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Rationale: Growing evidence has demonstrated that miRNA-21 (miR-21) upregulation is closely associated with tumor pathogenesis. However, the mechanisms by which miR-21 inhibition modulates the immunosuppressive tumor microenvironment (TME) and improves tumor sensitivity to immune checkpoint blockade therapies remain largely unexplored. In this study, we demonstrate the precise delivery of anti-miR-21 using a PD-L1-targeting peptide conjugate (P21) to the PD-L1high TME. Methods: Investigating miR-21 inhibition mechanisms involved conducting quantitative real-time PCR, western blot, flow cytometry, and confocal microscopy analyses. The antitumor efficacy and immune profile of P21 monotherapy, or combined with anti-PD-L1 immune checkpoint inhibitors, were assessed in mouse models bearing CT26.CL25 tumors and 4T1 breast cancer. Results Inhibition of oncogenic miR-21 in cancer cells by P21 efficiently activates tumor suppressor genes, inducing autophagy and endoplasmic reticulum stress. Subsequent cell-death-associated immune activation (immunogenic cell death) is initiated via the release of damage-associated molecular patterns. The in vivo results also illustrated that the immunogenic cell death triggered by P21 could effectively sensitize the immunosuppressive TME. That is, P21 enhances CD8+ T cell infiltration in tumor tissues by conferring immunogenicity to dying cancer cells and promoting dendritic cell maturation. Meanwhile, combining P21 with an anti-PD-L1 immune checkpoint inhibitor elicits a highly potent antitumor effect in a CT26.CL25 tumor-bearing mouse model and 4T1 metastatic tumor model. Conclusions: Collectively, we have clarified a miR-21-related immunogenic cell death mechanism through the precise delivery of anti-miR-21 to the PD-L1high TME. These findings highlight the potential of miR-21 as a target for immunotherapeutic interventions.
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Antígeno B7-H1 , Muerte Celular Inmunogénica , Inmunoterapia , MicroARNs , Microambiente Tumoral , MicroARNs/genética , MicroARNs/metabolismo , Animales , Ratones , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Muerte Celular Inmunogénica/efectos de los fármacos , Línea Celular Tumoral , Inmunoterapia/métodos , Femenino , Ratones Endogámicos BALB C , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Autofagia/efectos de los fármacos , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/terapia , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/genéticaRESUMEN
Lipid nanoparticles (LNPs) exhibit remarkable mRNA delivery efficiency, yet their majority accumulate in the liver or spleen after injection. Tissue-specific mRNA delivery can be achieved through modulating LNP properties, such as tuning PEGylation or varying lipid components systematically. In this paper, a streamlined method is used for incorporating tumor-targeting peptides into the LNPs; the programmed death ligand 1 (PD-L1) binding peptides are conjugated to PEGylated lipids via a copper-free click reaction, and directly incorporated into the LNP composition (Pep LNPs). Notably, Pep LNPs display robust interaction with PD-L1 proteins, which leads to the uptake of LNPs into PD-L1 overexpressing cancer cells both in vitro and in vivo. To evaluate anticancer immunotherapy mediated by restoring tumor suppressor, mRNA encoding phosphatase and tensin homolog (PTEN) is delivered via Pep LNPs to PTEN-deficient triple-negative breast cancers (TNBCs). Pep LNPs loaded with PTEN mRNA specifically promotes autophagy-mediated immunogenic cell death in 4T1 tumors, resulting in effective anticancer immune responses. This study highlights the potential of tumor-targeted LNPs for mRNA-based cancer therapy.
