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The authors have requested that this preprint be removed from Research Square.
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Severe heterogeneity within glioblastoma has spurred the notion that disrupting the interplay between multiple elements on immunosuppression is at the core of meaningful anti-tumor responses. T cell immunoreceptor with Ig and ITIM domains (TIGIT) and its glioblastoma-associated antigen, CD155, form a highly immunosuppressive axis in glioblastoma and other solid tumors, yet targeting of TIGIT, a functionally heterogeneous receptor on tumor-infiltrating immune cells, has largely been ineffective as monotherapy, suggesting that disruption of its inhibitory network might be necessary for measurable responses. It is within this context that we show that the usurpation of the TIGIT - CD155 axis via engineered synNotch-mediated activation of induced pluripotent stem cell-derived natural killer (NK) cells promotes transcription factor-mediated activation of a downstream signaling cascade that results in the controlled, localized blockade of CD73 to disrupt purinergic activity otherwise resulting in the production and accumulation of immunosuppressive extracellular adenosine. Such "decoy" receptor engages CD155 binding to TIGIT, but tilts inhibitory TIGIT/CD155 interactions toward activation via downstream synNotch signaling. Usurping activities of TIGIT and CD73 promotes the function of adoptively transferred NK cells into intracranial patient-derived models of glioblastoma and enhances their natural cytolytic functions against this tumor to result in complete tumor eradication. In addition, targeting both receptors, in turn, reprograms the glioblastoma microenvironment via the recruitment of T cells and the downregulation of M2 macrophages. This study demonstrates that TIGIT/CD155 and CD73 are targetable receptor partners in glioblastoma. Our data show that synNotch-engineered pluripotent stem cell-derived NK cells are not only effective mediators of anti-glioblastoma responses within the setting of CD73 and TIGIT/CD155 co-targeting, but represent a powerful allogeneic treatment option for this tumor.
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Glioblastoma , Células-Tronco Pluripotentes Induzidas , Células Matadoras Naturais , Humanos , Glioblastoma/terapia , Glioblastoma/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos T/metabolismo , Microambiente Tumoral , 5'-Nucleotidase/imunologia , 5'-Nucleotidase/metabolismoRESUMO
TIGIT is a receptor on human natural killer (NK) cells. Here, we report that TIGIT does not spontaneously induce inhibition of NK cells in glioblastoma (GBM), but rather acts as a decoy-like receptor, by usurping binding partners and regulating expression of NK activating ligands and receptors. Our data show that in GBM patients, one of the underpinnings of unresponsiveness to TIGIT blockade is that by targeting TIGIT, NK cells do not lose an inhibitory signal, but gains the potential for new interactions with other, shared, TIGIT ligands. Therefore, TIGIT does not define NK cell dysfunction in GBM. Further, in GBM, TIGIT+ NK cells are hyperfunctional. In addition, we discovered that 4-1BB correlates with TIGIT expression, the agonism of which contributes to TIGIT immunotherapy. Overall, our data suggest that in GBM, TIGIT acts as a regulator of a complex network, and provide new clues about its use as an immunotherapeutic target.
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Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) prodrug is a clinically tried and proven treatment modality for surface-level lesions. However, its use for deep-seated tumors has been limited due to the poor penetration depth of visible light needed to activate the photosensitizer protoporphyrin IX (PPIX), which is produced from ALA metabolism. Herein, we report the usage of poly(ethylene glycol-b-lactic acid) (PEG-PLA)-encapsulated calcium tungstate (CaWO4, CWO for short) nanoparticles (PEG-PLA/CWO NPs) as energy transducers for X-ray-activated PDT using ALA. Owing to the spectral overlap between radioluminescence afforded by the CWO core and the absorbance of PPIX, these NPs can serve as an in situ visible light activation source during radiotherapy (RT), thereby mitigating the limitation of penetration depth. We demonstrate that this effect is observed across different cell lines with varying radio-sensitivity. Importantly, both PPIX and PEG-PLA/CWO NPs exhibit no significant toxicities at therapeutic doses in the absence of radiation. To assess the efficacy of this approach, we conducted a study using a syngeneic mouse model subcutaneously implanted with inherently radio-resistant 4T1 tumors. The results show a significantly improved prognosis compared to conventional RT, even with as few as 2 fractions of 4 Gy X-rays. Taken together, these results suggest that PEG-PLA/CWO NPs are promising agents for application of ALA-PDT in deep-seated tumors, thereby significantly expanding the utility of the already established treatment strategy.
