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Understanding how antibody responses to SARS-CoV-2 evolve during infection may provide important insight into therapeutic approaches and vaccination for COVID-19. Here we profile the antibody responses of 162 COVID-19 symptomatic patients in the COVID-BioB cohort followed longitudinally for up to eight months from symptom onset to find SARS-CoV-2 neutralization, as well as antibodies either recognizing SARS-CoV-2 spike antigens and nucleoprotein, or specific for S2 antigen of seasonal beta-coronaviruses and hemagglutinin of the H1N1 flu virus. The presence of neutralizing antibodies within the first weeks from symptoms onset correlates with time to a negative swab result (p = 0.002), while the lack of neutralizing capacity correlates with an increased risk of a fatal outcome (p = 0.008). Neutralizing antibody titers progressively drop after 5–8 weeks but are still detectable up to 8 months in the majority of recovered patients regardless of age or co-morbidities, with IgG to spike antigens providing the best correlate of neutralization. Antibody responses to seasonal coronaviruses are temporarily boosted, and parallel those to SARS-CoV-2 without dampening the specific response or worsening disease progression. Our results thus suggest compromised immune responses to the SARS-CoV-2 spike to be a major trait of COVID-19 patients with critical conditions, and thereby inform on the planning of COVID-19 patient care and therapy prioritization. Antibody responses are critical for protection from developing severe COVID-19 following SARS-CoV-2 infection. Here the authors show that antibody responses against SARS-CoV-2 spike protein correlate with neutralizing capacity and protection, are not affected by heterologous boosting of influenza or common cold immunity, and can last up to 8 months.

The pressure towards innovation and creation of new model systems in regenerative medicine and cancer research has fostered the development of novel potential therapeutic applications. Kidney injuries provoke a high request of organ transplants making it the most demanding system in the field of regenerative medicine. Furthermore, renal cancer frequently threaten patients’ life and aggressive forms still remain difficult to treat. Ethical issues related to the use of embryonic stem cells, has fueled research on adult, patient-specific pluripotent stem cells as a model for discovery and therapeutic development, but to date, normal and cancerous renal experimental models are lacking. Several research groups are focusing on the development of organoid cultures. Since organoids mimic the original tissue architecture in vitro, they represent an excellent model for tissue engineering studies and cancer therapy testing. We established normal and tumor renal cell carcinoma organoids previously maintained in a heterogeneous multi-clone stem cell-like enriching medium. Starting from adult normal kidney specimens, we were able to isolate and propagate organoid 3D-structures composed of both differentiated and undifferentiated cells while expressing nephron specific markers. Furthermore, we were capable to establish organoids derived from cancer tissues although with a success rate inferior to that of their normal counterpart. Cancer cultures displayed epithelial and mesenchymal phenotype while retaining tumor specific markers. Of note, tumor organoids recapitulated neoplastic masses when orthotopically injected into immunocompromised mice. Our data suggest an innovative approach of long-term establishment of normal- and cancer-derived renal organoids obtained from cultures of fleshly dissociated adult tissues. Our results pave the way to organ replacement pioneering strategies as well as to new models for studying drug-induced nephrotoxicity and renal diseases. Along similar lines, deriving organoids from renal cancer patients opens unprecedented opportunities for generation of preclinical models aimed at improving therapeutic treatments.

Severe coronavirus disease 2019 (COVID-19) causes a hyperactivation of immune cells, resulting in lung inflammation. Recent studies showed that COVID-19 induces the production of factors previously implicated in the reawakening of dormant breast cancer cells such as neutrophil extracellular traps (NETs). The presence of NETs and of a pro-inflammatory microenvironment may therefore promote breast cancer reactivation, increasing the risk of pulmonary metastasis. Further studies will be required to confirm the link between COVID-19 and cancer recurrence. However, an increased awareness on the potential risks for breast cancer patients with COVID-19 may lead to improved treatment strategies to prevent metastatic relapse.

