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Targeting single tumor antigens makes it difficult to provide sufficient tumor selectivity for T cell engagers (TCEs), leading to undesirable toxicity and even treatment failure, which is particularly serious in solid tumors. Here, we designed novel trispecific TCEs (TriTCEs) to improve the tumor selectivity of TCEs by logic-gated dual tumor-targeting. TriTCE can effectively redirect and activate T cells to kill tumor cells (â¼18 pM EC50) by inducing the aggregation of dual tumor antigens, which was â¼70- or 750- fold more effective than the single tumor-targeted isotype controls, respectively. Further in vivo experiments indicated that TriTCE has the ability to accumulate in tumor tissue and can induce circulating T cells to infiltrate into tumor sites. Hence, TriTCE showed a stronger tumor growth inhibition ability and significantly prolonged the survival time of the mice. Finally, we revealed that this concept of logic-gated dual tumor-targeted TriTCE can be applied to target different tumor antigens. Cumulatively, we reported novel dual tumor-targeted TriTCEs that can mediate a robust T cell response by simultaneous recognition of dual tumor antigens at the same cell surface. TriTCEs allow better selective T cell activity on tumor cells, resulting in safer TCE treatment.
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Neoplasias , Linfocitos T , Ratones , Animales , Neoplasias/metabolismo , Antígenos de NeoplasiasRESUMEN
Bevacizumab is an FDA-approved class of monoclonal antibodies used to inhibit angiogenesis and promote normalization of blood vessels. It is usually combined with chemotherapeutic agents to treat a variety of solid tumors. However, the whole-body toxicities and toxicity associated with chemotherapy greatly limit the clinical use of this combination therapy. Antibody-drug conjugates (ADCs) couple monoclonal antibodies to cytotoxic molecules via a linker, utilizing the high specificity of monoclonal antibodies to tumor surface antigens to act as a "biological missile" to deliver chemotherapeutic drugs to the tumor site. Herein, we designed a bevacizumab-based ADC, Bevacizumab Vedotin, conjugating bevacizumab to the microtubulin inhibitor MMAE via a tissue protease-specific linker. Biological studies showed strong stability and good tumor cell targeting of our constructed ADCs; rapid drug release was achieved in the presence of exogenous histone protease B. In addition, Bevacizumab Vedotin exhibited good anti-proliferative, apoptosis-promoting and cell cycle-stalling effects on glioma (U87), hepatocellular carcinoma (HepG2), and breast cancer (MCF-7) cell lines. Further in vitro assays demonstrated the enhanced anti-migration activity against MCF-7, potent anti-angiogenic effects, and blockade of the VEGF/VEGFR pathway of Bevacizumab Vedotin.
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Antineoplásicos , Neoplasias de la Mama , Inmunoconjugados , Humanos , Femenino , Inmunoconjugados/farmacología , Bevacizumab/farmacología , Bevacizumab/uso terapéutico , Péptido Hidrolasas , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Anticuerpos Monoclonales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Antígenos de NeoplasiasRESUMEN
Photodynamic therapy (PDT) is a clinically approved treatment for cancer due to its high spatiotemporal selectivity and non-invasive modality. However, its therapeutic outcomes are always limited to the severe hypoxia environment of the solid tumor. Herein, two novel photosensitizers HY and HYM based on naturally antitumor alkaloids ß-carboline were designed and synthesized. Through a series of experiments, we found HY and HYM can produce type II ROS (singlet oxygen) after light irradiation. HYM had higher singlet oxygen quantum yield and molar extinction coefficient than HY, as well as type I PDT behavior, which further let us find that HYM could exhibit robust phototoxicity activities in both normoxia and hypoxia. Meanwhile, HYM showed tumor-selective cytotoxicity with minimal toxicity toward normal cells. Notably, thanks to HYM's hypoxia-tolerant type I/II PDT and tumor selective chemotherapy, HYM showed synergistic inhibitory effect on tumor growth (inhibition rate > 91%). Our research provides a promising photosensitizer for hypoxia-tolerant chemo-photodynamic therapy, and may also give a novel molecular skeleton for photosensitizer design.
