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Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Background: This study investigates whether a histone deacetylase subtype 6 (HDAC6) inhibitor could be used in the treatment of solid tumours. Methods: We evaluated the effect of a novel inhibitor, C1A, on HDAC6 biochemical activity and cell growth. We further examined potential of early noninvasive imaging of cell proliferation by [ 18 F]fluorothymidine positron emission tomography ([ 18 F]FLT-PET) to detect therapy response. Results: C1A induced sustained acetylation of HDAC6 substrates, α -tubulin and HSP90, compared with current clinically approved HDAC inhibitor SAHA. C1A induced apoptosis and inhibited proliferation of a panel of human tumour cell lines from different origins in the low micromolar range. Systemic administration of the drug inhibited the growth of colon tumours in vivo by 78%. The drug showed restricted activity on gene expression with <0.065% of genes modulated during 24 h of treatment. C1A treatment reduced tumour [ 18 F]FLT uptake by 1.7-fold at 48 h, suggesting that molecular imaging could provide value in future studies of this compound. Conclusion: C1A preferentially inhibits HDAC6 and modulates HDAC6 downstream targets leading to growth inhibition of a diverse set of cancer cell lines. This property together with the favourable pharmacokinetics and efficacy in vivo makes it a candidate for further pre-clinical and clinical development.

Cells exposed to low-oxygen conditions (hypoxia) alter their metabolism to survive. This response, although vital during development and high-altitude survival, is now known to be a major factor in the selection of cells with a transformed metabolic phenotype during tumorigenesis. It is thought that hypoxia-selected cells have increased invasive capacity and resistance to both chemo- and radiotherapies, and therefore represent an attractive target for antitumor therapy. Hypoxia inducible factors (HIFs) are responsible for the majority of gene expression changes under hypoxia, and are themselves controlled by the oxygen-sensing HIF prolyl hydroxylases (PHDs). It was previously shown that mutations in succinate dehydrogenase lead to the inactivation PHDs under normoxic conditions, which can be overcome by treatment with α-ketoglutarate derivatives. Given that solid tumors contain large regions of hypoxia, the reactivation of PHDs in these conditions could induce metabolic catastrophe and therefore prove an effective antitumor therapy. In this report we demonstrate that derivatized α-ketoglutarate can be used as a strategy for maintaining PHD activity under hypoxia. By increasing intracellular α-ketoglutarate and activating PHDs we trigger PHD-dependent reversal of HIF1 activation, and PHD-dependent hypoxic cell death. We also show that derivatized α-ketoglutarate can permeate multiple layers of cells, reducing HIF1α levels and its target genes in vivo .

Background: Cyclin-dependent kinases (CDKs) control cell cycle progression, RNA transcription and apoptosis, making them attractive targets for anticancer drug development. Unfortunately, CDK inhibitors developed to date have demonstrated variable efficacy. Methods: We generated drug-resistant cells by continuous low-dose exposure to a model pyrazolo[1,5- a ]pyrimidine CDK inhibitor and investigated potential structural alterations for optimal efficacy. Results: We identified induction of the ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2, in resistant cells. Assessment of features involved in the ABC transporter substrate specificity from a compound library revealed high polar surface area (>100 Å 2 ) as a key determinant of transporter interaction. We developed ICEC-0782 that preferentially inhibited CDK2, CDK7 and CDK9 in the nanomolar range. The compound inhibited phosphorylation of CDK substrates and downregulated the short-lived proteins, Mcl-1 and cyclin D1. ICEC-0782 induced G2/M arrest and apoptosis. The permeability and cytotoxicity of ICEC-0782 were unaffected by ABC transporter expression. Following daily oral dosing, the compound inhibited growth of human colon HCT-116 and human breast MCF7 tumour xenografts in vivo by 84% and 94%, respectively. Conclusion: We identified a promising pyrazolo[1,5- a ]pyrimidine compound devoid of ABC transporter interaction, highly suitable for further preclinical and clinical evaluation for the treatment of cancer.