Colorectal cancer stem cell (CSC)‐enriched cultures were obtained efficiently from primary tumor specimens by optimizing a CSC‐isolation protocol. Based on in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, panels of CSC lines were generated with defined patterns of genetic mutations and therapy sensitivity. An analysis of CSC response to EGFR‐targeted therapy in vivo and an overview of factors implicated in therapy response/resistance are presented. Colorectal cancer (CRC) therapy mainly relies on the use of conventional chemotherapeutic drugs combined, in a subset of patients, with epidermal growth factor receptor [EGFR]‐targeting agents. Although CRC is considered a prototype of a cancer stem cell (CSC)‐driven tumor, the effects of both conventional and targeted therapies on the CSC compartment are largely unknown. We have optimized a protocol for colorectal CSC isolation that allowed us to obtain CSC‐enriched cultures from primary tumor specimens, with high efficiency. CSC isolation was followed by in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, thus generating panels of CSC lines with defined patterns of genetic mutations and therapy sensitivity. Colorectal CSC lines were polyclonal and maintained intratumor heterogeneity in terms of somatically acquired mutations and differentiation state. Such CSC‐enriched cultures were used to investigate the effects of both conventional and targeted therapies on the CSC compartment in vivo and to generate a proteomic picture of signaling pathways implicated in sensitivity/resistance to anti‐EGFR agents. We propose CSC lines as a sound preclinical framework to test the effects of therapies in vitro and in vivo and to identify novel determinants of therapy resistance. Significance Colorectal cancer stem cells (CSCs) have been shown to be responsible for tumor propagation, metastatic dissemination, and relapse. However, molecular pathways present in CSCs, as well as mechanisms of therapy resistance, are mostly unknown. Taking advantage of genetically characterized CSC lines derived from colorectal tumors, this study provides an extensive analysis of CSC response to EGFR‐targeted therapy in vivo and an overview of factors implicated in therapy response or resistance. Furthermore, the implementation of a biobank of molecularly annotated CSC lines provides an innovative resource for future investigations in colorectal cancer.

Background Circulating tumor cells (CTCs) are responsible for the metastatic dissemination of colorectal cancer (CRC) to the liver, lungs and lymph nodes. CTCs rarity and heterogeneity strongly limit the elucidation of their biological features, as well as preclinical drug sensitivity studies aimed at metastasis prevention. Methods We generated organoids from CTCs isolated from an orthotopic CRC xenograft model. CTCs-derived organoids (CTCDOs) were characterized through proteome profiling, immunohistochemistry, immunofluorescence, flow cytometry, tumor-forming capacity and drug screening assays. The expression of intra- and extracellular markers found in CTCDOs was validated on CTCs isolated from the peripheral blood of CRC patients. Results CTCDOs exhibited a hybrid epithelial-mesenchymal transition (EMT) state and an increased expression of stemness-associated markers including the two homeobox transcription factors Goosecoid and Pancreatic Duodenal Homeobox Gene-1 (PDX1), which were also detected in CTCs from CRC patients. Functionally, CTCDOs showed a higher migratory/invasive ability and a different response to pathway-targeted drugs as compared to xenograft-derived organoids (XDOs). Specifically, CTCDOs were more sensitive than XDOs to drugs affecting the Survivin pathway, which decreased the levels of Survivin and X-Linked Inhibitor of Apoptosis Protein (XIAP) inducing CTCDOs death. Conclusions These results indicate that CTCDOs recapitulate several features of colorectal CTCs and may be used to investigate the features of metastatic CRC cells, to identify new prognostic biomarkers and to devise new potential strategies for metastasis prevention.

ObjectiveCancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies.DesignTo discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents.ResultsThe drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368.ConclusionsLY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

Background Quiescent/slow cycling cells have been identified in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown. Methods A population of chemoresistant quiescent/slow cycling cells was isolated through PKH26 staining (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global gene expression and pathway activation analyses. Factors expressed by the quiescent/slow cycling population were analyzed through lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets. Results Untreated colorectal tumors contain a population of quiescent/slow cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined mesenchymal-like chemoresistant phenotype. QCSCs expressed increased levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting in increased chemoresistance, enrichment of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy treatment of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype and activation of the ZEB2/pCRAF/pASK1 axis, resulting in a chemotherapy-unresponsive state. In CRC patients, increased ZEB2 levels correlated with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized by dismal prognosis, decreased proliferative rates and upregulation of EMT genes. Conclusions These results show that chemotherapy-naive tumors contain a cell population characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment and is responsible for chemotherapy resistance, thus representing a key target for more effective therapeutic approaches.