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Nanopartículas , Neoplasias , Fotoquimioterapia , Pró-Fármacos , Animais , Camundongos , Ácido Aminolevulínico/farmacologia , Ácido Aminolevulínico/uso terapêutico , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Neoplasias/tratamento farmacológico , Nanopartículas/uso terapêutico , Linhagem Celular TumoralRESUMO
Glioblastoma (GBM) is one of the most aggressive and lethal solid tumors in human. While efficacious therapeutics, such as emerging chimeric antigen receptor (CAR)-T cells and chemotherapeutics, have been developed to treat various cancers, their effectiveness in GBM treatment has been hindered largely by the blood-brain barrier and blood-brain-tumor barriers. Human neutrophils effectively cross physiological barriers and display effector immunity against pathogens but the short lifespan and resistance to genome editing of primary neutrophils have limited their broad application in immunotherapy. Here we genetically engineer human pluripotent stem cells with CRISPR/Cas9-mediated gene knock-in to express various anti-GBM CAR constructs with T-specific CD3ζ or neutrophil-specific γ-signaling domains. CAR-neutrophils with the best anti-tumor activity are produced to specifically and noninvasively deliver and release tumor microenvironment-responsive nanodrugs to target GBM without the need to induce additional inflammation at the tumor sites. This combinatory chemo-immunotherapy exhibits superior and specific anti-GBM activities, reduces off-target drug delivery and prolongs lifespan in female tumor-bearing mice. Together, this biomimetic CAR-neutrophil drug delivery system is a safe, potent and versatile platform for treating GBM and possibly other devastating diseases.
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Neoplasias Encefálicas , Glioblastoma , Nanopartículas , Camundongos , Feminino , Humanos , Animais , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Imunoterapia Adotiva , Neutrófilos , Linfócitos T , Microambiente Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Imunoterapia , Nanopartículas/uso terapêuticoRESUMO
Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92 cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor ß-chain (ΔIL-2Rß) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.
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Neutrophils, the most abundant white blood cells in circulation, are closely related to cancer development and progression. Healthy primary neutrophils present potent cytotoxicity against various cancer cell lines through direct contact and via generation of reactive oxygen species. However, due to their short half-life and resistance to genetic modification, neutrophils have not yet been engineered with chimeric antigen receptors (CARs) to enhance their antitumor cytotoxicity for targeted immunotherapy. Here, we genetically engineered human pluripotent stem cells with synthetic CARs and differentiated them into functional neutrophils by implementing a chemically defined platform. The resulting CAR neutrophils present superior and specific cytotoxicity against tumor cells both in vitro and in vivo. Collectively, we established a robust platform for massive production of CAR neutrophils, paving the way to myeloid cell-based therapeutic strategies that would boost current cancer-treatment approaches.
