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The integration of the lipid nanoparticle (LNP)-protein corona as a pioneering approach for the development of vaccines against the present and future SARS-CoV-2 variants of concern marks a significant shift in the field. This concept holds great promise, offering a universal platform that can be adaptable to combat future pandemics caused by unknown viruses. Understanding the complex interactions among the protein corona, LNPs, and receptors is crucial for harnessing its potential. This knowledge will allow optimal vaccine formulations and improve their effectiveness. Safety assessments are essential to ensure suitability for human use, compliance with regulatory standards, and rigorous quality control in manufacturing. This transformative workflow requires collaborative efforts, expanding our foundational knowledge and translating advancements from the laboratory to clinical reality. The LNP-protein corona approach represents a paradigmatic shift with far-reaching implications. Its principles and insights can be leveraged beyond specific applications against SARS-CoV-2, enabling a universal platform for addressing viral threats, cancer, and genetic diseases.
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Coroa de Proteína , Vacinas , Humanos , Lipossomos , Pandemias/prevenção & controleRESUMO
tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully understood. We have identified a new tRF (named tRF3E), derived from mature tRNAGlu, that is specifically expressed in healthy mammary glands but not in breast cancer (BC). Consistently, tRF3E levels significantly decrease in the blood of patients with epidermal growth factor receptor 2 (HER2)-positive BC reflecting tumor status (control > early cancer > metastatic cancer). tRF3E down-regulation was recapitulated in Δ16HER2 transgenic mice, representing a BC preclinical model. Pulldown assays, used to search for proteins capable to selectively bind tRF3E, have shown that this tRF specifically interacts with nucleolin (NCL), an RNA-binding protein overexpressed in BC and able to repress the translation of p53 mRNA. The binding properties of NCL-tRF3E complex, predicted in silico and analyzed by EMSA assays, are congruent with a competitive displacement of p53 mRNA by tRF3E, leading to an increased p53 expression and consequently to a modulation of cancer cell growth. Here, we provide evidence that tRF3E plays an important role in the pathogenesis of BC displaying tumor-suppressor functions through a NCL-mediated mechanism.-Falconi, M., Giangrossi, M., Elexpuru Zabaleta, M., Wang, J., Gambini, V., Tilio, M., Bencardino, D., Occhipinti, S., Belletti, B., Laudadio, E., Galeazzi, R., Marchini, C., Amici, A. A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.
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Neoplasias da Mama/metabolismo , Fosfoproteínas/metabolismo , RNA de Transferência de Ácido Glutâmico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Western Blotting , Neoplasias da Mama/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Fosfoproteínas/genética , RNA de Transferência de Ácido Glutâmico/genética , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , NucleolinaRESUMO
Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts'ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.
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Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/prevenção & controle , Antagonistas do Ácido Fólico/farmacologia , Lithospermum , Receptor ErbB-2/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Antraquinonas/isolamento & purificação , Antraquinonas/farmacocinética , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Antagonistas do Ácido Fólico/isolamento & purificação , Antagonistas do Ácido Fólico/farmacocinética , Humanos , Lithospermum/química , Camundongos Transgênicos , Raízes de Plantas , Receptor ErbB-2/genética , Transdução de Sinais , Distribuição Tecidual , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent advances in biochemical and biophysical research have been achieved through the employment of microfluidic devices. Microfluidic mixing of therapeutic agents with biomaterials yields systems with finely tuned physical-chemical properties for applications in drug and gene delivery. Here, we investigate the role of preparation technology (microfluidic mixing vs. bulk self-assembly) on the transfection efficiency (TE) and cytotoxicity of multicomponent cationic liposome/DNA complexes (lipoplexes) in live Chinese hamster ovarian (CHO) cells. Decoupling TE and cytotoxicity allowed us to combine them in a unique coherent vision. While bulk self-assembly produces highly efficient and highly toxic MC lipoplexes, microfluidics manufacture leads to less efficient, but less cytotoxic complexes. This discrepancy is ascribed to two main factors controlling lipid-mediated cell transfection, i.e. the lipoplex concentration at the cell surface and the lipoplex arrangement at the nanoscale. Further research is required to optimize microfluidic manufacturing of lipoplexes to obtain highly efficient and not cytotoxic gene delivery systems.
