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

3'-Deoxy-3'-[$^{18}$F]fluorothymidine positron emission tomography ([$^{18}$F]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 [$^{18}$F]FLT was significantly reduced 6 hours after drug administration. On the basis of the gemcitabine concentration and [$^{18}$F]FLT excretion measured, this was presumably related to a direct competition of gemcitabine with the radiotracer for cellular uptake. On day 1 after therapy, [$^{18}$F]FLT uptake was increased in both models, which was correlated with thymidine kinase 1 (TK1) expression. Two and 3 days after drug administration, [$^{18}$F]FLT uptake as well as TK1 and Ki67 expression were unchanged. A reduction in [$^{18}$F]FLT in the responsive H1975 xenografts could only be noted on day 5 of therapy. Changes in ADC$_{mean}$ 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 [$^{18}$F]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 [$^{18}$F]FLT-PET imaging of tumor response to gemcitabine is of crucial importance.The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (www.imi.europa.eu) under grant agreement number 115151, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution. This work was also supported by the Deutsche Forschungsgemeinschaft (DFG), Cells-in-Motion Cluster of Excellence (EXC1003 – CiM), University of Munster (Münster, Germany)

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'-[(¹⁸)F]fluorothymidine ([(¹⁸)F]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 [(¹⁸)F]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, [(¹⁸)F]FLT SUV_mean and SUV_max were significantly increased at days 4 and 7 after treatment with 5-FU (60 mg/kg) and returned to baseline at day 14 (SUV_max 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 [(¹⁸)F]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 [(¹⁸)F]FLT uptake of responsive CC531 tumors in the liver, while the ADC_mean did not change significantly. Future studies in larger groups are warranted to further investigate whether [(¹⁸)F]FLT PET can discriminate between disease progression and treatment response.The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (www.imi.europa.eu) under grant agreement number 115151, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution

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.The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (www.imi.europa.eu) under grant agreement number 115151, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution

Thymidine Metabolism as Confounding Factor of 3'-Deoxy-3'-[18F]Fluorothymidine Uptake after Therapy in a Colorectal Cancer Model.

Non-invasive monitoring of tumor therapy response helps in developing personalized treatment strategies. Here, we performed sequential positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI) to evaluate changes induced by a FOLFOX-like combination chemotherapy in colorectal cancer (CRC) xenografts, to identify the cellular and molecular determinants of these imaging biomarkers. Methods: Tumor bearing CD1 nude mice, engrafted with FOLFOX-sensitive Colo205 CRC xenografts, were treated with FOLFOX (5 fluorouracil, leucovorin and oxaliplatin) in weekly intervals. On d1, d2, d6, d9 and d13 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 d1 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), which was accompanied by increases in markers for proliferation (Ki67, TK1) and for activated DNA damage response (DDR; γH2AX), whereas the effect on cell death was minimal. As tracer uptake was unaltered in the HCT116 model, these changes appear to be specific for tumor response. Conclusion: We demonstrate that [18F]FLT PET can non-invasively monitor molecular alterations induced by a cancer treatment, including thymidine metabolism and DDR. The cellular or imaging changes may not, however, be directly related to therapy response as assessed by volumetric measurements

Correction to: The relationship between endogenous thymidine concentrations and [18F]FLT uptake in a range of preclinical tumour models.

Unfortunately, the original version of Figs. 4, 5 and 6b in the article [1] contained errors in the n numbers as indicated on the columns. Please note that column heights and error bars in the original figures and data in the ESM tables are correct and statistical tests are valid. These corrections do not affect any results or conclusions in this article

Investigating the effects or AKT/PKB inhibitors on [18F]-FDG uptake in cancer cells

