarcomas (RIS). Methods: We examined the medical and radiological records of 186 patients with histologically proven soft tissue and bone sarcomas, which referred to IRCCS CROB Centro di Riferimento Oncologico della Basilicata from January 2009 to May 2022. Among them, seven patients received a histological diagnosis of secondary RIS, according to Cahan's criteria. Clinicopathological features and treatment follow-up data of RIS patients were retrospectively analyzed. Results: Among these secondary RIS, five arose in irradiated breast cancer (5/2,570, 0.19%) and two in irradiated head and neck cancer (2/1,986, 0.10%) patients, with a mean onset latency time of 7.3 years. The histology of RIS was one desmoid tumor, two angiosarcomas, one chondrosarcoma, two leiomyosarcomas, and one undifferentiated pleomorphic sarcoma. Out of the seven RIS, one received radiotherapy, one received electrochemotherapy (ECT), one received a second-line chemotherapy, three were subjected to three lines of chemotherapy, and one underwent radiofrequency ablation, chemotherapy, and ECT. Median survival time is 36 months. No significant survival differences were found stratifying patients for age at RT, latency time, and age at RIS diagnosis. Conclusions: RIS represents a possible complication for long-survivor cancer patients. Therefore, adherence to a strict follow-up after the radiation treatment is recommended to allow early diagnosis and optimal management of RIS patients. After the planned follow-up period, considering the long-term risk to develop a RIS, a specific multispecialty survivorship care plan could be of benefit for patients.

Microsatellite Instability: From the Implementation of the Detection to a Prognostic and Predictive Role in Cancers.

Microsatellite instability (MSI) has been identified in several tumors arising from either germline or somatic aberration. The presence of MSI in cancer predicts the sensitivity to immune checkpoint inhibitors (ICIs), particularly PD1/PD-L1 inhibitors. To date, the predictive role of MSI is currently used in the selection of colorectal cancer patients for immunotherapy; moreover, the expansion of clinical trials into other cancer types may elucidate the predictive value of MSI for non-colorectal tumors. In clinical practice, several assays are used for MSI testing, including immunohistochemistry (IHC), polymerase chain reaction (PCR) and next-generation sequencing (NGS). In this review, we provide an overview of MSI in various cancer types, highlighting its potential predictive/prognostic role and the clinical trials performed. Finally, we focus on the comparison data between the different assays used to detect MSI in clinical practice.

Tools in the Era of Multidrug Resistance in Bacteria: Applications for New Antimicrobial Peptides Discovery.

Antimicrobial peptides (AMPs) are small molecules belonging to innate immunity that act against bacteria, fungi, and viruses. With the spread of bacterial strains resistant to current antibiotics, the scientific community is deeply committed to the identification and study of new molecules with putative antimicrobial activity. In this context, AMPs represent a promising alternative to overcome this problem. To date, several databases have been built up to provide information on the AMPs identified so far and their physico-chemical properties. Moreover, several tools have been developed and are available online that allow to highlight sequences with putative antimicrobial activity and predict their biological activity. These tools can also predict the secondary and tertiary structures of putative AMPs, thus allowing molecular docking studies to evaluate potential interactions with proteins/ligands. In this paper, we focused our attention on online available AMPs databases and computational tools for biological activity and tertiary structure prediction, highlighting some papers in which the computational approach was successfully used. As the identification of peptides starts from the analysis of a large amount of data, we show that bioinformatics predictions are the best starting point for the identification of new sequences of interest that can be subsequently produced and tested.

The pathogenesis of cryptoglandular anal fistula: New insight into the immunological profile.

AIM: The aetiology of cryptoglandular anal fistula (AF) is poorly understood. Evidence suggests that persistence and/or recurrence of the disease is more related to inflammatory than infectious factors. The aim of this study was to investigate the immune profile of cryptoglandular AF and to perform a histopathological characterization. METHOD: Fistulectomy was performed in all patients; healthy ischioanal fat from the same patients was used as a control. Samples were evaluated by the Luminex xMAP system for the detection of 27 analytes. AF tissues were analysed using immunofluorescence. Staining was performed using primary antibodies to identify M1 inflammatory and M2 anti-inflammatory macrophages. Selective staining of total T lymphocytes and different T lymphocyte subsets was performed. RESULTS: Twenty patients with AF underwent a fistulectomy. Specific cytokine pathways differentiated AF from healthy tissue: pro-inflammatory cytokines interleukin (IL)-1ß, IL-4, IL-8 and IL-17 and the anti-inflammatory cytokine IL-10 were overexpressed in AF compared with controls. Chemokines involved in macrophage recruitment (CCL2, CCL3, CCL4) were higher in AF than in healthy fatty tissue. Moreover, we showed that Tc17 cells characterize AF patients, thus confirming the enzyme-linked immunosorbent assay data. Furthermore, elevated infiltration of CD68+ myeloid cells and a reduction of the M1/M2 ratio characterize AF patients. CONCLUSION: A combination of inflammatory cytokines, chemokines and growth factors reside in the wound microenvironment of AF patients. For the first time an important prevalence of Tc17 cells and a reduction in the M1/M2 ratio was observed, thus suggesting new insights into the immunological characterization of AF patients.

