In order to explore a pre-clinical method to evaluate if [18F]FDG is valid for monitoring early response, we investigated the uptake of FDG in Multicellular tumour spheroids (MTS) without and with treatment with five routinely used chemotherapy agents in breast cancer.
The response to each anticancer treatment was evaluated by measurement of the [18F]FDG uptake and viable volume of the MTSs after 2 and 3 days of treatment.
The effect of Paclitaxel and Docetaxel on [18F]FDG uptake per viable volume was more evident in BT474 (up to 55% decrease) than in MCF-7 (up to 25% decrease).
Doxorubicin reduced the [18F]FDG uptake per viable volume more noticeable in MCF-7 (25%) than in BT474 MTSs.
Tamoxifen reduced the [18F]FDG uptake per viable volume only in MCF-7 at the highest dose of 1 μM.
No effect of Imatinib was observed.
MTS was shown to be appropriate to investigate the potential of FDG-PET for early breast cancer treatment monitoring; the treatment effect can be observed before any tumour size changes occur.
The combination of PET radiotracers and image analysis in MTS provides a good model to evaluate the relationship between tumour volume and the uptake of metabolic tracer before and after chemotherapy. This feature could be used for screening and selecting PET-tracers for early assessment of treatment response.
In addition, this new method gives a possibility to assess quickly, and in vitro, a good preclinical profile of existing and newly developed anti-cancer drugs.
Increased 2′-deoxy-2′-[18F]fluoro-D-glucose (FDG) uptake is the most commonly used marker for positron emission tomography in oncology. However, a proliferation tracer such as 3′-deoxy-3′-[18F]fluorothymidine (FLT) might be more specific for cancer. 3′-deoxy-3′-[18F]fluorothymidine uptake is dependent on thymidine kinase 1 (TK) activity, but the effects of chemotherapeutic agents are unknown. The aim of this study was to characterise FDG and FLT uptake mechanisms in vitro before and after exposure to chemotherapeutic agents. The effects of 5-fluorouracil (5-FU), doxorubicin and paclitaxel on FDG and FLT uptake were measured in MDA MB231 human breast cancer cells in relation to cell cycle distribution, expression and enzyme activity of TK-1. At IC50 concentrations, 5-FU resulted in accumulation in the G1 phase, but doxorubicin and paclitaxel induced a G2/M accumulation. Compared with untreated cells, 5-FU and doxorubicin increased TK-1 levels by >300. At 72 h, 5-FU decreased FDG uptake by 50% and FLT uptake by 54%, whereas doxorubicin increased FDG and FLT uptake by 71 and 173%, respectively. Paclitaxel increased FDG uptake with >100% after 48 h, whereas FLT uptake hardly changed. In conclusion, various chemotherapeutic agents, commonly used in the treatment of breast cancer, have different effects on the time course of uptake of both FDG and FLT in vitro. This might have implications for interpretation of clinical findings.
[18F]FLT; [18F]FDG; breast cancer cells; thymidine kinase; PET; chemotherapy
The basis for the use of nucleoside tracers in PET is that activity of the cell-growth–dependent enzyme thymidine kinase 1 is the rate-limiting factor driving tracer retention in tumors. Recent publications suggest that nucleoside transporters might influence uptake and thereby affect the tracer signal in vivo. Understanding transport mechanisms for different nucleoside PET tracers is important for evaluating clinical results. This study examined the relative role of different nucleoside transport mechanisms in uptake and retention of [methyl-3H]-3′-deoxy-3′-fluorothymidine (3H-FLT), [methyl-3H]-thymidine (3H-thymidine), and 3H-1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-methyluracil (3H-FMAU).
Transport of 3H-FLT, 3H-thymidine, and 3H-FMAU was examined in a single human adenocarcinoma cell line, A549, under both nongrowth and exponential-growth conditions.
3H-Thymidine transport was dominated by human equilibrative nucleoside transporter 1 (hENT1) under both growth conditions. 3H-FLT was also transported by hENT1, but passive diffusion dominated its transport. 3H-FMAU transport was dominated by human equilibrative nucleoside transporter 2. Cell membrane levels of hENT1 increased in cells under exponential growth, and this increase was associated with a more rapid rate of uptake for both 3H-thymidine and 3H-FLT. 3H-FMAU transport was not affected by changes in growth conditions. All 3 tracers concentrated in the plateau phase, nonproliferating cells at levels many-fold greater than their concentration in buffer, in part because of low levels of nucleoside metabolism, which inhibited tracer efflux.
