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Triple negative breast cancer (TNBC) is a highly aggressive form of cancer. Detecting TNBC early is crucial for improving disease prognosis and optimizing treatment. Unfortunately, conventional imaging techniques fall short in providing a comprehensive differentiation of TNBC subtypes due to their limited sensitivity and inability to capture subcellular details. In this study, we present a multimodal imaging platform that integrates heavy water (D2O)-probed stimulated Raman scattering (DO-SRS), two-photon fluorescence (TPF), and second harmonic generation (SHG) imaging. This platform allows us to directly visualize and quantify the metabolic activities of TNBC subtypes at a subcellular level. By utilizing DO-SRS imaging, we were able to identify distinct levels of de novo lipogenesis, protein synthesis, cytochrome c metabolic heterogeneity, and lipid unsaturation rates in various TNBC subtype tissues. Simultaneously, TPF imaging provided spatial distribution mapping of NAD[P]H and flavin signals in TNBC tissues, revealing a high redox ratio and significant lipid turnover rate in TNBC BL2 (HCC1806) samples. Furthermore, SHG imaging enabled us to observe diverse orientations of collagen fibers in TNBC tissues, with higher anisotropy at the tissue boundary compared to the center. Our multimodal imaging platform offers a highly sensitive and subcellular approach to characterizing not only TNBC, but also other tissue subtypes and cancers.
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BACKGROUND: Insulin feedback is a critical mechanism responsible for the poor clinical efficacy of phosphatidylinositol 3-kinase (PI3K) inhibition in cancer, and hyperglycemia is an independent factor associated with poor prognosis in glioblastoma (GBM). We investigated combination anti-hyperglycemic therapy in a mouse model of GBM and evaluated the association of glycemic control in clinical trial data from patients with GBM. METHODS: The effect of the anti-hyperglycemic regimens, metformin and the ketogenic diet, was evaluated in combination with PI3K inhibition in patient-derived GBM cells and in an orthotopic GBM mouse model. Insulin feedback and the immune microenvironment were retrospectively evaluated in blood and tumor tissue from a Phase 2 clinical trial of buparlisib in patients with recurrent GBM. RESULTS: We found that PI3K inhibition induces hyperglycemia and hyperinsulinemia in mice and that combining metformin with PI3K inhibition improves the treatment efficacy in an orthotopic GBM xenograft model. Through examination of clinical trial data, we found that hyperglycemia was an independent factor associated with poor progression-free survival in patients with GBM. We also found that PI3K inhibition increased insulin receptor activation and T-cell and microglia abundance in tumor tissue from these patients. CONCLUSION: Reducing insulin feedback improves the efficacy of PI3K inhibition in GBM in mice, and hyperglycemia worsens progression-free survival in patients with GBM treated with PI3K inhibition. These findings indicate that hyperglycemia is a critical resistance mechanism associated with PI3K inhibition in GBM and that anti-hyperglycemic therapy may enhance PI3K inhibitor efficacy in GBM patients.
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Neoplasias Encefálicas , Glioblastoma , Hiperglucemia , Metformina , Humanos , Animales , Ratones , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Fosfatidilinositol 3-Quinasa/farmacología , Fosfatidilinositol 3-Quinasa/uso terapéutico , Fosfatidilinositol 3-Quinasas , Insulina/farmacología , Insulina/uso terapéutico , Retroalimentación , Estudios Retrospectivos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Proliferación Celular , Hiperglucemia/tratamiento farmacológico , Metformina/farmacología , Metformina/uso terapéutico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Línea Celular Tumoral , Microambiente TumoralRESUMEN
Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells1. By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis1. Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8+ T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (Treg) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of Treg cells is dependent on Treg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with Treg cell function in the presence of glucose.