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Neoplasias , Células-Tronco Pluripotentes , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Neutrófilos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Receptores de Antígenos Quiméricos/metabolismoRESUMO
Immunometabolic reprogramming due to adenosine produced by CD73 (encoded by the 5'-ectonucleotidase gene NT5E) is a recognized immunosuppressive mechanism contributing to immune evasion in solid tumors. Adenosine is not only known to contribute to tumor progression, but it has specific roles in driving dysfunction of immune cells, including natural killer (NK) cells. Here, we engineered human NK cells to directly target the CD73-adenosine axis by blocking the enzymatic activity of CD73. In doing so, the engineered NK cells not only impaired adenosinergic metabolism driven by the hypoxic uptake of ATP by cancer cells in a model of non-small-cell lung cancer, but also mediated killing of tumor cells due to the specific recognition of overexpressed CD73. This resulted in a 'single agent' immunotherapy that combines antibody specificity, blockade of purinergic signaling, and killing of targets mediated by NK cells. We also showed that CD73-targeted NK cells are potent in vivo and result in tumor arrest, while promoting NK cell infiltration into CD73+ tumors and enhancing intratumoral activation.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Adenosina/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Humanos , Imunoterapia/métodos , Células Matadoras Naturais , Neoplasias Pulmonares/metabolismoRESUMO
Human hematopoietic stem cells (HSCs), which arise from aorta-gonad-mesonephros (AGM), are widely used to treat blood diseases and cancers. However, a technique for their robust generation in vitro is still missing. Here we show temporal manipulation of Wnt signaling is sufficient and essential to induce AGM-like hematopoiesis from human pluripotent stem cells. TGFß inhibition at the stage of aorta-like SOX17+CD235a- hemogenic endothelium yielded AGM-like hematopoietic progenitors, which closely resembled primary cord blood HSCs at the transcriptional level and contained diverse lineage-primed progenitor populations via single cell RNA-sequencing analysis. Notably, the resulting definitive cells presented lymphoid and myeloid potential in vitro; and could home to a definitive hematopoietic site in zebrafish and rescue bloodless zebrafish after transplantation. Engraftment and multilineage repopulating activities were also observed in mouse recipients. Together, our work provided a chemically-defined and feeder-free culture platform for scalable generation of AGM-like hematopoietic progenitor cells, leading to enhanced production of functional blood and immune cells for various therapeutic applications.
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Hemangioblastos , Animais , Diferenciação Celular/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas , Humanos , Mesonefro , Camundongos , Peixe-ZebraRESUMO
We have recently discovered that pulmonary administration of nanoparticles (micelles) formed by amphiphilic poly(styrene-block-ethylene glycol) (PS-PEG) block copolymers has the potential to treat a lung disorder involving lung surfactant (LS) dysfunction (called acute respiratory distress syndrome (ARDS)), as PS-PEG nanoparticles are capable of reducing the surface tension of alveolar fluid, while they are resistant to deactivation caused by plasma proteins/inflammation products unlike natural LS. Herein, we report studies of the clearance pathways and kinetics of PS-PEG nanoparticles from the lung, which are essential for designing further preclinical IND-enabling studies. Using fluorescently labeled PS-PEG nanoparticles, we found that, following pharyngeal aspiration in mice, the retention of these nanoparticles in the lungs extends over 2 weeks, while their transport into other (secondary) organs is relatively insignificant. An analysis based on a multicompartmental pharmacokinetic model suggests a biphasic mechanism involving a fast mucociliary escalator process through the conducting airways and much slower alveolar clearance processes by the action of macrophages and also via direct translocation into the circulation. An excessive dose of PS-PEG nanoparticles led to prolonged retention in the lungs due to saturation of the alveolar clearance capacity.