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Neoplasias , Fotoquimioterapia , Humanos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Oxígeno Singlete , Hipoxia/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Carbolinas/farmacología , Carbolinas/uso terapéutico , Línea Celular TumoralRESUMEN
Membrane lytic peptides (MLP) are widely explored as cellular delivery vehicles or antitumor/antibacterial agents. However, the poor selectivity between cancer and normal cells slims their prospects as potential anti-tumor drugs. Herein, we have developed a rationally designed self-assembly strategy to enhance tumor selectivity of MLP-based conjugates, incorporating a hydrophobic triphenylphosphonium (TPP) group for mitochondria targeting, and a hydrophilic arginine-glycine-aspartic acid (RGD) sequence targeting integrins. The self-assembly nanoparticles can enhance the stability of the peptides inâ vitro plasma and be endocytosed selectively into the cancer cells. The histidine-rich lytic peptide component assists the disruption of endosomal/lysosomal membranes and subsequent the mitochondria membrane, which leads to apoptosis. This rational design of MLP-based conjugates provides a practical strategy to increase the application prospects of lytic peptides in cancer treatment.
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Antineoplásicos , Nanopartículas , Neoplasias , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Humanos , Mitocondrias , Neoplasias/tratamiento farmacológico , Péptidos/uso terapéuticoRESUMEN
Poly (ADP-ribose) polymerase (PARP) inhibitors show potent antiproliferative activity in treatment with triple-negative breast cancer (TNBC) when combined with chemotherapeutic drugs. However, the emergence of safety issues and drug-resistance of PARP inhibitors prompt us to search for new strategies. It was proved that Proteolysis Targeting Chimeras (PROTACs) is more effective than traditional small molecule which can induce target proteins degradation rather than inhibition. In this article, based on the Olaparib derivatives and cereblon (CRBN) E3 ligase ligands, a series of PARP1 degraders, with linkers bearing different length and type were designed and synthesized. Among them, compound LB23 showed efficacious antiproliferative activity in various human cancer cells and can induce PARP1 protein degradation effectively. Moreover, LB23 showed 60-fold degradation selectivity in tumor cells with low degradation toxicity in normal cells. This study shows that the PROTAC tumor selectivity can be optimized by tuning the length and composition of the linker.
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Inhibidores de Poli(ADP-Ribosa) Polimerasas , Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Humanos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Two novel bioisosteres of cabozantinib, 3 and 4, were designed and synthesized. The benzene ring in the center of the cabozantinib structure was replaced by trimethylpyridine (3) and pyridine (4), respectively. Surprisingly, the two compounds showed extremely contrasting mesenchymal-epithelial transition factor (c-Met) inhibitory activities at 1 µM concentration (4% inhibition of 3 vs. 94% inhibition of 4). The IC50 value of compound 4 was 4.9 nM, similar to that of cabozantinib (5.4 nM). A ligand-based docking study suggested that 4 includes the preferred conformation for the binding to c-Met in the conformational ensemble, but 3 does not. The anti-proliferative activity of compound 4 against hepatocellular carcinoma (Hep3B and Huh7) and non-small-cell lung cancer (A549 and H1299) cell lines was better than that of cabozantinib, whereas 3 did not show a significant anti-proliferative activity. Moreover, the tumor selectivity of compound 4 toward hepatocellular carcinoma cell lines was higher than that of cabozantinib. In the xenograft chick tumor model, compound 4 inhibited Hep3B tumor growth to a much greater extent than cabozantinib. The present study suggests that compound 4 may be a good therapeutic candidate against hepatocellular carcinoma.
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Anilidas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Piridinas/farmacología , Anilidas/síntesis química , Animales , Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Piridinas/síntesis química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PEG coating was regarded as one effective method to improve the tumor-targeting efficiency of hyaluronic acid-based nanoparticles (HBN). However, the research of interaction between PEG coating and different receptors such as stabilin-2 and CD44 was limited. Herein, we synthesized a series of PEGylated hyaluronic acid with Curcumin (PHCs) to evaluate the role of PEG coating density in the interaction between HA and its receptors, which influenced tissues targeting activity, pharmacokinetic profiles and therapeutic efficacy of HBN. Compared with other counterparts, PHC HBN with about 5% PEG coating density preferably accumulated in the tumor mass, rather than in the liver, and hold desirable anti-cancer effect. These results indicated that to obtain optimized anticancer effect of HBN, the cellular uptake efficiency between different types of the cells should be carefully balanced by different PEG densities.