Modulation of Peripheral Immune Cell Subpopulations After RapidArc/Moderate Hypofractionated Radiotherapy for Localized Prostate Cancer: Findings and Comparison With 3D Conformal/Conventional Fractionation Treatment.

Radiotherapy (RT) is an important therapeutic option in patients with localized prostate cancer (PC). Unfortunately, radiation treatment causes a decrease in peripheral lymphocytes and, consequently, influences the patients' immune status. Our aim was to study changes in peripheral blood immune cell subpopulations after RT and during 6 months' follow-up in 2 groups of PC patients irradiated with different techniques and dose fractions with curative intent. We also investigated the presence of correlation between immune cell modulation and genitourinary or gastrointestinal toxicity. We enrolled 44 patients treated with curative RT (RapidArc/hypofractionation regimen or 3D conformal/conventional fractionation) for localized PC. Total white blood cell (WBC), absolute lymphocyte counts (ALCs), and peripheral immune cell subpopulations were analyzed at baseline, at the end of RT, and 3 and 6 months after the end of RT. WBC and ALC greatly decreased at the end of RT with a trend to recover at 6 months' follow-up in the hypofractionation group but not in the conventional one. Furthermore, B, total T, T CD4+, T CD8+, and NK cell values dropped significantly in both groups at the end of RT, with a minor decrease detectable in the hypofractionation group for B, total T, and T CD4+ lymphocytes with respect to the other technique/fractionation group. Double-negative T (DNT), double-positive T (DPT), and NKT cells significantly decreased at the end of RT with a slight tendency to recover values during follow-up, particularly in the hypofractionation group. No correlation with genitourinary or gastrointestinal toxicity was found. In this study, we showed, for the first time, the effects of RapidArc/moderate hypofractionation RT on immune cell subsets in patients treated for localized PC. Due to the growing interest in minority T-cell subpopulations for immunotherapy, we also reported longitudinal monitoring of the effects of RT on DNT, DPT, and NKT, which was never studied before. Our preliminary data highlight the importance of considering the effects of different RT techniques/fractionation regimens on peripheral immune cells, in the era of RT and immunotherapy combination.

Flow cytometric evaluation of measurable residual disease in chronic lymphocytic leukemia: Where do we stand?

Measurable residual disease (MRD) has emerged as a relevant parameter of response to therapy in chronic lymphocytic leukemia (CLL). Although several methods have been developed, flow cytometry has emerged as the most useful and standardized approach to measure and quantify MRD. The improved sensitivity of MRD measurements has been paralleled by the development of more effective therapeutic strategies for CLL, increasing the applicability of MRD detection in this setting. Chemotherapy and chemoimmunotherapy have firstly demonstrated their ability to obtain a deep MRD. Combined targeted therapies are also demonstrating a high molecular response rate and prospective trials are exploring the role of MRD to guide the duration of treatment in this setting. In this review we briefly summarize what we have learned about MRD with emphasis on its flow cytometric detection.

Acute Myeloid Leukemia Cells Functionally Compromise Hematopoietic Stem/Progenitor Cells Inhibiting Normal Hematopoiesis Through the Release of Extracellular Vesicles.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal disorder of hematopoietic stem/progenitor cells (HSPCs). It is not well known how leukemia cells alter hematopoiesis promoting tumor growth and leukemic niche formation. In this study, we investigated how AML deregulates the hematopoietic process of HSPCs through the release of extracellular vesicles (EVs). First, we found that AML cells released a heterogeneous population of EVs containing microRNAs involved in AML pathogenesis. Notably, AML-EVs were able to influence the fate of HSPCs modifying their transcriptome. In fact, gene expression profile of AML-EV-treated HSPCs identified 923 down- and 630 up-regulated genes involved in hematopoiesis/differentiation, inflammatory cytokine production and cell movement. Indeed, most of the down-regulated genes are targeted by AML-EV-derived miRNAs. Furthermore, we demonstrated that AML-EVs were able to affect HSPC phenotype, modifying several biological functions, such as inhibiting cell differentiation and clonogenicity, activating inflammatory cytokine production and compromising cell movement. Indeed, a redistribution of HSPC populations was observed in AML-EV treated cells with a significant increase in the frequency of common myeloid progenitors and a reduction in granulocyte-macrophage progenitors and megakaryocyte-erythroid progenitors. This effect was accompanied by a reduction in HSPC colony formation. AML-EV treatment of HSPCs increased the levels of CCL3, IL-1B and CSF2 cytokines, involved in the inflammatory process and in cell movement, and decreased CXCR4 expression associated with a reduction of SDF-1 mediated-migration. In conclusion, this study demonstrates the existence of a powerful communication between AML cells and HSPCs, mediated by EVs, which suppresses normal hematopoiesis and potentially contributes to create a leukemic niche favorable to neoplastic development.