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Polietilenoglicóis , Polímeros , Animais , Pulmão , Camundongos , Micelas , Polietilenoglicóis/farmacocinética , TensoativosRESUMO
Hepatocellular carcinoma (HCC) is mostly triggered by environmental and life-style factors and may involve epigenetic aberrations. However, a comprehensive documentation of the link between the dysregulated epigenome, transcriptome, and liver carcinogenesis is lacking. In the present study, Fischer-344 rats were fed a choline-deficient (CDAA, cancer group) or choline-sufficient (CSAA, healthy group) L-amino acid-defined diet. At the end of 52 weeks, transcriptomic alterations in livers of rats with HCC tumours and healthy livers were investigated by RNA sequencing. DNA methylation and gene expression were assessed by pyrosequencing and quantitative reverse-transcription PCR (qRT-PCR), respectively. We discovered 1,848 genes that were significantly differentially expressed in livers of rats with HCC tumours (CDAA) as compared with healthy livers (CSAA). Upregulated genes in the CDAA group were associated with cancer-related functions, whereas macronutrient metabolic processes were enriched by downregulated genes. Changes of highest magnitude were detected in numerous upregulated genes that govern key oncogenic signalling pathways, including Notch, Wnt, Hedgehog, and extracellular matrix degradation. We further detected perturbations in DNA methylating and demethylating enzymes, which was reflected in decreased global DNA methylation and increased global DNA hydroxymethylation. Four selected upregulated candidates, Mmp12, Jag1, Wnt4, and Smo, demonstrated promoter hypomethylation with the most profound decrease in Mmp12. MMP12 was also strongly overexpressed and hypomethylated in human HCC HepG2 cells as compared with primary hepatocytes, which coincided with binding of Ten-eleven translocation 1 (TET1). Our findings provide comprehensive evidence for gene expression changes and dysregulated epigenome in HCC pathogenesis, potentially revealing novel targets for HCC prevention/treatment.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Ratos , Aminoácidos/genética , Aminoácidos/metabolismo , Carcinoma Hepatocelular/patologia , Colina , DNA/metabolismo , Metilação de DNA , Epigênese Genética , Expressão Gênica , Neoplasias Hepáticas/metabolismo , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , Ratos Endogâmicos F344RESUMO
The 3H-pyrazolo[4,3-f]quinoline moiety has been recently shown to be a privileged kinase inhibitor core with potent activities against acute myeloid leukemia (AML) cell lines in vitro. Herein, various 3H-pyrazolo[4,3-f]quinoline-containing compounds were rapidly assembled via the Doebner-Povarov multicomponent reaction from the readily available 5-aminoindazole, ketones, and heteroaromatic aldehydes in good yields. The most active compounds potently inhibit the recombinant FLT3 kinase and its mutant forms with nanomolar IC50 values. Docking studies with the FLT3 kinase showed a type I binding mode, where the 3H-pyrazolo group interacts with Cys694 in the hinge region. The compounds blocked the proliferation of AML cell lines harboring oncogenic FLT3-ITD mutations with remarkable IC50 values, which were comparable to the approved FLT3 inhibitor quizartinib. The compounds also inhibited the growth of leukemia in a mouse-disseminated AML model, and hence, the novel 3H-pyrazolo[4,3-f]quinoline-containing kinase inhibitors are potential lead compounds to develop into anticancer agents, especially for kinase-driven cancers.
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Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Switch/sucrose-nonfermentable (SWI/SNF) chromatin-remodeling complexes are critical regulators of chromatin dynamics during transcription, DNA replication, and DNA repair. A recently identified SWI/SNF subcomplex termed GLTSCR1/1L-BAF (GBAF; or "noncanonical BAF", ncBAF) uniquely contains bromodomain-containing protein BRD9 and glioma tumor suppressor candidate region 1 (GLTSCR1) or its paralog GLTSCR1-like (GLTSCR1L). Recent studies have identified a unique dependency on GBAF (ncBAF) complexes in synovial sarcoma and malignant rhabdoid tumors, both of which possess aberrations in canonical BAF (cBAF) and Polybromo-BAF (PBAF) complexes. Dependencies on GBAF in malignancies without SWI/SNF aberrations, however, are less defined. Here, we show that GBAF, particularly its BRD9 subunit, is required for the viability of prostate cancer cell lines in vitro and for optimal xenograft tumor growth in vivo. BRD9 interacts with androgen receptor (AR) and CCCTC-binding factor (CTCF), and modulates AR-dependent gene expression. The GBAF complex exhibits overlapping genome localization and transcriptional targets as bromodomain and extraterminal domain-containing (BET) proteins, which are established AR coregulators. Our results demonstrate that GBAF is critical for coordinating SWI/SNF-BET cooperation and uncover a new druggable target for AR-positive prostate cancers, including those resistant to androgen deprivation or antiandrogen therapies. SIGNIFICANCE: Advanced prostate cancers resistant to androgen receptor antagonists are still susceptible to nontoxic BRD9 inhibitors, making them a promising alternative for halting AR signaling in progressed disease.