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Curcumina , Sistemas de Liberación de Medicamentos , Receptores de Hialuranos/metabolismo , Nanopartículas , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales , Animales , Curcumina/química , Curcumina/farmacocinética , Curcumina/farmacología , Femenino , Ácido Hialurónico/química , Ácido Hialurónico/farmacocinética , Ácido Hialurónico/farmacología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Nanopartículas/química , Nanopartículas/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Polietilenglicoles/farmacologíaRESUMEN
A series of new 4-(3-(4-substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl) benzenesulfonamides (7-12) was synthesized starting from 2-(4-substitutedbenzylidene)-2,3-dihydro-1H-inden-1-one (1-6) and 4-hydrazinobenzenesulfonamide. The substituted benzaldehydes from which the key intermediate was prepared by introducing 2- or 4-substituents such as fluorine, hydroxy, methoxy, or the 3,4,5-trimethoxy moieties. The compounds were tested for their cytotoxicity, tumor-specificity and potential as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The 3,4,5-trimethoxy and the 4-hydroxy derivatives showed interesting cytotoxic activities, which may be crucial for further anti-tumor activity studies, whereas some of these sulfonamides strongly inhibited both human (h) cytosolic isoforms hCA I and II.
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Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Espectroscopía de Resonancia Magnética con Carbono-13 , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Ionización de Electrospray , BencenosulfonamidasRESUMEN
A series of polymethoxylated-pyrazoline benzene sulfonamides were synthesized, investigated for their cytotoxic activities on tumor and non-tumor cell lines and inhibitory effects on carbonic anhydrase isoenzymes (hCA I and hCA II). Although tumor selectivity (TS) of the compounds were less than the reference compounds 5-Fluorouracil and Melphalan, trimethoxy derivatives 4, 5, and 6 were more selective than dimethoxy derivatives 2 and 3 as judged by the cytotoxicity assay with the cells both types originated from the gingival tissue. The compound 6 (4-[3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl] benzene sulfonamide) showed the highest TS values and can be considered as a lead molecule of the series for further investigations. All compounds synthesized showed superior CA inhibitory activity than the reference compound acetazolamide on hCA I, and II isoenzymes, with inhibition constants in the range of 26.5-55.5 nM against hCA I and of 18.9-28.8 nM against hCA II, respectively.
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Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica I/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/farmacología , Pirazoles/farmacología , Anhidrasa Carbónica I/metabolismo , Anhidrasa Carbónica II/metabolismo , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Relación Estructura-ActividadRESUMEN
Introduction: Magnetic resonance imaging (MRI) is an important tool for the accurate diagnosis of malignant tumors in clinical settings. However, the lack of tumor-specific MRI contrast agents limits diagnostic accuracy. Methods: Herein, we developed αv integrin receptor-targeting multi-crystalline manganese oxide (MCMO) as a novel MRI contrast agent for accurate diagnosis of tumors by coupling iRGD cyclopeptide PEGylation polymer onto the surface of MCMO (iRGD-pMCMO). Results: The MCMO consisted of numerous small crystals and exhibited an oval structure of 200 nm in size. The iRGD-pMCMO actively recognizes tumor cells and effectively accumulates at the tumor site, consequently releasing abundant Mn2+ ions in a weakly acidic and high-GSH-expressing tumor microenvironment. Subsequently, Mn2+ ions interact with cellular GSH to form Mn-GSH chelates, enabling efficient T1-weighted MR contrast imaging. In vivo experiments indicated that iRGD-pMCMO significantly improved T1-weighted images, achieving an accurate diagnosis of subcutaneous and orthotopic tumors. The results verified that the T1 contrast effect of iRGD-pMCMO was closely associated with the expression of GSH in tumor cells. Conclusion: Altogether, the novel tumor-targeting, highly sensitive MRI contrast agent developed in this study can improve the accuracy of MRI for tumor diagnosis.