Transport mechanisms are not the same for 3H-thymidine, 3H-FLT, and 3H-FMAU. Levels of hENT1, an important transporter of 3H-FLT and 3H-thymidine, increase as proliferating cells enter the cell cycle.
proliferation; nucleoside transport; nucleoside metabolism; 3H-FLT; 3H-FMAU
Many cancers can be diagnosed using positron emission tomography (PET) and PET can also be used to monitor how effective various treatments are in individual patients. Tumor spheroids are cancer cells grown on agar coated dishes forming a 3D structure. They are widely used in preclinical cancer research, where the multicellular tumor spheroid model is considered biologically and physiologically similar to in vivo grown tumors. In this study we have used Two-dimensional Difference Gel Electrophoresis (2-D DIGE) analysis to gain more insight in differences in protein expression as a result of drug treatment of multicellular tumor spheroids. 2-D DIGE can be used to identify possible new biomarkers for development of new PET tracers and drug targets. Combining 2-D DIGE and PET results can be used for improving diagnosis and treatment.
3′-deoxy-3′-[18F]fluorothymidine (18F-FLT) is a tracer used to assess cell proliferation in vivo. The aim of the study was to use 18F-FLT positron emission tomography (PET) to study treatment responses to a new anti-cancer compound. To do so, we studied early anti-proliferative effects of the experimental chemotherapy Top216 non-invasively by PET.
In vivo uptake of 18F-FLT in human ovary cancer xenografts in mice (A2780) was studied at various time points after Top216 treatment (50 mg/kg i.v. at 0 and 48 hours) was initiated. Baseline 18F-FLT scans were made before either Top216 (n = 7–10) or vehicle (n = 5–7) was injected and repeated after 2 and 6 hours and 1 and 5 days of treatment. A parallel study was made with 2′-deoxy-2′-[18F]fluoro-D-glucose (18F-FDG) (n = 8). Tracer uptake was quantified using small animal PET/CT. Imaging results were validated by tumor volume changes and gene-expression of Ki67 and TK1. Top216 (50 mg/kg 0 and 48 hours) inhibited the growth of the A2780 tumor compared to the control group (P<0.001). 18F-FLT uptake decreased significantly at 2 hours (−52%; P<0.001), 6 hours (−49%; P = 0.002) and Day 1 (−47%; P<0.001) after Top216 treatment. At Day 5 18F-FLT uptake was comparable to uptake in the control group. Uptake of 18F-FLT was unchanged in the control group during the experiment. In the treatment group, uptake of 18F-FDG was significantly decreased at 6 hours (−21%; P = 0.003), Day 1 (−29%; P<0.001) and Day 5 (−19%; P = 0.05) compared to baseline.
One injection with Top216 initiated a fast and significant decrease in cell-proliferation assessable by 18F-FLT after 2 hours. The early reductions in tumor cell proliferation preceded changes in tumor size. Our data indicate that 18F-FLT PET is promising for the early non-invasive assessment of chemotherapy effects in both drug development and for tailoring therapy in patients.
Fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging demonstrated the change of glucose consumption of tumor cells, but problems with specificity and difficulties in early detection of tumor response to chemotherapy have led to the development of new PET tracers. Fluorine-18-fluorothymidine (18F-FLT) images cellular proliferation by entering the salvage pathway of DNA synthesis. In this study, we evaluate the early response of colon carcinoma to the chemotherapeutic drug, lipo-Dox, in C26 murine colorectal carcinoma-bearing mice by 18F-FDG and 18F-FLT. The male BALB/c mice were bilaterally inoculated with 1 × 105 and 1 × 106 C26 tumor cells per flank. Mice were intravenously treated with 10 mg/kg lipo-Dox at day 8 after 18F-FDG and 18F-FLT imaging. The biodistribution of 18F-FDG and 18F-FLT were followed by the microPET imaging at day 9. For the quantitative measurement of microPET imaging at day 9, 18F-FLT was superior to
18F-FDG for early detection of tumor response to Lipo-DOX at various tumor sizes (P < 0.05). The data of biodistribution showed similar results with those from the quantification of SUV (standard uptake value) by microPET imaging. The study indicates that 18F-FLT/microPET is a useful imaging modality for early detection of chemotherapy in the colorectal mouse model.
3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) is a tracer used to assess cell proliferation in vivo. The aim of the study was to use [18F]FLT positron emission tomography (PET) to study non-invasively early anti-proliferative effects of the experimental chemotherapeutic agent TP202377 in both sensitive and resistant tumors.