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Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Fatores de Transcrição/fisiologia , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Nus , Células PC-3 , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores Androgênicos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Photodynamic therapy (PDT) has shown potential as a cancer treatment modality, but its clinical application is limited due to its visible-light activation since visible wavelengths of light cannot penetrate tissues well. Additionally, combination therapies utilizing PDT and radiotherapy have shown clinical promise in several cancers but are limited again by light penetration and the need for selective photosensitization of the treatment area. Herein, we report the development of bilirubin-photodynamic nanoparticles (PEGylated bilirubin-encapsulated CaWO4 nanoparticles or "PEG-BR/CWO NPs"). PEG-BR/CWO NPs are a formulation of PEGylated bilirubin micelles encapsulating CaWO4 nanoparticles. These particles are capable of activating PDT via X-ray irradiation within deep tissues due to the radioluminescence properties of their CaWO4 nanoparticle cores. PEG-BR/CWO NPs facilitate a combination of photodynamic and radiation therapy and represent a previously unexplored application of PEG-bilirubin conjugates as photosensitizing agents. When irradiated by X-rays, PEG-BR/CWO NPs emit UV-A and visible light from their CaWO4 cores, which excites bilirubin and leads to the production of singlet oxygen. PEG-BR/CWO NPs exhibit improvements over X-ray therapy alone in vitro and in murine xenograft models of head and neck cancer. The data presented in this study indicate that PEG-BR/CWO NPs are promising agents for facilitating combined radio-photodynamic therapy in deep tissue tumors.
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The present work demonstrates a novel concept for intratumoral chemo-radio combination therapy for locally advanced solid tumors. For some locally advanced tumors, chemoradiation is currently standard of care. This combination treatment can cause acute and long term toxicity that can limit its use in older patients or those with multiple medical comorbidities. Intratumoral chemotherapy has the potential to address the problem of systemic toxicity that conventional chemotherapy suffers, and may, in our view, be a better strategy for treating certain locally advanced tumors. The present study proposes how intratumoral chemoradiation can be best implemented. The enabling concept is the use of a new chemotherapeutic formulation in which chemotherapy drugs (e.g., paclitaxel (PTX)) are co-encapsulated with radioluminecsnt nanoparticles (e.g., CaWO4 (CWO) nanoparticles (NPs)) within protective capsules formed by biocompatible/biodegradable polymers (e.g., poly(ethylene glycol)-poly(lactic acid) or PEG-PLA). This drug-loaded polymer-encapsulated radioluminescent nanoparticle system can be locally injected in solution form into the patient's tumor before the patient receives normal radiotherapy (e.g., 30-40 fractions of 2-3 Gy daily X-ray dose delivered over several weeks for locally advanced head and neck tumors). Under X-ray irradiation, the radioluminescent nanoparticles produce UV-A light that has a radio-sensitizing effect. These co-encapsulated radioluminescent nanoparticles also enable radiation-triggered release of chemo drugs from the polymer coating layer. The non-toxic nature (absence of dark toxicity) of this drug-loaded polymer-encapsulated radioluminescent nanoparticle ("PEG-PLA/CWO/PTX") formulation was confirmed by the MTT assay in cancer cell cultures. A clonogenic cell survival assay confirmed that these drug-loaded polymer-encapsulated radioluminescent nanoparticles significantly enhance the cancer cell killing effect of radiation therapy. In vivo study validated the efficacy of PEG-PLA/CWO/PTX-based intratumoral chemo-radio therapy in mouse tumor xenografts (in terms of tumor response and mouse survival). Results of a small-scale NP biodistribution (BD) study demonstrate that PEG-PLA/CWO/PTX NPs remained at the tumor sites for a long period of time (> 1â¯month) following direct intratumoral administration. A multi-compartmental pharmacokinetic model (with rate constants estimated from in vitro experiments) predicts that this radiation-controlled drug release technology enables significant improvements in the level and duration of drug availability within the tumor (throughout the typical length of radiation treatment, i.e., > 1â¯month) over conventional delivery systems (e.g., PEG-PLA micelles with no co-encapsulated CaWO4, or an organic liquid, e.g., a 50:50 mixture of Cremophor EL and ethanol, as in Taxol), while it is capable of maintaining the systemic level of the chemo drug far below the toxic threshold limit over the entire treatment period. This technology thus has the potential to offer a new therapeutic option that has not previously been available for patients excluded from conventional chemoradiation protocols.
