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Background: Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL, Acly ), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear. Methods: We utilized human heart tissue samples from healthy donor hearts and patients with hypertrophic cardiomyopathy. Further, we used CRISPR/Cas9 gene editing to inactivate Acly in cardiomyocytes of MyH6-Cas9 mice. In vivo, positron emission tomography and ex vivo stable isotope tracer labeling were used to quantify metabolic flux changes in response to the loss of ACL. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics and proteomics. Experimental data were integrated into computational modeling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level. Results: Here, we show that in mice, ACL drives metabolic adaptation in the heart to sustain contractile function, histone acetylation, and lipid modulation. Notably, we show that loss of ACL increases glucose oxidation while maintaining fatty acid oxidation. Ex vivo isotope tracing experiments revealed a reduced efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. We demonstrate that YAP inactivation facilitates ACL deficiency. Computational flux analysis and integrative multi-omics analysis indicate that loss of ACL induces alternative isocitrate dehydrogenase 1 flux to compensate. Conclusions: This study mechanistically delineates how cardiac metabolism compensates for suppressed citrate metabolism in response to ACL loss and uncovers metabolic vulnerabilities in the heart.
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B7-H3 (CD276) has two isoforms (2Ig and 4Ig), no confirmed cognate receptor, and physiological functions that remain elusive. While differentially expressed on many solid tumors correlating with poor survival, mechanisms of how B7-H3 signals in cis (tumor cell) versus in trans (immune cell co-regulator) to elicit pro-tumorigenic phenotypes remain poorly defined. Herein, we characterized a tumorigenic and signaling role for tumor cell-expressed 4Ig-B7-H3, the dominant human isoform, in gynecological cancers that could be abrogated upon CRISPR/Cas9 knockout of B7-H3; tumorigenesis was rescued upon re-expression of 4Ig-B7-H3. Size exclusion chromatography revealed dimerization states for the extracellular domains of both human 4Ig- and murine 2Ig-B7-H3. mEGFP lifetimes of expressed 4Ig-B7-H3-mEGFP fusions determined by FRET-FLIM assays confirmed close-proximity interactions of 4Ig-B7-H3 and identified two distinct homo-FRET lifetime populations, consistent with monomeric and homo-dimer interactions. In live cells, bioluminescence imaging of 4Ig-B7-H3-mediated split luciferase complementation showed dimerization of 4Ig-B7-H3. To separate basal from dimer state activities in the absence of a known receptor, C-terminus (cytosolic) chemically-induced dimerization of 4Ig-B7-H3 increased tumor cell proliferation and cell activation signaling pathways (AKT, Jak/STAT, HIF1α, NF-κß) significantly above basal expression of 4Ig-B7-H3 alone. These results revealed a new, dimerization-dependent intrinsic tumorigenic signaling role for 4Ig-B7-H3, likely acting in cis, and provide a therapeutically-actionable target for intervention of B7-H3-dependent tumorigenesis.
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Antígenos B7 , Carcinogénesis , Proliferación Celular , Transducción de Señal , Animales , Humanos , Ratones , Antígenos B7/genética , Dimerización , Polímeros , Isoformas de Proteínas/genética , Factores de TranscripciónRESUMEN
We previously showed that ablation of tumor hypoxia can sensitize tumors to immune checkpoint blockade (ICB). Here, we used a Kras+/G12D TP53+/R172H Pdx1-Cre-derived (KPC-derived) model of pancreatic adenocarcinoma to examine the tumor response and adaptive resistance mechanisms involved in response to 2 established methods of hypoxia-reducing therapy: the hypoxia-activated prodrug TH-302 and vascular endothelial growth factor receptor 2 (VEGFR-2) blockade. The combination of both modalities normalized tumor vasculature, increased DNA damage and cell death, and delayed tumor growth. In contrast with prior cancer models, the combination did not alleviate overall tissue hypoxia or sensitize these KPC tumors to ICB therapy despite qualitative improvements to the CD8+ T cell response. Bulk tumor RNA sequencing, flow cytometry, and adoptive myeloid cell transfer suggested that treated tumor cells increased their capacity to recruit granulocytic myeloid-derived suppressor cells (G-MDSCs) through CCL9 secretion. Blockade of the CCL9/CCR1 axis could limit G-MDSC migration, and depletion of Ly6G-positive cells could sensitize tumors to the combination of TH-302, anti-VEGFR-2, and ICB. Together, these data suggest that pancreatic tumors modulate G-MDSC migration as an adaptive response to vascular normalization and that these immunosuppressive myeloid cells act in a setting of persistent hypoxia to maintain adaptive immune resistance.