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DNA/administração & dosagem , Lipídeos/química , Lipossomos/química , Transfecção/métodos , Animais , Células CHO , Cátions/química , Cricetulus , DNA/química , DNA/genética , Desenho de Equipamento , Dispositivos Lab-On-A-ChipRESUMO
To date, efficiency upon non-viral DNA delivery remains low and this implies the existence of unidentified transfection barriers. Here we explore the mechanisms of action of multicomponent (MC) cationic liposome/DNA complexes (lipoplexes) by a combination of reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), fluorescence activated cell sorting (FACS) analysis and laser scanning confocal microscopy (LSCM) in live cells. Lipofectamine - the gold standard among transfection reagents - was used as a reference. On the basis of our results, we suggest that an additional transfection barrier impairs transfection efficiency, that is: low lipoplex concentration at the cell surface. Based on the acquired knowledge we propose an optimized transfection protocol that allowed us to efficiently transfect DND41, JURKAT, MOLT3, P12-ICHIKAWA, ALL-SILL, TALL-1 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines known to be difficult-to-transfect by using non-viral vectors and where LFN-based technologies fail to give satisfactory results.
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Lipossomos , Transfecção , Animais , Linhagem Celular , DNA , Humanos , Lipídeos , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favorable toxicity and clearance properties. Here, we show that the ruthenium(II) complex [Ru(p-cymene)(bis(3,5-dimethylpyrazol-1-yl)methane)Cl]Cl (UNICAM-1) exhibits potent in vivo antitumor effects. When administered as four-dose course, by repeating a single dose (52.4mgkg-1) every three days, UNICAM-1 significantly reduces the growth of A17 triple negative breast cancer cells transplanted into FVB syngeneic mice. Pharmacokinetic studies indicate that UNICAM-1 is rapidly eliminated from kidney, liver and bloodstream thanks to its high hydrosolubility, exerting excellent therapeutic activity with minimal side effects. Immunohistological analysis revealed that the efficacy of UNICAM-1, mainly relies on its capacity to reverse tumor-associated immune suppression by significantly reducing the number of tumor-infiltrating regulatory T cells. Therefore, UNICAM-1 appears very promising for the treatment of TNBC.
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Antineoplásicos/uso terapêutico , Compostos Organometálicos/uso terapêutico , Rutênio/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Compostos Organometálicos/sangue , Compostos Organometálicos/farmacocinética , Compostos Organometálicos/farmacologia , Rutênio/sangue , Rutênio/farmacocinética , Rutênio/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacosRESUMO
Mature adipocytes have shown dynamic plasticity to be converted into fibroblast-like and lipid-free cells. After the dedifferentiation process, these cells re-entered the cell cycle and acquired a high proliferation potential, becoming a valid source of stem cells. However, many aspects of the cellular biosafety about dedifferentiated fat cells remained unclear. This study aimed to elucidate their potential susceptibility to malignant transformation and to ascertain the safety of these cells for clinical use. To evaluate the genomic stability of dedifferentiated adipocytes, telomere length, hTERT gene transcription, the capacity of these cells to grow in an anchorage-independent manner and the presence of DNA damage by single cell gel electrophoresis assay were studied. Spontaneous chromosomal alterations were excluded by cytogenetic analysis and the expression level of c-myc and p53, tumor associated genes, were assessed, evaluating also p53 loss of function mutations. Despite the high proliferation capacity of dedifferentiated adipocytes, these cells showed stable telomere length compared with mature adipocytes, no hTERT transcriptions and consequently no telomerase activity, suggesting that both transformation and senescence were avoided. A constant expression level of c-myc and p53, the inability of dedifferentiated adipocytes to grow in an anchorage-independent manner, the absence of DNA damage suggested the safety of these cells. Moreover, a normal karyotype was preserved throughout the dedifferentiation process. Data in vivo showed that dedifferentiated adipocytes analyzed for tumorigenicity did not develop tumors. In conclusion, our data indicated that dedifferentiated adipocytes could be a relatively easily accessible resource for cell therapy and regenerative medicine.