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Antígeno CTLA-4/antagonistas & inhibidores , Glucólisis , Neoplasias/inmunología , Neoplasias/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Melanoma/genética , Melanoma/inmunología , Melanoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
To enhance human prostate-specific membrane antigen (hPSMA)-specific chimeric antigen receptor (CAR) T cell therapy in a hPSMA+ MyC-CaP tumor model, we studied and imaged the effect of lactate dehydrogenase A (LDH-A) depletion on the tumor microenvironment (TME) and tumor progression. Effective LDH-A short hairpin RNA (shRNA) knockdown (KD) was achieved in MyC-CaP:hPSMA+ Renilla luciferase (RLuc)-internal ribosome entry site (IRES)-GFP tumor cells, and changes in tumor cell metabolism and in the TME were monitored. LDH-A downregulation significantly inhibited cell proliferation and subcutaneous tumor growth compared to control cells and tumors. However, total tumor lactate concentration did not differ significantly between LDH-A knockdown and control tumors, reflecting the lower vascularity, blood flow, and clearance of lactate from LDH-A knockdown tumors. Comparing treatment responses of MyC-CaP tumors with LDH-A depletion and/or anti-hPSMA CAR T cells showed that the dominant effect on tumor growth was LDH-A depletion. With anti-hPSMA CAR T cell treatment, tumor growth was significantly slower when combined with tumor LDH-A depletion and compared to control tumor growth (p < 0.0001). The lack of a complete tumor response in our animal model can be explained in part by (1) the lower activity of human CAR T cells against hPSMA-expressing murine tumors in a murine host, and (2) a loss of hPSMA antigen from the tumor cell surface in progressive generations of tumor cells.
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Triple-negative breast cancer (TNBC) is an aggressive subset of breast cancer that is more common in African-American and Hispanic women. Early detection followed by intensive treatment is critical to improving poor survival rates. The current standard to diagnose TNBC from histopathology of biopsy samples is invasive and time-consuming. Imaging methods such as mammography and magnetic resonance (MR) imaging, while covering the entire breast, lack the spatial resolution and specificity to capture the molecular features that identify TNBC. Two nonlinear optical modalities of second harmonic generation (SHG) imaging of collagen, and resonance Raman spectroscopy (RRS) potentially offer novel rapid, label-free detection of molecular and morphological features that characterize cancerous breast tissue at subcellular resolution. In this study, we first applied MR methods to measure the whole-tumor characteristics of metastatic TNBC (4T1) and nonmetastatic estrogen receptor positive breast cancer (67NR) models, including tumor lactate concentration and vascularity. Subsequently, we employed for the first time in vivo SHG imaging of collagen and ex vivo RRS of biomolecules to detect different microenvironmental features of these two tumor models. We achieved high sensitivity and accuracy for discrimination between these two cancer types by quantitative morphometric analysis and nonnegative matrix factorization along with support vector machine. Our study proposes a new method to combine SHG and RRS together as a promising novel photonic and optical method for early detection of TNBC.
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Neoplasias de la Mama , Microscopía de Generación del Segundo Armónico , Neoplasias de la Mama Triple Negativas , Mama , Neoplasias de la Mama/diagnóstico por imagen , Femenino , Humanos , Mamografía , Espectrometría Raman , Neoplasias de la Mama Triple Negativas/diagnóstico por imagenRESUMEN
Iron deposits are a phenotypic trait of tumor-associated macrophages (TAMs). Histological iron imaging and contrast-agent free magnetic resonance imaging (MRI) can detect these deposits, but their presence in human cancer, and correlation with immunotherapeutic response is largely untested. Here, primarily using these iron imaging approaches, we evaluated the spatial distribution of polarized macrophage populations containing high endogenous levels of iron in preclinical murine models and human breast cancer, and used them as metabolic biomarkers to correlate TAM infiltration with response to immunotherapy in preclinical trials. Macrophage-targeted inhibition of the colony stimulating factor 1 receptor (CSF1R) by immunotherapy was confirmed to inhibit macrophage accumulation and slow mammary tumor growth in mouse models while also reducing hemosiderin iron-laden TAM accumulation as measured by both iron histology and in vivo iron MRI (FeMRI). Spatial profiling of TAM iron deposit infiltration defined regions of maximal accumulation and response to the CSF1R inhibitor, and revealed differences between microenvironments of human cancer according to levels of polarized macrophage iron accumulation in stromal margins. We therefore demonstrate that iron deposition serves as an endogenous metabolic imaging biomarker of TAM infiltration in breast cancer that has high translational potential for evaluation of immunotherapeutic response.