Xenografts in mice from 3 human cancer cell lines were used: the TP202377 sensitive A2780 ovary cancer cell line (n = 8–16 tumors/group), the induced resistant A2780/Top216 cell line (n = 8–12 tumors/group) and the natural resistant SW620 colon cancer cell line (n = 10 tumors/group). In vivo uptake of [18F]FLT was studied at baseline and repeated 6 hours, Day 1, and Day 6 after TP202377 treatment (40 mg/kg i.v.) was initiated. Tracer uptake was quantified using small animal PET/CT.
TP202377 (40 mg/kg at 0 hours) caused growth inhibition at Day 6 in the sensitive A2780 tumor model compared to the control group (P<0.001). In the A2780 tumor model TP202377 treatment caused significant decrease in uptake of [18F]FLT at 6 hours (-46%; P<0.001) and Day 1 (-44%; P<0.001) after treatment start compared to baseline uptake. At Day 6 uptake was comparable to baseline. Treatment with TP202377 did not influence tumor growth or [18F]FLT uptake in the resistant A2780/Top216 and SW620 tumor models. In all control groups uptake of [18F]FLT did not change. Ki67 gene expression paralleled [18F]FLT uptake.
Treatment of A2780 xenografts in mice with TP202377 (single dose i.v.) caused a significant decrease in cell proliferation assessed by [18F]FLT PET after 6 hours. Inhibition persisted at Day 1; however, cell proliferation had returned to baseline at Day 6. In the resistant A2780/Top216 and SW620 tumor models uptake of [18F]FLT did not change after treatment. With [18F]FLT PET it was possible to distinguish non-invasively between sensitive and resistant tumors already 6 hours after treatment initiation.
Considering the width and importance of using Multicellular Tumor Spheroids (MTS) in oncology research, size determination of MTSs by an accurate and fast method is essential. In the present study an effective, fast and semi-automated method, SASDM, was developed to determinate the size of MTSs. The method was applied and tested in MTSs of three different cell-lines. Frozen section autoradiography and Hemotoxylin Eosin (H&E) staining was used for further confirmation.
SASDM was shown to be effective, user-friendly, and time efficient, and to be more precise than the traditional methods and it was applicable for MTSs of different cell-lines. Furthermore, the results of image analysis showed high correspondence to the results of autoradiography and staining.
The combination of assessment of metabolic condition and image analysis in MTSs provides a good model to evaluate the effect of various anti-cancer treatments.
Molecular imaging biomarkers of proliferation hold great promise for quantifying response to personalized medicine. One such approach utilizes the positron emission tomography (PET) tracer 3′-deoxy-3′ [18F]-fluorothymidine ([18F]FLT), an investigational agent whose uptake reflects thymidine salvage-dependent DNA synthesis. The goal of this study was to evaluate [18F]FLT-PET in the setting of Ménétrier’s disease (MD), a rare, premalignant hyperproliferative disorder of the stomach treatable with cetuximab therapy. Over 15 months, a patient with confirmed MD underwent cetuximab therapy and was followed with sequential [18F]FLT-PET. For comparison to MD, an [18F]FLT-PET study was conducted in another patient to quantify uptake in a normal stomach.Prior to cetuximab therapy, stomach tissue in MD was easily visualized with [18F]FLT-PET, with pre-treatment uptake levels exceeding normal stomach uptake by approximately 4-fold. Diminished [18F]FLT-PET in MD was observed following the initial and subsequent doses of cetuximab and correlated with clinical resolution of the disease. To our knowledge, this study reports the first clinical use of [18F]FLT-PET to assess proliferation in a premalignant disorder. We illustrate that the extent of MD involvement throughout the stomach could be easily visualized using [18F]FLT-PET, and that response to cetuximab could be followed quantitatively and non-invasively in sequential [18F]FLT-PET studies. Thus, [18F]FLT-PET appears to have potential to monitor response to treatment in this and potentially other hyperproliferative disorders.
FLT; proliferation; treatment response; EGFR; Ménétrier’s disease
Positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG) has been established as a useful tool in the management of patients with non-small cell lung cancer and promises to be as valuable in the clinical management of other cancers. PET imaging with FDG allows the assessment of tumor glucose metabolism in vivo; however, a number of other PET tracers are being used in oncologic research to assess changes in other cellular processes associated with malignant transformation of the cell. [11C]-Labeled methionine and choline are being used to assess changes in cell membrane synthesis; however, small studies have not shown the added information from these tracers to be clinically useful. DNA synthesis can be assessed by measuring the uptake of the thymidine analog 3′-deoxy-3′-[18F]fluorothymidine, which may be more specific for evaluating malignancy without the problem of false-positive results from inflammatory lesions, as seen with FDG. Tumor hypoxia imaging with copper-labeled diacetyl-bis(N(4)-methylthiosemicarbazone) or [18F]fluoromisonidazole may provide a better method of predicting which tumors will respond best to conventional therapy. The role of PET will continue to evolve with further clinical studies using these and other new tracers.
