Heightened Virulence of Yersinia Is Associated with Decreased Function of the YopJ Protein.

Despite the maintenance of YopP/J alleles throughout the human-pathogenic Yersinia lineage, the benefit of YopP/J-induced phagocyte death for Yersinia pathogenesis in animals is not obvious. To determine how the sequence divergence of YopP/J has impacted Yersinia virulence, we examined protein polymorphisms in this type III secreted effector protein across 17 Yersinia species and tested the consequences of polymorphism in a murine model of subacute systemic yersiniosis. Our evolutionary analysis revealed that codon 177 has been subjected to positive selection; the Yersinia enterocolitica residue had been altered from a leucine to a phenylalanine in nearly all Yersinia pseudotuberculosis and Yersinia pestis strains examined. Despite this change being minor, as both leucine and phenylalanine have hydrophobic side chains, reversion of YopJF177 to the ancestral YopJL177 variant yielded a Y. pseudotuberculosis strain with enhanced cytotoxicity toward macrophages, consistent with previous findings. Surprisingly, expression of YopJF177L in the mildly attenuated ksgA- background rendered the strain completely avirulent in mice. Consistent with this hypothesis that YopJ activity relates indirectly to Yersinia pathogenesis in vivo, ksgA- strains lacking functional YopJ failed to kill macrophages but actually regained virulence in animals. Also, treatment with the antiapoptosis drug suramin prevented YopJ-mediated macrophage cytotoxicity and enhanced Y. pseudotuberculosis virulence in vivo. Our results demonstrate that Yersinia-induced cell death is detrimental for bacterial pathogenesis in this animal model of illness and indicate that positive selection has driven YopJ/P and Yersinia evolution toward diminished cytotoxicity and increased virulence, respectively.

Human O6 -methylguanine-DNA methyltransferase containing C145A does not prevent hepatocellular carcinoma in C3HeB/FeJ transgenic mice.

The prevalence of hepatocellular carcinoma (HCC) was diminished from 60% to 18% at 15 months of age in C3HeB/FeJ male transgenic mice expressing hMGMT in our previous studies. To directly test if the methyltransferase activity is required for diminished tumor prevalence, two separate lines of transgenic mice bearing an enzymatically inactive form of hMGMT were used. In these lines, cysteine 145 was substituted with alanine (C145A). Expression of the hMGMT C145A transgene in liver was demonstrated by Northern blots and Western blots. Immunohistochemistry revealed predominantly nuclear localization of the hMGMT C145A protein. hMGMT C145A transgenic mice were crossed with lacI transgenic mice to assess mutant frequencies in the presence of the mutant protein. Mutant frequencies were similar among livers of lacI × hMGMT C145A bi-transgenic mice and lacI × wild-type (WT) mice. DNA sequence analysis of recovered lacI mutants revealed similar mutation spectra for hMGMT C145A and WT mice. The prevalence of HCC was also similar for the two tested lines of hMGMT C145A mice, 45% and 48% prevalence with median tumor sizes of 11 and 8 mm, and WT mice, 40% prevalence and median tumor size of 10 mm. These results provide evidence that residue C145 in hMGMT is required to reduce the prevalence of HCC in C3HeB/FeJ mice transgenic for hMGMT.

Size discrimination in rat and mouse gastric emptying.