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Antineoplásicos/uso terapéutico , Biomarcadores Farmacológicos/metabolismo , Neoplasias de la Mama/inmunología , Inmunoterapia/métodos , Hierro/metabolismo , Macrófagos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/terapia , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Diagnóstico por Imagen , Femenino , Hemosiderina/metabolismo , Humanos , Espacio Intracelular , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Microambiente TumoralRESUMEN
Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.
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Recombinación Homóloga , Neoplasias , Daño por Reperfusión , Transducción de Señal , Animales , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Humanos , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/radioterapia , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Magnetic resonance imaging applications utilizing nanoparticle agents for polarized macrophage detection are conventionally analyzed according to iron-dependent parameters averaged over large regions of interest (ROI). However, contributions from macrophage iron deposits are usually obscured in these analyses due to their lower spatial frequency and smaller population size compared with the bulk of the tumor tissue. We hypothesized that, by addressing MRI and histological pixel contrast heterogeneity using computer vision image analysis approaches rather than statistical ROI distribution averages, we could enhance our ability to characterize deposits of polarized tumor-associated macrophages (TAMs). We tested this approach using in vivo iron MRI (FeMRI) and histological detection of macrophage iron in control and ultrasmall superparamagnetic iron oxide (USPIO) enhanced mouse models of breast cancer. Automated spatial profiling of the number and size of iron-containing macrophage deposits according to localized high-iron FeMRI or Prussian blue pixel clustering performed better than using distribution averages to evaluate the effects of contrast agent injections. This analysis was extended to characterize subpixel contributions to the localized FeMRI measurements with histology that confirmed the association of endogenous and nanoparticle-enhanced iron deposits with macrophages in vascular regions and further allowed us to define the polarization status of the macrophage iron deposits detected by MRI. These imaging studies demonstrate that characterization of TAMs in breast cancer models can be improved by focusing on spatial distributions of iron deposits rather than ROI averages and indicate that nanoparticle uptake is dependent on the polarization status of the macrophage populations. These findings have broad implications for nanoparticle-enhanced biomedical imaging especially in cancer.
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Neoplasias de la Mama/diagnóstico por imagen , Medios de Contraste/química , Hierro/análisis , Macrófagos/metabolismo , Imagen por Resonancia Magnética/métodos , Nanopartículas/uso terapéutico , Animales , Neoplasias de la Mama/patología , Humanos , Procesamiento de Imagen Asistido por Computador , Macrófagos/patología , Ratones , Análisis EspacialRESUMEN
Previous studies show that LDH-A knockdown reduces orthotopic 4T1 breast tumor lactate and delays tumor growth and the development of metastases in nude mice. Here, we report significant changes in the tumor microenvironment (TME) and a more robust anti-tumor response in immune competent BALB/c mice. 4T1 murine breast cancer cells were transfected with shRNA plasmids directed against LDH-A (KD) or a scrambled control plasmid (NC). Cells were also transduced with dual luciferase-based reporter systems to monitor HIF-1 activity and the development of metastases by bioluminescence imaging, using HRE-sensitive and constitutive promoters, respectively. The growth and metastatic profile of orthotopic 4T1 tumors developed from these cell lines were compared and a primary tumor resection model was studied to simulate the clinical management of breast cancer. Primary tumor growth, metastasis formation and TME phenotype were significantly different in LDH-A KD tumors compared with controls. In LDH-A KD cells, HIF-1 activity, hexokinase 1 and 2 expression and VEGF secretion were reduced. Differences in the TME included lower HIF-1α expression that correlated with lower vascularity and pimonidazole staining, higher