FDG; cell proliferation; cell hypoxia
This study investigates whether a histone deacetylase subtype 6 (HDAC6) inhibitor could be used in the treatment of solid tumours.
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 [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to detect therapy response.
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 [18F]FLT uptake by 1.7-fold at 48 h, suggesting that molecular imaging could provide value in future studies of this compound.
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.
tumour; HDAC; PET; biomarkers; pharmacology
Advanced endometrial cancer often shows resistance to clinical chemotherapy although potencies of anticancer drugs in vitro are promising. The disparity suggests that in vivo microenvironments are not recapitulated by in vitro models used for preclinical testing. However, spheroids replicate some important properties of tumours in vivo. Therefore, for the first time, we compared effects of doxorubicin and cisplatin on 3D multicellular structures and 2D cell monolayers of endometrial cancer cells.
3D multicellular structures were generated by culturing cancer cells on non-adherent surfaces; and for comparison cell monolayers were cultured on adherent culture plates. Ishikawa, RL95-2, and KLE cell lines were studied. Morphologies of 3D multicellular structures were examined. After 48 hours treatment with anticancer drugs, apoptosis, proliferation, glucose metabolism and vascular endothelial growth factor (VEGF) were analysed. Immunostaining of PCNA, Glut-1, p-Erk1/2, SOD-1 and p-Akt1/2/3 was also performed.
Distinct 3D multicellular morphologies were formed by three different endometrial cancer cell lines. Doxorubicin induced less apoptosis in 3D multicellular structures of high grade cancer cells (RL95-2 and KLE cell lines) than in cell monolayers. Parallel alterations in Erk1/2 phosphorylation and cell proliferation might suggest they were linked and again doxorubicin had less effect on 3D multicellular structures than cell monolayers. On the other hand, there was no correlation between altered glucose metabolism and proliferation. The responses depended on cancer cell lines and were apparently not mediated by altered Glut-1 levels. The level of SOD-1 was high in 3D cell cultures. The effects on VEGF secretion were various and cancer cell line dependent. Importantly, both doxorubicin and cisplatin had selective paradoxical stimulatory effects on VEGF secretion. The microenvironment within 3D multicellular structures sustained Akt phosphorylation, consistent with it having a role in anchorage-independent pathways.
The cancer cells responded to microenvironments in a distinctive manner. 3D multicellular structures exhibited greater resistance to the agents than 2D monolayers, and the differences between the culture formats were dependent on cancer cell lines. The effects of anticancer drugs on the intracellular mediators were not similar in 3D and 2D cultures. Therefore, using 3D cell models may have a significant impact on conclusions derived from screening drugs for endometrial carcinomas.
Paclitaxel (Taxol), an anti-cancer drug derived from Taxus species, was tested for its anti-migrational, anti-invasive and anti-proliferative effect on two human glioma cell lines (GaMg and D-54Mg) grown as multicellular tumour spheroids. In addition, the direct effect of paclitaxel on glioma cells was studied using flow cytometry and scanning confocal microscopy. Both cell lines showed a dose-dependent growth and migratory response to paclitaxel. The GaMg cells were found to be 5-10 times more sensitive to paclitaxel than D-54Mg cells. Paclitaxel also proved to be remarkably effective in preventing invasion in a co-culture system in which tumour spheroids were confronted with fetal rat brain cell aggregates. Control experiments with Cremophor EL (the solvent of paclitaxel for clinical use) in this study showed no effect on tumour cell migration, cell proliferation or cell invasion. Scanning confocal microscopy of both cell lines showed an extensive random organization of the microtubules in the cytoplasm. After paclitaxel exposure, the GaMg and the D-54Mg cells exhibited a fragmentation of the nuclear material, indicating a possible induction of apoptosis. In line with this, flow cytometric DNA histograms showed an accumulation of cells in the G2/M phase of the cell cycle after 24 h of paclitaxel exposure. After 48 h, a deterioration of the DNA histograms was observed indicating nuclear fragmentation.