OBJECTIVES: To investigate the relationship between particle size and gastric emptying in rodents using radiolabeled insoluble polymethyl methacrylate (PMMA) microcapsules/beads. METHODS: PMMA microcapsules (50-500 µm) and beads (0.5-3 mm) loaded with technetium-99 m diethylenetriamine pentaacetic acid ((99m) Tc-DTPA) were administered to ICR mice or Sprague Dawley (SD) rats by oral gavage. Gamma scintiscans were acquired initially following administration and then at hourly intervals to 4 hours. RESULTS: Scintiscans revealed that the smallest PMMA microcapsules (50-100 µm) or beads (0.5-1 mm) were impeded in the stomach and emptied slower than large particles in both rodent species. In mice, no significant difference in gastric emptying was found with microcapsules between 100 and 300 µm in diameter (p = 0.25) and particles more than 300 µm could not be administered. In rats, capsules containing 0.5-3 mm beads were stuck to the esophagus (up to 1 hour), this was a limitation of dosing beads of this size because they cannot be suspended in a liquid media for oral gavage purposes. Beads with diameters of 2-3 mm stayed in the stomach for up to 4 hours. CONCLUSIONS: The cut-off emptying size in ICR mice could not be determined, due to the limitation of current available dosing methods. The cut-off emptying size in SD rats was between 1.5 and 2 mm. Therefore, particles with a diameter greater than 2 mm should not be used for gastric emptying studies of intact particles in SD rats, as their emptying is retarded in the stomach.

Chemoradionuclide therapy with 186Re-labeled liposomal doxorubicin in combination with radiofrequency ablation for effective treatment of head and neck cancer in a nude rat tumor xenograft model.

PURPOSE: To determine the therapeutic efficacy of rhenium 186 ((186)Re)-labeled PEGylated liposomal doxorubicin ((186)Re-liposomal doxorubicin) in combination with radiofrequency (RF) ablation of human head and neck squamous cell carcinoma (HNSCC) xenograft in nude rats. MATERIALS AND METHODS: This investigation was approved by the animal care committee. Sixty nude rats with subcutaneously implanted HNSCC xenografts (six per group) were treated with (a) RF ablation (70 °C for 5 minutes), (b) PEGylated liposomes, (c) liposomal doxorubicin, (d) (186)Re-PEGylated liposomes (1295 MBq/kg), (e) (186)Re-liposomal doxorubicin (555 MBq/kg), (f) PEGylated liposomes plus RF ablation, (g) liposomal doxorubicin plus RF ablation, (h) (186)Re-PEGylated liposomes plus RF ablation, or (i) (186)Re-liposomal doxorubicin plus RF ablation. Six rats did not receive any treatment (control group). Tumor uptake in (186)Re therapy groups was monitored with small-animal single photon emission computed tomography for 5 days. Therapeutic efficacy was monitored for 6 weeks with measurement of tumor volume, calculation of the percentage injected dose of fluorine 18 fluorodeoxyglucose (FDG) in tumor from small-animal positron emission tomography (PET) images, and determination of viable tumor volume at histopathologic examination. Significant differences between groups were determined with analysis of variance. RESULTS: The average tumor volume (± standard deviation) on the day of therapy was 1.32 cm(3) ± 0.17. At 6 weeks after therapy, control of tumor growth was better with (186)Re-liposomal doxorubicin than with liposomal doxorubicin alone (tumor volume, 2.26 cm(3) ± 0.89 vs 5.43 cm(3) ± 0.93, respectively; P < .01). The use of RF ablation with liposomal doxorubicin and (186)Re-liposomal doxorubicin further improved tumor control (tumor volume, 2.05 cm(3) ± 1.36 and 1.49 cm(3) ± 1.47, respectively). The tumor growth trend correlated with change in percentage of injected dose of FDG in tumor for all groups (R(2) = 0.85, P < .001). Viable tumor volume was significantly decreased in the group treated with (186)Re-liposomal doxorubicin plus RF ablation (0.54 cm(3) ± 0.38; P < .001 vs all groups except (186)Re-liposomal doxorubicin alone). CONCLUSION: Triple and dual therapies had an observable trend ((186)Re-liposomal doxorubicin plus RF ablation > (186)Re-liposomal doxorubicin > liposomal doxorubicin plus RF ablation > liposomal doxorubicin) of improved tumor growth control and decreased viable tumor compared with other therapies. FDG PET could be used as a noninvasive surrogate marker for tumor growth and viability in this tumor model.

Novel asparagine-derived lipid enhances distearoylphosphatidylcholine bilayer resistance to acidic conditions.