infiltration of CD3+ and CD4+ T cells and less infiltration of TAMs. These changes resulted in a greater delay in metastases formation and 40% long-term survivors (>20 weeks) in the LDH-A KD cohort following surgical resection of the primary tumor. We show for the first time that LDH-depletion inhibits the formation of metastases and prolongs survival of mice through changes in tumor microenvironment that modulate the immune response. We attribute these effects to diminished HIF-1 activity, vascularization, necrosis formation and immune suppression in immune competent animals. Gene-expression analyses from four human breast cancer datasets are consistent with these results, and further demonstrate the link between glycolysis and immune suppression in breast cancer.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/metabolismo , Microambiente Tumoral/inmunología , Microambiente Tumoral/fisiología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/genética , Lactato Deshidrogenasa 5 , Ácido Láctico/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia/inmunología , Metástasis de la Neoplasia/patología , Neovascularización Patológica , Transducción de SeñalRESUMEN
PURPOSE: To automate dynamic contrast-enhanced MRI (DCE-MRI) data analysis by unsupervised pattern recognition (PR) to enable spatial mapping of intratumoral vascular heterogeneity. METHODS: Three steps were automated. First, the arrival time of the contrast agent at the tumor was determined, including a calculation of the precontrast signal. Second, four criteria-based algorithms for the slice-specific selection of number of patterns (NP) were validated using 109 tumor slices from subcutaneous flank tumors of five different tumor models. The criteria were: half area under the curve, standard deviation thresholding, percent signal enhancement, and signal-to-noise ratio (SNR). The performance of these criteria was assessed by comparing the calculated NP with the visually determined NP. Third, spatial assignment of single patterns and/or pattern mixtures was obtained by way of constrained nonnegative matrix factorization. RESULTS: The determination of the contrast agent arrival time at the tumor slice was successfully automated. For the determination of NP, the SNR-based approach outperformed other selection criteria by agreeing >97% with visual assessment. The spatial localization of single patterns and pattern mixtures, the latter inferring tumor vascular heterogeneity at subpixel spatial resolution, was established successfully by automated assignment from DCE-MRI signal-versus-time curves. CONCLUSION: The PR-based DCE-MRI analysis was successfully automated to spatially map intratumoral vascular heterogeneity. Magn Reson Med 79:1736-1744, 2018. © 2017 International Society for Magnetic Resonance in Medicine.
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Interpretación de Imagen Asistida por Computador/métodos , Imagen por Resonancia Magnética/métodos , Neoplasias/diagnóstico por imagen , Neovascularización Patológica/diagnóstico por imagen , Reconocimiento de Normas Patrones Automatizadas/métodos , Algoritmos , Medios de Contraste/química , Medios de Contraste/farmacocinética , Humanos , Neoplasias/irrigación sanguínea , Análisis de Componente PrincipalRESUMEN
Iron-deposition is a metabolic biomarker of macrophages in both normal and pathological situations, but the presence of iron in tumor and metastasis-associated macrophages is not known. Here we mapped and quantified hemosiderin-laden macrophage (HLM) deposits in murine models of metastatic breast cancer using iron and macrophage histology, and in vivo MRI. Iron MRI detected high-iron pixel clusters in mammary tumors, lung metastasis, and brain metastasis as well as liver and spleen tissue known to contain the HLMs. Iron histology showed these regions to contain clustered macrophages identified by their common iron status and tissue-intrinsic association with other phenotypic macrophage markers. The in vivo MRI and ex vivo histological images were further processed to determine the frequencies and sizes of the iron deposits, and measure the number of HLMs in each deposit to estimate the in vivo MRI sensitivity for these cells. Hemosiderin accumulation is a macrophage biomarker and intrinsic contrast source for cellular MRI associated with the innate function of macrophages in iron metabolism systemically, and in metastatic cancer.