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Adipócitos/citologia , Desdiferenciação Celular/fisiologia , Adipócitos/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese , Linhagem Celular Tumoral , Ensaio Cometa , Regulação da Expressão Gênica/fisiologia , Humanos , Cariótipo , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Experimentais , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
INTRODUCTION: The availability of mice transgenic for the human HER2 gene (huHER2) and prone to the development of HER2-driven mammary carcinogenesis (referred to as FVB-huHER2 mice) prompted us to study active immunopreventive strategies targeting the human HER2 molecule in a tolerant host. METHODS: FVB-huHER2 mice were vaccinated with either IL-12-adjuvanted human HER2-positive cancer cells or DNA vaccine carrying chimeric human-rat HER2 sequences. Onset and number of mammary tumors were recorded to evaluate vaccine potency. Mice sera were collected and passively transferred to xenograft-bearing mice to assess their antitumor efficacy. RESULTS: Both cell and DNA vaccines significantly delayed tumor onset, leading to about 65% tumor-free mice at 70 weeks, whereas mock-vaccinated FVB-huHER2 controls developed mammary tumors at a median age of 45 weeks. In the DNA vaccinated group, 65% of mice were still tumor-free at about 90 weeks of age. The number of mammary tumors per mouse was also significantly reduced in vaccinated mice. Vaccines broke the immunological tolerance to the huHER2 transgene, inducing both humoral and cytokine responses. The DNA vaccine mainly induced a high and sustained level of anti-huHER2 antibodies, the cell vaccine also elicited interferon (IFN)-γ production. Sera of DNA-vaccinated mice transferred to xenograft-carrying mice significantly inhibited the growth of human HER2-positive cancer cells. CONCLUSIONS: Anti-huHER2 antibodies elicited in the tolerant host exert antitumor activity.
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Vacinas Anticâncer/imunologia , Neoplasias Mamárias Animais/imunologia , Receptor ErbB-2/imunologia , Vacinas de DNA/imunologia , Transferência Adotiva , Animais , Anticorpos/sangue , Formação de Anticorpos/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Interferon gama/biossíntese , Interleucina-12/imunologia , Células MCF-7 , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/prevenção & controle , Camundongos , Camundongos Transgênicos , Receptor ErbB-2/genética , Baço/citologia , Baço/transplanteRESUMO
Plastic pollution poses a significant threat to both ecosystems and human health, as fragments of microscale size are daily inhaled and ingested. Such tiny specks are defined as microplastics (MPs), and although their presence as environmental contaminants is ubiquitous in the world, their possible effects at biological and physiological levels are still not clear. To explore the potential impacts of MP exposure, we produced and characterized polyethylene terephthalate (PET) micro-fragments, then administered them to living cells. PET is widely employed in the production of plastic bottles, and thus represents a potential source of environmental MPs. However, its potential effects on public health are hardly investigated, as the current bio-medical research on MPs mainly utilizes different models, such as polystyrene particles. This study employed cell viability assays and Western blot analysis to demonstrate cell-dependent and dose-dependent cytotoxic effects of PET MPs, as well as a significant impact on HER-2-driven signaling pathways. Our findings provide insight into the biological effects of MP exposure, particularly for a widely used but poorly investigated material such as PET.