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Adenocarcinoma , Células Supresoras de Origen Mieloide , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patología , Adenocarcinoma/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Hipoxia/metabolismoRESUMEN
Lipid metabolism plays a central role in prostate cancer. To date, the major focus has centered on de novo lipogenesis and lipid uptake in prostate cancer, but inhibitors of these processes have not benefited patients. A better understanding of how cancer cells access lipids once they are created or taken up and stored could uncover more effective strategies to perturb lipid metabolism and treat patients. Here, we identified that expression of adipose triglyceride lipase (ATGL), an enzyme that controls lipid droplet homeostasis and a previously suspected tumor suppressor, correlates with worse overall survival in men with advanced, castration-resistant prostate cancer (CRPC). Molecular, genetic, or pharmacologic inhibition of ATGL impaired human and murine prostate cancer growth in vivo and in cell culture or organoids under conditions mimicking the tumor microenvironment. Mass spectrometry imaging demonstrated that ATGL profoundly regulates lipid metabolism in vivo, remodeling membrane composition. ATGL inhibition induced metabolic plasticity, causing a glycolytic shift that could be exploited therapeutically by cotargeting both metabolic pathways. Patient-derived phosphoproteomics identified ATGL serine 404 as a target of CAMKK2-AMPK signaling in CRPC cells. Mutation of serine 404 did not alter the lipolytic activity of ATGL but did decrease CRPC growth, migration, and invasion, indicating that noncanonical ATGL activity also contributes to disease progression. Unbiased immunoprecipitation/mass spectrometry suggested that mutation of serine 404 not only disrupts existing ATGL protein interactions but also leads to new protein-protein interactions. Together, these data nominate ATGL as a therapeutic target for CRPC and provide insights for future drug development and combination therapies. SIGNIFICANCE: ATGL promotes prostate cancer metabolic plasticity and progression through both lipase-dependent and lipase-independent activity, informing strategies to target ATGL and lipid metabolism for cancer treatment.
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Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Ratones , Animales , Lipólisis/genética , Metabolismo de los Lípidos , Lipasa/genética , Lipasa/metabolismo , Serina/metabolismo , Microambiente Tumoral , Quinasa de la Proteína Quinasa Dependiente de Calcio-CalmodulinaRESUMEN
The alpha emitter astatine-211 (211At) is a promising candidate for cancer treatment based on Targeted Alpha (α) Therapy (TAT). A small number of facilities, distributed across the United States, are capable of accelerating α-particle beams to produce 211At. However, challenges remain regarding strategic methods for shipping 211At in a form adaptable to advanced radiochemistry reactions and other uses of the radioisotope. PURPOSE: Our method allows shipment of 211At in various quantities in a form convenient for further radiochemistry. PROCEDURES: For this study, a 3-octanone impregnated Amberchrom CG300M resin bed in a column cartridge was used to separate 211At from the bismuth matrix on site at the production accelerator (Texas A&M) in preparation for shipping. Aliquots of 6 M HNO3 containing up to ≈2.22 GBq of 211At from the dissolved target were successfully loaded and retained on columns. Exempt packages (<370 MBq) were shipped to a destination radiochemistry facility, University of Texas MD Anderson Cancer Center, in the form of a convenient air-dried column. Type A packages have been shipped overnight to University of Alabama at Birmingham. MAIN FINDINGS: Air-dried column hold times of various lengths did not inhibit simple and efficient recovery of 211At. Solution eluted from the column was sufficiently high in specific activity to successfully radiolabel a model compound, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1), with 211At. The method to prepare and ship 211At described in this manuscript has also been used to ship larger quantities of 211At a greater distance to University of Alabama at Birmingham. PRINCIPAL CONCLUSIONS: The successful proof of this method paves the way for the distribution of 211At from Texas A&M University to research institutions and clinical oncology centers in Texas and elsewhere. Use of this simple method at other facilities has the potential increase the overall availability of 211At for preclinical and clinical studies.
