FACS-based protocol to assess cytotoxicity and clonogenic potential of colorectal cancer stem cells using a Wnt/β-catenin signaling pathway reporter

Cancer stem cells (CSCs) play a key role in tumor initiation and progression. A real-time tool to evaluate the activation of CSC-specific signaling pathways is crucial for the study of this cancer cell subset. Here, we present a protocol to monitor, in vitro, the activation of Wnt/β-catenin signaling pathway, which is considered a functional biomarker for colorectal CSCs (CR-CSCs). This flow-cytometry-based protocol allows it to isolate CR-CSCs and to evaluate their cytotoxicity upon anti-tumor treatments

Human Wharton’s jelly-derived mesenchymal stem cells express several immunomodulatory molecules both in their naïve state and hepatocyte-like differentiated progeny: prospects for their use in liver diseases.

Wharton’s jelly (WJ), the main constituent of umbilical cord, is a reliable source of mesenchymal stem cells (MSC). WJ-MSC show unique ability in crossing lineage borders. As other extraembryonic mesenchymal populations (placenta and amnionderived cells), WJ-MSC express several immunomodulatory molecules, essential during the initial phases of human development. Indeed, our recent work pointed out the expression of non-classical HLA molecules as HLA-G in such cells, together with a favorable combination of B7 costimulators. Very few data in literature suggest that some of the immune features of the naïve cells are maintained after performing differentiation. The aim of this work was extending the knowledge on the expression of immunomodulatory molecules by naïve and differentiated WJ-MSC. To this purpose, WJMSC underwent differentiation to osteoblasts, adipocytes and hepatocyte-like cells. Differentiated cells were characterized, by both RT-PCR, ICC and histological stains for the acquisistion of the desired phenotypical features. RT-PCR and ICC were used to investigate the differential expression of immune-related molecules in control and differentiated cells. WJ-MSC resulted expressing diverse immunomodulatory molecules which spans from non-classical type I HLAs (i.e. HLA-E, -F, -G) , to further members of the B7 family, and of the CEA superfamily, for all of which in vivo immunomodulating functions are known. In addition, we demonstrated for the first time that the expression of these molecules is maintained after performing osteogenic, adipogenic or hepatogenic differentiation. Further experiments are undergoing to better evaluating the implications of these findings in the evolving field of liver regenerative medicine

Destroying the Shield of Cancer Stem Cells: Natural Compounds as Promising Players in Cancer Therapy

In a scenario where eco-sustainability and areduction in chemotherapeutic drug waste are certainly a prerogative to safeguard the biosphere, the use of natural products (NPs) represents an alternative therapeutic approach to counteract cancer diseases. The presence of a heterogeneous cancer stem cell (CSC) population within a tumor bulk is related to disease recurrence and therapy resistance. For this reason, CSC targeting presents a promising strategy for hampering cancer recurrence. Increasing evidence shows that NPs can inhibit crucial signaling pathways involved in the maintenance of CSC stemness and sensitize CSCs to standard chemotherapeutic treatments. Moreover, their limited toxicity and low costs for large-scale production could accelerate the use of NPs in clinical settings. In this review, we will summarize the most relevant studies regarding the effects of NPs derived from major natural sources, e.g., food, botanical, and marine species, on CSCs, elucidating their use in pre-clinical and clinical studies