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Microplásticos , Poluentes Químicos da Água , Humanos , Microplásticos/toxicidade , Plásticos/toxicidade , Polietilenotereftalatos/toxicidade , Ecossistema , Poliestirenos/toxicidade , Poluentes Químicos da Água/toxicidade , Monitoramento AmbientalRESUMO
The histone deacetylase sirtuin 6 (SIRT6) has been endowed with anti-cancer capabilities in many tumor types. Here, we investigate the impact of SIRT6-overexpression (SIRT6-OE) in Delta16HER2 mice, which are a bona fide model of HER2-positive breast cancer. After an initial delay in the tumor onset, SIRT6-OE induces a more aggressive phenotype of Delta16HER2 tumors promoting the formation of higher number of tumor foci and metastases than controls. This phenotype of SIRT6-OE tumors is associated with cancer stem cell (CSC)-like features and tumor dormancy, and low senescence and oxidative DNA damage. Accordingly, a sub-set of HER2-positive breast cancer patients with concurrent SIRT6-OE has a significant poorer relapse-free survival (RFS) probability than patients with low expression of SIRT6. ChIP-seq, RNA-seq and RT-PCR experiments indicate that SIRT6-OE represses the expression of the T-box transcription factor 3 (Tbx3) by deacetylation of H3K9ac. Accordingly, loss-of-function mutations of TBX3 or low TBX3 expression levels are predictive of poor prognosis in HER2-positive breast cancer patients. Our work indicates that high levels of SIRT6 are indicative of poor prognosis and high risk of metastasis in HER2-positive breast cancer and suggests further investigation of TBX3 as a downstream target of SIRT6 and co-marker of poor-prognosis. Our results point to a breast cancer subtype-specific effect of SIRT6 and warrant future studies dissecting the mechanisms of SIRT6 regulation in different breast cancer subtypes.
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Neoplasias da Mama , Sirtuínas , Humanos , Animais , Camundongos , Feminino , Neoplasias da Mama/patologia , Recidiva Local de Neoplasia , Sirtuínas/metabolismo , Doença CrônicaRESUMO
INTRODUCTION: Intrinsic plasticity of breast carcinoma cells allows them to undergo a transient and reversible conversion into mesenchymal cells to disseminate into distant organs, where they can re-differentiate to an epithelial-like status to form a cohesive secondary mass. The p130Cas scaffold protein is overexpressed in human ER+ and HER2+ breast cancer where it contributes to cancer progression, invasion and resistance to therapy. However, its role in regulating mesenchymal aggressive breast cancer cells remains to be determined. The aim of this study was to investigate the molecular and functional involvement of this adaptor protein in breast cancer cell plasticity. METHODS: We used silencing strategies and rescue experiments to evaluate phenotypic and biochemical changes from mesenchymal to epithelial traits in breast tumor cell lines. In the mouse A17 cell model previously related to mesenchymal cancer stem cells and basal-like breast cancer, we biochemically dissected the signaling pathways involved and performed functional in vivo tumor growth ability assays. The significance of the signaling platform was assessed in a human setting through the use of specific inhibitors in aggressive MDA-MB-231 subpopulation LM2-4175 cells. To evaluate the clinical relevance of the results, we analyzed publicly available microarray data from the Netherlands Cancer Institute and from the Koo Foundation Sun Yat-Sen Cancer Center. RESULTS: We show that p130Cas silencing induces loss of mesenchymal features, by downregulating Vimentin, Snail, Slug and Twist transcriptional factors, resulting in the acquirement of epithelial-like traits. Mechanistically, p130Cas controls Cyclooxygenase-2 transcriptional expression, which in turn contributes to p130Cas-dependent maintenance of mesenchymal phenotype. This cascade of events also compromises in vivo tumor growth through inhibition of cell signaling controlling cell cycle progression. c-Src and JNK kinases are sequential players in p130Cas/ Cyclooxygenase-2 axis and their pharmacological inhibition is sufficient to downregulate Cyclooxygenase-2 leading to an epithelial phenotype. Finally, in silico microarray data analysis indicates that p130Cas and Cyclooxygenase-2 concomitant overexpression predicts poor survival and high probability of breast tumor recurrence. CONCLUSIONS: Overall, these data identify a new p130Cas/Cyclooxygenase-2 axis as a crucial element in the control of breast tumor plasticity, opening new therapeutic strategies leading to inhibition of these pathways in aggressive breast carcinoma.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Substrato Associada a Crk/metabolismo , Ciclo-Oxigenase 2/metabolismo , Animais , Neoplasias da Mama/genética , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/genética , Ciclo-Oxigenase 2/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Modelos Biológicos , Fenótipo , Característica Quantitativa Herdável , Quinases da Família src/metabolismoRESUMO
The effect of a low dose of the insecticide permethrin administered during early-life was evaluated on leukocytes inflammation mediators on 300- and 500-day-old rats. Nurr1, NF-κB-p65, Nrf2, lipid peroxidation and GSH levels increased with age but compared to the control group, treatment with permethrin induced a significant increase only of Nurr1 and lipid peroxidation in oldest rats. TNF-α and Rantes increased, while IL-1ß, IL-2, IL-13 decreased in oldest treated rats. The results propose Nurr1, TNF-α, Rantes, GSH and plasma lipid peroxidation as peripheral biomarkers for monitoring the impact of early-life environmental exposure to xenobiotics in old age.