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Astato , Humanos , Astato/uso terapéutico , Astato/química , Radioisótopos/química , Partículas alfa/uso terapéutico , Radioquímica/métodosRESUMEN
The worldwide incidence of hepatocellular carcinoma (HCC) continues to rise, in part due to poor diet, limited exercise, and alcohol abuse. Numerous studies have suggested that the loss or mutation of PTEN plays a critical role in HCC tumorigenesis through the activation of the PI3K/Akt signaling axis. The homozygous knockout of PTEN in the livers of mice results in the accumulation of fat (steatosis), inflammation, fibrosis, and eventually progression to HCC. This phenotype bears a striking similarity to non-alcoholic steatohepatitis (NASH) which is thought to occupy an intermediate stage between non-alcoholic fatty liver disease (NAFLD), fibrosis, and HCC. The molecular and physiological phenotypes that manifest during the transition to HCC suggest that molecular imaging could provide a non-invasive screening platform to identify the hallmarks of HCC initiation prior to the presentation of clinical disease. We have carried out longitudinal imaging studies on the liver-specific PTEN knockout mouse model using CT, MRI, and multi-tracer PET to interrogate liver size, steatosis, inflammation, and apoptosis. In male PTEN knockout mice, significant steatosis was observed as early as 3 months using both magnetic resonance spectroscopy (MRS) and computed tomography (CT). Enhanced uptake of the apoptosis tracer 18F-TBD was also observed in the livers of male PTEN homozygous knockout mice between 3 and 4 months of age relative to heterozygous knockout controls. Liver uptake of the inflammation tracer [18F]4FN remained relatively low and constant over 7 months in male PTEN homozygous knockout mice, suggesting the suppression of high-energy ROS/RNS with PTEN deletion relative to heterozygous males where the [18F]4FN liver uptake was elevated at early and late time points. All male PTEN homozygous mice developed HCC lesions by month 10. In contrast to the male cohort, only 20% (2 out of 10) of female PTEN homozygous knockout mice developed HCC lesions by month 10. Steatosis was significantly less pronounced in the female PTEN homozygous knockout mice relative to males and could not accurately predict the eventual occurrence of HCC. As with the males, the [18F]4FN uptake in female PTEN homozygous knockout mice was low and constant throughout the time course. The liver uptake of 18F-TBD at 3 and 4.5 months was higher in the two female PTEN knockout mice that would eventually develop HCC and was the most predictive imaging biomarker for HCC in the female cohort. These studies demonstrate the diagnostic and prognostic role of multi-modal imaging in HCC mouse models and provide compelling evidence that disease progression in the PTEN knockout model is highly dependent on gender.
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Programmed death ligand 1 (PD-L1) is a type 1 transmembrane immunosuppressive protein that is expressed on a wide range of cell types, including cancer cells. Anti-PD-L1 antibodies have revolutionized cancer therapy and have led to improved outcomes for subsets of cancer patients, including triple-negative breast cancer (TNBC) patients. As a result, PET imaging of PD-L1 protein expression in cancer patients has been explored for noninvasive detection of PD-L1 expressing tumors as well as monitoring response to anti-PD-L1 immune checkpoint therapy. Previous studies have indicated that the in vivo stability and in vivo target detection of antibody-based radio-conjugates can be dramatically affected by the chelator used. These reports demonstrated that the chelator HOPO diminishes 89Zr de-chelation compared to DFO. Herein, we report an improved HOPO synthesis and evaluated a series of novel analogues for thermal stability, serum stability, PD-L1-specific binding using the BT-549 TNBC cell line, PET imaging in vivo, as well as biodistribution of 89Zr-labeled anti-PD-L1 antibodies in BT-549 xenograft murine models. A new chelator, C5HOPO, demonstrated high stability in vitro and afforded effective PD-L1 targeting in vivovia immuno-PET. These results demonstrated that an improved HOPO chelator is an effective chelating agent that can be utilized to image therapeutically relevant targets in vivo.
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One of the major obstacles to treating pancreatic ductal adenocarcinoma (PDAC) is its immunoresistant microenvironment. The functional importance and molecular mechanisms of Schwann cells in PDAC remains largely elusive. We characterized the gene signature of tumor-associated nonmyelinating Schwann cells (TASc) in PDAC and indicated that the abundance of TASc was correlated with immune suppressive tumor microenvironment and the unfavorable outcome of patients with PDAC. Depletion of pancreatic-specific TASc promoted the tumorigenesis of PDAC tumors. TASc-expressed long noncoding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) was triggered by the tumor cell-produced interleukin-6. Mechanistically, PVT1 modulated RAF proto-oncogene serine/threonine protein kinase-mediated phosphorylation of tryptophan 2,3-dioxygenase in TASc, facilitating its enzymatic activities in catalysis of tryptophan to kynurenine. Depletion of TASc-expressed PVT1 suppressed PDAC tumor growth. Furthermore, depletion of TASc using a small-molecule inhibitor effectively sensitized PDAC to immunotherapy, signifying the important roles of TASc in PDAC immune resistance.
