Elena Trombetta

Giant Lysosomes as a Chemotherapy Resistance Mechanism in Hepatocellular Carcinoma Cells

Despite continuous improvements in therapeutic protocols, cancer-related mortality is still one of the main problems facing public health. The main cause of treatment failure is multi-drug resistance (MDR: simultaneous insensitivity to different anti-cancer agents), the underlying molecular and biological mechanisms of which include the activity of ATP binding cassette (ABC) proteins and drug compartmentalisation in cell organelles. We investigated the expression of the main ABC proteins and the role of cytoplasmic vacuoles in the MDR of six hepatocellular carcinoma (HCC) cell lines, and confirmed the accumulation of the yellow anti-cancer drug sunitinib in giant (four lines) and small cytoplasmic vacuoles of lysosomal origin (two lines). ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes. MTT assays showed that the cell lines with giant lysosomes were more resistant to sorafenib treatment than those with small lysosomes (p<0.01), and that verapamil incubation can revert this resistance, especially if it is administered after drug pre-incubation. The findings of this study demonstrate the involvement of PGP-positive lysosomes in drug sequestration and MDR in HCC cell lines. The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment.

Modulation of gamma globin genes expression by histone deacetylase inhibitors: an in vitro study

Induction of fetal haemoglobin (HbF) is a promising therapeutic approach for the treatment of β‐thalassaemia and sickle cell disease (SCD). Several pharmacological agents, such as hydroxycarbamide (HC) and butyrates, have been shown to induce the γ‐globin genes (HBG1, HBG2). However, their therapeutic use is limited due to weak efficacy and an inhibitory effect on erythroid differentiation. Thus, more effective agents are needed. The histone deacetylase (HDAC) inhibitors are potential therapeutic haemoglobin (Hb) inducers able to modulate gene expression through pleiotropic mechanisms. We investigated the effects of a HDAC inhibitor, Givinostat (GVS), on erythropoiesis and haemoglobin synthesis and compared it with sodium butyrate and HC. We used an in vitro erythropoiesis model derived from peripheral CD34+ cells of healthy volunteers and SCD donors. GVS effects on erythroid proliferation and differentiation and on Hb synthesis were investigated. We found that GVS at high concentrations delayed erythroid differentiation with no specific effect on HBG1/2 transcription. At a low concentration (1 nmol/l), GVS induced Hb production with no effects on cells proliferation and differentiation. The efficacy of GVS 1 mol/l in Hb induction in vitro was comparable to that of HC and butyrate. Our results support the evaluation of GVS as a new candidate molecule for the treatment of the haemoglobinophathies due to its positive effects on haemoglobin production at low and non‐toxic concentrations.

iPSC-derived LewisX+CXCR4+β1-integrin+ neural stem cells improve the amyotrophic lateral sclerosis phenotype by preserving motor neurons and muscle innervation in human and rodent models

Amyotrophic lateral sclerosis (ALS) is a fatal incurable neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs), leading to relentless muscle paralysis. In the early stage of the disease, MN loss and consequent muscle denervation are compensated by axonal sprouting and reinnervation by the remaining MNs, but this mechanism is insufficient in the long term. Here, we demonstrate that induced pluripotent stem cell-derived neural stem cells (NSCs), in particular the subpopulation positive for LewisX-CXCR4-β1-integrin, enhance neuronal survival and axonal growth of human ALS-derived MNs co-cultured with toxic ALS astrocytes, acting on both autonomous and non-autonomous ALS disease features. Transplantation of this NSC fraction into transgenic SOD1G93A ALS mice protects MNs in vivo, promoting their ability to maintain neuromuscular junction integrity, inducing novel axonal sprouting and reducing macro- and microgliosis. These effects result in a significant increase in survival and an improved neuromuscular phenotype in transplanted SOD1G93A mice. Our findings suggest that effective protection of MN functional innervation can be achieved by modulation of multiple dysregulated cellular and molecular pathways in both MNs and glial cells. These pathways must be considered in designing therapeutic strategies for ALS patients.

Multi-center harmonization of flow cytometers in the context of the European “PRECISESADS” project

The innovative medicine initiative project called PRECISESADS will study 2.500 individuals affected by systemic autoimmune diseases (SADs) and controls. Among extensive OMICS approaches, multi-parameter flow cytometry analyses will be performed in eleven different centers. Therefore, the integration of all data in common bioinformatical and biostatistical investigations requires a fine mirroring of all instruments. We describe here the procedure elaborated to achieve this prerequisite. One flow cytometer chosen as reference instrument fixed the mean fluorescence intensities (MFIs) of 8 different fluorochrome-conjugated antibodies (Abs) using VersaComp Ab capture beads. The ten other centers adjusted their own PMT voltages to reach the same MFIs. Subsequently, all centers acquired Rainbow 8-peak beads data on a daily basis to follow the stability of their instrument overtime. One blood sample has been dispatched and concomitantly stained in all centers. Comparison of leukocytes frequencies and cell surface marker MFIs demonstrated the close sensitivity of all flow cytometers, allowing a multicenter analysis. The effective multi-center harmonization enables the constitution of a workable wide flow cytometry database for the identification of specific molecular signatures in individuals with SADs.