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Hemosiderina/metabolismo , Hierro/metabolismo , Macrófagos/metabolismo , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Animales , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/patología , Femenino , Macrófagos/patología , Imagen por Resonancia Magnética , Neoplasias Mamarias Experimentales/patología , Ratones , Metástasis de la NeoplasiaRESUMEN
Immune cells such as macrophages are drivers and biomarkers of most cancers. Scoring macrophage infiltration in tumor tissue provides a prognostic assessment that is correlated with disease outcome and therapeutic response, but generally requires invasive biopsy. Routine detection of hemosiderin iron aggregates in macrophages in other settings histologically and in vivo by MRI suggests that similar assessments in cancer can bridge a gap in our ability to assess tumor macrophage infiltration. Quantitative histological and in vivo MRI assessments of non-heme cellular iron revealed that preclinical prostate tumor models could be differentiated according to hemosiderin iron accumulation-both in tumors and systemically. Monitoring cellular iron levels during "off-label" administration of the FDA-approved iron chelator deferiprone evidenced significant reductions in tumor size without extensive perturbation to these iron deposits. Spatial profiling of the iron-laden infiltrates further demonstrated that higher numbers of infiltrating macrophage iron deposits was associated with lower anti-tumor chelation therapy response. Imaging macrophages according to their innate iron status provides a new phenotypic window into the immune tumor landscape and reveals a prognostic biomarker associated with macrophage infiltration and therapeutic outcome.
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Quelantes del Hierro/farmacología , Hierro/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Biomarcadores , Modelos Animales de Enfermedad , Humanos , Quelantes del Hierro/uso terapéutico , Macrófagos/patología , Imagen por Resonancia Magnética , Masculino , Ratones , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/tratamiento farmacológico , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PBRM1 is the second most commonly mutated gene after VHL in clear cell renal cell carcinoma (ccRCC). However, the biological consequences of PBRM1 mutations for kidney tumorigenesis are unknown. Here, we find that kidney-specific deletion of Vhl and Pbrm1, but not either gene alone, results in bilateral, multifocal, transplantable clear cell kidney cancers. PBRM1 loss amplified the transcriptional outputs of HIF1 and STAT3 incurred by Vhl deficiency. Analysis of mouse and human ccRCC revealed convergence on mTOR activation, representing the third driver event after genetic inactivation of VHL and PBRM1. Our study reports a physiological preclinical ccRCC mouse model that recapitulates somatic mutations in human ccRCC and provides mechanistic and therapeutic insights into PBRM1 mutated subtypes of human ccRCC.
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Carcinoma de Células Renales/metabolismo , Proteínas HMGB/metabolismo , Neoplasias Renales/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Proteínas de Unión al ADN , Regulación hacia Abajo/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas HMGB/deficiencia , Humanos , Hidronefrosis/genética , Hidronefrosis/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Integrasas/metabolismo , Riñón/metabolismo , Riñón/patología , Neoplasias Renales/genética , Neoplasias Renales/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Fosforilación Oxidativa , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
Cancer growth and proliferation rely on intracellular iron availability. We studied the effects of Deferiprone (DFP), a chelator of intracellular iron, on three prostate cancer cell lines: murine, metastatic TRAMP-C2; murine, non-metastatic Myc-CaP; and human, non-metastatic 22rv1. The effects of DFP were evaluated at different cellular levels: cell culture proliferation and migration; metabolism of live cells (time-course multi-nuclear magnetic resonance spectroscopy cell perfusion studies, with 1-13 C-glucose, and extracellular flux analysis); and expression (Western blot) and activity of mitochondrial aconitase, an iron-dependent enzyme. The 50% and 90% inhibitory concentrations (IC50 and IC90 , respectively) of DFP for the three cell lines after 48 h of incubation were within the ranges 51-67 µM and 81-186 µM, respectively. Exposure to 100 µM DFP led to: (i) significant inhibition of cell migration after different exposure times, ranging from 12 h (TRAMP-C2) to 48 h (22rv1), in agreement with the respective cell doubling times; (ii) significantly decreased glucose consumption and glucose-driven tricarboxylic acid cycle activity in metastatic TRAMP-C2 cells, during the first 10 h of exposure, and impaired cellular bioenergetics and membrane phospholipid turnover after 23 h of exposure, consistent with a cytostatic effect of DFP. At this time point, all cell lines studied showed: (iii) significant decreases in mitochondrial functional parameters associated with the oxygen consumption rate, and (iv) significantly lower mitochondrial aconitase expression and activity. Our results indicate the potential of DFP to inhibit prostate cancer proliferation at clinically relevant doses and plasma concentrations.