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Antígeno B7-H1 , Nanopartículas , Fosfohidrolasa PTEN , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Nanopartículas/química , Animales , Ratones , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Femenino , Modelos Animales de Enfermedad , Lípidos/química , Humanos , Línea Celular Tumoral , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/terapia , Ratones Endogámicos BALB C , Inmunoterapia/métodos , LiposomasRESUMEN
BACKGROUND: Recently, increased attention has been given on exosomes as ideal nanocarriers of drugs owing to their intrinsic properties that facilitate the transport of biomolecular cargos. However, large-scale exosome production remains a major challenge in the clinical application of exosome-based drug delivery systems. Considering its biocompatibility and stability, bovine milk is a suitable natural source for large-scale and stable exosome production. Because the active-targeting ability of drug carriers is essential to maximize therapeutic efficacy and minimize side effects, precise membrane functionalization strategies are required to enable tissue-specific delivery of milk exosomes with difficulty in post-isolation modification. METHODS: In this study, the membrane functionalization of a milk exosome platform modified using a simple post-insertion method was examined comprehensively. Exosomes were engineered from bovine milk (mExo) with surface-tunable modifications for the delivery of tumor-targeting doxorubicin (Dox). The surface modification of mExo was achieved through the hydrophobic insertion of folate (FA)-conjugated lipids. RESULTS: We have confirmed the stable integration of functionalized PE-lipid chains into the mExo membrane through an optimized post-insertion technique, thereby effectively enhancing the surface functionality of mExo. Indeed, the results revealed that FA-modified mExo (mExo-FA) improved cellular uptake in cancer cells via FA receptor (FR)-mediated endocytosis. The designed mExo-FA selectively delivered Dox to FR-positive tumor cells and triggered notable tumor cell death, as confirmed by in vitro and in vivo analyses. CONCLUSIONS: This simple and easy method for post-isolation modification of the exosomal surface may be used to develop milk-exosome-based drug delivery systems.
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Messenger RNA (mRNA) is now in the limelight as a powerful tool for treating various human diseases, especially malignant tumors, thanks to the remarkable clinical outcomes of mRNA vaccines using lipid nanoparticle technology during the COVID-19 pandemic. Recent promising preclinical and clinical results that epitomize the advancement in mRNA and nanoformulation-based delivery technologies have highlighted the tremendous potential of mRNA in cancer immunotherapy. mRNAs can be harnessed for cancer immunotherapy in forms of various therapeutic modalities, including cancer vaccines, adoptive T-cell therapies, therapeutic antibodies, and immunomodulatory proteins. This review provides a comprehensive overview of the current state and prospects of mRNA-based therapeutics, including numerous delivery and therapeutic strategies.
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COVID-19 , Neoplasias , Humanos , ARN Mensajero , Pandemias , COVID-19/terapia , Inmunoterapia/métodosRESUMEN
In vitro transcribed mRNA for the synthesis of any given protein has shown great potential in cancer gene therapy, especially in cancer vaccines for immunotherapy. To overcome physiological barriers, such as rapid degradation by enzymatic attack and poor cellular uptake due to their large size and hydrophilic properties, many delivery carriers for mRNAs are being investigated for improving the bioavailability of mRNA. Recently, cell-penetrating peptides (CPPs) have received attention as promising tools for gene delivery. In terms of their biocompatibility and the ability to target specific cells with the versatility of peptide sequences, they may provide clues to address the challenges of conventional delivery systems for cancer mRNA delivery. In this study, optimal conditions for the CPP/mRNA complexes were identified in terms of complexation capacity and N/P ratio, and protection against RNase was confirmed. When cancer cells were treated at a concentration of 6.8 nM, which could deliver the highest amount of mRNA without toxicity, the amphipathic CPP/mRNA complexes with a size less than 200 nm showed high cellular uptake and protein expression. With advances in our understanding of CPPs, CPPs designed to target tumor tissues will be promising for use in developing a new class of mRNA delivery vehicles in cancer therapy.
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The tumor suppressor protein p53 is frequently inactivated in human malignancies, in which it is associated with cancer aggressiveness and metastasis. Because p53 is heavily involved in epithelial-mesenchymal transition (EMT), a primary step in cell migration, p53 regulation is important for preventing cancer metastasis. p53 function can be modulated by diverse post-translational modifications including neddylation, a reversible process that conjugates NEDD8 to target proteins and inhibits the transcriptional activity of p53. However, the role of p53 in cancer migration by neddylation has not been fully elucidated. In this study, we reported that neddylation blockade induces cell migration depending on p53 status, specifically via the EMT-promoting transcription factor Slug. In cancer cell lines expressing wild type p53, neddylation blockade increased the transcriptional activity of p53 and expression of its downstream genes p21 and MDM2, eventually promoting proteasomal degradation of Slug. In the absence of p53, neddylation blockade increased cell migration by activating the PI3K/Akt/mTOR/Slug signaling axis. Because mutant p53 was transcriptionally inactivated but maintained the ability to bind to Slug, neddylation blockade did not affect the migration of cells expressing mutant p53. Our findings highlight how the p53 expression status influences neddylation-mediated cell migration in multiple cancer cell lines via Slug.