Thymidine Metabolism as a Confounding Factor for 3'-Deoxy-3'-18F-Fluorothymidine Uptake After Therapy in a Colorectal Cancer Model.

Noninvasive monitoring of tumor therapy response helps in developing personalized treatment strategies. Here, we performed sequential PET and diffusion-weighted MRI to evaluate changes induced by a FOLFOX-like combination chemotherapy in colorectal cancer xenografts, to identify the cellular and molecular determinants of these imaging biomarkers. Methods: Tumor-bearing CD1 nude mice, engrafted with FOLFOX-sensitive Colo205 colorectal cancer xenografts, were treated with FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin) weekly. On days 1, 2, 6, 9, and 13 of therapy, tumors were assessed by in vivo imaging and ex vivo analyses. In addition, HCT116 xenografts, which did not respond to the FOLFOX treatment, were imaged on day 1 of therapy. Results: In Colo205 xenografts, FOLFOX induced a profound increase in uptake of the proliferation PET tracer 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) accompanied by increases in markers for proliferation (Ki-67, thymidine kinase 1) and for activated DNA damage response (γH2AX), whereas the effect on cell death was minimal. Because tracer uptake was unaltered in the HCT116 model, these changes appear to be specific for tumor response. Conclusion: We demonstrated that 18F-FLT PET can noninvasively monitor cancer treatment-induced molecular alterations, including thymidine metabolism and DNA damage response. The cellular or imaging changes may not, however, be directly related to therapy response as assessed by volumetric measurements.

3'-Deoxy-3'-[18F]Fluorothymidine Uptake Is Related to Thymidine Phosphorylase Expression in Various Experimental Tumor Models.

PURPOSE: We recently reported that high thymidine phosphorylase (TP) expression is accompanied by low tumor thymidine concentration and high 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) uptake in four untreated lung cancer xenografts. Here, we investigated whether this relationship also holds true for a broader range of tumor models. PROCEDURES: Lysates from n = 15 different tumor models originating from n = 6 institutions were tested for TP and thymidylate synthase (TS) expression using western blots. Results were correlated to [18F]FLT accumulation in the tumors as determined by positron emission tomography (PET) measurements in the different institutions and to previously published thymidine concentrations. RESULTS: Expression of TP correlated positively with [18F]FLT SUVmax (ρ = 0.549, P < 0.05). Furthermore, tumors with high TP levels possessed lower levels of thymidine (ρ = - 0.939, P < 0.001). CONCLUSIONS: In a broad range of tumors, [18F]FLT uptake as measured by PET is substantially influenced by TP expression and tumor thymidine concentrations. These data strengthen the role of TP as factor confounding [18F]FLT uptake.

The action of ß-hydroxybutyrate on the growth, metabolism and global histone H3 acetylation of spontaneous mouse mammary tumours: evidence of a ß-hydroxybutyrate paradox.

BACKGROUND: Ketone bodies have both metabolic and epigenetic roles in cancer. In several studies, they showed an anti-cancer effect via inhibition of histone deacetylases; however, other studies observed faster tumour growth. The related molecule butyrate also inhibits growth of some cancer cells and accelerates it in others. This "butyrate paradox" is thought to be due to butyrate mediating histone acetylation and thus inhibiting cell proliferation in cancers that preferentially utilise glucose (the Warburg effect); whereas in cells that oxidise butyrate as a fuel, it fails to reach inhibitory concentrations and can stimulate growth. METHODS: We treated transgenic mice bearing spontaneous MMTV-NEU-NT mammary tumours with the ketone body ß-hydroxybutyrate (ß-OHB) and monitored tumour growth, metabolite concentrations and histone acetylation. In a cell line derived from these tumours, we also measured uptake of ß-OHB and glucose, and lactate production, in the absence and presence of ß-OHB. RESULTS: ß-OHB administration accelerated growth of MMTV-NEU-NT tumours, and their metabolic profile showed significant increases in ATP, glutamine, serine and choline-related metabolites. The ß-OHB concentration within the treated tumours, 0.46 ± 0.05 µmol/g, had no effect on histone acetylation as shown by western blots. Cultured tumour cells incubated with 0.5 mM ß-OHB showed ß-OHB uptake that would be equivalent to 54% of glycolytic ATP phosphorylation and no significant change in glucose consumption or lactate production. CONCLUSIONS: These results suggest that a ß-OHB paradox may occur in these mammary tumours in a manner analogous to the butyrate paradox. At low ß-OHB concentrations (<1 mM, as observed in our tumour model post-treatment), and in the absence of a Warburg effect, ß-OHB is consumed and thus acts as an oxidative energy source and not as an epigenetic factor. This would explain the increase in tumour growth after treatment, the metabolic profiles and the absence of an effect on histone H3 acetylation.