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Exposição Ambiental , Inseticidas/toxicidade , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Permetrina/toxicidade , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Citocinas/sangue , Feminino , Glutationa/sangue , Leucócitos/metabolismo , Peroxidação de Lipídeos , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/sangue , Fator de Transcrição RelA/metabolismoRESUMO
Breast cancers (BCs) may present dramatic diagnoses, both for ineffective therapies and for the limited outcomes in terms of lifespan. For these types of tumors, the search for new drugs is a primary necessity. It is widely recognized that gold compounds are highly active and extremely potent as anticancer agents against many cancer cell lines. The presence of the metal plays an essential role in the activation of the cytotoxicity of these coordination compounds, whose activity, if restricted to the ligands alone, would be non-existent. On the other hand, gold exhibits a complex biochemistry, substantially variable depending on the chemical environments around the central metal. In this review, the scientific findings of the last 6-7 years on two classes of gold(I) compounds, containing phosphane or carbene ligands, are reviewed. In addition to this class of Au(I) compounds, the recent developments in the application of Auranofin in regards to BCs are reported. Auranofin is a triethylphosphine-thiosugar compound that, being a drug approved by the FDA-therefore extensively studied-is an interesting lead gold compound and a good comparison to understand the activities of structurally related Au(I) compounds.
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Antineoplásicos , Auranofina , Neoplasias da Mama , Ouro , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Auranofina/química , Auranofina/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Feminino , Ouro/química , Ouro/uso terapêutico , Humanos , Relação Estrutura-AtividadeRESUMO
In the last decade, graphene oxide (GO)-based nanomaterials have attracted much attention for their potential anti-cancer properties against various cancer cell types. However, while in vitro studies are promising, following in vivo investigations fail to show any relevant efficacy. Recent research has clarified that the wide gap between benchtop discoveries and clinical practice is due to our limited knowledge about the physical-chemical transformation of nanomaterials in vivo. In physiological environments, nanomaterials are quickly coated by a complex dress of biological molecules referred to as the protein corona. Mediating the interaction between the pristine material and the biological system the protein corona controls the mechanisms of action of nanomaterials up to the sub-cellular level. Here we investigate the anticancer ability of GO in SK-BR-3 human breast cancer cells over-expressing the human epidermal growth factor receptor 2 (HER-2), which is functionally implicated in the cell growth and proliferation through the activation of downstream pathways, including the PI3K/AKT/mTOR and MAPK/ERK signaling cascades. Western blot analysis demonstrated that GO treatment resulted in a marked decrease in total HER-2, associated with a down-regulation of the expression and activation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) thus indicating that GO may act as a potent HER-2 inhibitor. On the other side, the protein corona reverted the effects of GO on HER-2 expression and molecular downstream events to the control level. Our findings may suggest a mechanistic explanation of the reduced anticancer properties of GO-based nanomaterials in vivo. These results may also represent a good prediction strategy for the anticancer activity of nanomaterials designed for biomedical purposes, reaffirming the necessity of exploring their effectiveness under physiologically relevant conditions before moving on to the next in vivo studies.
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Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines). Despite this, the delivery of plasmid DNA (pDNA) by LNPs in preclinical studies is still unsatisfactory, mainly due to the lack of systematic structural and functional studies on DNA-loaded LNPs. To tackle this issue, we developed, characterized, and tested a library of 16 multicomponent DNA-loaded LNPs which were prepared by microfluidics and differed in lipid composition, surface functionalization, and manufacturing factors. 8 out of 16 formulations exhibited proper size and zeta potential and passed to the validation step, that is, the simultaneous quantification of transfection efficiency and cell viability in human embryonic kidney cells (HEK-293). The most efficient formulation (LNP15) was then successfully validated both in vitro, in an immortalized adult keratinocyte cell line (HaCaT) and in an epidermoid cervical cancer cell line (CaSki), and in vivo as a nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice. Finally, by a combination of confocal microscopy, transmission electron microscopy and synchrotron small-angle X-ray scattering, we were able to show that the superior efficiency of LNP15 can be linked to its disordered nanostructure consisting of small-size unoriented layers of pDNA sandwiched between closely apposed lipid membranes that undergo massive destabilization upon interaction with cellular lipids. Our results provide new insights into the structure-activity relationship of pDNA-loaded LNPs and pave the way to the clinical translation of this gene delivery technology.
