Monitoring Tumor Response after Liposomal Doxorubicin in Combination with Liposomal Vinorelbine Treatment Using 3'-Deoxy-3'-[18F]Fluorothymidine PET.

PURPOSE: Surgical resection is the standard treatment for localized colorectal cancer, which is the most common type of gastrointestinal cancer. However, over 40 % cases are diagnosed metastasized and apparently inoperable. Systemic chemotherapy provides an alternative to these patients. This study aims to evaluate the therapeutic potential of liposomal doxorubicin (lipoDox) in combination with liposomal vinorelbine (lipoVNB) in a CT-26 colon carcinoma-bearing mouse model. PROCEDURES: The in vitro cytotoxicity of Dox and VNB on CT-26 cancer cells was determined by MTT and colony formation assays. Mice were subcutaneously inoculated with 2 × 105 of CT-26 cells in the right hind flank. When tumor size reached 200 ± 50 mm3, mice were assigned to receive different treatment protocols. The pharmacokinetics, micro single-photon emission computed tomography/x-ray computed tomography imaging, biodistribution, and immunohistochemical staining studies were performed to survey the therapeutic efficacy of each regimen. RESULTS: Based on the results of pharmacokinetic study, co-administration of lipoDox and lipoVNB did not affect their individual systemic distribution, while lipoDox retained longer in blood than lipoVNB did. Superior tumor growth retardation was observed in the group received lipoDox plus lipoVNB administration (1 mg/kg each, namely D1V1) than those injected with lipoDox plus VNB (1 mg/kg each, namely D1fV1). No severe side effects were detected in each group. The tumor-to-muscle ratio (T/M) derived from 3'-dexoy-3'-[18F]fluorothymidine ([18F]FLT) micro positron emission tomography (PET) images of D1V1- and D1fV1-treated mice and the controls on day 7 was 6.88 ± 0.54, 7.50 ± 0.84, and 9.87 ± 0.73, respectively, suggesting that D1V1 is a more efficacious regimen against CT-26 xenografts. The results of proliferating cell nuclear antigen (PCNA) immunohistochemical staining were consistent with those findings obtained from [18F]FLT microPET imaging. CONCLUSION: This study demonstrated that lipoDox in combination with lipoVNB was more efficacious than clinically used regimen, lipoDox plus VNB, in the treatment of colon carcinoma and [18F]FLT-PET is a promising approach in monitoring the treatment outcome at early stage.

Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine.

BACKGROUND: The enzyme-prodrug system is considered a promising tool for tumor treatment when conjugated with a targeting molecule. The asparagine-glycine-arginine (NGR) motif is a developing and interesting targeting peptide that could specifically bind to aminopeptidase N (APN), which is an NGR receptor expressed on the cell membrane of angiogenic endothelial cells and a number of tumor cells within the tumor tissues. The objective of this study was to develop a novel targeted enzyme-prodrug system using 5-fluorocytosine (5-FC) and an NGR-containing peptide fused with yeast cytosine deaminase (yCD), i.e. CNGRC-yCD fusion protein, to target APN-expressing cells within the tumor tissues and to convert 5-FC into 5-fluorouracil (5-FU) to kill tumors. RESULTS: Both yCD and CNGRC-yCD proteins were cloned into the pET28a vector and expressed by an Escherichia coli host. Both yCD and CNGRC-yCD proteins were purified and the yields were approximately 20 mg/L with over 95 % purity. The binding assay demonstrated that the CNGRC-yCD fusion protein had specific binding affinity toward purified APN recombinant protein and high-APN-expressing cells, including human endothelial cells (HUVECs) and various types of human tumor cell lines, but not low-APN-expressing tumor cell lines. Moreover, the enzyme activity and cell viability assay showed that the CNGRC-yCD fusion protein could effectively convert 5-FC into 5-FU and resulted in significant cell death in both high-APN-expressing tumor cells and HUVECs. CONCLUSIONS: This study successfully constructs a new targeting enzyme-prodrug system, CNGRC-yCD fusion protein/5-FC. Systematic experiments demonstrated that the CNGRC-yCD protein retained both the APN-binding affinity of NGR and the enzyme activity of yCD to convert 5-FC into 5-FU. The combined treatment of the CNGRC-yCD protein with 5-FC resulted in the significantly increased cell death of high-APN-expressing cells as compared to that of low-APN-expressing cells.