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Medios de Contraste , Compuestos de Manganeso , Neoplasias , Humanos , Neoplasias/diagnóstico por imagen , Óxidos , Imagen por Resonancia Magnética , Microambiente TumoralRESUMEN
BACKGROUND: The antiphagocytic molecule CD47 is overexpressed in a wide variety of cancer cells, and antibodies targeting CD47 for cancer therapies are currently under intensive investigation. However, owing to the ubiquitous expression of CD47 on healthy cells, anti-CD47 therapies often achieve only weak therapeutic benefits and can induce severe side effects. Here, we report the generation of a pH-dependent anti-CD47 antibody (BC31M4) which selectively binds to tumors under the acidic solid tumor microenvironment. METHODS: BC31M4 was generated using antibody phage display and a pH-dependent selection strategy. The pH-dependent binding and blocking activities of BC31M4 were verified using in vitro assays, and the structural basis of the pH-dependent binding property was characterized. BC31M4's antitumor effect was confirmed by both phagocytosis assays and studies in xenograft models. The tumor selectivity, mechanism of action, PK properties, side effects, and therapeutic efficacy were further evaluated in humanized (hCD47 and its receptor hSIRPα) immunocompetent syngeneic mouse models. RESULTS: The crystal structure reveals that two histidines locate within the CDRs of the light chain directly contribute to the pH-dependent binding of BC31M4. BC31M4 promotes macrophage phagocytosis of tumor cells more potently at acidic-pH than at physiological-pH. Our hCD47/hSIRPα humanized syngeneic mouse model results demonstrated that BC31M4 selectively accumulates in tumors but not in normal tissues. BC31M4 causes minimal side effects and exhibits superior PK properties as compared to the other examined anti-CD47 antibodies. When combined with adoptive T cell transfer, BC31M4 efficiently promotes adaptive immune responses against tumors and also induces immune memory. Moreover, we show that BC31M4's antitumor effects rely on an Fc that mediates strong effector functions. CONCLUSIONS: Our study illustrates that the development of a tumor-selective, pH-dependent anti-CD47 antibody safely confers strong therapeutic effects against solid tumors, thus providing a promising therapeutic strategy to overcome the challenges of anti-CD47 therapy.
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Antígeno CD47 , Neoplasias , Animales , Humanos , Ratones , Anticuerpos/uso terapéutico , Modelos Animales de Enfermedad , Concentración de Iones de Hidrógeno , Macrófagos/metabolismo , Neoplasias/patología , Fagocitosis , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Polyethylene glycol (PEG)-doxorubicin (DOX) conjugation is an important strategy to improve toxicity and enhance clinically therapeutic efficacy. However, with the frequent use of PEG-modified drugs, the accumulation of anti-PEG antibodies has become a tough issue, which limits the application of PEG-drug conjugation. As an alternative solution, poly(2-oxazoline) (POX)-DOX conjugation has shown great potential in the anti-tumor field, but the reported conjugation process of POX with DOX has drawbacks such as complex synthetic steps and purification. Herein, we propose a convenient and controllable strategy for the synthesis of POX-DOX conjugation with different chain lengths and narrow dispersity by N-boc-2-bromoacetohydrazide-initiated 2-ethyl-oxazoline polymerization and the subsequent deprotection of the N-Boc group and direct reaction with DOX. The DOX-PEtOx conjugates were firstly purified, and the successful conjugations were confirmed through various characterization methods. The synthetic DOX-PEtOxn conjugates reduce the toxicity of DOX and increase the selectivity to tumor cells, reflecting the promising application of this POX-DOX conjugation strategy in drug modification and development.