Background Prevention and treatment of metastatic breast cancer (BC) is an unmet clinical need. The retinoic acid derivative fenretinide (FeR) was previously evaluated in Phase I-III clinical trials but, despite its excellent tolerability and antitumor activity in preclinical models, showed limited therapeutic efficacy due to poor bioavailability. We recently generated a new micellar formulation of FeR, Bionanofenretinide (Bio-nFeR) showing enhanced bioavailability, low toxicity, and strong antitumor efficacy on human lung cancer, colorectal cancer, and melanoma xenografts. In the present study, we tested the effect of Bio-nFeR on a preclinical model of metastatic BC. Methods We used BC cell lines for in vitro analyses of cell viability, cell cycle and migratory capacity. For in vivo studies, we used HER2/neu transgenic mice (neuT) as a model of spontaneously metastatic BC. Mice were treated orally with Bio-nFeR and at sacrifice primary and metastatic breast tumors were analyzed by histology and immunohistochemistry. Molecular pathways activated in primary tumors were analyzed by immunoblotting. Stem cell content was assessed by flow cytometry, immunoblotting and functional assays such as colony formation ex vivo and second transplantation assay in immunocompromised mice. Results Bio-nFeR inhibited the proliferation and migration of neuT BC cells in vitro and showed significant efficacy against BC onset in neuT mice. Importantly, Bio-nFeR showed the highest effectiveness against metastatic progression, counteracting both metastasis initiation and expansion. The main mechanism of Bio-nFeR action consists of promoting tumor dormancy through a combined induction of antiproliferative signals and inhibition of the mTOR pathway. Conclusion The high effectiveness of Bio-nFeR in the neuT model of mammary carcinogenesis, coupled with its low toxicity, indicates this formulation as a potential candidate for the treatment of metastatic BC and for the adjuvant therapy of BC patients at high risk of developing metastasis.

Europe is experiencing a third wave of COVID-19 due to the spread of highly transmissible SARS-CoV-2 variants. A number of positive and negative factors constantly shape the rates of COVID-19 infections, hospitalization, and mortality. Among these factors, the rise in increasingly transmissible variants on one side and the effect of vaccinations on the other side create a picture deeply different from that of the first pandemic wave. Starting from the observation that in several European countries the number of COVID-19 infections in the second and third pandemic wave increased without a proportional rise in disease severity and mortality, we hypothesize the existence of an additional factor influencing SARS-CoV-2 dynamics. This factor consists of an immune defence against severe COVID-19, provided by SARS-CoV-2-specific T cells progressively developing upon natural exposure to low virus doses present in populated environments. As suggested by recent studies, low-dose viral particles entering the respiratory and intestinal tracts may be able to induce T cell memory in the absence of inflammation, potentially resulting in different degrees of immunization. In this scenario, non-pharmaceutical interventions would play a double role, one in the short term by reducing the detrimental spreading of SARS-CoV-2 particles, and one in the long term by allowing the development of a widespread (although heterogeneous and uncontrollable) form of immune protection.

Treatment of lung cancer is an unmet need as it accounts for the majority of cancer deaths worldwide. The development of new therapies urges the identification of potential targets. MicroRNAs’ expression is often deregulated in cancer and their modulation has been proposed as a successful strategy to interfere with tumor cell growth and spread. We recently reported on an unbiased high-content approach to identify miRNAs regulating cell proliferation and tumorigenesis in non-small cell lung cancer (NSCLC). Here we studied the oncogenic role of miR-663 in NSCLC biology and analyzed the therapeutic potential of miR-663 targeting. We found that miR-663 regulates apoptosis by controlling mitochondrial outer membrane permeabilization (MOMP) through the expression of two novel direct targets PUMA/BBC3 and BTG2. Specifically, upon miR-663 knockdown the BH3-only protein PUMA/BBC3 directly activates mitochondrial depolarization and cell death, while BTG2 accumulation further enhances this effect by triggering p53 mitochondrial localization. Moreover, we show that miR-663 depletion is sufficient to elicit cell death in NSCLC cells and to impair tumor growth in vivo.