Quantitative and textural analysis of magnetization transfer and diffusion images in the early detection of brain metastases.

PURPOSE: The sensitivity of the magnetization transfer ratio (MTR) and apparent diffusion coefficient (ADC) for early detection of brain metastases was investigated in mice and humans. METHODS: Mice underwent MRI twice weekly for up to 31 d following intracardiac injection of the brain-homing breast cancer cell line MDA-MB231-BR. Patients with small cell lung cancer underwent quarterly MRI for 1 year. MTR and ADC were measured in regions of metastasis and matched contralateral tissue at the final time point and in registered regions at earlier time points. Texture analysis and linear discriminant analysis were performed to detect metastasis-containing slices. RESULTS: Compared with contralateral tissue, mouse metastases had significantly lower MTR and higher ADC at the final time point. Some lesions were visible at earlier time points on the MTR and ADC maps: 24% of these were not visible on corresponding T2 -weighted images. Texture analysis using the MTR maps showed 100% specificity and 98% sensitivity for metastasis at the final time point, with 77% sensitivity 2-4 d earlier and 46% 5-8 d earlier. Only 2 of 16 patients developed metastases, and their penultimate scans were normal. CONCLUSIONS: Some brain metastases may be detected earlier on MTR than conventional T2 ; however, the small gain is unlikely to justify "predictive" MRI. Magn Reson Med 77:1987-1995, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Response Monitoring with [18F]FLT PET and Diffusion-Weighted MRI After Cytotoxic 5-FU Treatment in an Experimental Rat Model for Colorectal Liver Metastases.

PURPOSE: The aim of the study was to investigate the potential of diffusion-weighted magnetic resonance imaging (DW-MRI) and 3'-dexoy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography (PET) as early biomarkers of treatment response of 5-fluorouracil (5-FU) in a syngeneic rat model of colorectal cancer liver metastases. PROCEDURES: Wag/Rij rats with intrahepatic syngeneic CC531 tumors were treated with 5-FU (15, 30, or 60 mg/kg in weekly intervals). Before treatment and at days 1, 3, 7, and 14 after treatment rats underwent DW-MRI and [18F]FLT PET. Tumors were analyzed immunohistochemically for Ki67, TK1, and ENT1 expression. RESULTS: 5-FU inhibited the growth of CC531 tumors in a dose-dependent manner. Immunohistochemical analysis did not show significant changes in Ki67, TK1, and ENT1 expression. However, [18F]FLT SUVmean and SUVmax were significantly increased at days 4 and 7 after treatment with 5-FU (60 mg/kg) and returned to baseline at day 14 (SUVmax at days -1, 4, 7, and 14 was 1.1 ± 0.1, 2.3 ± 0.5, 2.3 ± 0.6, and 1.5 ± 0.4, respectively). No changes in [18F]FLT uptake were observed in the nontreated animals. Furthermore, the apparent diffusion coefficient (ADCmean) did not change in 5-FU-treated rats compared to untreated rats. CONCLUSION: This study suggests that 5-FU treatment induces a flare in [18F]FLT uptake of responsive CC531 tumors in the liver, while the ADCmean did not change significantly. Future studies in larger groups are warranted to further investigate whether [18F]FLT PET can discriminate between disease progression and treatment response.

Gemcitabine Mechanism of Action Confounds Early Assessment of Treatment Response by 3'-Deoxy-3'-[18F]Fluorothymidine in Preclinical Models of Lung Cancer.

3'-Deoxy-3'-[18F]fluorothymidine positron emission tomography ([18F]FLT-PET) and diffusion-weighted MRI (DW-MRI) are promising approaches to monitor tumor therapy response. Here, we employed these two imaging modalities to evaluate the response of lung carcinoma xenografts in mice after gemcitabine therapy. Caliper measurements revealed that H1975 xenografts responded to gemcitabine treatment, whereas A549 growth was not affected. In both tumor models, uptake of [18F]FLT was significantly reduced 6 hours after drug administration. On the basis of the gemcitabine concentration and [18F]FLT excretion measured, this was presumably related to a direct competition of gemcitabine with the radiotracer for cellular uptake. On day 1 after therapy, [18F]FLT uptake was increased in both models, which was correlated with thymidine kinase 1 (TK1) expression. Two and 3 days after drug administration, [18F]FLT uptake as well as TK1 and Ki67 expression were unchanged. A reduction in [18F]FLT in the responsive H1975 xenografts could only be noted on day 5 of therapy. Changes in ADCmean in A549 xenografts 1 or 2 days after gemcitabine did not seem to be of therapy-related biological relevance as they were not related to cell death (assessed by caspase-3 IHC and cellular density) or tumor therapy response. Taken together, in these models, early changes of [18F]FLT uptake in tumors reflected mechanisms, such as competing gemcitabine uptake or gemcitabine-induced thymidylate synthase inhibition, and only reflected growth-inhibitory effects at a later time point. Hence, the time point for [18F]FLT-PET imaging of tumor response to gemcitabine is of crucial importance. Cancer Res; 76(24); 7096-105. ©2016 AACR.