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Targeting folate receptors is a potential solution to low tumor selectivity concerning conventional chemotherapeutics. Apart from antibody-drug conjugates, folate-functionalized nanoparticle drug delivery systems are interesting to be explored due to many advantages, yet currently, none seems to enter the clinical trials. Multiple in vitro evidence is available to support its efficacy compared to the non-targeting carrier and free drug formulation. Additionally, several studies pointed out factors affecting its effectiveness, including surface properties and endosomal trapping. However, in vivo biodistribution studies revealed issues that may arise from folate receptor targeting, including rapid liver uptake, subsequently reducing the nanoparticles' tumor uptake. This issue may be due to the folate receptor ß expressed by the activated macrophages in the liver; route of administration and tumor location might also influence the targeting effectiveness. Moreover, it is perplexing to generalize nanoparticles reported from various publications, primarily due to the different formulations, lack of characterization, and experimental settings, making it harder to determine the accurate factor influencing targeting effectiveness.
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Vascular disrupting agents (VDAs) have great potential in antitumor therapy, while the efficiency is limited by cardiovascular toxicity. In this study, a self-activating nanoized plinabulin (poly (l-glutamic acid) grafted Azo-Plinabulin, AzoP-NP) was constructed. The AzoP-NPs can selectively be activated to an amino derivative of plinabulin (AmP) by intrinsic tumor hypoxia, disrupting tumor vessels and amplifying hypoxia, whilst be activated by self-amplified tumor hypoxia, then selectively inhibit tumor growth. In 4T1 tumor model, the AzoP-NPs had a selective biodistribution in tumor, as the free AmP in tumors at 24 h after AzoP-NPs treatment was 18.6 fold of that after AmP treatment and significantly higher than that in other tissues. Accordingly, AzoP-NPs resulted in no obvious acute cardiovascular toxicity (plasma von Willebrand factor in PBS, AzoP-NPs and AmP group: 143.1, 184.0 and 477.6 ng/mL) and a significantly stronger tumor inhibition than AmP. And the sustained release of drug in AzoP-NPs led to a higher maximum tolerated dose (MTD) (MTD of AzoP-NPs and AmP: > 80 vs 20 mg/kg). In addition, AzoP-NPs amplified tumor hypoxic, and synergized the anti-tumor effect of Tirapazamine (TPZ), a hypoxia-activated drug in clinical trials, with an inhibition rate of 97.7% and Q value of 1.89. Therefore, our findings provide new insights into next generation VDAs and their application in tumor therapy.
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Antineoplásicos , Hipoxia , Animales , Ratones , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Ratones Endogámicos BALB C , Distribución TisularRESUMEN
The clinical use of anti-EGFR antibody-based cancer therapy has been limited by antibody-EGFR binding in normal tissues, so developing pH-dependent anti-EGFR antibodies that selectively bind with EGFR in tumors-by taking advantage of the acidity of tumor microenvironment relative to normal tissues-may overcome these limitations. Here, we generated pH-dependent anti-EGFR antibodies with cross-species reactivity for human and mouse EGFR, and we demonstrate that pH-dependent antibodies exhibit tumor-selective binding by binding strongly to EGFR under acidic conditions (pH 6.5) but binding weakly under neutral (pH 7.4) conditions. Based on screening a non-immune human antibody library and antibody affinity maturation, we initially generated antibodies with cross-species reactivity for human and mouse EGFR. A structure model was subsequently constructed and interrogated for hotspots affecting pH-dependent binding, which supported development of a cross-reactive pH-dependent anti-EGFR antibody, G532. Compared with its non-pH-dependent antibody variant, G532 exhibits improved tumor selectivity, tumor penetration, and antitumor activity. Thus, beyond showing that pH-dependent anti-EGFR antibodies can overcome multiple limitations with antibody-based cancer therapies targeting EGFR, our study illustrates a structure-guided antibody-antigen binding pH-dependency engineering strategy to enhance antibody tumor selectivity and tumor penetration, which can inform the future development of antibody-based cancer therapies targeting other ubiquitously expressed molecules.
