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Three murine glioma cell lines (GL261, CT2A, and ALTS1C1) were modified to downregulate the expression of the murine LDH-A gene using shRNA, and compared to shRNA scrambled control (NC) cell lines. Differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles, were observed in the six cell lines, and confirmed successful LDH-A KD. LDH-A KD (knock-down) resulted in metabolic changes in cells with a reduction in glycolysis (GlycoPER) and an increase in basal respiratory rate (mitoOCR). GL261 cells had a more limited ATP production capacity compared to CT2A and ALTS1C1 cells. An analysis of mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines do not have this capacity. These two observations suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment, and that specific nutrient limitations have a variable impact on tumor cell metabolism and proliferation. The phenotypic effects of LDH-A KD were compared to those in control (NC) cells and tumors. LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice, whereas GL261 NC tumors had a prolonged growth delay in C57BL/6 mice. In nude mice, both LDH-A KD and NC GL261 tumors grew rapidly (more rapidly than GL261 NC tumors in C57BL/6 mice), demonstrating the impact of an intact immune system on GL261 tumor growth. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors, despite the small changes to their LDH isoenzyme profiles. These results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed to have the capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion. We observed that the combined impact of LDH-A depletion and the immune system had a significant impact on the growth of subcutaneous-located GL261 tumors.
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The effects of the LDH-A depletion via shRNA knockdown on three murine glioma cell lines and corresponding intracranial (i.c.) tumors were studied and compared to pharmacologic (GNE-R-140) inhibition of the LDH enzyme complex, and to shRNA scrambled control (NC) cell lines. The effects of genetic-shRNA LDH-A knockdown and LDH drug-targeted inhibition (GNE-R-140) on tumor-cell metabolism, tumor growth, and animal survival were similar. LDH-A KD and GNE-R-140 unexpectedly increased the aggressiveness of GL261 intracranial gliomas, but not CT2A and ALTS1C1 i.c. gliomas. Furthermore, the bioenergetic profiles (ECAR and OCR) of GL261 NC and LDH-A KD cells under different nutrient limitations showed that (a) exogenous pyruvate is not a major carbon source for metabolism through the TCA cycle of native GL261 cells; and (b) the unique upregulation of LDH-B that occurs in GL261 LDH-A KD cells results in these cells being better able to: (i) metabolize lactate as a primary carbon source through the TCA cycle, (ii) be a net consumer of lactate, and (iii) showed a significant increase in the proliferation rate following the addition of 10 mM lactate to the glucose-free media (only seen in GL261 KD cells). Our study suggests that inhibition of LDH-A/glycolysis may not be a general strategy to inhibit the i.c. growth of all gliomas, since the level of LDH-A expression and its interplay with LDH-B can lead to complex metabolic interactions between tumor cells and their environment. Metabolic-inhibition treatment strategies need to be carefully assessed, since the inhibition of glycolysis (e.g., inhibition of LDH-A) may lead to the unexpected development and activation of alternative metabolic pathways (e.g., upregulation of lipid metabolism and fatty-acid oxidation pathways), resulting in enhanced tumor-cell survival in a nutrient-limited environment and leading to increased tumor aggressiveness.
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Triple negative breast cancer (TNBC) is an aggressive disease that is difficult to treat and portends a poor prognosis in many patients. Recent efforts to implement immune checkpoint inhibitors into the treatment landscape of TNBC have led to improved outcomes in a subset of patients both in the early stage and metastatic settings. However, a large portion of patients with TNBC remain resistant to immune checkpoint inhibitors and have limited treatment options beyond cytotoxic chemotherapy. The interplay between the anti-tumor immune response and tumor metabolism contributes to immunotherapy response in the preclinical setting, and likely in the clinical setting as well. Specifically, tumor glycolysis and lactate production influence the tumor immune microenvironment through creation of metabolic competition with infiltrating immune cells, which impacts response to immune checkpoint blockade. In this review, we will focus on how glucose metabolism within TNBC tumors influences the response to immune checkpoint blockade and potential ways of harnessing this information to improve clinical outcomes.
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Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter-membrane-anchored Cypridina luciferase (maCLuc)-paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d-luciferin- or coelenterazine-based luciferase reporters. We compare maCLuc with several established luciferase-based reporter systems (firefly, click beetle, Renilla, and Gaussia luciferases), using both in vitro and in vivo models. We demonstrate the different imaging characteristics of these reporter systems, which allow for multiplexed-luciferase imaging of 3 and 4 separate targets concurrently in the same animal within 24 h. The imaging paradigms described here can be directly applied for simultaneous in vivo monitoring of multiple cell populations, the activity of selected signal transduction pathways, or a combination of both constitutive and inducible reporter imaging.