Rapid multi-tracer PET aims to image two or more tracers in a single scan, simultaneously characterizing multiple aspects of physiology and function without the need for repeat imaging visits. Using dynamic imaging with staggered injections, constraints on the kinetic behavior of each tracer are applied to recover individual-tracer measures from the multi-tracer PET signal. The ability to rapidly and reliably image both 18F-fluorodeoxyglucose (FDG) and 18F-fluorothymidine (FLT) would provide complementary measures of tumor metabolism and proliferative activity, with important applications in guiding oncologic treatment decisions and assessing response. However, this tracer combination presents one of the most challenging dual-tracer signal-separation problems—both tracers have the same radioactive half-life, and the injection delay is short relative to the half-life and tracer kinetics. This work investigates techniques for single-scan dual-tracer FLT+FDG PET tumor imaging, characterizing the performance of recovering static and dynamic imaging measures for each tracer from dual-tracer datasets. Simulation studies were performed to characterize dual-tracer signal-separation performance for imaging protocols with both injection orders and injection delays of 10–60 min. Better performance was observed when FLT was administered first, and longer delays before administration of FDG provided more robust signal-separation and recovery of the single-tracer imaging measures. An injection delay of 30 min led to good recovery (R > 0.96) of static image values (e.g. SUV), Knet, and K1 as compared to values from separate, single-tracer time-activity curves. Recovery of higher order rate parameters (k2, k3) was less robust, indicating that information regarding these parameters was harder to recover in the presence of statistical noise and dual-tracer effects. Performance of the dual-tracer FLT(0 min)+FDG(32 min) technique was further evaluated using PET/CT imaging studies in five patients with primary brain tumors where the data from separate scans of each tracer were combined to synthesize dual-tracer scans with known single-tracer components; results demonstrated similar dual-tracer signal recovery performance. We conclude that rapid dual-tracer FLT+FDG tumor imaging is feasible and can provide quantitative tumor imaging measures comparable to those from conventional separate-scan imaging.
Docetaxel is an important chemotherapeutic agent used for the treatment of several cancer types. As radiolabelled anticancer agents provide a potential means for personalized treatment planning, docetaxel was labelled with the positron emitter 11C. Non-invasive measurements of [11C]docetaxel uptake in organs and tumours may provide additional information on pharmacokinetics and pharmacodynamics of the drug docetaxel. The purpose of the present study was to determine the biodistribution and radiation absorbed dose of [11C]docetaxel in humans.
Biodistribution of [11C]docetaxel was measured in seven patients (five men and two women) with solid tumours using PET/CT. Venous blood samples were collected to measure activity in blood and plasma. Regions of interest (ROI) for various source organs were defined on PET (high [11C]docetaxel uptake) or CT (low [11C]docetaxel uptake). ROI data were used to generate time-activity curves and to calculate percentage injected dose and residence times. Radiation absorbed doses were calculated according to the MIRD method using OLINDA/EXM 1.0 software.
Gall bladder and liver demonstrated high [11C]docetaxel uptake, whilst uptake in brain and normal lung was low. The percentage injected dose at 1 h in the liver was 47 ± 9%. [11C]docetaxel was rapidly cleared from plasma and no radiolabelled metabolites were detected. [11C]docetaxel uptake in tumours was moderate and highly variable between tumours.
The effective dose of [11C]docetaxel was 4.7 µSv/MBq. As uptake in normal lung is low, [11C]docetaxel may be a promising tracer for tumours in the thoracic region.
[11C]docetaxel; Biodistribution; Radiation dose; Cancer; PET/CT
We have previously developed 11C-erlotinib as a new positron emission tomography (PET) tracer and shown that it accumulates in epidermal growth factor receptor (EGFR)-positive lung cancer xenografts in mice. Here, we present a study in patients with non-small cell lung cancer (NSCLC) investigating the feasibility of 11C-erlotinib PET as a potential method for the identification of lung tumours accumulating erlotinib.
Thirteen patients with NSCLC destined for erlotinib treatment were examined by contrast-enhanced computed tomography (CT), 11C-erlotinib PET/low-dose CT and 18F-fluoro-2-deoxy--glucose (18F-FDG) PET/low-dose CT before start of the erlotinib treatment. After 12 weeks treatment, they were examined by 18F-FDG PET/contrast-enhanced CT for the assessment of clinical response.
Of the 13 patients included, 4 accumulated 11C-erlotinib in one or more of their lung tumours or lymph-node metastases. Moreover, 11C-erlotinib PET/CT identified lesions that were not visible on 18F-FDG PET/CT. Of the four patients with accumulation of 11C-erlotinib, one died before follow-up, whereas the other three showed a positive response to erlotinib treatment. Three of the nine patients with no accumulation died before follow-up, four showed progressive disease while two had stable disease after 12 weeks of treatment.