A novel asparagine-derived lipid analogue (ALA(11,17)) bearing a tetrahydropyrimidinone headgroup and two fatty chains (11 and 17 indicate the lengths of linear alkyl groups) was synthesized in high yield and purity. The thin film hydration of formulations containing 5 mol % or greater ALA(11,17) in distearoylphosphatidylcholine (DSPC) generated multilamellar vesicles (MLVs) that remained unaggregated according to optical microscopy, while those formed from DSPC only were highly clustered. The MLVs were processed into unilamellar liposomes via extrusion and were characterized by dynamic light scattering (DLS), zeta potential, turbidity, and scanning electron microscopy (SEM) analysis. Results show that the presence of ALA(11,17) in DSPC liposomes significantly alters the morphology, colloidal stability, and retention of encapsulated materials in both acidic and neutral conditions. The ability of ALA(11,17)-hybrid liposomes to encapsulate and retain inclusions under neutral and acidic conditions (pH < 2) was demonstrated by calcein dequenching experiments. DLS and SEM confirmed that ALA(11,17)/DSPC liposomes remained intact under these conditions. The bilayer integrity observed under neutral and acidic conditions and the likely biocompatibility of these fatty amino acid analogues suggest that ALA(11,17) is a promising additive for modulating phosphatidylcholine lipid bilayer properties.

A colorful approach towards developing new nano-based imaging contrast agents for improved cancer detection.

Providing physicians with new imaging agents to help detect cancer with better sensitivity and specificity has the potential to significantly improve patient outcomes. Development of new imaging agents could offer improved early cancer detection during routine screening or help surgeons identify tumor margins for surgical resection. In this study, we evaluate the optical properties of a colorful class of dyes and pigments that humans routinely encounter. The pigments are often used in tattoo inks and the dyes are FDA approved for the coloring of foods, drugs, and cosmetics. We characterized their absorption, fluorescence and Raman scattering properties in the hopes of identifying a new panel of dyes that offer exceptional imaging contrast. We found that some of these coloring agents, coined as "optical inks", exhibit a multitude of useful optical properties, outperforming some of the clinically approved imaging dyes on the market. The best performing optical inks (Green 8 and Orange 16) were further incorporated into liposomal nanoparticles to assess their tumor targeting and optical imaging potential. Mouse xenograft models of colorectal, cervical and lymphoma tumors were used to evaluate the newly developed nano-based imaging contrast agents. After intravenous injection, fluorescence imaging revealed significant localization of the new "optical ink" liposomal nanoparticles in all three tumor models as opposed to their neighboring healthy tissues (p < 0.05). If further developed, these coloring agents could play important roles in the clinical setting. A more sensitive imaging contrast agent could enable earlier cancer detection or help guide surgical resection of tumors, both of which have been shown to significantly improve patient survival.

Combination radiofrequency ablation and intravenous radiolabeled liposomal Doxorubicin: imaging and quantification of increased drug delivery to tumors.