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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
B-cell non-Hodgkin lymphomas (B-NHLs) are highly heterogenous by genetic, phenotypic, and clinical appearance. Next-generation sequencing technologies and multi-dimensional data analyses have further refined the way these diseases can be more precisely classified by specific genomic, epigenomic, and transcriptomic characteristics. The molecular and genetic heterogeneity of B-NHLs may contribute to the poor outcome of some of these diseases, suggesting that more personalized precision-medicine approaches are needed for improved therapeutic efficacy. The germinal center (GC) B-cell like diffuse large B-cell lymphomas (GCB-DLBCLs) and follicular lymphomas (FLs) share specific epigenetic programs. These diseases often remain difficult to treat and surprisingly do not respond advanced immunotherapies, despite arising in secondary lymphoid organs at sites of antigen recognition. Epigenetic dysregulation is a hallmark of GCB-DLBCLs and FLs, with gain-of-function (GOF) mutations in the histone methyltransferase EZH2, loss-of-function (LOF) mutations in histone acetyl transferases CREBBP and EP300, and the histone methyltransferase KMT2D representing the most prevalent genetic lesions driving these diseases. These mutations have the common effect to disrupt the interactions between lymphoma cells and the immune microenvironment, via decreased antigen presentation and responsiveness to IFN-γ and CD40 signaling pathways. This indicates that immune evasion is a key step in GC B-cell lymphomagenesis. EZH2 inhibitors are now approved for the treatment of FL and selective HDAC3 inhibitors counteracting the effects of CREBBP LOF mutations are under development. These treatments can help restore the immune control of GCB lymphomas, and may represent optimal candidate agents for more effective combination with immunotherapies. Here, we review recent progress in understanding the impact of mutant chromatin modifiers on immune evasion in GCB lymphomas. We provide new insights on how the epigenetic program of these diseases may be regulated at the level of metabolism, discussing the role of metabolic intermediates as cofactors of epigenetic enzymes. In addition, lymphoma metabolic adaptation can negatively influence the immune microenvironment, further contributing to the development of immune cold tumors, poorly infiltrated by effector immune cells. Based on these findings, we discuss relevant candidate epigenetic/metabolic/immune targets for rational combination therapies to investigate as more effective precision-medicine approaches for GCB lymphomas.
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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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The first reporter systems were developed in the early 1980s and were based on measuring the activity of an enzyme-as a surrogate measure of promoter-driven transcriptional activity-which is now known as a reporter gene system. The initial objective and application of reporter techniques was to analyze the activity of a specific promoter (namely, the expression of a gene that is under the regulation of the specific promoter that is linked to the reporter gene). This system allows visualization of specific promoter activity with great sensitivity. In general, there are 2 classes of reporter systems: constitutively expressed (always-on) reporter constructs used for cell tracking, and inducible reporter systems sensitive to endogenous signaling molecules and transcription factors that characterize specific tissues, tumors, or signaling pathways.This review traces the development of different reporter systems, using fluorescent and bioluminescent proteins as well as radionuclide-based reporter systems. The development and application of radionuclide-based reporter systems is the focus of this review. The question at the end of the review is whether the "promise" of reporter gene imaging has been realized. What is required for moving forward with radionuclide-based reporter systems, and what is required for successful translation to clinical applications?