Over-expression of cofilin-1 suppressed growth and invasion of cancer cells is associated with up-regulation of let-7 microRNA.

Cofilin-1, a non-muscle isoform of actin regulatory protein that belongs to the actin-depolymerizing factor (ADF)/cofilin family is known to affect cancer development. Previously, we found that over-expression of cofilin-1 suppressed the growth and invasion of human non-small cell lung cancer (NSCLC) cells in vitro. In this study, we further investigated whether over-expression of cofilin-1 can suppress tumor growth in vivo, and performed a microRNA array analysis to better understand whether specific microRNA would be involved in this event. The results showed that over-expression of cofilin-1 suppressed NSCLC tumor growth using the xenograft tumor model with the non-invasive reporter gene imaging modalities. Additionally, cell motility and invasion were significantly suppressed by over-expressed cofilin-1, and down-regulation of matrix metalloproteinase (MMPs) -1 and -3 was concomitantly detected. According to the microRNA array analysis, the let-7 family, particularly let-7b and let-7e, were apparently up-regulated among 248 microRNAs that were affected after over-expression of cofilin-1 up to 7 days. Knockdown of let-7b or let-7e using chemical locked nucleic acid (LNA) could recover the growth rate and the invasion of cofilin-1 over-expressing cells. Next, the expression of c-myc, LIN28 and Twist-1 proteins known to regulate let-7 were analyzed in cofilin-1 over-expressing cells, and Twist-1 was significantly suppressed under this condition. Up-regulation of let-7 microRNA by over-expressed cofilin-1 could be eliminated by co-transfected Twist-1 cDNA. Taken together, current data suggest that let-7 microRNA would be involved in over-expression of cofilin-1 mediated tumor suppression in vitro and in vivo.

Monitoring Tumor Response After Histone Deacetylase Inhibitor Treatment Using 3'-Deoxy-3'-[18F]-fluorothymidine PET.

PURPOSE: This study employed 3'-deoxy-3'-[(18)F]-fluorothymidine ([(18)F]FLT) microPET scanning to assess the treatment response of histone deacetylase inhibitors (HDACi), e.g., N1-hydroxy-N8-phenyloctanediamide (SAHA) and its iodinated derivative ISAHA, in a hepatoma mouse model. PROCEDURES: The in vitro cytotoxicity of HDACi in various hepatoma cell lines was determined by MTT assay and flow cytometry. ISAHA and SAHA were used to treat HepG2 hepatoma xenograft-bearing mice. The treatment responses were characterized in terms of tumor burden, microPET imaging, and immunohistochemical staining of tumor sections. RESULTS: ISAHA effectively inhibited HepG2 hepatoma cell survival and tumor growth. A significantly reduced tumor uptake during HDACi treatment was noticed in [(18)F]FLT microPET imaging, which was consistent with the findings in immunohistochemical staining. CONCLUSIONS: ISAHA can suppress tumor cell proliferation both in vitro and in vivo. [(18)F]FLT PET is a promising modality for evaluating the in vivo therapeutic efficacy of HDACi at the early stage of treatment.

Pulsed-focused ultrasound enhances boron drug accumulation in a human head and neck cancer xenograft-bearing mouse model.

PURPOSE: This study aims to demonstrate that pulsed high-intensity focused ultrasound (pulsed-HIFU) may enhance the fructose-conjugated 4-borono-L-phenylalanine (BPA-Fr) accumulation in tumor lesion using (18)F-FBPA-Fr microPET scans. PROCEDURES: To the mice bearing orthotopic SASC03 human tongue squamous carcinoma xenograft, a 2-min pulsed-HIFU was applied to tumor. Immediately after pulsed-HIFU treatment, (18)F-FBPA-Fr was intravenously injected, and biological characterizations including microPET imaging and biodistribution were conducted. RESULTS: Both biodistribution studies and microPET imaging performed after intravenous injection of (18)F-FBPA-Fr revealed higher tumor uptake in HIFU-treated mice than that of the control. CD31 and Ki-67 histochemical staining of tumor sections and H&E staining of nearby normal tissues revealed no significant difference between the pulsed-HIFU-treated mice and the control. CONCLUSION: This study demonstrated that pulsed-HIFU was beneficial to the accumulation of boron drug in the head and neck tumor lesion and may enhance the therapeutic efficacy of clinical BNCT.

