Biology subjects

Gagliardi, A.

Publications and source records attributed to Gagliardi, A..

9 recordsLinked to original sources

bioRxiv2026

Cytokine-bearing Bacterial Outer Membrane Vesicles with Empowered Efficacy in Intratumoral Immunotherapy

Bacterial Outer Membrane Vesicles (OMVs), spherical bilayered nanoparticles naturally released by all Gram-negative bacteria, are gaining increasing interest not only in the design of prophylactic vaccines but also in cancer immunotherapy. In particular, thanks to their potent built-in adjuvanticity and to their intrinsic capacity to directly kill tumor cells, OMVs have been successfully tested in intratumoral in situ vaccination (ISV), a strategy in which immunostimulatory formulations are injected directly into tumors to convert the tumor microenvironment (TME) into an immune-reactive state. Previous studies have shown that OMVs induce robust inflammation and a Th1-skewed immune response, resulting in complete tumor remission in a substantial fraction of mice bearing syngeneic tumors. Here, we show that OMVs from our Escherichia coli {Delta}60 strain can be efficiently engineered with multiple cytokines and chemokines. Moreover, CCL3, Flt3L, TNF, and IL-2 not only accumulated on the OMV surface but also retained their in vitro biological activity. Furthermore, OMVs displaying these cytokines exhibited potent antitumor activity, and in particular the intratumoral injection of the combined TNF- and IL-2-engineered OMVs eradicated tumors in over 95% of mice across several syngeneic models. Immunostaining and flow cytometry analyses revealed that injection of engineered OMVs markedly remodeled the TME, promoting the recruitment of inflammatory myeloid cells and {gamma}{delta} T cells, the persistence of local CD8 and CD4 {beta} T cells, and the reduction of regulatory T cells. Overall, these results highlight cytokine-bearing OMVs as a versatile and highly effective platform for intratumoral immunotherapy.

cancer biology↗

bioRxiv2025

Mammalian Cell Culture and Transfection in a Low-Cost, CO2-Independent Environment

Traditional mammalian cell culture requires CO2 incubation to maintain physiological pH, creating infrastructure costs that limit accessibility. We demonstrate successful HEK293T/17 culture using HEPES-buffered CO2-independent medium in a repurposed consumer-grade egg incubator, reducing incubator equipment costs by >98% while maintaining viability through 10+ passages. To assess feasibility for recombinant protein production, we performed plasmid transfection with pcDNA3.1(+)-EGFP via electroporation. Fluorescence microscopy confirmed robust EGFP expression within 48 hours. These results demonstrate that mammalian cell culture and genetic engineering for recombinant protein production can be achieved using cost-effective, widely available equipment rather than conventional high-cost laboratory infrastructure.

cell biology↗

bioRxiv2024

Mapping in silico genetic networks of the KMT2D tumour suppressor gene to uncover novel functional associations and cancer cell vulnerabilities

Loss-of-function (LOF) alterations in tumour suppressor genes cannot be directly targeted. Approaches characterising gene function and vulnerabilities conferred by such mutations are required. Here, we computationally map genetic networks of KMT2D, a tumour suppressor gene frequently mutated in several cancer types. Using KMT2D loss-of-function (KMT2DLOF) mutations as a model, we illustrate the utility of in silico genetic networks in uncovering novel functional associations and vulnerabilities in cancer cells with LOF alterations affecting tumour suppressor genes. We revealed genetic interactors with functions in histone modification, metabolism, and immune response, and synthetic lethal (SL) candidates, including some encoding existing therapeutic targets. Analysing patient data from The Cancer Genome Atlas and the Personalized OncoGenomics Project, we showed, for example, elevated immune checkpoint response markers in KMT2DLOF cases, possibly supporting KMT2DLOF as an immune checkpoint inhibitor biomarker. Our study illustrates how tumour suppressor gene LOF alterations can be exploited to reveal potentially targetable cancer cell vulnerabilities.

bioinformatics↗

bioRxiv2023

Anti-tumor Efficacy of in situ Vaccination Using Bacterial Outer Membrane Vesicles

In situ vaccination (ISV) is a promising cancer immunotherapy strategy, consists in the intratumoral administration of immunostimulatory molecules (adjuvants). The rationale is that tumor antigens are abundant at the tumor site and therefore to elicit an effective anti-tumor immune response all is needed is an adjuvant, which can turn the immunosuppressive environment into an immunologically active one. Bacterial Outer Membrane Vesicles (OMVs) are potent adjuvants since they contain a number of microbe-associated molecular patterns (MAMPs) naturally present in the outer membrane and in the periplasmic space of Gram-negative bacteria. Therefore, they appear particularly indicted for ISV. In this work we first show that the OMVs from E. coli BL21(DE3){Delta}60 strain promote a strong anti-tumor activity when intratumorally injected into the tumors of two different mouse models. Tumor inhibition correlates with a rapid infiltration of DCs and NK cells. We also show that the addition of neo-epitopes to OMVs synergizes with the vesicle adjuvanticity, as judged by a two-tumor mouse models. Overall, our data support the use of the OMVs in ISV and suggests that ISV efficacy could benefit from the addition of properly selected tumor-specific neo-antigens.