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Genes Reporteros , Imagen Molecular/métodos , Animales , Humanos , CintigrafíaRESUMEN
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
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
Chimeric antigen receptor (CAR) T cell therapy in hematologic malignancies has shown remarkable responses, but the same level of success has not been observed in solid tumors. A new prostate cancer model (Myc-CaP:PSMA(+)) and a second-generation anti-hPSMA human CAR T cells expressing a Click Beetle Red luciferase reporter) were used to study hPSMA targeting and assess CAR T cell trafficking and persistence by bioluminescence imaging (BLI). We investigated the antitumor efficacy of human CAR T cells targeting human prostate-specific membrane antigen (hPSMA), in the presence and absence of the target antigen; first alone and then combined with a monoclonal antibody targeting the human programmed death receptor 1 (anti-hPD1 mAb). PDL-1 expression was detected in Myc-CaP murine prostate tumors growing in immune competent FVB/N and immune-deficient SCID mice. Endogenous CD3+ T cells were restricted from the centers of Myc-CaP tumor nodules growing in FVB/N mice. Following anti-programmed cell death protein 1 (PD-1) treatment, the restriction of CD3+ T cells was reversed, and a tumor-treatment response was observed. Adoptive hPSMA-CAR T cell immunotherapy was enhanced when combined with PD-1 blockade, but the treatment response was of comparatively short duration, suggesting other immune modulation mechanisms exist and restrict CAR T cell targeting, function, and persistence in hPSMA expressing Myc-CaP tumors. Interestingly, an "inverse pattern" of CAR T cell BLI intensity was observed in control and test tumors, which suggests CAR T cells undergo changes leading to a loss of signal and/or number following hPSMA-specific activation. The lower BLI signal intensity in the hPSMA test tumors (compared with controls) is due in part to a decrease in T cell mitochondrial function following T cell activation, which may limit the intensity of the ATP-dependent Luciferin-luciferase bioluminescence signal.
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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
INTRODUCTION: Chemokine receptor-4 (CXCR4, fusin, CD184) is expressed on several tissues involved in immune regulation and is upregulated in many diseases including malignant gliomas. A radiolabeled small molecule that readily crosses the blood-brain barrier can aid in identifying CXCR4-expressing gliomas and monitoring CXCR4-targeted therapy. In the current work, we have synthesized and evaluated an [(18)F]-labeled small molecule based on a pyrimidine-pyridine amine for its ability to target CXCR4. EXPERIMENTAL: The nonradioactive standards and the nitro precursor used in this study were prepared using established methods. An HPLC method was developed to separate the nitro-precursor from the nonradioactive standard and radioactive product. The nitro-precursor was radiolabeled with (18)F under inert, anhydrous conditions using the [(18)F]-kryptofix 2.2.2 complex to form the desired N-(4-(((6-[(18)F]fluoropyridin-2-yl)amino)methyl)benzyl)pyrimidin-2-amine ([(18)F]-3). The purified radiolabeled compound was used in serum stability, partition coefficient, cellular uptake, and in vivo cancer targeting studies. RESULTS: [(18)F]-3 was synthesized in 4-10% decay-corrected yield (to start of synthesis). [(18)F]-3 (tR ≈ 27 min) was separated from the precursor (tR ≈ 30 min) using a pentafluorophenyl column with an isocratic solvent system. [(18)F]-3 displayed acceptable serum stability over 2 h. The amount of [(18)F]-3 bound to the plasma proteins was determined to be > 97%. The partition coefficient (LogD7.4) is 1.4 ± 0.5. Competitive in vitro inhibition indicated 3 does not inhibit uptake of (67)Ga-pentixafor. Cell culture media incubation and ex vivo urine analysis indicate rapid metabolism of [(18)F]-3 into hydrophilic metabolites. Thus, in vitro uptake of [(18)F]-3 in CXCR4 overexpressing U87 cells (U87 CXCR4) and U87 WT indicated no specific binding. In vivo studies in mice bearing U87 CXCR4 and U87 WT tumors on the left and right shoulders were carried out using [(18)F]-3 and (68)Ga-pentixafor on consecutive days. The CXCR4 positive tumor was clearly visualized in the PET study using (68)Ga-pentixafor, but not with [(18)F]-3. CONCLUSIONS: We have successfully synthesized both a radiolabeled analog to previously reported CXCR4-targeting molecules and a nitro precursor. Our in vitro and in vivo studies indicate that [(18)F]-3 is rapidly metabolized and, therefore, does not target CXCR4-expressing tumors. Optimization of the structure to improve the in vivo (and in vitro) stability, binding, and solubility could lead to an appropriate CXCR4-targeted radiodiagnositic molecule.