Monitoring tumor response with [18F]FMAU in a sarcoma-bearing mouse model after liposomal vinorelbine treatment.

OBJECTIVE: Previous studies have shown that the accumulation level of FMAU in tumor is proportional to its proliferation rate. This study demonstrated that 2'-deoxy-2'-[(18)F]fluoro-ß-d-arabinofuranosyluracil ([(18)F]FMAU) is a promising PET probe for noninvasively monitoring the therapeutic efficacy of 6% PEGylated liposomal vinorelbine (lipo-VNB) in a subcutaneous murine NG4TL4 sarcoma mouse model. METHODS: Female syngenic FVB/N mice were inoculated with NG4TL4 cells in the right flank. After tumor size reached 150 ± 50 mm(3) (day 0), lipo-VNB (5mg/kg) was intravenously administered on days 0, 3 and 6. To monitor the therapeutic efficacy of lipo-VNB, [(18)F]FMAU PET was employed to evaluate the proliferation rate of tumor, and it was compared with that observed from [(18)F]FDG/[(18)F]fluoroacetate PET. The expression of proliferating cell nuclear antigen (PCNA) in tumor during treatment was determined by semiquantitative analysis of immunohistochemical staining. RESULTS: A significant inhibition (p<0.001) in tumor growth was observed on day 3 after a single dose treatment. The tumor-to-muscle ratio (T/M) derived from [(18)F]FMAU-PET images of lipo-VNB-treated group declined from 2.33 ± 0.16 to 1.26 ± 0.03 after three doses of treatment, while that of the control remained steady. The retarded proliferation rate of lipo-VNB-treated sarcoma was confirmed by PCNA immunohistochemistry staining. However, both [(18)F]FDG and [(18)F]fluoroacetate microPET imaging did not show significant difference in T/M between the therapeutic and the control groups throughout the entire experimental period. CONCLUSION: Lipo-VNB can effectively impede the growth of NG4TL4 sarcoma. [(18)F]FMAU PET is an appropriate modality for early monitoring of the tumor response during the treatment course of lipo-VNB.

Evaluation of (131/123)I-5-iodo-2'-deoxycytidine as a novel proliferation probe in a tumor mouse model.

This study evaluated a radioiodinated deoxycytidine analog, (131)I-5-iodo-2'-deoxycytidine ([(131)I]ICdR), as a novel proliferation probe and compared it with (131)I-5-iodo-2'-deoxyuridine ([(131)I]IUdR) in a NG4TL4 sarcoma-bearing mouse model. As an imaging agent, the biological characteristics of [(123)I]IUdR is not satisfactory due to its metabolic instability and short biological half-life in vivo. With [(123)I]ICdR/SPECT it was possible to clearly delineate the tumor lesion at 1h post-injection (tumor-to-muscle ratio 7.74) in tumor-bearing mice. The results of biodistribution were consistent with those observed in scintigraphic imaging. This study demonstrated that [(131)I]ICdR is a more promising SPECT probe than [(131)I]IUdR for imaging proliferation.

Monitoring tumor response with radiolabeled nucleoside analogs in a hepatoma-bearing mouse model early after doxisome(®) treatment.