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Antineoplásicos Fitogênicos/administração & dosagem , Compostos de Cálcio/administração & dosagem , Sistemas de Liberação de Medicamentos , Substâncias Luminescentes/administração & dosagem , Nanopartículas/administração & dosagem , Paclitaxel/administração & dosagem , Polietilenoglicóis/administração & dosagem , Compostos de Tungstênio/administração & dosagem , Animais , Antineoplásicos Fitogênicos/química , Compostos de Cálcio/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimiorradioterapia , Liberação Controlada de Fármacos , Feminino , Humanos , Substâncias Luminescentes/química , Camundongos , Nanopartículas/química , Neoplasias/terapia , Paclitaxel/química , Polietilenoglicóis/química , Compostos de Tungstênio/químicaRESUMO
BACKGROUND: Acute myeloid leukemia (AML) remains one of the most lethal, rarely cured cancers, despite decades of active development of AML therapeutics. Currently, the 5-year survival of AML patients is about 30% and for elderly patients, the rate drops to <10%. About 30% of AML patients harbor an activating mutation in the tyrosine kinase domain (TKD) of Fms-Like Tyrosine kinase 3 (FLT3) or a FLT3 internal tandem duplication (FLT3-ITD). Inhibitors of FLT3, such as Rydapt that was recently approved by the FDA, have shown good initial response but patients often relapse due to secondary mutations in the FLT3 TKD, like D835Y and F691â¯L mutations. METHODS: Alkynyl aminoisoquinoline and naphthyridine compounds were synthesized via Sonogashira coupling. The compounds were evaluated for their in vitro and in vivo effects on leukemia growth. FINDINGS: The compounds inhibited FLT3 kinase activity at low nanomolar concentrations. The lead compound, HSN431, also inhibited Src kinase activity. The compounds potently inhibited the viability of MV4-11 and MOLM-14 AML cells with IC50 values <1â¯nM. Furthermore, the viability of drug-resistant AML cells harboring the D835Y and F691â¯L mutations were potently inhibited. In vivo efficacy studies in mice demonstrated that the compounds could drastically reduce AML proliferation in mice. INTERPRETATION: Compounds that inhibit FLT3 and downstream targets like Src (for example HSN431) are good leads for development as anti-AML agents. FUND: Purdue University, Purdue Institute for Drug Discovery (PIDD), Purdue University Center for Cancer Research, Elks Foundation and NIH P30 CA023168.