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COVID-19 , Nanopartículas , Animais , Camundongos , Humanos , Vacinas contra COVID-19 , Células HEK293 , Lipídeos/química , Camundongos Endogâmicos C57BL , DNA/química , Nanopartículas/química , RNA Interferente PequenoRESUMO
DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure-activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies.
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The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient's immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment.
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The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2-dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202-1A cells), and by its reexpression in ErbB2-transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth, and in vivo growth of orthotopic N202-1A tumors. Moreover, intranipple injection of p130Cas-stabilized siRNAs in the mammary gland of Balbc-NeuT mice decreases the growth of spontaneous tumors. In ErbB2-transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c-Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202-1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.
Assuntos
Transformação Celular Neoplásica , Proteína Substrato Associada a Crk/fisiologia , Genes erbB-2 , Neoplasias Mamárias Experimentais/patologia , Animais , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/genética , Feminino , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , RNA Interferente Pequeno , Transdução de SinaisRESUMO
In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.
RESUMO
BACKGROUND: Tumor endothelial marker 1 (TEM1) is a protein expressed in the tumor-associated endothelium and/or stroma of various types of cancer. We previously demonstrated that immunization with a plasmid-DNA vaccine targeting TEM1 reduced tumor progression in three murine cancer models. Radiation therapy (RT) is an established cancer modality used in more than 50% of patients with solid tumors. RT can induce tumor-associated vasculature injury, triggering immunogenic cell death and inhibition of the irradiated tumor and distant non-irradiated tumor growth (abscopal effect). Combination treatment of RT with TEM1 immunotherapy may complement and augment established immune checkpoint blockade. METHODS: Mice bearing bilateral subcutaneous CT26 colorectal or TC1 lung tumors were treated with a novel heterologous TEM1-based vaccine, in combination with RT, and anti-programmed death-ligand 1 (PD-L1) antibody or combinations of these therapies, tumor growth of irradiated and abscopal tumors was subsequently assessed. Analysis of tumor blood perfusion was evaluated by CD31 staining and Doppler ultrasound imaging. Immunophenotyping of peripheral and tumor-infiltrating immune cells as well as functional analysis was analyzed by flow cytometry, ELISpot assay and adoptive cell transfer (ACT) experiments. RESULTS: We demonstrate that addition of RT to heterologous TEM1 vaccination reduces progression of CT26 and TC1 irradiated and abscopal distant tumors as compared with either single treatment. Mechanistically, RT increased major histocompatibility complex class I molecule (MHCI) expression on endothelial cells and improved immune recognition of the endothelium by anti-TEM1 T cells with subsequent severe vascular damage as measured by reduced microvascular density and tumor blood perfusion. Heterologous TEM1 vaccine and RT combination therapy boosted tumor-associated antigen (TAA) cross-priming (ie, anti-gp70) and augmented programmed cell death protein 1 (PD-1)/PD-L1 signaling within CT26 tumor. Blocking the PD-1/PD-L1 axis in combination with dual therapy further increased the antitumor effect and gp70-specific immune responses. ACT experiments show that anti-gp70 T cells are required for the antitumor effects of the combination therapy. CONCLUSION: Our findings describe novel cooperative mechanisms between heterologous TEM1 vaccination and RT, highlighting the pivotal role that TAA cross-priming plays for an effective antitumor strategy. Furthermore, we provide rationale for using heterologous TEM1 vaccination and RT as an add-on to immune checkpoint blockade as triple combination therapy into early-phase clinical trials.