Our data show a potential for 11C-erlotinib PET/CT for visualizing NSCLC lung tumours, including lymph nodes not identified by 18F-FDG PET/CT. Large clinical studies are now needed to explore to which extent pre-treatment 11C-erlotinib PET/CT can predict erlotinib treatment response.
erlotinib; EGFR; tarceva; lung cancer; PET imaging
Three-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening.
Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids.
IC50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.
The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.
The purpose of this study was to investigate the kinetic behavior of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) before and early after initiation of chemoradiation therapy in patients with squamous cell head and neck cancer.
A total of 8 patients with head and neck cancer underwent 18F-FLT PET scans (7 patients at baseline and after 5 d [10 Gy] of radiation therapy given with concomitant chemotherapy and 1 patient only at baseline). Dynamic PET images were obtained with concurrent arterial or venous blood sampling. Kinetic parameters including the flux constant of 18F-FLT based on compartmental analysis (K-FLT), the Patlak influx constant (K-Patlak), and standardized uptake value (SUV) were calculated for the primary tumor and 18F-FLT–avid cervical lymph nodes for all scans.
Mean pretreatment values of uptake for the primary tumor and cervical nodes were 0.075 ± 0.006 min−1, 0.042 ± 0.004 min−1, and 3.4 ± 0.5 (mean ± SD) for K-FLT, K-Patlak, and SUV, respectively. After 10 Gy of radiation therapy, these values were 0.040 ± 0.01 min−1, 0.018 ± 0.016 min−1, and 1.8 ± 1.1 for K-FLT, K-Patlak, and SUV, respectively. For all lesions seen on pretherapy and midtherapy scans, the correlation was 0.90 between K-FLT and K-Patlak, 0.91 between K-FLT and SUV, and 0.99 between K-Patlak and SUV.
The initial 18F-FLT uptake and change early after treatment in squamous head and neck tumors can be adequately characterized with SUV obtained at 45–60 min, which demonstrates excellent correlation with influx parameters obtained from compartmental and Patlak analyses.
3′-deoxy-3′-18F-fluorothymidine; 18F-FLT; head and neck cancer
In previous clinical Positron Emission Tomography (PET) studies novel approaches for application of Principal Component Analysis (PCA) on dynamic PET images such as Masked Volume Wise PCA (MVW-PCA) have been introduced. MVW-PCA was shown to be a feasible multivariate analysis technique, which, without modeling assumptions, could extract and separate organs and tissues with different kinetic behaviors into different principal components (MVW-PCs) and improve the image quality.
In this study, MVW-PCA was applied to 14 dynamic 11C-metomidate-PET (MTO-PET) examinations of 7 patients with small adrenocortical tumours. MTO-PET was performed before and 3 days after starting per oral cortisone treatment. The whole dataset, reconstructed by filtered back projection (FBP) 0–45 minutes after the tracer injection, was used to study the tracer pharmacokinetics.
Early, intermediate and late pharmacokinetic phases could be isolated in this manner. The MVW-PC1 images correlated well to the conventionally summed image data (15–45 minutes) but the image noise in the former was considerably lower. PET measurements performed by defining "hot spot" regions of interest (ROIs) comprising 4 contiguous pixels with the highest radioactivity concentration showed a trend towards higher SUVs when the ROIs were outlined in the MVW-PC1 component than in the summed images. Time activity curves derived from "50% cut-off" ROIs based on an isocontour function whereby the pixels with SUVs between 50 to 100% of the highest radioactivity concentration were delineated, showed a significant decrease of the SUVs in normal adrenal glands and in adrenocortical adenomas after cortisone treatment.
In addition to the clear decrease in image noise and the improved contrast between different structures with MVW-PCA, the results indicate that the definition of ROIs may be more accurate and precise in MVW-PC1 images than in conventional summed images. This might improve the precision of PET measurements, for instance in therapy monitoring as well as for delineation of the tumour in radiation therapy planning.