Variability of Proliferation and Diffusion in Different Lung Cancer Models as Measured by 3'-Deoxy-3'-¹8F-Fluorothymidine PET and Diffusion-Weighted MR Imaging.

UNLABELLED: Molecular imaging allows the noninvasive assessment of cancer progression and response to therapy. The aim of this study was to investigate molecular and cellular determinants of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET and diffusion-weighted (DW) MR imaging in lung carcinoma xenografts. METHODS: Four lung cancer cell lines (A549, HTB56, EBC1, and H1975) were subcutaneously implanted in nude mice, and growth was followed by caliper measurements. Glucose uptake and tumor proliferation were determined by (18)F-FDG and (18)F-FLT PET, respectively. T2-weighted MR imaging was performed, and the apparent diffusion coefficient (ADC) was determined by DW MR imaging as an indicator of cell death. Imaging findings were correlated to histology with markers for tumor proliferation (Ki67, 5-bromo-2'-deoxyuridine [BrdU]) and cell death (caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). The expression of human equilibrative nucleoside transporter 1 (hENT1), thymidine kinase 1 (TK1), thymidylate synthase, and thymidine phosphorylase (TP) were analyzed by Western blot and immunohistochemistry. Thymidine levels were determined by liquid chromatography-mass spectrometry. RESULTS: Xenografts varied with respect to in vivo growth rates. MR imaging and PET revealed intratumoral heterogeneities, which were confirmed by histology. (18)F-FLT uptake differed significantly between tumor lines, with A549 and H1975 demonstrating the highest radiotracer accumulation (A549, 8.5 ± 3.2; HTB56, 4.4 ± 0.7; EBC1, 4.4 ± 1.2; and H1975, 12.1 ± 3.5 maximal percentage injected dose per milliliter). In contrast, differences in (18)F-FDG uptake were only marginal. No clear relationship between (18)F-FLT accumulation and immunohistochemical markers for tumor proliferation (Ki67, BrdU) as well as hENT1, TK1, or TS expression was detected. However, TP was highly expressed in A549 and H1975 xenografts, which was accompanied by low tumor thymidine concentrations, suggesting that tumor thymidine levels influence (18)F-FLT uptake in the tumor models investigated. MR imaging revealed higher ADC values within proliferative regions of H1975 and A549 tumors than in HTB56 and EBC1. These ADC values were negatively correlated with cell density but not directly related to cell death. CONCLUSION: A direct relationship of (18)F-FLT with proliferation or ADC with cell death might be complicated by the interplay of multiple processes at the cellular and physiologic levels in untreated tumors. This issue must be considered when using these imaging modalities in preclinical or clinical settings.

In vitro and in vivo evaluations of a hydrophilic 64Cu-bis(thiosemicarbazonato)-glucose conjugate for hypoxia imaging.

UNLABELLED: A water-soluble glucose conjugate of the hypoxia tracer 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) was synthesized and radiolabeled (64Cu-ATSE/A-G). Here we report our initial biological experiments with 64Cu-ATSE/A-G and compare the results with those obtained for 64Cu-ATSM and 18F-FDG. METHODS: The uptake of 64Cu-ATSE/A-G and 64Cu-ATSM into HeLa cells in vitro was investigated at a range of dissolved oxygen concentrations representing normoxia, hypoxia, and anoxia. Small-animal PET with 64Cu-ATSE/A-G was performed in male BDIX rats implanted with P22 syngeneic carcinosarcomas. Images of 64Cu-ATSM and 18F-FDG were obtained in the same model for comparison. RESULTS: 64CuATSE/A-G showed oxygen concentration-dependent uptake in vitro and, under anoxic conditions, showed slightly lower levels of cellular uptake than 64Cu-ATSM; uptake levels under hypoxic conditions were also lower. Whereas the normoxic uptake of 64Cu-ATSM increased linearly over time, 64Cu-ATSE/A-G uptake remained at low levels over the entire time course. In the PET study, 64CuATSE/A-G showed good tumor uptake and a biodistribution pattern substantially different from that of each of the controls. In marked contrast to the findings for 64Cu-ATSM, renal clearance and accumulation in the bladder were observed. 64Cu-ATSE/A-G did not display the characteristic brain and heart uptake of 18F-FDG. CONCLUSION: The in vitro cell uptake studies demonstrated that 64Cu-ATSE/A-G retained hypoxia selectivity and had improved characteristics when compared with 64Cu-ATSM. The in vivo PET results indicated a difference in the excretion pathways, with a shift from primarily hepatointestinal for 64Cu-ATSM to partially renal with 64Cu-ATSE/A-G. This finding is consistent with the hydrophilic nature of the glucose conjugate. A comparison with 18F-FDG PET results revealed that 64Cu-ATSE/A-G was not a surrogate for glucose metabolism. We have demonstrated that our method for the modification of Cu-bis(thiosemicarbazonato) complexes allows their biodistribution to be modified without negating their hypoxia selectivity or tumor uptake properties.