Development of hypoxia-targeted therapies has stimulated the search for clinically applicable noninvasive markers of tumour hypoxia. Here, we describe the validation of [ 18 F]fluoroetanidazole ([ 18 F]FETA) as a tumour hypoxia marker by positron emission tomography (PET). Cellular transport and retention of [ 18 F]FETA were determined in vitro under air vs nitrogen. Biodistribution and metabolism of the radiotracer were determined in mice bearing MCF-7, RIF-1, EMT6, HT1080/26.6, and HT1080/1-3C xenografts. Dynamic PET imaging was performed on a dedicated small animal scanner. [ 18 F]FETA, with an octanol–water partition coefficient of 0.16±0.01, was selectively retained by RIF-1 cells under hypoxia compared to air (3.4- to 4.3-fold at 60–120 min). The radiotracer was stable in the plasma and distributed well to all the tissues studied. The 60-min tumour/muscle ratios positively correlated with the percentage of p O 2 values <5 mmHg ( r =0.805, P =0.027) and carbogen breathing decreased [ 18 F]FETA-derived radioactivity levels ( P =0.028). In contrast, nitroreductase activity did not influence accumulation. Tumours were sufficiently visualised by PET imaging within 30–60 min. Higher fractional retention of [ 18 F]FETA in HT1080/1-3C vs HT1080/26.6 tumours determined by dynamic PET imaging ( P =0.05) reflected higher percentage of p O 2 values <1 mmHg ( P =0.023), lower vessel density ( P =0.026), and higher radiobiological hypoxic fraction ( P =0.008) of the HT1080/1-3C tumours. In conclusion, [ 18 F]FETA shows hypoxia-dependent tumour retention and is, thus, a promising PET marker that warrants clinical evaluation.

Summary The National Cancer Imaging Translational Accelerator (NCITA) is creating a UK national coordinated infrastructure for accelerated translation of imaging biomarkers for clinical use. Through the development of standardised protocols, data integration tools and ongoing training programmes, NCITA provides a unique scalable infrastructure for imaging biomarker qualification using multicentre clinical studies.

The availability of a noninvasive method to detect and quantify apoptosis in tumours will enable tumour response to several cancer therapies to be assessed. We have synthesised two radiotracers, annexin V and the N -succinimidyl-3-iodobenzoic acid (SIB) derivative of annexin V, labelled with radio-iodine ( 124 I and 125 I) and provided proof of the concept by assessing specific binding and biodistribution of these probes to apoptotic cells and tumours. We have also assessed the tumour uptake of [ 124 I]annexin V in a mouse model of apoptosis. RIF-1 cells induced to undergo apoptosis in vitro showed a drug concentration-dependent increased binding of [ 125 I]annexin V and [ 125 I]SIB–annexin V. In the same model system, there was an increase in terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL)-positive cells and a decrease in clonogenic survival. Radiotracer binding was completely inhibited by preincubation with unlabelled annexin V. In RIF-1 tumour-bearing mice, rapid distribution of [ 125 I]SIB–annexin V-derived radioactivity to kidneys was observed and the radiotracer accumulated in urine. The binding of [ 125 I]SIB–annexin V to RIF-1 tumours increased by 2.3-fold at 48 h after a single intraperitoneal injection of 5-fluorouracil (165 mg kg −1 body weight), compared to a 4.4-fold increase in TUNEL-positive cells measured by immunostaining. Positron emission tomography images with both radiotracers demonstrated intense localisation in the kidneys and bladder. Unlike [ 124 I]SIB–annexin V, [ 124 I]annexin V also showed localisation in the thyroid region presumably due to deiodination of the radiolabel. [ 124 I]SIB–annexin V is an attractive candidate for in vivo imaging of apoptosis by PET.