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Organoids are complex three-dimensional structures, which contain different cell types and help to overcome many limitations of conventional 2D cell culture techniques. Here, we present a protocol for the cultivation of murine matched-pairs of small intestinal and colonic epithelial organoids, and colonic tumor organoids derived from the chemical colorectal cancer (CRC) AOM/DSS mouse model. Therefore, intestinal crypts or tumor tissue containing stem cells are isolated from the same donor mouse and cultivated in Matrigel®. The culture medium is supplemented with different growth factors to model the intestinal stem cell niche, allowing their self-renewal and differentiation. Matched-pair organoids enable the analysis of pharmacological effects and the tumor selectivity of drugs. Graphic abstract: Schematic overview of colonic matched pair organoid preparation, generated from the chemical AOM/DSS colorectal cancer mouse model. Please note that normal colon-derived organoids (green) differ in their morphology from tumor-derived organoids (red). Normal colonic-derived organoids display a thicker and crypt-like epithelial layer, whereas tumor-derived organoids are round with a thin epithelial layer.
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Poly (ADP-ribose) polymerase (PARP) enzymes play an important role in the cellular response to DNA damage and the inhibition of PARP causes synthetic lethality in homologous recombination (HR)-deficient cancer. Multiple PARP inhibitors have been developed and have shown remarkable clinical benefits. However, treatment-related toxicities, especially the hematologic toxicities, are common and restrict the clinical applications of PARP inhibitors. In this study, we designed the first glucuronide prodrug of PARP inhibitor, TSL-1502, based on a novel and highly potent PARP inhibitor TSL-1502M. TSL-1502M exhibited promising inhibitory activity on PARP1/2, significantly induced DNA double strand breaks, G2/M arrest and apoptosis in HR-deficient cells, selectively inhibited the proliferation of HR-deficient cancer cells and sensitized both HR-deficient and HR-proficient cancer cells to conventional chemotherapy. Notably, TSL-1502M was superior to olaparib, the first-in-class PARP inhibitor, in all these processes. TSL-1502 had no inhibitory effects on PARP1/2 itself, but could selectively liberate the active drug TSL-1502M in tumor after administration in nude mice. Moreover, TSL-1502 elicited significant more potent inhibitory effects than olaparib in HR-deficient tumors, and sensitized chemotherapy in both HR-deficient and HR-proficient tumors. No severe toxicities were caused by TSL-1502 in this study. Based on the encouraging preclinical antitumor activity and the selective decomposition characteristic of TSL-1502, a clinical phase I study was initiated in China, and an Investigational New Drug (IND) was granted by the US FDA. TSL-1502 could represent a new potential therapeutic choice of PARP inhibitors.
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Alphavirus M1 is a promising oncolytic virus for cancer therapy. Here, we constructed a fluorescent reporter virus for real-time visualization and quantification of M1 virus both in vitro and in vivo. The reporter-encoding M1 virus maintained the characteristics of parental virus in the aspects of structure, replication capacity, the feature to induce cytopathic cell death, and the property of tumor targeting. The fluorescence is positively correlated with virus replication both in vitro and in vivo. More importantly, the reporter can be stably expressed for at least 10 generations in a serial passage assay. In summary, we successfully constructed stable and authentic reporter viruses for studying M1 virus and provided a feasible technical route for gene modification of oncolytic virus M1.