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Aminas/química , Radioisótopos de Flúor , Glioma/diagnóstico por imagen , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Piridinas/química , Pirimidinas/química , Receptores CXCR4/metabolismo , Aminas/síntesis química , Aminas/metabolismo , Animales , Transporte Biológico , Línea Celular Tumoral , Técnicas de Química Sintética , Estabilidad de Medicamentos , Glioma/metabolismo , Glioma/patología , Halogenación , Humanos , Marcaje Isotópico , Masculino , Ratones , RadioquímicaRESUMEN
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
UNLABELLED: Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging. METHODS: Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodium-iodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 10(5) to 3 × 10(6) reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (background) was measured. RESULTS: The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-(18)F-fluorobenzylguanidine ((18)F-MFBG) reporter system could detect less than 1 × 10(5) T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/(124)I-iodide reporter system could detect approximately 1 × 10(6) T cells; (124)I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2'-(18)F-fluoro-5-ethyl-1-ß-d-arabinofuranosyluracil ((18)F-FEAU) and hsvTK/(18)F-FEAU reporter systems detected approximately 3 × 10(5) T cells, respectively. (18)F-FEAU was a more efficient probe (higher uptake, lower background) than (124)I-1-(2-deoxy-2-fluoro-1-d-arabinofuranosyl)-5-iodouracil for both hdCKDM and hsvTK. CONCLUSION: A comparison of different reporter gene-reporter probe systems for imaging of T cell number was performed, and the hNET/(18)F-MFBG PET reporter system was found to be the most sensitive and capable of detecting approximately 35-40 × 10(3) T cells at the site of T cell injection in the animal model.
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Genes Reporteros , Linfocitos T/citología , Animales , Arabinofuranosil Uracilo/análogos & derivados , Arabinofuranosil Uracilo/química , Supervivencia Celular , Radioisótopos de Flúor/química , Fluorobencenos/química , Guanidinas/química , Herpesvirus Humano 1/enzimología , Humanos , Inmunoterapia , Masculino , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Tomografía de Emisión de Positrones , Retroviridae/genética , Retroviridae/metabolismo , Simportadores/química , Timidina Quinasa/metabolismo , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
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.
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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
The biology of cells transplanted with bone grafts is incompletely understood. Focusing on the early angiogenic response postgrafting, we report a mouse femur graft model in which grafts were derived from mice transgenic for a firefly luciferase (FLuc) bioluminescence reporter gene driven by a promoter for the angiogenic signaling molecule vascular endothelial growth factor (VEGF). Upon transplantation into wild-type (wt) mice, in vivo bioluminescence imaging (BLI) permitted longitudinal visualization and measurements of VEGF promoter activity in the transplanted graft cells and demonstrated a lag period of 7 days posttransplantation prior to robust induction of the promoter. To determine cellular mediators of VEGF induction in graft bone, primary graft-derived osteoblastic cells (GDOsts) were characterized. In vitro BLI on GDOsts showed hypoxia-induced VEGF expression and that this induction depended on PI3K signaling and, to a lesser degree, on the MEK pathway. This transcriptional regulation correlated with VEGF protein production and was validated in GDOsts seeded on demineralized bone matrix (DBM), a bone graft substitute material. Together, combined imaging of VEGF expression in living animals and in live cells provided clues about the regulation of VEGF in cells post-bone grafting. These data are particularly significant toward the development of future smart bone graft substitutes.
Asunto(s)
Fémur/citología , Imagen Molecular , Osteoblastos/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Diferenciación Celular , Hipoxia de la Célula , Femenino , Fémur/metabolismo , Fémur/patología , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Ratones , Osteoblastos/metabolismo , Osteoblastos/trasplante , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Cicatrización de HeridasRESUMEN
PURPOSE: Both (131)I- and (123)I-labeled meta-iodobenzylguanidine (MIBG) have been widely used in the clinic for targeted imaging of the norepinephrine transporter (NET). The human NET (hNET) gene has been imaged successfully with (124)I-MIBG positron emission tomography (PET) at time points of >24 h post-injection (p.i.). (18)F-labeled MIBG analogs may be ideal to image hNET expression at time points of <8 h p.i. We developed improved methods for the synthesis of known MIBG analogs, [(18)F]MFBG and [(18)F]PFBG and evaluated them in hNET reporter gene-transduced C6 rat glioma cells and xenografts. METHODS: [(18)F]MFBG and [(18)F]PFBG were synthesized manually using a three-step synthetic scheme. Wild-type and hNET reporter gene-transduced C6 rat glioma cells and xenografts were used to comparatively evaluate the (18)F-labeled analogs with [(123)I]/[(124)I]MIBG. RESULTS: The fluorination efficacy on benzonitrile was predominantly determined by the position of the trimethylammonium group. The para-isomer afforded higher yields (75 ± 7%) than meta-isomer (21 ± 5%). The reaction of [(18)F]fluorobenzylamine with 1H-pyrazole-1-carboximidamide was more efficient than with 2-methyl-2-thiopseudourea. The overall radiochemical yields (decay-corrected) were 11 ± 2% (n = 12) for [(18)F]MFBG and 41 ± 12% (n = 5) for [(18)F]PFBG, respectively. The specific uptakes of [(18)F]MFBG and [(18)F]PFBG were similar in C6-hNET cells, but 4-fold less than that of [(123)I]/[(124)I]MIBG. However, in vivo [(18)F]MFBG accumulation in C6-hNET tumors was 1.6-fold higher than that of [(18)F]PFBG at 1 h p.i., whereas their uptakes were similar at 4 h. Despite [(18)F]MFBG having a 2.8-fold lower affinity to hNET and approximately 4-fold lower cell uptake in vitro compared to [(123)I]/[(124)I]MIBG, PET imaging demonstrated that [(18)F]MFBG was able to visualize C6-hNET xenografts better than [(124)I]MIBG. Biodistribution studies showed [(18)F]MFBG and (123)I-MIBG had a similar tumor accumulation, which was lower than that of no-carrier-added [(124)I]MIBG, but [(18)F]MFBG showed a significantly more rapid body clearance and lower uptake in most non-targeting organs. CONCLUSION: [(18)F]MFBG and [(18)F]PFBG were synthesized in reasonable radiochemical yields under milder conditions. [(18)F]MFBG is a better PET ligand to image hNET expression in vivo at 1-4 h p.i. than both [(18)F]PFBG and [(123)I]/[(124)I]MIBG.