Positron emission tomography (PET) imaging has become a useful tool for assessing early biologic response to cancer therapy and may be particularly useful in the development of new cancer therapeutics. RAF265, a novel B-Raf/vascular endothelial growth factor receptor-2 inhibitor, was evaluated in the preclinical setting for its ability to inhibit the uptake of PET tracers in the A375M(B-RafV600E) human melanoma cell line. RAF265 inhibited 2-deoxy-2-[18F]fluoro-d-glucose (FDG) accumulation in cell culture at 28 hours in a dose-dependent manner. RAF265 also inhibited FDG accumulation in tumor xenografts after 1 day of drug treatment. This decrease persisted for the remaining 2 weeks of treatment. DNA microarray analysis of treated tumor xenografts revealed significantly decreased expression of genes regulating glucose and thymidine metabolism and revealed changes in apoptotic genes, suggesting that the imaging tracers FDG, 3-deoxy-3-[18F]fluorothymidine, and annexin V could serve as potential imaging biomarkers for RAF265 therapy monitoring. We concluded that RAF265 is highly efficacious in this xenograft model of human melanoma and decreases glucose metabolism as measured by DNA microarray analysis, cell culture assays, and small animal FDG PET scans as early as 1 day after treatment. Our results support the use of FDG PET in clinical trials with RAF265 to assess early tumor response. DNA microarray analysis and small animal PET studies may be used as complementary technologies in drug development. DNA microarray analysis allows for analysis of drug effects on multiple pathways linked to cancer and can suggest corresponding imaging tracers for further analysis as biomarkers of tumor response.
With the dismal prognosis for malignant glioma patients, survival predictions become key elements in patient management. This study compares the value of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) PET and MRI for early outcome predictions in patients with recurrent malignant glioma on bevacizumab therapy.
Thirty patients treated with bevacizumab combination therapy underwent 18F-FLT PET immediately before and at 2 and 6 wk after the start of treatment. A metabolic treatment response was defined as a decrease of equal to or greater than 25% in tumor 18F-FLT uptake (standardized uptake values) from baseline using receiver-operating-characteristic analysis. MRI treatment response was assessed at 6 wk according to the Response Assessment in Neurooncology criteria. 18F-FLT responses at different times were compared with MRI response and correlated with progression-free survival and overall survival using Kaplan–Meier analysis. Metabolic response based on 18F-FLT was further compared with other outcome predictors using Cox regression analysis.
Early and late changes in tumor 18F-FLT uptake were more predictive of overall survival than MRI criteria (P < 0.001 and P = 0.01, respectively). 18F-FLT uptake changes were also predictive of progression-free survival (P < 0.001). The median overall survival for responders was 3.3 times longer than for nonresponders based on 18F-FLT PET criteria (12.5 vs. 3.8 mo, P < 0.001) but only 1.4 times longer using MRI assessment (12.9 vs. 9.0 mo, P = 0.05). On the basis of the 6-wk 18F-FLT PET response, there were 16 responders (53%) and 14 nonresponders (47%), whereas MRI identified 9 responders (7 partial response, 2 complete response, 31%) and 20 nonresponders (13 stable disease, 7 progressive disease, 69%). In 7 of the 8 discrepant cases between MRI and PET, 18F-FLT PET was able to demonstrate response earlier than MRI. Among various outcome predictors, multivariate analysis identified 18F-FLT PET changes at 6 wk as the strongest independent survival predictor (P < 0.001; hazard ratio, 10.051).
Changes in tumor 18F-FLT uptake were highly predictive of progression-free and overall survival in patients with recurrent malignant glioma on bevacizumab therapy. 18F-FLT PET seems to be more predictive than MRI for early treatment response.
18F-FLT PET; bevacizumab; malignant glioma; survival prediction
Positron Emission Tomography (PET) is a nuclear medicine imaging technique that is widely used in early detection and treatment follow up of many diseases, including cancer. This modality requires positron-emitting isotope labeled biomolecules, which are synthesized prior to perform imaging studies. Fluorine-18 is one of the several isotopes of fluorine that is routinely used in radiolabeling of biomolecules for PET; because of its positron emitting property and favorable half-life of 109.8 min. The biologically active molecule most commonly used for PET is 2-deoxy-2-18F-fluoro-β-D-glucose (18F-FDG), an analogue of glucose, for early detection of tumors. The concentrations of tracer accumulation (PET image) demonstrate the metabolic activity of tissues in terms of regional glucose metabolism and accumulation. Other tracers are also used in PET to image the tissue concentration. In this review, information on fluorination and radiofluorination reactions, radiofluorinating agents, and radiolabeling of various compounds and their application in PET imaging is presented.