HDAC6 is emerging as an important therapeutic target for cancer. We investigated mechanisms responsible for survival of tumor cells treated with a HDAC6 inhibitor. Expression of the 20 000 genes examined did not change following HDAC6 treatment in vivo . We found that HDAC6 inhibition led to an increase of AKT activation (P-AKT) in vitro , and genetic knockdown of HDAC6 phenocopied drug-induced AKT activation. The activation of AKT was not observed in PTEN null cells; otherwise, PTEN/PIK3CA expression per se did not predict HDAC6 inhibitor sensitivity. Interestingly, HDAC6 inhibitor treatment led to inactivating phosphorylation of PTEN (P-PTEN Ser380), which likely led to the increased P-AKT in cells that express PTEN. Synergy was observed with phosphatidylinositol 3’-kinases (PI3K) inhibitor treatment in vitro, accompanied by increased caspase 3/7 activity. Furthermore, combination of HDAC6 inhibitor with a PI3K inhibitor caused substantial tumor growth inhibition in vivo compared with either treatment alone, also detectable by Ki-67 immunostaining and 18 F-FLT positron emission tomography (PET). In aggregate AKT activation appears to be a key survival mechanism for HDAC6 inhibitor treatment. Our findings indicate that dual inhibition of HDAC6 and P-AKT may be necessary to substantially inhibit growth of solid tumors.

An understanding of epigenetic drivers of tumorigenesis has developed rapidly during the last years. The identification of these changes including DNA methylation and histone modifications in gastroenteropancreatic neuroendocrine tumours (GEP-NETs) is a step forward in trying to define underlying biologic processes in this heterogeneous disease. The reversible nature of these changes represents a potential therapeutic target. We present an overview of the current knowledge of epigenetic alterations related to GEP-NETs, focusing on the influence and impact these changes have on pathogenesis and prognosis. The potential role of demethylating agents in the management of this patient population is discussed.

Purpose This study aimed to evaluate the utility of plasma pharmacokinetic analyses of anti-cancer agents from data obtained during positron emission tomography (PET) oncology studies of radiolabelled anti-cancer agents. Patients and methods Thirteen patients were administered fluorine-18 radiolabelled 5-FU ([ 18 F]5-FU) admixed with 5-FU, corresponding to a total 5-FU dose of 380–407 mg/m 2 (eight patients) and 1 mg/m 2 (five patients). Nine patients received 2.2–19.2 μg/m 2 of carbon-11 radiolabelled N -[2-(dimethylamino)ethyl]acridine-4-carboxamide ([ 11 C]DACA) at 1/1,000th of phase I dose, as part of phase 0 microdosing study. Radioactivity of parent drug obtained from arterial blood samples, the injected activity of the radiolabelled drug, and the total dose of injected drug were used to obtain plasma drug concentrations. Plasma pharmacokinetic parameters were estimated using model-dependent and model-independent methods. Results 5-FU plasma concentrations at therapeutic doses were above the Km and a single compartment kinetic model was best used to fit the kinetics, with a mean half-life of 8.6 min. Clearance and volumes of distribution ( V d ) obtained using both model-dependent and model-independent methods were similar. Mean (SE) clearance was 1,421(144), ml min −1 and 1,319 (119) ml min −1 and the mean (SE) V d was 17.3 (1.8) l and 16.3 (1.9) l by the model-independent method and model-dependent methods, respectively. In contrast, with 1 mg/m 2 , plasma concentrations of 5-FU were less than the Km and a two-compartment model was used to best fit the kinetics, with the mean 5-FU half-life of 6.5 min. The mean (SE) clearances obtained by the model-independent method and model-dependent methods were 3,089 (314) ml min −1 and 2,225 (200) ml min −1 , respectively and the mean (SE) V d were 27.9 (7.0) l and 2.3 (0.4) l, by the model independent and dependent methods, respectively. Extrapolation of AUC 0–Clast to AUC 0–∞ was less than 3% in both these cohort of patients. A two-compartment model with a mean half-life of 42.1 min was used to best fit the kinetics of DACA; considerable extrapolation (mean 26%) was required to obtain AUC 0–∞ from AUC 0–Clast . Mean (SE) clearance of DACA was 1,920 (269) ml min −1 , with the model-independent method and 1,627 (287) ml min −1 with the model-dependent method. Similarly, V d [mean (SE)] of DACA with the model-independent and model-dependent methods were 118 (22) l and 50 (15) l, respectively. Conclusions Pharmacokinetic parameters can be estimated with confidence from PET studies for agents given at therapeutic doses, whose half-lives are significantly less than the total sampling time during the scan. Tracer studies performed alone, wherein plasma levels below the Km are expected, may also provide valuable information on drug clearance for the entire range of linear kinetics. However, drugs with half-lives longer than the sampling duration are inappropriate for PET plasma pharmacokinetic evaluation.