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Carcinoma Ductal Pancreático , Quinurenina , Neoplasias Pancreáticas , ARN Largo no Codificante , Humanos , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Quinurenina/genética , Quinurenina/metabolismo , Neoplasias Pancreáticas/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Microambiente Tumoral/genética , Neoplasias PancreáticasRESUMEN
BACKGROUND: The presence of a highly immunosuppressive tumor microenvironment has limited the success of immune checkpoint therapy (ICT). Immune suppressing myeloid cells with increased production of reactive oxygen species are critical drivers of this immunosuppressive tumor microenvironment. Strategies to limit these immune suppressing myeloid cells are needed to enhance response to ICT. METHODS: To evaluate the contribution of myeloperoxidase (MPO), a myeloid lineage-restricted enzyme and a major source of reactive oxygen species, to mediating ICT response, we compared treatment outcome and immune composition in wild-type, MPO-deficient (MPO -/- ), and MPO inhibitor-treated wild-type mice using established primary melanoma models. RESULTS: Tumor growth and survival studies demonstrated that either host deficiency (MPO -/- ) or pharmacological inhibition of MPO enhanced ICT response in two preclinical models of established primary melanoma in aged animals. The tumor microenvironment and systemic immune landscape underwent striking changes in infiltration of myeloid cells, T cells, B cells, and dendritic cells in MPO -/- mice; furthermore, a significant increase in myeloid cells was observed in ICT non-responders. The contribution of CD4+ T cells and NK cells during ICT response also changed in MPO -/- mice. Interestingly, MPO enzymatic activity, but not protein, was increased in CD11b+Ly6G+ myeloid cells isolated from marrow, spleen, and peritoneal cavities of mice bearing untreated melanoma, indicating systemic activation of innate immunity. Notably, repurposing MPO-specific inhibitors (verdiperstat, AZD5904) in combination with ICT pointedly enhanced response rates above ICT alone. Indeed, long-term survival was 100% in the YUMM3.3 melanoma model on treatment with verdiperstat plus ICT. CONCLUSION: MPO contributes to ICT resistance in established melanoma. Repurposing MPO-specific inhibitors may provide a promising therapeutic strategy to enhance ICT response.
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Melanoma , Peroxidasa , Animales , Ratones , Especies Reactivas de Oxígeno , Linfocitos B , Inmunidad Innata , Inmunosupresores , Microambiente TumoralRESUMEN
Primary and adaptive resistance to immune checkpoint therapies (ICT) represent a considerable obstacle to achieving enhanced overall survival. Innate immune activators have been actively pursued for their antitumor potential. Herein we report that a syngeneic 4T1 mammary carcinoma murine model for established highly-refractory triple negative breast cancer showed enhanced survival when treated intra-tumorally with either the TLR5 agonist flagellin or CBLB502, a flagellin derivative, in combination with antibodies targeting CTLA-4 and PD-1. Long-term survivor mice showed immunologic memory upon tumor re-challenge and a distinctive immune activating cytokine profile that engaged both innate and adaptive immunity. Low serum levels of G-CSF and CXCL5 (as well as high IL-15) were candidate predictive biomarkers correlating with enhanced survival. CBLB502-induced enhancement of ICT was also observed in poorly immunogenic B16-F10 melanoma tumors. Combination immune checkpoint therapy plus TLR5 agonists may offer a new therapeutic strategy to treat ICT-refractory solid tumors.
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Melanoma Experimental , Receptor Toll-Like 5 , Animales , Ratones , Inmunidad Adaptativa , Citocinas , Flagelina/farmacología , Melanoma Experimental/tratamiento farmacológico , Receptor Toll-Like 5/agonistasRESUMEN
KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma.
