RESUMEN
The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos , Ratones , Animales , Vectores Genéticos/genética , Ratones Transgénicos , Terapia Genética , Transgenes , Dependovirus/genética , Transducción GenéticaRESUMEN
High-dimensional immunoprofiling is essential for studying host response to immunotherapy, infection, and disease in murine model systems. However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40-color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer's patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co-stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre-clinical settings where treatments can modulate leukocyte abundance and/or function. Ultimately, this 40-color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.
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Inmunidad Innata , Tejido Linfoide , Ratones , Animales , Citometría de Flujo/métodos , Linfocitos T , Células Asesinas Naturales , InmunofenotipificaciónRESUMEN
Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and â¼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-ß, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-ß). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-ß plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.
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Inmunoterapia/métodos , Interferón beta/genética , Neoplasias Experimentales/terapia , Linfocitos T/inmunología , Transfección/métodos , Ondas Ultrasónicas , Animales , Línea Celular Tumoral , Membrana Celular/efectos de la radiación , Movimiento Celular , Humanos , Interferón beta/metabolismo , Ratones , Microburbujas/uso terapéutico , Linfocitos T/fisiologíaRESUMEN
We report on results from the comparison of two algorithms designed to estimate the attenuation coefficient from ultrasonic B-mode scans obtained from a numerical phantom simulating an ultrasound breast scan. It is well documented that this parameter significantly diverges between normal tissue and malignant lesions. To improve the diagnostic accuracy it is of great importance to devise and test algorithms that facilitate the accurate, low variance and spatially resolved estimation of the tissue's attenuation properties. A numerical phantom is realized using k-Wave, which is an open source Matlab toolbox for the time-domain simulation of acoustic wave fields that facilitates both linear and nonlinear wave propagation in homogeneous and heterogeneous tissue, as compared to strictly linear ultrasound simulation tools like Field II. k-Wave allows to simulate arbitrary distributions, resolved down to single voxel sizes, of parameters including the speed of sound, mass density, scattering strength and to include power law acoustic absorption necessary for simulation tasks in medical diagnostic ultrasound. We analyze the properties and the attainable accuracy of both the spectral-log-difference technique, and a statistical moments based approach and compare the results to known reference values from the sound field simulation.
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Algoritmos , Ultrasonido , Simulación por Computador , Fantasmas de Imagen , UltrasonografíaRESUMEN
Ligament injuries occur frequently, substantially hindering routine daily activities and sports participation in patients. Surgical reconstruction using autogenous or allogeneic tissues is the gold standard treatment for ligament injuries. Although surgeons routinely perform ligament reconstructions, the integrity of these reconstructions largely depends on adequate biological healing of the interface between the ligament graft and the bone. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would lead to significantly improved ligament graft integration. To test this hypothesis, an anterior cruciate ligament reconstruction procedure was performed in Yucatan mini-pigs. A collagen scaffold was implanted in the reconstruction sites to facilitate recruitment of endogenous mesenchymal stem cells. Ultrasound-mediated reporter gene delivery successfully transfected 40% of cells recruited to the reconstruction sites. When BMP-6 encoding DNA was delivered, BMP-6 expression in the reconstruction sites was significantly enhanced. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to significantly enhanced osteointegration in all animals 8 weeks after surgery. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively improve ligament reconstruction in large animals, thereby addressing a major unmet orthopedic need and offering new possibilities for translation to the clinical setting.