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Neoplasias de la Próstata/patología , Piridonas/farmacología , Aconitato Hidratasa/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Deferiprona , Humanos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Factores de TiempoRESUMEN
Human breast tumors contain significant amounts of stromal cells. There exists strong evidence that these stromal cells support cancer development and progression by altering various pathways (e.g. downregulation of tumor suppressor genes or autocrine signaling loops). Here, we suggest that stromal carcinoma-associated fibroblasts (CAFs), shown to be generated from bone marrow-derived mesenchymal stem cells, may (i) recycle tumor-derived lactate for their own energetic requirements, thereby sparing glucose for neighboring glycolytic tumor cells, and (ii) subsequently secrete surplus energetically and biosynthetically valuable metabolites of lactate oxidation, such as pyruvate, to support tumor growth. Lactate, taken up by stromal CAFs, is converted to pyruvate, which is then utilized by CAFs for energy needs as well as excreted and shared with tumor cells. We have interrogated lactate oxidation in CAFs to determine what metabolites may be secreted, and how they may affect the metabolism and growth of MDA-MB-231 breast cancer cells. We found that CAFs secrete pyruvate as a metabolite of lactate oxidation. Further, we show that pyruvate is converted to lactate to promote glycolysis in MDA-MB-231 cells and helps to control elevated ROS levels in these tumor cells. Finally, we found that inhibiting or interfering with ROS management, using the naturally occurring flavonoid phloretin (found in apple tree leaves), adds to the cytotoxicity of the conventional chemotherapeutic agent doxorubicin. Our work demonstrates that a lactate-pyruvate, reciprocally-supportive metabolic relationship may be operative within the tumor microenvironment (TME) to support tumor growth, and may be a useful drug target.
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Neoplasias de la Mama/metabolismo , Fibroblastos/metabolismo , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Células del Estroma/metabolismo , Microambiente Tumoral , Comunicación Autocrina , Neoplasias de la Mama/patología , Radioisótopos de Carbono/metabolismo , Comunicación Celular , Células Cultivadas , Femenino , Fibroblastos/patología , Glucólisis , Humanos , Redes y Vías Metabólicas , Células del Estroma/patologíaRESUMEN
Bortezomib, a novel proteasome inhibitor, has been approved for treating multiple myeloma and mantle cell lymphoma and studied pre-clinically and clinically for solid tumors. Preferential cytotoxicity of bortezomib was found toward hypoxic tumor cells and endothelial cells in vitro. The purpose of this study is to investigate the role of a pretreatment hypoxic tumor microenvironment on the effects of bortezomib in vitro and ex vivo, and explore the feasibility of dynamic contrast enhanced magnetic resonance imaging (DCE MRI) to noninvasively evaluate the biological effects of bortezomib. It was shown in vitro by Western blot, flow cytometry, and ELISA that bortezomib accumulated HIF-1α in non-functional forms and blocks its hypoxia response in human colorectal cancer cell lines. Ex vivo experiments were performed with fluorescent immunohistochemical staining techniques using multiple endogenous and exogenous markers to identify hypoxia (pimonidazole, HRE-TKeGFP), blood flow/permeability (Hoechst 33342), micro-vessels (CD31 and SMA), apoptosis (cleaved caspase 3) and hypoxia response (CA9). After bortezomib administration, overall apoptosis index was significantly increased and blood perfusion was dramatically decreased in tumor xenografts. More importantly, apoptosis signals were found preferentially located in moderate and severe pretreatment hypoxic regions in both tumor and endothelial cells. Meanwhile, DCE MRI examinations showed that the tumor blood flow and permeability decreased significantly after bortezomib administration. The present study revealed that bortezomib reduces tumor hypoxia response and blood perfusion, thus, presenting antivascular properties. It will be important to determine the hypoxic/perfusion status pre- and during treatment at further translational studies.