Fluorine-18; positron emission tomography (PET); PET radiopharmaceuticals
APO866 is a new anti-tumor compound inhibiting nicotinamide phosphoribosyltransferase (NAMPT). APO866 has an anti-tumor effect in several pre-clinical tumor models and is currently in several clinical phase II studies. 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) is a tracer used to assess cell proliferation in vivo. The aim of this study was non-invasively to study effect of APO866 treatment on [18F]FLT and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake.
In vivo uptake of [18F]FLT and [18F]FDG in human ovary cancer xenografts in mice (A2780) was studied at various time points after APO866 treatment. Baseline [18F]FLT or [18F]FDG scans were made before treatment and repeated after 24 hours, 48 hours and 7 days. Tumor volume was followed with computed tomography (CT). Tracer uptake was quantified using small animal PET/CT. One hour after iv injection of tracer, static PET scans were performed. Imaging results were compared with Ki67 immunohistochemistry.
Tumors treated with APO866 had volumes that were 114% (24 h), 128% (48 h) and 130% (Day 7) relative to baseline volumes at Day 0. In the control group tumor volumes were 118% (24 h), 145% (48 h) and 339% (Day 7) relative to baseline volumes Day 0. Tumor volume between the treatment and control group was significantly different at Day 7 (P = 0.001). Compared to baseline, [18F]FLT SUVmax was significantly different at 24 h (P<0.001), 48 h (P<0.001) and Day 7 (P<0.001) in the APO866 group. Compared to baseline, [18F]FDG SUVmax was significantly different at Day 7 (P = 0.005) in the APO866 group.
APO866 treatment caused a significant decrease in [18F]FLT uptake 24 and 48 hours after treatment initiation. The early reductions in tumor cell proliferation preceded decrease in tumor volume. The results show the possibility to use [18F]FLT and [18F]FDG to image treatment effect early following treatment with APO866 in future clinical studies.
Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to multidrug resistance in tumours. However, the physiological role of P-gp in tumours growing as multicellular spheroids is not well understood. Recent evidence suggests that P-gp activity may be modulated by cellular components such as membrane proteins, membrane-anchoring proteins or membrane-lipid composition. Since, multicellular spheroids studies have evidenced alterations in numerous cellular components, including those related to the plasma membrane function, result plausible that some of these changes might modulate P-gp function and be responsible for the acquisition of multicellular drug resistance. In the present study, we asked if a human lung cancer cell line (INER-51) grown as multicellular spheroids can modify the P-gp activity to decrease the levels of doxorubicin (DXR) retained and increase their drug resistance.
Our results showed that INER-51 spheroids retain 3-folds lower doxorubicin than the same cells as monolayers however; differences in retention were not observed when the P-gp substrate Rho-123 was used. Interestingly, neither the use of the P-gp-modulating agent cyclosporin-A (Cs-A) nor a decrease in ATP-pools were able to increase DXR retention in the multicellular spheroids. Only the lack of P-gp expression throughout the pharmacological selection of a P-gp negative (P-gpneg) mutant clone (PSC-1) derived from INER-51 cells, allow increase of DXR retention in spheroids.
Thus, multicellular arrangement appears to alter the P-gp activity to maintain lower levels of DXR. However, the non expression of P-gp by cells forming multicellular spheroids has only a minor impact in the resistance to chemotherapeutic agents.
P-Glycoprotein; Multicellular spheroids; Multicellular drug resistance; NSCLC
Evaluation and comparison of 3’-[18F]-fluoro-3’-deoxy-L-thymidine (FLT) and 2-[18F]-fluoro-2-deoxyglucose (FDG)-PET to monitor early response following both cyclophosphamide and temsirolimus treatment in a mouse model of Burkitt lymphoma.
Daudi xenograft mice were treated with either cyclophosphamide or temsirolimus and imaged with FLT-PET and FDG-PET on appropriate days post therapy inititiation. Immunohistochemical (IHC) studies (H&E, TUNEL, CD20, PCNA and ki-67) and DNA flow cytometry studies were performed.
FDG tumor uptake decreased immediately after cyclophosphamide treatment while FLT-PET showed only a late and less pronounced decrease. A fast induction of apoptosis was observed together with an early accumulation of cells in the S-phase of the cell cycle, suggesting DNA repair. Temsirolimus treatment reduced both FDG and FLT tumor uptake immediately after therapy and resulted in a fast induction of apoptosis and G0-G1 phase accumulation.
FLT response was less distinct than FDG response and may be controlled by DNA repair early after cyclophosphamide. Nevertheless, FLT-PET was able to reflect decreased proliferation following temsirolimus.
FDG-PET; FLT-PET; Burkitt lymphoma; cyclophosphamide; mTOR inhibition; therapy response