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Aloinjertos/citología , Reconstrucción del Ligamento Cruzado Anterior/métodos , Ligamentos/citología , Tendones/citología , Aloinjertos/metabolismo , Animales , Proteína Morfogenética Ósea 6/metabolismo , Colágeno/metabolismo , Técnicas de Transferencia de Gen , Ligamentos/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Porcinos , Porcinos Enanos , Tendones/metabolismo , Trasplante Homólogo/métodos , Ultrasonografía/métodos , Microtomografía por Rayos X/métodosRESUMEN
Personalized cancer vaccines (PCVs) are receiving attention as an avenue for cancer immunotherapy. PCVs employ immunogenic peptide epitopes capable of stimulating the immune system to destroy cancer cells with great specificity. Challenges associated with effective delivery of these peptides include poor solubility of hydrophobic sequences, rapid clearance, and poor immunogenicity, among others. The incorporation of peptides into nanoparticles has the potential to overcome these challenges, but the broad range of functionalities found in amino acids presents a challenge to conjugation due to possible interferences and lack of reaction specificity. Herein, a facile and versatile approach to generating nanosized PCVs under mild nonstringent conditions is reported. Following a simple two-step semibatch synthetic approach, amphiphilic hyperbranched polymer-peptide conjugates were prepared by the conjugation of melanoma antigen peptides, either TRP2 (hydrophobic) or MUT30 (hydrophilic), to an alkyne functionalized core via strain-promoted azide-alkyne click chemistry. Self-assembly of the amphiphiles gave spherical nanovaccines (by transmission electron microscopy) with sizes in the range of 10-30 nm (by dynamic light scattering). Fluorescently labeled nanovaccines were prepared to investigate the cellular uptake by antigen presenting cells (dendritic cells), and uptake was confirmed by flow cytometry and microscopy. The TRP2 nanovaccine was taken up the most followed by MUT30 nanoparticles and, finally, nanoparticles without peptide. The nanovaccines showed good biocompatibility against B16-F10 cells, yet the TRP2 peptide showed signs of toxicity, possibly due to its hydrophobicity. A test for immunogenicity revealed that the nanovaccines were poorly immunogenic, implying the need for an adjuvant when administered in vivo. Treatment of mice with melanoma tumors showed that in combination with adjuvant, CpG, groups with the peptide nanovaccines slowed tumor growth and improved survival (up to 24 days, TRP2) compared to the untreated group (14 days).
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Vacunas contra el Cáncer/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Proteínas de la Membrana/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Péptidos/uso terapéutico , Alquinos/química , Animales , Azidas/química , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Química Clic , Femenino , Inmunoterapia , Melanoma Experimental/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Nanopartículas/uso terapéutico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Péptidos/química , Péptidos/inmunología , Medicina de PrecisiónRESUMEN
Prostaglandins derived from the cyclooxygenase (COX) pathway and epoxyeicosatrienoic acids (EETs) from the cytochrome P450/soluble epoxide hydrolase (sEH) pathway are important eicosanoids that regulate angiogenesis and tumorigenesis. COX-2 inhibitors, which block the formation of prostaglandins, suppress tumor growth, whereas sEH inhibitors, which increase endogenous EETs, stimulate primary tumor growth and metastasis. However, the functional interactions of these two pathways in cancer are unknown. Using pharmacological inhibitors as probes, we show here that dual inhibition of COX-2 and sEH synergistically inhibits primary tumor growth and metastasis by suppressing tumor angiogenesis. COX-2/sEH dual pharmacological inhibitors also potently suppress primary tumor growth and metastasis by inhibiting tumor angiogenesis via selective inhibition of endothelial cell proliferation. These results demonstrate a critical interaction of these two lipid metabolism pathways on tumorigenesis and suggest dual inhibition of COX-2 and sEH as a potential therapeutic strategy for cancer therapy.
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Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/metabolismo , Epóxido Hidrolasas , Proteínas de Neoplasias/farmacología , Neoplasias Experimentales , Animales , Antineoplásicos , Sinergismo Farmacológico , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Masculino , Ratones , Metástasis de la Neoplasia , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/patologíaRESUMEN
The purpose of this pilot study was to assess the feasibility of Cadence contrast pulse sequencing ultrasound to predict clinical and angiogenic tumor response in dogs undergoing chemotherapy. Contrast ultrasound facilitated visualization of bladder tumors but failed to identify a straightforward relationship between ultrasound measures and clinical outcome.