PURPOSE: This study aims to demonstrate that 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) positron emission tomography (PET) is a promising modality for noninvasively monitoring the therapeutic efficacy of Doxisome(®) in a subcutaneous hepatoma mouse model. PROCEDURES: Male BALB/c nu/nu mice were inoculated with HepG2 hepatoma xenograft in the right flank. Doxisome(®) (5 mg/kg, three times a week for 2 weeks) was intravenously administrated for treatment. (18)F-FLT-microPET, biodistribution studies, and immunohistochemistry of Ki-67 were performed. RESULTS: A significant difference (p < 0.05) in tumor volume was observed on day 5 between treated and control groups. The tumor-to-muscle ratio derived from (18)F-FLT-PET and (123)I-ICdR-microSPECT images of Doxisome(®)-treated mice dropped from 12.55 ± 0.76 to 3.81 ± 0.31 and from 2.48 ± 0.42 to 1.59 ± 0.08 after a three-dose treatment, respectively, while that of the control group remained steady. The retarded proliferation rate of treated xenograft was confirmed by Ki-67 immunohistochemistry staining. CONCLUSIONS: This study clearly demonstrated that Doxisome(®) is an effective anti-cancer drug against the growth of HepG2 hepatoma and that (18)F-FLT-PET could provide early information of tumor response during treatment.

Evaluation of F-18-labeled 5-iodocytidine (18F-FIAC) as a new potential positron emission tomography probe for herpes simplex virus type 1 thymidine kinase imaging.

OBJECTIVE: Herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene in combination with radiolabeled nucleoside substrates is the most widely used reporter system. This study characterized 1-(2'-deoxy-2'-[(18)F]fluoro-ß-D-arabinofuranosyl)-5-iodocytosine ((18)F-FIAC) as a new potential positron emission tomography (PET) probe for HSV1-tk gene imaging and compared it with 2'-deoxy-2'-[(18)F]fluoro-5-iodo-1-ß-D-arabinofuranosyluracil ((18)F-FIAU) and 2'-deoxy-2'-[(18)F]fluoro-5-ethyl-1-ß-D-arabinofuranosyluracil((18)F-FEAU) (thymidine analogues) in an NG4TL4-WT/STK sarcoma-bearing mouse model. METHODS: A cellular uptake assay, biodistribution study, radioactive metabolites assay and microPET imaging of NG4TL4-WT/STK tumor-bearing mice post administration of (18)F-FIAC, (18)F-FIAU and (18)F-FEAU were conducted to characterize the biological properties of these tracers. RESULTS: Highly specific uptake of (18)F-FIAC, (18)F-FIAU and (18)F-FEAU in tk-transfected [tk(+)] cells was observed. The tk(+)-to-tk(-) cellular uptake ratio after a 2-h incubation was 66.6±25.1, 76.3±18.2 and 247.2±37.2, respectively. In biodistribution studies, (18)F-FIAC showed significant tk(+) tumor specificity (12.6; expressed as the tk(+)-to-tk(-) tumor uptake ratio at 2 h postinjection) comparable with (18)F-FIAU (15.8) but lower than (18)F-FEAU (48.0). The results of microPET imaging also revealed the highly specific accumulation of these three radioprobes in the NG4TL4-tk(+) tumor. CONCLUSION: Our findings suggested that the cytidine analogue (18)F-FIAC is a new potential PET probe for the imaging of HSV1-tk gene expression. (18)F-FIAC may be regarded as the prodrug of (18)F-FIAU in vivo.

Radiosynthesis of F-18 labeled cytidine analog 2'-fluoro-5-iodo-l-beta-d-arabinofuranosylcytosine ([(18)F]FIAC).

We reported the synthesis of 2'-deoxy-2'-[(18)F]fluoro-5-iodo-1-beta-d-arabinofuranosyl-5-iodo-cytosine ([(18)F]FIAC) with 15-20% radiochemical yield (decay corrected) in 3.5h. 2-deoxy-2-[(18)F]fluoro-1,3,5-tri-O-benzoyl-alpha-d-arabinofuranose was prepared following literature procedures with some modifications (yield>70%). The (18)F-fluorosugar was converted to 1-bromo-(18)F-fluorosugar, and then coupled with 5-iodocytocine silyl ether. A mixture of acetonitrile (ACN) and 1,2-dichloroethane (DCE) were employed to achieve optimum radiochemical yield and acceptable beta-anomer selectivity (alpha/beta=1/3). After hydrolyzed with sodium methoxide, the crude product was purified using HPLC to afford the beta-[(18)F]FIAC with high radiochemical purity (>or=98%).