Cancer cell targeting by CAR-T cells: A matter of stemness

Chimeric antigen receptor (CAR)-T cell therapy represents one of the most innovative immunotherapy approaches. The encouraging results achieved by CAR-T cell therapy in hematological disorders paved the way for the employment of CAR engineered T cells in different types of solid tumors. This adoptive cell therapy represents a selective and efficacious approach to eradicate tumors through the recognition of tumor-associated antigens (TAAs). Binding of engineered CAR-T cells to TAAs provokes the release of several cytokines, granzyme, and perforin that ultimately lead to cancer cells elimination and patient’s immune system boosting. Within the tumor mass a subpopulation of cancer cells, known as cancer stem cells (CSCs), plays a crucial role in drug resistance, tumor progression, and metastasis. CAR-T cell therapy has indeed been exploited to target CSCs specific antigens as an effective strategy for tumor heterogeneity disruption. Nevertheless, a barrier to the efficacy of CAR-T cell-based therapy is represented by the poor persistence of CAR-T cells into the hostile milieu of the CSCs niche, the development of resistance to single targeting antigen, changes in tumor and T cell metabolism, and the onset of severe adverse effects. CSCs resistance is corroborated by the presence of an immunosuppressive tumor microenvironment (TME), which includes stromal cells, cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and immune cells. The relationship between TME components and CSCs dampens the efficacy of CAR-T cell therapy. To overcome this challenge, the double strategy based on the use of CAR-T cell therapy in combination with chemotherapy could be crucial to evade immunosuppressive TME. Here, we summarize challenges and limitations of CAR-T cell therapy targeting CSCs, with particular emphasis on the role of TME and T cell metabolic demands

Novel in vitro and in vivo data on the cellular localization of Hsp10 in smokers affected by COPD and in lung-derived cell lines exposed to cigarette smoke extract as stressor

Cigarette smoke is a potent stressor for the respiratory system, contributing to pathogenesis, for instance in chronic obstructive pulmonary disease (COPD), but its effects on the expression, function, and cellular localization of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and characterized the effects of non-lethal doses of cigarette smoke extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial (16HBE). We applied various in vitro methods: immunohistochemistry (IHC), subcellular fractionation analyses (SFA), Western blotting (WB), immunocytochemistry (ICC), and transmission electron microscopy (TEM) immunogold, and used bioinformatics and databases searches to gather structural in silico data for interpreting and complementing the in vitro results. IHC showed that in smokers and non-smokers COPD patients Hsp10 was localized in both, the cytoplasm and the nucleus of epithelial and lamina propria cells, while Hsp60 was present only in the cytosol. ICC, SFA, and WB on both CSE-exposed cell lines confirmed the presence of nuclear Hsp10, with an increasing trend in parallel to CSE concentration. TEM immunogold further confirmed Hsp10 in the nucleus, in addition to its presence in the cytoplasm and mitochondria, on both cell lines. Bioinformatics and in silico structural analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals in its primary sequence. Our data suggest a link between exposure to exogenous oxidative stress and cell response, involving Hsp10, which would play roles different from its canonical functions. It is known that Hsp10 can display an array of functions depending on its location: cytoplasm, mitochondria, or extracellular. Here, we show for the first time the presence of Hsp10 in the nucleus of epithelial and stromal human-lung cell lines, paralleling the observations in vivo in COPD patients, and indicating that intranuclear Hsp10 levels are affected by oxidative stress due to an exogenous stressor like cigarette-smoke. The questions now are by what mechanism Hsp10 becomes a resident of the nucleus and what are its functions there.

Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

: Breast cancer (BC) is the second cause of cancer-related deceases in the worldwide female population. Despite the successful treatment advances, 25% of BC develops resistance to current therapeutic regimens, thereby remaining a major hurdle for patient management. Current therapies, targeting the molecular events underpinning the adaptive resistance, still require effort to improve BC treatment. Using BC sphere cells (BCSphCs) as a model, here we showed that BC stem-like cells express high levels of Myc, which requires the presence of the multifunctional DNA/RNA binding protein Sam68 for the DNA-damage repair. Analysis of a cohort of BC patients displayed that Sam68 is an independent negative factor correlated with the progression of the disease. Genetic inhibition of Sam68 caused a defect in PARP-induced PAR chain synthesis upon DNA-damaging insults, resulting in cell death of TNBC cells. In contrast, BC stem-like cells were able to survive due to an upregulation of Rad51. Importantly, the inhibition of Rad51 showed synthetic lethal effect with the silencing of Sam68, hampering the cell viability of patient-derived BCSphCs and stabilizing the growth of tumor xenografts, including those TNBC carrying BRCA mutation. Moreover, the analysis of Myc, Sam68 and Rad51 expression demarcated a signature of a poor outcome in a large cohort of BC patients. Thus, our findings suggest the importance of targeting Sam68-PARP1 axis and Rad51 as potential therapeutic candidates to counteract the expansion of BC cells with an aggressive phenotype

CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

Limited therapeutic options are available for advanced colorectal cancer (CRC). Herein, we report that exposure to a neo-synthetic bis(indolyl)thiazole alkaloid analog, nortopsentin 234 (NORA234), leads to an initial reduction of proliferative and clonogenic potential of CRC sphere cells (CR-CSphCs), followed by an adaptive response selecting the CR-CSphC-resistant compartment. Cells spared by the treatment with NORA234 express high levels of CD44v6, associated with a constitutive activation of Wnt pathway. In CR-CSphC-based organoids, NORA234 causes a genotoxic stress paralleled by G2-M cell cycle arrest and activation of CHK1, driving the DNA damage repair of CR-CSphCs, regardless of the mutational background, microsatellite stability, and consensus molecular subtype. Synergistic combination of NORA234 and CHK1 (rabusertib) targeting is synthetic lethal inducing death of both CD44v6-negative and CD44v6-positive CRC stem cell fractions, aside from Wnt pathway activity. These data could provide a rational basis to develop an effective strategy for the treatment of patients with CRC

Nuclear localization and new isoforms detection give new insights on Hsp10 functions in normal and cigarette smoke-stressed lung cells

Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular dis- tribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is a potent stressor for the respiratory system, but its effects on the expression, function, and cellular locali- zation of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and charac- terized the effects of non-lethal doses of CS extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial cells (16HBE). We applied various in vitro methods: IHC, subcellular fractionation analyses (SFA), western blotting (WB), ICC, transmission electron microscopy (TEM) immunogold, chromati protein extracts (CPE), as well as 2D-gel based proteomics analyses. Bioinformatics was used to gather structural in silico data. IHC showed that Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers. ICC, SFA, and WB showed that 16HBE and HFL-1 cells featured nuclear Hsp10, before and after CSE exposure; TEM immunogold further confirmed this observation. Proteomics data showed that CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts. Bioinformatics analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals. Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein as suggested by CPE/gel retardation experiments. Data reported here indicate that in human cells of the respiratory mucosa there are at least three different intracellular locales for Hsp10: mitochondrial, nuclear, and cyto- solic. Further experiments are en route for the definition of the mechanisms underlying the transfer of Hsp10 to the nucleus and other cellular/extracellular compartments. This work was supported by grants from University of Palermo (FFR 2012) to GLR

EBF1, MYO6 and CALR expression levels predict therapeutic response in diffuse large B-cell lymphomas

BackgroundDiffuse large B-cell lymphoma (DLBCL) is a hematological malignancy representing one-third of non-Hodgkin’s lymphoma cases. Notwithstanding immunotherapy in combination with chemotherapy (R-CHOP) is an effective therapeutic approach for DLBCL, a subset of patients encounters treatment resistance, leading to low survival rates. Thus, there is an urgent need to identify predictive biomarkers for DLBCL including the elderly population, which represents the fastest-growing segment of the population in Western countries.MethodsGene expression profiles of n=414 DLBCL biopsies were retrieved from the public dataset GSE10846. Differentially expressed genes (DEGs) (fold change >1.4, p-value <0.05, n=387) have been clustered in responder and non-responder patient cohorts. An enrichment analysis has been performed on the top 30 up-regulated genes of responder and non-responder patients to identify the signatures involved in gene ontology (MSigDB). The more significantly up-regulated DEGs have been validated in our independent collection of formalin-fixed paraffin-embedded (FFPE) biopsy samples of elderly DLBCL patients, treated with R-CHOP as first-line therapy.ResultsFrom the analysis of two independent cohorts of DLBCL patients emerged a gene signature able to predict the response to R-CHOP therapy. In detail, expression levels of EBF1, MYO6, CALR are associated with a significant worse overall survival.ConclusionsThese results pave the way for a novel characterization of DLBCL biomarkers, aiding the stratification of responder versus non-responder patients