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Antineoplásicos/farmacología , Bortezomib/farmacología , Hipoxia de la Célula/efectos de los fármacos , Neoplasias Experimentales/irrigación sanguínea , Microambiente Tumoral/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Medios de Contraste , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Aumento de la Imagen , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Imagen por Resonancia Magnética/métodos , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer cells adapt their metabolism during tumorigenesis. We studied two isogenic breast cancer cells lines (highly metastatic 4T1; nonmetastatic 67NR) to identify differences in their glucose and glutamine metabolism in response to metabolic and environmental stress. Dynamic magnetic resonance spectroscopy of (13)C-isotopomers showed that 4T1 cells have higher glycolytic and tricarboxylic acid (TCA) cycle flux than 67NR cells and readily switch between glycolysis and oxidative phosphorylation (OXPHOS) in response to different extracellular environments. OXPHOS activity increased with metastatic potential in isogenic cell lines derived from the same primary breast cancer: 4T1 > 4T07 and 168FARN (local micrometastasis only) > 67NR. We observed a restricted TCA cycle flux at the succinate dehydrogenase step in 67NR cells (but not in 4T1 cells), leading to succinate accumulation and hindering OXPHOS. In the four isogenic cell lines, environmental stresses modulated succinate dehydrogenase subunit A expression according to metastatic potential. Moreover, glucose-derived lactate production was more glutamine dependent in cell lines with higher metastatic potential. These studies show clear differences in TCA cycle metabolism between 4T1 and 67NR breast cancer cells. They indicate that metastases-forming 4T1 cells are more adept at adjusting their metabolism in response to environmental stress than isogenic, nonmetastatic 67NR cells. We suggest that the metabolic plasticity and adaptability are more important to the metastatic breast cancer phenotype than rapid cell proliferation alone, which could 1) provide a new biomarker for early detection of this phenotype, possibly at the time of diagnosis, and 2) lead to new treatment strategies of metastatic breast cancer by targeting mitochondrial metabolism.
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Adaptación Fisiológica , Glucosa/metabolismo , Glutamina/metabolismo , Microambiente Tumoral , Animales , Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Glucosa/farmacología , Glutamina/farmacología , Glucólisis/efectos de los fármacos , Concentración de Iones de Hidrógeno , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Fosforilación Oxidativa/efectos de los fármacos , Fosfolípidos/metabolismoRESUMEN
Generally, solid tumors (>400 mm(3)) are inherently acidic, with more aggressive growth producing greater acidity. If the acidity could be targeted as a biomarker, it would provide a means to gauge the pace of tumor growth and degree of invasiveness, as well as providing a basis for predicting responses to pH-dependent chemotherapies. We have developed a (64)Cu pH (low) insertion peptide (pHLIP) for targeting, imaging, and quantifying acidic tumors by PET, and our findings reveal utility in assessing prostate tumors. The new pHLIP version limits indiscriminate healthy tissue binding, and we demonstrate its targeting of extracellular acidification in three different prostate cancer models, each with different vascularization and acid-extruding protein carbonic anhydrase IX (CAIX) expression. We then describe the tumor distribution of this radiotracer ex vivo, in association with blood perfusion and known biomarkers of acidity, such as hypoxia, lactate dehydrogenase A, and CAIX. We find that the probe reveals metabolic variations between and within tumors, and discriminates between necrotic and living tumor areas.
Asunto(s)
Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico por imagen , Radiofármacos/farmacología , Animales , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/metabolismo , Línea Celular Tumoral , Quelantes/farmacología , Radioisótopos de Galio/farmacología , Compuestos Heterocíclicos con 1 Anillo/farmacología , Humanos , Concentración de Iones de Hidrógeno , Hipoxia , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5 , Masculino , Proteínas de la Membrana/farmacología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , FenotipoRESUMEN
This review focuses on the applications of high magnetic field magnetic resonance imaging (MRI) and spectroscopy (MRS) to cancer studies in small animals. High-field MRI can provide information about tumor physiology, the microenvironment, metabolism, vascularity and cellularity. Such studies are invaluable for understanding tumor growth and proliferation, response to treatment and drug development. The MR techniques reviewed here include (1)H, (31)P, chemical exchange saturation transfer imaging and hyperpolarized (13)C MRS as well as diffusion-weighted, blood oxygen level dependent contrast imaging and dynamic contrast-enhanced MRI. These methods have been proven effective in animal studies and are highly relevant to human clinical studies.