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Antineoplásicos/farmacologia , Isoquinolinas/farmacologia , Naftiridinas/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Isoquinolinas/química , Leucemia Mieloide Aguda , Camundongos , Estrutura Molecular , Mutação , Naftiridinas/química , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Radiation therapy is a primary treatment modality for many forms of cancer. Normally, the highest tolerable dose of ionizing radiation is used to treat tumors, but limitations imposed by normal tissue complications present challenges for local tumor control. In light of this, a class of compounds called radio-sensitizers have been developed to enhance the effectiveness of radiation. Many of these are small molecule drugs found to interact favorably with radiation therapy, but recent advances have been made using nanoparticles as radio-sensitizers. Herein, we report the utilization of radio-luminescent calcium tungstate nanoparticles that emit photoelectrons, UV-A, and visible light during X-ray irradiation, acting as effective radio-sensitizers ("Radio Luminescence Therapy"). In addition, a folic acid-functionalized form of these nanoparticles was shown to enhance radio-sensitization in vitro and in murine models of head and neck cancer. Folic acid-functionalized particles were found to decrease UV-A-induced clonogenic cell survival relative to nonfunctionalized particles. Several possible mechanisms were explored, and the folic acid-functionalized particles were found to mediate this increase in efficacy likely by activating pro-proliferative signaling through folate's innate mitogenic activity, leading to decreased repair of UV-A-induced DNA lesions. Finally, a clinical case study of a canine sarcoma patient demonstrated the initial safety and feasibility of translating these folic acid-functionalized particles into the clinic as radio-sensitizers in the treatment of spontaneous tumors.
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X-ray computed tomography (CT) is currently one of the most powerful, noninvasive, clinical in vivo imaging techniques, which has resulted from advances in both X-ray device and contrast enhancement technologies. The present study demonstrates, for the first time, that metal tungstates (such as CaWO4) are promising contrast agents for X-ray, radiation, and CT imaging, because of the high X-ray mass attenuation of tungsten (W). We have developed a method of formulation, in which CaWO4 (CWO) nanoparticles (NPs) are encapsulated within a biocompatible poly(ethylene glycol-b-d,l-lactic acid) (PEG-PLA) block copolymer (BCP) capsule. We show that these PEG-PLA-encapsulated CWO NPs (170 ± 10 nm hydrodynamic diameter) produce a higher CT contrast (by a factor of about 2) than commercial iodine-based radiocontrast agents (e.g., Iohexol) at identical molar concentrations of W or I atoms. PEG-PLA-coated CWO NPs are chemically stable and completely nontoxic. It was confirmed that the maximum tolerated dose (MTD) of this material in mice is significantly higher (250 ± 50 mg per kg body weight following a single intravenous (IV) administration) than, for instance, commercially available dextran-coated iron oxide nanoparticles that are currently used clinically as MRI contrast agents (MTD in mice ≈ 168 mg/kg per dose IV). IV-injected PEG-PLA/CWO NPs caused no histopathologic damage in major excretory organs (heart, liver, lungs, spleen, and kidney). When an IV dose of 100 mg/kg was given to mice, the blood circulation half-life was measured to be about 4 h, and more than 90% of the NPs were cleared from the mice within 24 h via the renal and hepatobiliary systems. When intratumorally administered, PEG-PLA-coated CWO NPs showed complete retention in a tumor-bearing mouse model (measurements were made up to 1 week). These results suggest that PEG-PLA-coated CWO NPs are promising materials for use in CT contrast.
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Meios de Contraste/química , Nanopartículas , Contagem de Cintilação , Tomografia Computadorizada por Raios X/métodos , Animais , Relação Dose-Resposta a Droga , CamundongosRESUMO
Polyrotaxanes, a family of rod-shaped nanomaterials comprised of noncovalent polymer/macrocycle assemblies, are being used in a growing number of materials and biomedical applications. Their physiochemical properties can vary widely as a function of composition, potentially leading to different in vivo performance outcomes. We sought to characterize the pharmacokinetic profiles, toxicities, and protein corona compositions of 2-hydroxypropyl-ß-cyclodextrin polyrotaxanes as a function of variations in macrocycle threading efficiency, molecular weight, and triblock copolymer core structure. We show that polyrotaxane fate in vivo is governed by the structure and dynamics of their rodlike morphologies, such that highly threaded polyrotaxanes are long circulating and deposit in the liver, whereas lung deposition and rapid clearance is observed for species bearing lower 2-hydroxypropyl-ß-cyclodextrin threading percentages. Architecture differences also promote recruitment of different serum protein classes and proportions; however, physiochemical differences have little or no influence on their toxicity. These findings provide important structural insights for guiding the development of polyrotaxanes as scaffolds for biomedical applications.