cancer biology↗

bioRxiv2021

Prolonged XPO1 inhibition is essential for optimal anti-leukemic activity in NPM1-mutated AML

NPM1 encodes for a nucleolar multifunctional protein and is the most frequently mutated gene in adult acute myeloid leukemia (AML). NPM1 mutations cause the aberrant accumulation of mutant NPM1 (NPM1c) in the cytoplasm of leukemic cells, that is mediated by the nuclear exporter Exportin-1 (XPO1). Recent work has demonstrated that the interaction between NPM1c and XPO1 promotes high homeobox (HOX) genes expression, which is critical for maintaining the leukemic state of NPM1-mutated cells. However, the XPO1 inhibitor Selinexor administered once or twice/week in early-phase clinical trials did not translate into clinical benefit for NPM1-mutated AML patients. Here, we demonstrate that this dosing strategy results in only temporary disruption of the XPO1-NPM1c interaction and transient HOX genes downregulation, limiting the efficacy of Selinexor in the context of NPM1-mutated AML. Since second-generation XPO1 inhibitors can be administered more frequently, we compared intermittent (twice/week) versus prolonged (5 days/week) XPO1 inhibition in NPM1-mutated AML models. Integrating in vitro and in vivo data, we show that only prolonged XPO1 inhibition results in stable HOX downregulation, cell differentiation and remarkable anti-leukemic activity. This study lays the groundwork for the accurate design of clinical trials with second-generation XPO1 inhibitors in NPM1-mutated AML.

cancer biology↗

bioRxiv2021

Outer Membrane Vesicles from the gut microbiome contribute to tumor immunity by eliciting cross-reactive T cells

The gut microbiome plays a key role in cancer immunity. One proposed mechanism is through the elicitation of T cells, which incidentally recognize neo-epitopes arising from cancer mutations ("molecular mimicry (MM)" hypothesis). To support MM, Escherichia coli Nissle was engineered with the SIINFEKL epitope (OVA) and orally administered to C57BL/6 mice. The treatment elicited OVA-specific CD8+ T cells in the lamina propria and inhibited the growth of OVA-B16F10 tumors. Importantly, the administration of Outer Membrane Vesicles (OMVs) engineered with different T cell epitopes elicited epitope-specific T cells and inhibited tumor growth. Microbiome shotgun sequencing and TCR sequencing provided evidence that cross-reacting T cells were induced at the mucosal level and subsequently reached the tumor site. Overall, our data support the role of MM in tumor immunity, assign a new role to OMVs and pave the way to new probiotics/OMV-based anti-cancer immunotherapies.

microbiology↗

bioRxiv2021

Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in immunized mice, resulting in the production of neutralizing antibodies. The immunity induced by the vaccine is sufficient to protect K18-hACE2 transgenic mice from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with RBM peptides derived from a different genetic variant of SARS-CoV-2, inducing a similarly potent neutralization activity in vaccinated mice. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

immunology↗

bioRxiv2020

Complex sex-specific age-related changes in DNA methylation including variability, epimutations and entropy

In humans, females live longer than males but experience a worse longevity, as genome-wide autosomal DNA methylation differences between males and females have been reported. So far, few studies have investigated if DNA methylation is differently affected by aging in males and females. We performed a meta-analysis of 4 large whole blood datasets, comparing 4 aspects of epigenetic age-dependent remodeling between the two sexes: differential methylation, variability, epimutations and entropy. We reported that a large fraction (43%) of sex-associated probes undergoes age-associated DNA methylation changes, and that a limited number of probes shows age-by-sex interaction. We experimentally validated 2 regions mapping in FIGN and PRR4 genes, and showed sex-specific deviations of their methylation patterns in models of decelerated (centenarians) and accelerated (Down syndrome) aging. While we did not find sex differences in the age-associated increase in epimutations and in entropy, we showed that the number of probes showing age-related increase in methylation variability is 15 times higher in males compared to females. Our results can offer new epigenetic tools to study the interaction between aging and sex and can pave the way to the identification of molecular triggers of sex differences in longevity and age-related diseases prevalence.

genomics↗

bioRxiv2019

Loss of CIC promotes mitotic dysregulation and chromosome segregation defects

CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CICs interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, and also observed novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines predominantly at 3 and 5 untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CICs functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CICs potential roles in cancers. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts. Simple summaryCapicua (CIC) is a gene that is frequently mutated in several cancer types, including stomach cancers and certain subtypes of brain tumours and sarcomas. CIC, the protein encoded by the CIC gene, has been shown to play a multitude of roles in both normal and cancer cell functions; however, most studies exploring these roles focus on a single aspect of CIC function and may therefore overlook complex interconnected activities in which CIC is involved. In this study, we have used multiple complementary approaches to obtain a broader view of CICs complex functional networks. We observed novel interactions (genetic or physical) between CIC and genes/proteins involved in various aspects of cellular function, including regulation of cell division and processing of RNA molecules. Altogether, our work characterises the complexity of CICs functional network and expands our understanding of its potential roles in cancer.

cancer biology↗