Faisabilité de l'échographie de contraste quantitative des tumeurs des reins chez les chiens. Cette étude pilote avait pour but d'évaluer la faisabilité de l'échographie de contraste par séquençage des pulsations (CadenceTM) pour prédire la réponse clinique et angiogénique de la tumeur chez les chiens subissant la chimiothérapie. L'échographie de contraste a facilité la visualisation des tumeurs rénales mais n'a pas réussi à identifier un lien direct entre les mesures de l'échographie et le résultat clinique.(Traduit par Isabelle Vallières).
Asunto(s)
Medios de Contraste/farmacología , Enfermedades de los Perros/diagnóstico por imagen , Ultrasonografía/veterinaria , Neoplasias de la Vejiga Urinaria/veterinaria , Animales , Antineoplásicos/uso terapéutico , Carcinoma de Células Transicionales/diagnóstico por imagen , Carcinoma de Células Transicionales/orina , Carcinoma de Células Transicionales/veterinaria , Enfermedades de los Perros/orina , Perros , Femenino , Masculino , Microburbujas , Proyectos Piloto , Ultrasonografía/métodos , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/orina , Factor A de Crecimiento Endotelial Vascular/orinaRESUMEN
Epidemiological and preclinical evidence supports that omega-3 dietary fatty acids (fish oil) reduce the risks of macular degeneration and cancers, but the mechanisms by which these omega-3 lipids inhibit angiogenesis and tumorigenesis are poorly understood. Here we show that epoxydocosapentaenoic acids (EDPs), which are lipid mediators produced by cytochrome P450 epoxygenases from omega-3 fatty acid docosahexaenoic acid, inhibit VEGF- and fibroblast growth factor 2-induced angiogenesis in vivo, and suppress endothelial cell migration and protease production in vitro via a VEGF receptor 2-dependent mechanism. When EDPs (0.05 mg · kg(-1) · d(-1)) are coadministered with a low-dose soluble epoxide hydrolase inhibitor, EDPs are stabilized in circulation, causing ~70% inhibition of primary tumor growth and metastasis. Contrary to the effects of EDPs, the corresponding metabolites derived from omega-6 arachidonic acid, epoxyeicosatrienoic acids, increase angiogenesis and tumor progression. These results designate epoxyeicosatrienoic acids and EDPs as unique endogenous mediators of an angiogenic switch to regulate tumorigenesis and implicate a unique mechanistic linkage between omega-3 and omega-6 fatty acids and cancers.
Asunto(s)
Carcinoma Pulmonar de Lewis/prevención & control , Transformación Celular Neoplásica/efectos de los fármacos , Ácidos Docosahexaenoicos/metabolismo , Compuestos Epoxi/farmacología , Ácidos Grasos Omega-3/química , Ácidos Grasos Insaturados/farmacología , Metástasis de la Neoplasia/prevención & control , Neovascularización Patológica/prevención & control , Animales , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Epóxido Hidrolasas/antagonistas & inhibidores , Compuestos Epoxi/metabolismo , Ácidos Grasos Insaturados/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , MicroscopíaRESUMEN
The ability to detect and quantify macrophage accumulation can provide important diagnostic and prognostic information for atherosclerotic plaque. We have previously shown that LyP-1, a cyclic 9-amino acid peptide, binds to p32 proteins on activated macrophages, facilitating the visualization of atherosclerotic plaque with PET. Yet, the in vivo plaque accumulation of monomeric [(18)F]FBA-LyP-1 was low (0.31 ± 0.05%ID/g). To increase the avidity of LyP-1 constructs to p32, we synthesized a dendritic form of LyP-1 on solid phase using lysine as the core structural element. Imaging probes (FAM or 6-BAT) were conjugated to a lysine or cysteine on the dendrimer for optical and PET studies. The N-terminus of the dendrimer was further modified with an aminooxy group in order to conjugate LyP-1 and ARAL peptides bearing a ketone. Oxime ligation of peptides to both dendrimers resulted in (LyP-1)4- and (ARAL)4-dendrimers with optical (FAM) and PET probes (6-BAT). For PET-CT studies, (LyP-1)4- and (ARAL)4-dendrimer-6-BAT were labeled with (64)Cu (t1/2 = 12.7 h) and intravenously injected into the atherosclerotic (ApoE(-/-)) mice. After two hours of circulation, PET-CT coregistered images demonstrated greater uptake of the (LyP-1)4-dendrimer-(64)Cu than the (ARAL)4-dendrimer-(64)Cu in the aortic root and descending aorta. Ex vivo images and the biodistribution acquired at three hours after injection also demonstrated a significantly higher uptake of the (LyP-1)4-dendrimer-(64)Cu (1.1 ± 0.26%ID/g) than the (ARAL)4-dendrimer-(64)Cu (0.22 ± 0.05%ID/g) in the aorta. Similarly, subcutaneous injection of the LyP-1-dendrimeric carriers resulted in preferential accumulation in plaque-containing regions over 24 h. In the same model system, ex vivo fluorescence images within aortic plaque depict an increased accumulation and penetration of the (LyP-1)4-dendrimer-FAM as compared to the (ARAL)4-dendrimer-FAM. Taken together, the results suggest that the (LyP-1)4-dendrimer can be applied for in vivo PET imaging of plaque and that LyP-1 could be further exploited for the delivery of therapeutics with multivalent carriers or nanoparticles.