Cell-of-Origin and Genetic, Epigenetic, and Microenvironmental Factors Contribute to the Intra-Tumoral Heterogeneity of Pediatric Intracranial Ependymoma.

Intra-tumoral heterogeneity (ITH) is a complex multifaceted phenomenon that posits major challenges for the clinical management of cancer patients. Genetic, epigenetic, and microenvironmental factors are concurrent drivers of diversity among the distinct populations of cancer cells. ITH may also be installed by cancer stem cells (CSCs), that foster unidirectional hierarchy of cellular phenotypes or, alternatively, shift dynamically between distinct cellular states. Ependymoma (EPN), a molecularly heterogeneous group of tumors, shows a specific spatiotemporal distribution that suggests a link between ependymomagenesis and alterations of the biological processes involved in embryonic brain development. In children, EPN most often arises intra-cranially and is associated with an adverse outcome. Emerging evidence shows that EPN displays large intra-patient heterogeneity. In this review, after touching on EPN inter-tumoral heterogeneity, we focus on the sources of ITH in pediatric intra-cranial EPN in the framework of the CSC paradigm. We also examine how single-cell technology has shed new light on the complexity and developmental origins of EPN and the potential impact that this understanding may have on the therapeutic strategies against this deadly pediatric malignancy.

Mutational status of plasma exosomal KRAS predicts outcome in patients with metastatic colorectal cancer.

Liquid biopsy has become a useful alternative in metastatic colorectal cancer (mCRC) patients when tissue biopsy of metastatic sites is not feasible. In this study we aimed to investigate the clinical utility of circulating exosomes DNA in the management of mCRC patients. Exosomes level and KRAS mutational status in exosomal DNA was assesed in 70 mCRC patients and 29 CRC primary tumor and were analysed at different disease steps evaluating serial blood samples (240 blood samples). There was a significant correlation between the extension of disease and exosomes level and the resection of primary localized tumor was correlated with a decrease of KRAS G12V/ D copies and fractional abundance in metastatic disease. CEA expression and liver metastasis correlated with a higher number of KRAS G12V/D copies/ml and a higher fractional abundance; in the subgroup of mCRC patients eligible for surgery, the size of tumor and the radiological response were related to exosomes level but only the size was related to the number of KRAS WT copies; both KRAS wild-type and mutated levels were identified as a prognostic factor related to OS. Finally, we found that 91% of mutated mCRC patients became wild type after the first line chemotherapy but this status reverted in mutated one at progression in 80% of cases. In a prospective cohort of mCRC patients, we show how longitudinal monitoring using exosome-based liquid biopsy provides clinical information relevant to therapeutic stratification.

Two Novel Precursors of the HIV-1 Protease Inhibitor Darunavir Target the UPR/Proteasome System in Human Hepatocellular Carcinoma Cell Line HepG2.

Background: Several pre-clinical and clinical reports suggest that HIV-1 protease inhibitors, in addition to the antiretroviral properties, possess pleiotropic pharmacological effects including anticancer action. Therefore, we investigated the pro-apoptotic activity in tumor cells of two molecules, RDD-19 and RDD-142, which are hydroxyethylamine derivatives' precursors of darunavir and several HIV-1 protease inhibitors. Methods: Three hepatoma cell lines and one non-pathological cell line were treated with RDD-19 and RDD-142, and cell viability was assessed. The expression levels of several markers for ER stress, autophagy, cellular ubiquitination, and Akt activation were quantified in HepG2 cells treated with RDD-19 and RDD-142 to evaluate apoptotic and non-apoptotic cell death. Results: RDD-19 and RDD-142 showed a greater dose-dependent cytotoxicity towards the hepatic tumor cell line HepG2 compared to the non-pathological hepatic cell line IHH. Both molecules caused two types of cell death, a caspase-dependent apoptosis, which was ascertained by a series of biochemical and morphological assays, and a caspase-independent death that was characterized by the induction of ER stress and autophagy. The strong increase of ubiquitinated proteins inside the cells suggested that the target of these molecules could be the proteasome and in silico molecular docking analysis that was used to support the plausibility of this hypothesis. Furthermore, cells treated with the two compounds displayed decreased levels of p-AKT, which interferes with cell survival and proliferation. Conclusions: These findings demonstrate that two compounds, RDD-19 and RDD-142, have pleiotropic effects and that they may represent promising anticancer candidates.