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N-Metilaspartato , Células Ganglionares de la Retina , Animales , Apoptosis , Caspasas/metabolismo , Fluoresceínas , Colorantes Fluorescentes , N-Metilaspartato/farmacología , Péptidos , Ratas , Células Ganglionares de la Retina/metabolismo , Ácidos SulfónicosRESUMEN
Mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2) is part of the malate-aspartate shuttle, a mechanism by which cells transfer reducing equivalents from the cytosol to the mitochondria. GOT2 is a key component of mutant KRAS (KRAS*)-mediated rewiring of glutamine metabolism in pancreatic ductal adenocarcinoma (PDA). Here, we demonstrate that the loss of GOT2 disturbs redox homeostasis and halts proliferation of PDA cells in vitro. GOT2 knockdown (KD) in PDA cell lines in vitro induced NADH accumulation, decreased Asp and α-ketoglutarate (αKG) production, stalled glycolysis, disrupted the TCA cycle, and impaired proliferation. Oxidizing NADH through chemical or genetic means resolved the redox imbalance induced by GOT2 KD, permitting sustained proliferation. Despite a strong in vitro inhibitory phenotype, loss of GOT2 had no effect on tumor growth in xenograft PDA or autochthonous mouse models. We show that cancer-associated fibroblasts (CAFs), a major component of the pancreatic tumor microenvironment (TME), release the redox active metabolite pyruvate, and culturing GOT2 KD cells in CAF conditioned media (CM) rescued proliferation in vitro. Furthermore, blocking pyruvate import or pyruvate-to-lactate reduction prevented rescue of GOT2 KD in vitro by exogenous pyruvate or CAF CM. However, these interventions failed to sensitize xenografts to GOT2 KD in vivo, demonstrating the remarkable plasticity and differential metabolism deployed by PDA cells in vitro and in vivo. This emphasizes how the environmental context of distinct pre-clinical models impacts both cell-intrinsic metabolic rewiring and metabolic crosstalk with the TME.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Aspartato Aminotransferasa Mitocondrial/genética , Aspartato Aminotransferasa Mitocondrial/metabolismo , Carcinoma Ductal Pancreático/patología , Proteínas de Unión a Ácidos Grasos , Humanos , Ratones , NAD/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ácido Pirúvico/metabolismo , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
PURPOSE: Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. PROCEDURES: We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [18F]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. RESULTS: In UMRC3 cells, [18F]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60-90% in the presence of excess glutamine in the media and by 20-50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [18F]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [18F]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [18F]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [18F]FGln retention did not change significantly upon treatment with CB-839. CONCLUSIONS: We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [18F]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [18F]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake.
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Carcinoma de Células Renales , Neoplasias Renales , Neoplasias Pulmonares , Animales , Ratones , Humanos , Carcinoma de Células Renales/diagnóstico por imagen , Glutamina/metabolismo , ARN Interferente Pequeño , Células HEK293 , Tomografía de Emisión de Positrones/métodos , Neoplasias Pulmonares/diagnóstico por imagen , Imagen por Resonancia Magnética , Neoplasias Renales/diagnóstico por imagenRESUMEN
High-redox-potential reactive oxygen species and reactive nitrogen species (ROS/RNS), generated by NADPH oxidase-2 (NOX2), myeloperoxidase (MPO) and related enzymes, are key effector molecules of innate immunity. High-redox-potential radicals are difficult to distinguish by imaging from less potent ROS/RNS functioning as background biological signaling molecules. Here we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a redox-tuned radiopharmaceutical that selectively binds proteins and cells when oxidized by products of human MPO plus H2O2, but not H2O2 alone, and can be detected using positron emission tomography (PET). Activating HL-60 neutrophil-like human cells with phorbol ester (PMA) caused [18F]4FN retention five-fold over unstimulated cells. An MPO-specific inhibitor (4-ABAH) blocked cellular retention by more than 95%. [18F]4FN PET/CT imaging discriminated inflammatory foci in vivo in three murine models of activated innate immunity: endotoxin-induced toxic shock, PMA-induced contact dermatitis and lipopolysaccharide-induced ankle arthritis. 4-ABAH and Cybb-/- (Nox2-/-) gene deletion strongly abrogated [18F]4FN retention in vivo. Thus, [18F]4FN shows promise as a robust reporter of innate immunity activation by PET/CT.