Increased AKT activity due to hyperactivated PI3K or inactivation of PTEN is frequently found in cancer. Thus the development of AKT inhibitors and pharmacodynamic (PO) biomarkers for AKT inhibition is of major interest for cancer therapy. [18F]-FDG PET is a non-invasive imaging technique which allows the isualization of glucose metabolism. As AKT activity has been associated with glucose uptake and phosphorylation, it has been hypothesized that CSF]-FOG PET is a potential PO biomarker for the assessment of AKT inhibitor action in the clinic. The primary aim of the work presented in this thesis was to investigate whether (18F]-FOG uptake can be used as a PO biomarker for AKT inhibition. An in vitro system was established including assembly of a gamma counter which was used to demonstrate that both ATP competitive and allosteric inhibitors of AKT block [18F]-FDG uptake in the PTEN null U87MG human glioblastoma cell line. These results led to a detailed investigation of the mechanism(s) by which the AKT inhibitors affect [18F]-FDG uptake. However, no change in expression or translocation of the glucose transporters GLUT-1 and GLUT-4, or in hexokinase phosphorylation or activity was seen after inhibitor treatment. Reduction of [18F]-FDG uptake was then confirmed with a later generation of AKT inhibitors and in a second cellular model, namely the human breast cancer cell line BT474. These results were also complimented by MRS studies of lactate production. AKT knock down studies using siRNA were also carried out. However, complete functional removal of AKT was not achieved, although AKT protein level was substantially reduced. In addition, in vivo studies demonstrated that [18F]-FDG uptake and AKT signalling were decreased in U87MG xenografts by the AKT inhibitor CCT129254. Hence, it was concluded that [18F]-FDG uptake can be used as a PD biomarker of AKT inhibition for the types of compound studied.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Preclinical Applications of 3'-Deoxy-3'-[18F]Fluorothymidine in Oncology - A Systematic Review

The positron emission tomography (PET) tracer 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) has been proposed to measure cell proliferation non-invasively in vivo. Hence, it should provide valuable information for response assessment to tumor therapies. To date, [18F]FLT uptake has found limited use as a response biomarker in clinical trials in part because a better understanding is needed of the determinants of [18F]FLT uptake and therapy-induced changes of its retention in the tumor. In this systematic review of preclinical [18F]FLT studies, comprising 174 reports, we identify the factors governing [18F]FLT uptake in tumors, among which thymidine kinase 1 plays a primary role. The majority of publications (83 %) report that decreased [18F]FLT uptake reflects the effects of anticancer therapies. 144 times [18F]FLT uptake was related to changes in proliferation as determined by ex vivo analyses. Of these approaches, 77 % describe a positive relation, implying a good concordance of tracer accumulation and tumor biology. These preclinical data indicate that [18F]FLT uptake holds promise as an imaging biomarker for response assessment in clinical studies. Understanding of the parameters which influence cellular [18F]FLT uptake and retention as well as the mechanism of changes induced by therapy is essential for successful implementation of this PET tracer. Hence, our systematic review provides the background for the use of [18F]FLT in future clinical studies

Evaluation of apoptosis imaging biomarkers in a genetic model of cell death

Abstract Purpose We have previously developed the caspase-based radiotracer, 18F-ICMT-11, for PET imaging to monitor treatment response. We further validated 18F-ICMT-11 specificity in a murine melanoma death-switch tumour model with conditional activation of caspase-3 induced by doxycycline. Methods Caspase-3/7 activity and cellular uptake of 18F-ICMT-11, 18F-ML-10 and 18F-FDG were assessed in B16ova and B16ovaRevC3 cells after death-switch induction. Death-switch induction was confirmed in vivo in xenograft tumours, and 18F-ICMT-11 and 18F-ML-10 biodistribution was assessed by ex vivo gamma counting of select tissues. PET imaging was performed with 18F-ICMT-11, 18F-ML-10 and 18F-FDG. Caspase-3 activation was confirmed by immunohistochemistry. Results Significantly increased caspase-3/7 activity was observed only in B16ovaRevC3 cells after death-switch induction, accompanied by significantly increased 18F-ICMT-11 (p < 0.001) and 18F-ML-10 (p < 0.05) and decreased 18F-FDG (p < 0.001) uptake compared with controls. B16ova and B16ovaRevC3 tumours had similar growth in vivo; however, B16ovaRevC3 growth was significantly reduced with death-switch induction (p < 0.01). Biodistribution studies showed significantly increased 18F-ICMT-11 tumour uptake following death-switch induction (p < 0.01), but not for 18F-ML-10. Tumour uptake of 18F-ICMT-11 was higher than that of 18F-ML-10 after death-switch induction. PET imaging studies showed that 18F-ICMT-11 can be used to detect apoptosis after death-switch induction, which was accompanied by significantly increased expression of cleaved caspase-3. 18F-FDG signal decreased in tumours after death-switch induction. Conclusions We demonstrate that 18F-ICMT-11 can be used to detect caspase-3 activation in a death-switch tumour model, independent of the confounding effects of cancer therapeutics, thus confirming its specificity and supporting the development of this radiotracer for clinical use to monitor tumour apoptosis and therapy response