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Aterosclerosis/diagnóstico por imagen , Radioisótopos de Cobre/química , Dendrímeros/química , Imagen Multimodal , Péptidos Cíclicos/química , Tomografía de Emisión de Positrones/métodos , Tomografía Computarizada por Rayos X/métodos , Secuencia de Aminoácidos , Animales , Apolipoproteínas E/genética , Dendrímeros/farmacocinética , Ratones , Ratones Noqueados , Péptidos Cíclicos/farmacocinética , Distribución TisularRESUMEN
3-Helix micelles have demonstrated excellent in vitro and in vivo stability. Previous studies showed that the unique design of the peptide-polymer conjugate based on protein tertiary structure as the headgroup is the main design factor to achieve high kinetic stability. In this contribution, using amphiphiles with different alkyl tails, namely, C16 and C18, we quantified the effect of alkyl length on the stability of 3-helix micelles to delineate the contribution of the micellar core and shell on the micelle stability. Both amphiphiles form well-defined micelles, <20 nm in size, and show good stability, which can be attributed to the headgroup design. C18-micelles exhibit slightly higher kinetic stability in the presence of serum proteins at 37 °C, where the rate constant of subunit exchange is 0.20 h(-1) for C18-micelles vs 0.22 h(-1) for C16-micelles. The diffusion constant for drug release from C18-micelles is approximately half of that for C16-micelles. The differences between the two micelles are significantly more pronounced in terms of in vivo stability and extent of tumor accumulation. C18-micelles exhibit significantly longer blood circulation time of 29.5 h, whereas C16-micelles have a circulation time of 16.1 h. The extent of tumor accumulation at 48 h after injection is â¼43% higher for C18-micelles. The present studies underscore the importance of core composition on the biological behavior of 3-helix micelles. The quantification of the effect of this key design parameter on the stability of 3-helix micelles provides important guidelines for carrier selection and use in complex environment.
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Antineoplásicos , Portadores de Fármacos , Micelas , Neoplasias Experimentales/tratamiento farmacológico , Péptidos , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Femenino , Ratones , Neoplasias Experimentales/sangre , Tamaño de la Partícula , Péptidos/química , Péptidos/farmacocinética , Péptidos/farmacologíaRESUMEN
Epidemiological and pre-clinical studies support the anti-tumor effects of ω-3 PUFAs; however, the results from human trials are mixed, making it difficult to provide dietary guidelines or recommendations of ω-3 PUFAs for disease prevention or treatment. Understanding the molecular mechanisms by which ω-3 PUFAs inhibit cancer could lead to better nutritional paradigms and human trials to clarify their health effects. The ω-3 PUFAs exert their biological activities mainly through the formation of bioactive lipid metabolites. Here we discuss the biology of cyclooxygenase, lipoxygenase and cytochrome P450 enzymes-derived ω-3-series lipid metabolites on angiogenesis, inflammation and cancer.