Blood vessel maturation and response to vascular-disrupting therapy in single vascular endothelial growth factor-A isoform-producing tumors.

Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.

The vascular response of tumor and normal tissues in the rat to the vascular targeting agent, combretastatin A-4-phosphate, at clinically relevant doses.

The antivascular actions of disodium combretastatin A-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 carcinosarcoma after single doses of 10 or 30 mg x kg(-1). Pharmacokinetic data showed that 10 mg x kg(-1) in the rat gave a plasma exposure similar to that achieved in the clinic. Blood flow rate to the tumor and normal tissues was measured using the uptake of radiolabelled iodoantipyrine (IAP). Quantitative autoradiography was used to determine changes in spatial distribution of tumor blood flow. Both doses caused an increase in mean arterial blood pressure (MABP) and a reduction in heart rate 1 h after treatment. Blood flow rate to the tumor decreased to below 15% of control for both doses at 1 h, whereas the normal tissues were much less affected. A further reduction (to 2% of control at 6 h) was found for 30 mg x kg(-1). Recovery was essentially complete by 24 h for both doses. Vascular resistance increased 80-fold in tumor at 6 h after 30 mg x kg(-1), compared with a maximum 5-fold increase in normal tissues. Analysis of the spatial distribution of tumor blood flow illustrated an overall reduction in all areas of the tumor at 1 h after 10 mg x kg(-1), with a tendency for blood flow in the peripheral regions of the tumor to recover more quickly than in central regions. Tumor blood flow reduction was related to vascular damage including vessel distension, coagulation and haemorrhage, and tumor cell damage culminating in necrosis. No pathology was evident in any of the normal tissues following treatment. The data provide an insight into the mechanisms underlying tissue blood flow changes occurring after clinically relevant doses of CA-4-P. It is currently being used to aid interpretation of pharmacodynamic data obtained from phase I/II clinical trials of CA-4-P and is relevant for future drug development in this area.

Validation of the fluorinated 2-nitroimidazole SR-4554 as a noninvasive hypoxia marker detected by magnetic resonance spectroscopy.

PURPOSE: Tumor hypoxia is associated with poor prognosis and a more malignant tumor phenotype. SR-4554, a fluorinated 2-nitroimidazole, is selectively bioreduced and bound in hypoxic cells. We present validation studies of SR-4554 as a noninvasive hypoxia marker detected by fluorine-19 magnetic resonance spectroscopy ((19)F MRS) in the P22 carcinosarcoma, a tumor with clinically relevant hypoxia levels. EXPERIMENTAL DESIGN: Tumor-bearing female severe combined immunodeficient mice received SR-4554 at 180 mg/kg. Pharmacokinetic studies of parent SR-4554 in plasma and tumors were performed using high-performance liquid chromatography-UV. Total SR-4554 (parent SR-4554 and bioreduction products) was monitored in tumor by (19)F MRS using a 4.7 T spectrometer, with continuous acquisition for up to 5 h. A parameter of total SR-4554 retention, the 3-h (19)F retention index ((19)FRI) was determined. Tumor pO(2), assessed polarographically, was decreased (5 mg/kg hydralazine or 100 mg/kg combretastatin A-4 phosphate) or increased [1 l/min carbogen (5% CO(2), 95% O(2)) plus 500 mg/kg nicotinamide], and the corresponding (19)FRI was measured. RESULTS: Comparative HPLC-UV- and MRS-derived assessments of parent and total SR-4554, respectively, indicated that concentrations of total SR-4554 consistently exceeded parent SR-4554, the differential increasing with time. This indicates formation and retention of SR-4554 bioreduction products in tumor, confirming the presence of hypoxia. The (19)FRI was higher in hydralazine- and combretastatin-treated animals compared with unmodulated animals (P = 0.004 and 0.15, respectively) and animals receiving carbogen and nicotinamide (P = 0.0001 and 0.005, respectively). Significant correlations were demonstrated between mean (19)FRI and polarographic pO(2) parameters (P < 0.002). CONCLUSIONS: Retention of hypoxia-related SR-4554 bioreduction products can be detected in the clinically relevant P22 tumor by (19)F MRS, and the (19)FRI correlates with polarographically measured pO(2). These findings support the use of SR 4554 as a noninvasive hypoxia marker.