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Alphavirus , Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Alphavirus/genética , Línea Celular Tumoral , Humanos , Neoplasias/genética , Neoplasias/terapia , Virus Oncolíticos/genética , Replicación ViralRESUMEN
BACKGROUND: Gold nanoparticles (AuNPs) are commonly used in nanomedicine because of their unique spectral properties, chemical and biological stability, and ability to quench the fluorescence of organic dyes attached to their surfaces. However, the utility of spherical AuNPs for activatable fluorescence sensing of molecular processes have been confined to resonance-matched fluorophores in the 500 nm to 600 nm spectral range to maximize dye fluorescence quenching efficiency. Expanding the repertoire of fluorophore systems into the NIR fluorescence regimen with emission >800 nm will facilitate the analysis of multiple biological events with high detection sensitivity. OBJECTIVE: The primary goal of this study is to determine if spherical AuNP-induced radiative rate suppression of non-resonant near-infrared (NIR) fluorescent probes can serve as a versatile nanoconstruct for highly sensitive detection and imaging of activated caspase-3 in aqueous media and cancer cells. This required the development of activatable NIR fluorescence sensors of caspase-3 designed to overcome the nonspecific degradation and release of the surface coatings in aqueous media. METHOD: We harnessed the fluorescence-quenching properties and multivalency of spherical AuNPs to develop AuNP-templated activatable NIR fluorescent probes to detect activated caspase-3, an intracellular reporter of early cell death. Freshly AuNPs were coated with a multifunctional NIR fluorescent dye-labeled peptide (LS422) consisting of an RGD peptide sequence that targets αvß3-integrin protein (αvß3) on the surface of cancer cells to mediate the uptake and internalization of the sensors in tumor cells; a DEVD peptide sequence for reporting the induction of cell death through caspase-3 mediated NIR fluorescence enhancement; and a multidentate hexacysteine sequence for enhancing self-assembly and stabilizing the multifunctional construct on AuNPs. The integrin binding affinity of LS422 and caspase-3 kinetics were determined by a radioligand competitive binding and fluorogenic peptide assays, respectively. Detection of intracellular caspase-3, cell viability, and the internalization of LS422 in cancer cells were determined by confocal NIR fluorescence spectroscopy and microscopy. RESULTS: Narrow size AuNPs (13 nm) were prepared and characterized by transmission electron microscopy and dynamic light scattering. When assembled on the AuNPs, the binding constant of LS422 for αvß3 improved 11-fold from 13.2 nM to 1.2 nM. Whereas the catalytic turnover of caspase-3 by LS422-AuNPs was similar to the reference fluorogenic peptide, the binding affinity for the enzyme increased by a factor of 2. Unlike the αvß3 positive, but caspase-3 negative breast cancer MCF-7 cells, treatment of the αvß3 and caspase-3 positive lung cancer A549 cells with Paclitaxel showed significant fluorescence enhancement within 30 minutes, which correlated with caspase-3 specific activation of LS422-AuNPs fluorescence. Incorporation of a 3.5 mW NIR laser source into our spectrofluorometer increased the detection sensitivity by an order of magnitude (limit of detection ~0.1 nM of cypate) and significantly decreased the signal noise relative to a xenon lamp. This gain in sensitivity enabled the detection of substrate hydrolysis at a broad range of inhibitor concentrations without photobleaching the cypate dye. CONCLUSION: The multifunctional AuNPs demonstrate the use of a non-resonant quenching strategy to design activatable NIR fluorescence molecular probes. The nanoconstruct offers a selective reporting method for detecting activated caspase-3, imaging of cell viability, identifying dying cells, and visualizing the functional status of intracellular enzymes. Performing these tasks with NIR fluorescent probes creates an opportunity to translate the in vitro and cellular analysis of enzymes into in vivo interrogation of their functional status using deep tissue penetrating NIR fluorescence analytical methods.
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Pyropheophorbide a (Pyro) is a promising photosensitizer; however, it has no tumor selectivity and enrichment capability. In our former work, the prepared folic acid (FA)-Pyro conjugates showed considerably improved tumor accumulation and photodynamic therapy (PDT) activity in cell- and animal-based studies. However, the targeting capability, selectivity and water solubility of the conjugate remain problematic. Here, we evaluated the installation of hydrophilic polyethylene glycol chains as the linker between Pyro and FA, by avoiding direct conjugation of Pyro with FA, aiming to improve tumor selectivity and accumulation. However, PEGylation may have negative effects on the PDT activity and cutaneous phototoxicity. Therefore, we chose various lengths of PEGs as linkers to optimize the molecular weight, hydrophilicity, and PDT activity and, thus, to balance the tumor selectivity and biological function of the conjugate. One optimized conjugate, Pyro-PEG1K-FA, exhibited excellent tumor enrichment and was able to eradicate subcutaneous tumors at a considerably reduced dose. We report the synthesis and characterization of these conjugates as well as the evaluation of their tumor accumulation ability and the corresponding PDT efficiency through in vitro and in vivo experiments.