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Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-6/metabolismo , Inflamación/metabolismo , Neoplasias/metabolismo , Animales , Citocromos/metabolismo , Humanos , Inflamación/enzimología , Metabolismo de los Lípidos , Lipooxigenasas/metabolismo , Neoplasias/irrigación sanguínea , Neoplasias/enzimología , Neovascularización Patológica/enzimología , Neovascularización Patológica/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1(+) cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1-coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[(18)F]fluorobenzoic acid ([(18)F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [(18)F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [(18)F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.
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Apolipoproteínas E , Aterosclerosis/metabolismo , Compuestos Férricos/farmacocinética , Nanopartículas , Péptidos Cíclicos/farmacocinética , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/patología , Sistemas de Liberación de Medicamentos/métodos , Femenino , Compuestos Férricos/farmacología , Ratones , Ratones Mutantes , Oligopéptidos/farmacocinética , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacologíaRESUMEN
Induction of pyroptosis can promote anti-PD-L1 therapeutic efficacy due to the release of pro-inflammatory cytokines, but current approaches can cause off target toxicity. Herein, a phthalocyanine-conjugated mesoporous silicate nanoparticle (PMSN) is designed for amplifying sonodynamic therapy (SDT) to augment oxidative stress and induce robust pyroptosis in tumors. The sub-10 nm diameter structure and c(RGDyC)-PEGylated modification enhance tumor targeting and renal clearance. The unique porous architecture of PMSN doubles ROS yield and enhances pyroptotic cell populations in tumors (25.0%) via a cavitation effect. PMSN-mediated SDT treatment efficiently reduces tumor mass and suppressed residual tumors in treated and distant sites by synergizing with PD-L1 blockade (85.93% and 77.09%, respectively). Furthermore, loading the chemotherapeutic, doxorubicin, into PMSN intensifies SDT-pyroptotic effects and increased efficacy. This is the first report of the use of SDT regimens to induce pyroptosis in liver cancer. This noninvasive and effective strategy has potential for clinical translation.
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Neoplasias Hepáticas , Nanopartículas , Terapia por Ultrasonido , Humanos , Piroptosis , Antígeno B7-H1 , Línea Celular Tumoral , Nanopartículas/química , InmunoterapiaRESUMEN
We apply spatial transcriptomics and proteomics to select pancreatic cancer surface receptor targets for molecular imaging and theranostics using an approach that can be applied to many cancers. Selected cancer surfaceome epithelial markers were spatially correlated and provided specific cancer localization, whereas the spatial correlation between cancer markers and immune- cell or fibroblast markers was low. While molecular imaging of cancer-associated fibroblasts and integrins has been proposed for pancreatic cancer, our data point to the tight junction protein claudin-4 as a theranostic target. Claudin-4 expression increased â¼16 fold in cancer as compared with normal pancreas, and the tight junction localization conferred low background for imaging in normal tissue. We developed a peptide-based molecular imaging agent targeted to claudin-4 with accumulation to â¼25% injected activity per cc (IA/cc) in metastases and â¼18% IA/cc in tumors. Our work motivates a new approach for data-driven selection of molecular targets.
RESUMEN
Designing stable drug nanocarriers, 10-30 nm in size, would have significant impact on their transport in circulation, tumor penetration, and therapeutic efficacy. In the present study, biological properties of 3-helix micelles loaded with 8 wt % doxorubicin (DOX), ~15 nm in size, were characterized to validate their potential as a nanocarrier platform. DOX-loaded micelles exhibited high stability in terms of size and drug retention in concentrated protein environments similar to conditions after intravenous injections. DOX-loaded micelles were cytotoxic to PPC-1 and 4T1 cancer cells at levels comparable to free DOX. 3-Helix micelles can be disassembled by proteolytic degradation of peptide shell to enable drug release and clearance to minimize long-term accumulation. Local administration to normal rat striatum by convection enhanced delivery (CED) showed greater extent of drug distribution and reduced toxicity relative to free drug. Intravenous administration of DOX-loaded 3-helix micelles demonstrated improved tumor half-life and reduced toxicity to healthy tissues in comparison to free DOX. In vivo delivery of DOX-loaded 3-helix micelles through two different routes clearly indicates the potential of 3-helix micelles as safe and effective nanocarriers for cancer therapeutics.