The epigenetically regulated miR-494 associates with stem-cell phenotype and induces sorafenib resistance in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, especially in patients not amenable for curative treatments. The multi-kinase inhibitor sorafenib represents the first-line treatment option for advanced HCC; nevertheless, its effectiveness is limited due to tumor heterogeneity as well as innate or acquired drug resistance, raising the need for new therapeutic strategies. MicroRNAs (miRNAs) involvement in treatment response as well as their safety and efficacy in preclinical models and clinical trials have been widely documented in the oncologic field, including HCC. Here, we identified miR-494 upregulation in a subgroup of human and rat HCCs with stem cell-like characteristics, as well as multiple epigenetic mechanisms involved in its aberrant expression in HCC cell lines and patients. Moreover, we identified p27, puma and pten among miR-494 targets, contributing to speed up cell cycle progression, enhance survival potential in stressful conditions and increase invasive and clonogenic capabilities. MiR-494 overexpression increased sorafenib resistance via mTOR pathway activation in HCC cell lines and, in line, high miR-494 levels associated with decreased sorafenib response in two HCC animal models. A sorafenib-combined anti-miR-494-based strategy revealed an enhanced anti-tumor potential with respect to sorafenib-only treatment in our HCC rat model. In conclusion, our findings suggested miR-494 as a possible therapeutic target as well as a candidate biomarker for patient stratification in advanced HCC.

Angiogenesis in human brain tumors: screening of drug response through a patient-specific cell platform for personalized therapy

Gliomas are the most common brain tumors, with diverse biological behaviour. Glioblastoma (GBM), the most aggressive and with the worst prognosis, is characterized by an intense and aberrant angiogenesis, which distinguishes it from low-grade gliomas (LGGs) and benign expansive lesions, as meningiomas (MNGs). With increasing evidence for the importance of vascularization in tumor biology, we focused on the isolation and characterization of endothelial cells (ECs) from primary GBMs, LGGs and MNGs. Gene expression analysis by Real-Time PCR, immunofluorescence and flow cytometry analysis, tube-like structures formation and vascular permeability assays were performed. Our results showed a higher efficiency of ECs to form a complex vascular architecture, as well as a greater impairment of a brain blood barrier model, and an overexpression of pro-angiogenic mediators in GBM than in LGG and MNG. Furthermore, administration of temozolomide, bevacizumab, and sunitinib triggered a different proliferative, apoptotic and angiogenic response, in a dose and time-dependent manner. An increased resistance to temozolomide was observed in T98G cells co-cultured in GBM-EC conditioned media. Therefore, we developed a novel platform to reproduce tumor vascularization as “disease in a dish”, which allows us to perform screening of sensitivity/resistance to drugs, in order to optimize targeted approaches to GBM therapy.

Diagnostic value of cell bound and circulating neutrophil antibody detection in pediatric neutropenia

Chronic benign neutropenia of infancy includes primary autoimmune neutropenia (pAIN) and chronic idiopathic neutropenia (CIN). A diagnosis of CIN is supported by the absence of free and/or cell‐bound neutrophil autoantibodies, which can be detected by flow cytometry with the indirect‐granulocyte immunofluorescence test (I‐GIFT) and direct‐granulocyte immunofluorescence test (D‐GIFT), respectively. Conclusive evidence is lacking on the diagnostic value of the D‐GIFT, whose performance requires specific laboratory expertise, may be logistically difficult, and hampered by very low neutrophil count in patient samples. This study investigated whether the evaluation of D‐GIFT improves the diagnostic accuracy of pediatric neutropenia.

Expression of C19MC miRNAs in HCC associates with stem-cell features and the cancer-testis genes signature

BACKGROUND Intratumor heterogeneity of hepatocellular carcinoma (HCC) and, among HCC cell subsets, the cancer stem cell population (hCSC), is responsible for therapeutic resistance and disease relapse. AIMS To characterize hCSC-enriched HCCs at the molecular level. METHODS Side population (SP) was used to identify the hCSCs in multiple tumor sampling from different patients and primary HCCs cultures. FACS was used to immunoprofile cultures. miRNAs were profiled in samples and correlated to SP. The Cancer Genome Atlas (TCGA) HCC dataset was analyzed to search for signatures associated with C19MC miRNAs expression. Results were confirmed by immunohistochemistry. RESULTS The miRNA cluster on chromosome 19 (C19MC) was enriched in SP and in HCCs with a high SP fraction. At the molecular level, an elevated C19MC was correlated with expression of precursor transcripts. In TCGA-HCC series, high C19MC expression identified a subset of patients with poorer prognosis, advanced disease and overexpression of the cancer-testis (CT) antigens. These data were confirmed in an independent cohort of HCCs and at the protein level. CONCLUSION C19MC miRNAs and CT antigens overexpression represents a novel oncogenic pathway in a subset of hCSC-enriched HCCs with dismal prognosis. CT antigens are promising immunotherapy targets. Therefore, these molecular signatures could identify HCCs who could benefit from immunotherapy.