Asunto(s)
Fluorobencenos/síntesis química , Guanidinas/síntesis química , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Radiofármacos/síntesis química , Animales , Línea Celular Tumoral , Fluorobencenos/farmacocinética , Fluorobencenos/farmacología , Glioma/diagnóstico por imagen , Guanidinas/farmacocinética , Guanidinas/farmacología , Humanos , Ratones , Tomografía de Emisión de Positrones , Radiofármacos/farmacocinética , Radiofármacos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To investigate the relationship between lactate dehydrogenase A (LDH-A) expression, lactate concentration, cell metabolism, and metastases in murine 4T1 breast tumors. EXPERIMENTAL DESIGN: Inhibition of LDH-A expression and protein levels were achieved in a metastatic breast cancer cell line (4T1) using short hairpin RNA (shRNA) technology. The relationship between tumor LDH-A protein levels and lactate concentration (measured by magnetic resonance spectroscopic imaging, MRSI) and metastases was assessed. RESULTS: LDH-A knockdown cells (KD9) showed a significant reduction in LDH-A protein and LDH activity, less acid production, decreased transwell migration and invasion, lower proliferation, reduced glucose consumption and glycolysis, and increase in oxygen consumption, reactive oxygen species (ROS), and cellular ATP levels, compared with control (NC) cells cultured in 25 mmol/L glucose. In vivo studies showed lower lactate levels in KD9, KD5, and KD317 tumors than in NC or 4T1 wild-type tumors (P < 0.01), and a linear relationship between tumor LDH-A protein expression and lactate concentration. Metastases were delayed and primary tumor growth rate decreased. CONCLUSIONS: We show for the first time that LDH-A knockdown inhibited the formation of metastases, and was accompanied by in vivo changes in tumor cell metabolism. Lactate MRSI can be used as a surrogate to monitor targeted inhibition of LDH-A in a preclinical setting and provides a noninvasive imaging strategy to monitor LDH-A-targeted therapy. This imaging strategy can be translated to the clinic to identify and monitor patients who are at high risk of developing metastatic disease.
Asunto(s)
Neoplasias de la Mama/patología , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/metabolismo , Neoplasias Pulmonares/secundario , Adenosina Trifosfato/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Adhesión Celular , Movimiento Celular , Proliferación Celular , Femenino , Glucosa/metabolismo , Glucólisis , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/genética , Lactato Deshidrogenasa 5 , Neoplasias Pulmonares/metabolismo , Ratones , Consumo de Oxígeno , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno , Células Tumorales CultivadasRESUMEN
Noninvasive reporter gene imaging is a component of molecular imaging. Reporter imaging can provide noninvasive assessments of endogenous biologic processes in living subjects and can be performed using different imaging modalities. This review will focus on radionuclide-based reporter gene imaging as developed and applied in preclinical and clinical studies. Examples of different reporter systems are presented, with a focus on human reporter systems. Selected applications are discussed, including adoptive cell therapies, gene and oncoviral therapies, oncogenesis, signal pathway monitoring, and imaging drug treatment. Molecular imaging, and noninvasive reporter gene imaging in particular, are making important contributions to our understanding of disease development, progression, and treatment in our current era of molecular medicine and individualized patient care.