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Antineoplásicos/farmacología , Doxorrubicina/farmacología , Portadores de Fármacos/farmacología , Nanoestructuras/química , Neoplasias Experimentales/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inyecciones Intravenosas , Ratones , Ratones Transgénicos , Micelas , Modelos Moleculares , Neoplasias Experimentales/patología , Tamaño de la Partícula , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
Optical imaging involves the propagation of light through tissue. Current optical breast imaging technologies, including diffuse optical spectroscopy, diffuse optical tomography, and photoacoustic imaging, capitalize on the selective absorption of light in the near-infrared spectrum by deoxygenated and oxygenated hemoglobin. They provide information on the morphological and functional characteristics of different tissues based on their varied interactions with light, including physiologic information on lesion vascular content and anatomic information on tissue vascularity. Fluorescent contrast agents, such as indocyanine green, are used to visualize specific tissues, molecules, or proteins depending on how and where the agent accumulates. In this review, we describe the physical principles, spectrum of technologies, and clinical applications of the most common optical systems currently being used or developed for breast imaging. Most notably, US co-registered photoacoustic imaging and US-guided diffuse optical tomography have demonstrated efficacy in differentiating benign from malignant breast masses, thereby improving the specificity of diagnostic imaging. Diffuse optical tomography and diffuse optical spectroscopy have shown promise in assessing treatment response to preoperative systemic therapy, and photoacoustic imaging and diffuse optical tomography may help predict tumor phenotype. Lastly, fluorescent imaging using indocyanine green dye performs comparably to radioisotope mapping of sentinel lymph nodes and appears to improve the outcomes of autologous tissue flap breast reconstruction.
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Deep abdominal images suffer from poor diffraction-limited lateral resolution. Extending the aperture size can improve resolution. However, phase distortion and clutter can limit the benefits of larger arrays. Previous studies have explored these effects using numerical simulations, multiple transducers, and mechanically swept arrays. In this work, we used an 8.8-cm linear array transducer to investigate the effects of aperture size when imaging through the abdominal wall. We acquired channel data in fundamental and harmonic modes using five aperture sizes. To avoid motion and increase the parameter sampling, we decoded the full-synthetic aperture data and retrospectively synthesized nine apertures (2.9-8.8 cm). We imaged a wire target and a phantom through ex vivo porcine abdominal samples and scanned the livers of 13 healthy subjects. We applied bulk sound speed correction to the wire target data. Although point resolution improved from 2.12 to 0.74 mm at 10.5 cm depth, contrast resolution often degraded with aperture size. In subjects, larger apertures resulted in an average maximum contrast degradation of 5.5 dB at 9-11 cm depth. However, larger apertures often led to visual detection of vascular targets unseen with conventional apertures. An average 3.7-dB contrast improvement over fundamental mode in subjects showed that the known benefits of tissue-harmonic imaging extend to larger arrays.
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Hígado , Transductores , Animales , Porcinos , Ultrasonografía/métodos , Estudios Retrospectivos , Fantasmas de Imagen , Hígado/diagnóstico por imagenRESUMEN
Microbubbles are currently approved for diagnostic ultrasound imaging and are under evaluation in therapeutic protocols. Here, we present a protocol for in vitro sonoporation validation using non-targeted microbubbles for gene delivery. We describe steps for computational simulation, experimental calibration, reagent preparation, ultrasound treatment, validation, and gene expression analysis. This protocol uses approved diagnostic microbubbles and parameters that are applicable for human use. For complete details on the use and execution of this protocol, please refer to Bez et al. (2017).1.
Asunto(s)
Sistemas de Liberación de Medicamentos , Microburbujas , Humanos , Sistemas de Liberación de Medicamentos/métodos , Ultrasonografía/métodos , Técnicas de Transferencia de GenRESUMEN
Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.