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NADPH Oxidasas , Tomografía Computarizada por Tomografía de Emisión de Positrones , Animales , Humanos , Peróxido de Hidrógeno , Inmunidad Innata , Ratones , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
PURPOSE: Macropinocytosis serves as a highly conserved endocytotic process that has recently been shown as a critical mechanism by which RAS-transformed cells transport extracellular protein into intracellular amino acid pathways to support their unique metabolic needs. We developed NIR fluorescently labeled molecular imaging probes to monitor macropinocytosis-mediated uptake of albumin in a K-RAS-dependent manner. PROCEDURES: Using western blot analysis, immunofluorescence, and flow cytometry, albumin retention was characterized in vitro across several RAS-activated lung and pancreatic cancer cell lines. AF790-albumin was synthesized and administered to mice bearing K-RAS mutant xenograft tumors of H460 (K-RAS p.Q61H) and H358 (K-RAS p.G12C) non-small cell lung cancers on each flank. Mice were treated daily with 2 mg/kg of ARS-1620, a targeted RAS p.G12C inhibitor, for 2 days and imaged following each treatment. Subsequently, the mice were then treated daily with 10 mg/kg of amiloride, a general inhibitor of macropinocytosis, for 2 days and imaged. Intratumoral distribution of AF790-albumin was assessed in vivo using near-infrared (NIR) fluorescence imaging. RESULTS: Albumin retention was observed as a function of K-RAS activity and macropinocytosis across several lung and pancreatic cancer cell lines. We documented that ARS-1620-induced inhibition of K-RAS activity or amiloride-mediated inhibition of macropinocytosis significantly reduced albumin uptake. Tumor retention in vivo of AF790-albumin was both RAS inhibition-dependent as well as abrogated by inhibition of macropinocytosis. CONCLUSIONS: These data provide a novel approach using NIR-labeled human serum albumin to identify and monitor RAS-driven tumors as well as evaluate the on-target efficacy in vivo of inhibitors, such as ARS-1620.
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Antineoplásicos , Neoplasias Pulmonares , Neoplasias Pancreáticas , Albúminas/metabolismo , Albúminas/farmacología , Amilorida , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Dextranos , Humanos , Ratones , Mutación/genética , Imagen Óptica , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Piperazinas , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinazolinas , Neoplasias PancreáticasRESUMEN
Rapid diagnosis and therapeutic monitoring of aggressive diseases such as glioblastoma can improve patient survival by providing physicians the time to optimally deliver treatment. This research tested whether metabolic imaging with hyperpolarized MRI could detect changes in tumor progression faster than conventional anatomic MRI in patient-derived glioblastoma murine models. To capture the dynamic nature of cancer metabolism, hyperpolarized MRI, NMR spectroscopy, and immunohistochemistry were performed at several time-points during tumor development, regression, and recurrence. Hyperpolarized MRI detected significant changes of metabolism throughout tumor progression whereas conventional MRI was less sensitive. This was accompanied by aberrations in amino acid and phospholipid lipid metabolism and MCT1 expression. Hyperpolarized MRI can help address clinical challenges such as identifying malignant disease prior to aggressive growth, differentiating pseudoprogression from true progression, and predicting relapse. The individual evolution of these metabolic assays as well as their correlations with one another provides context for further academic research.
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Neoplasias Encefálicas/diagnóstico por imagen , Glioblastoma/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones DesnudosRESUMEN
In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B-two essential components of the autophagosome maturation machinery-with mid-nanomolar affinities and disrupted protein-protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.
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Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-13C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1-dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.
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L-Lactato Deshidrogenasa/antagonistas & inhibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ácido Pirúvico/farmacología , Simportadores/metabolismo , Ácidos/metabolismo , Tampones (Química) , Isótopos de Carbono , Extractos Celulares , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Espacio Extracelular/química , Glucólisis/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Cinética , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/biosíntesis , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Notch1 plays a key role in epithelial-mesenchymal transition (EMT) and in the maintenance of cancer stem cells. In the present study we tested whether high levels of activated Notch1 in oncogene-driven NSCLC can induce a reversible shift of driver dependence from EGFR to Notch1, and thus causing resistance to EGFR inhibitors. Adherent cells (parental) and tumor spheres (TS) from NSCLC H1975 cells and patient-derived CD133-positive cells were tested for EGFR and Notch1 signaling cascade. The Notch1-dependent modulation of EGFR, NCID, Hes1, p53, and Sp1 were then analyzed in parental cells by binding assays with a Notch1 agonist, DLL4. TS were more resistant than parental cells to EGFR inhibitors. A strong upregulation of Notch1 and a concomitant downregulation of EGFR were observed in TS compared to parental cells. Parental cell exposure to DLL4 showed a dose-dependent decrease of EGFR and a simultaneous increase of NCID, Hes1, p53, and Sp1, along with the dislocation of Sp1 from the EGFR promoter. Furthermore, an enhanced interaction between p53 and Sp1 was observed in TS. In NSCLC cells, high levels of active Notch1 can promote a reversible shift of driver dependence from EGFR to Notch1, leading to resistance to EGFR inhibitors.