PURPOSE: To identify, with noninvasive imaging, the zone of radiopharmaceutical uptake after combination therapy with radiofrequency (RF) ablation and intravenous administration of technetium 99m ((99m)Tc) liposomal doxorubicin in a small-animal tumor model, and to quantify and correlate the uptake by using imaging and tissue counting of intratumoral doxorubicin accumulation. MATERIALS AND METHODS: This study was approved by the animal care committee. Two phases of animal experiments were performed. In the first experiment, a single human head-and-neck squamous cell carcinoma tumor was grown in each of 10 male nude rats. Seven of these animals were treated with intravenous (99m)Tc-liposomal doxorubicin followed by RF tumor ablation at a mean temperature of 70 degrees C + or - 2 for 5 minutes, and three were treated with intravenous (99m)Tc-liposomal doxorubicin only. Combination single photon emission computed tomography-computed tomography (SPECT/CT) was performed at 15 minutes, 4 hours, and 20 hours after therapy. In the second experiment, two tumors each were grown in 11 rats, but only one of the tumors was ablated after intravenous administration of (99m)Tc-liposomal doxorubicin. SPECT/CT and planar scintigraphy were performed at the same posttreatment intervals applied in the first experiment, with additional planar imaging performed at 44 hours. After imaging, tissue counting in the excised tumors was performed. Radiotracer uptake, as determined with imaging and tissue counting, was quantified and compared. In a subset of three animals, intratumoral doxorubicin accumulation was determined with fluorimetry and correlated with the imaging and tissue-counting data. RESULTS: At both SPECT/CT and planar scintigraphy, increased uptake of (99m)Tc-liposomal doxorubicin was visibly apparent in the ablated tumors. Results of quantitative analysis with both imaging and tissue counting confirmed significantly greater uptake in the RF ablation-treated tumors (P < .001). Intratumoral doxorubicin accumulation correlated closely with imaging (r = 0.9185-0.9871) and tissue-counting (r = 0.995) results. CONCLUSION: Study results show that increased delivery of intravenous liposomal doxorubicin to tumors combined with RF ablation can be depicted and quantified with noninvasive imaging.

QA procedures for multimodality preclinical tumor drug response testing.

PURPOSE: There are growing expectations that imaging biomarkers for tumor therapeutic drug response assessment will speed up preclinical testing of anticancer drugs in rodent models. The only imaging biomarker presently approved by the U.S. Food and Drug Administration is tumor size measurement based on either World Health Organization (WHO) criteria or Response Evaluation Criteria in Solid Tumors (RECIST). Frequently, preclinical data are accumulated from multiple research centers on multiple continents using scanners from different manufacturers and sometimes even using different imaging modalities. Very expensive cancer drug response studies can be compromised by inadequate controls to assure precision and accuracy of tumor size measurements. This project was undertaken to develop standardized quality assurance (QA) procedures using a multimodality preclinical tumor response phantom to validate the accuracy of tumor size measurements based on WHO criteria, RECIST, or global tumor volume criteria for evaluation of cytostatic drugs. METHODS: A tumor response phantom containing five low contrast test objects designed to simulate animal tumor models was made of tissue-mimicking materials. Imaging of the phantom was performed using three modalities in two institutions to evaluate size measurement of tumor-simulating test objects. RESULTS: Evaluation of tumor measurements from the three commonly used imaging devices in two different institutions for monitoring tumor size changes showed that a single phantom for multiple modalities was feasible. The tumor response phantom validated precision and accuracy of tumor response data input from ultrasound, computed tomography, and/or magnetic resonance imaging devices. CONCLUSIONS: Measurement results show that the standardized QA procedures using the tumor response phantom can provide a rationale check of data that excludes input from poorly maintained instruments, inadequate measurement protocols, or random operator error that frequently introduce unacceptable variability or systematic error in multiple institutions trials.

Caspase-2 deficiency enhances aging-related traits in mice.

Alteration of apoptotic activity has been observed in a number of tissues in aging mammals, but it remains unclear whether and/or how apoptosis may affect aging. Caspase-2 is a member of the cysteine protease family that plays a critical role in apoptosis. To understand the impact of compromised apoptosis function on mammalian aging, we conducted a comparative study on caspase-2 deficient mice and their wild-type littermates with a specific focus on the aging-related traits at advanced ages. We found that caspase-2 deficiency enhanced a number of traits commonly seen in premature aging animals. Loss of caspase-2 was associated with shortened maximum lifespan, impaired hair growth, increased bone loss, and reduced body fat content. In addition, we found that the livers of caspase-2 deficient mice had higher levels of oxidized proteins than those of age-matched wild-type mice, suggesting that caspase-2 deficiency compromised the animal's ability to clear oxidatively damaged cells. Collectively, these results suggest that caspase-2 deficiency affects aging in the mice. This study thus demonstrates for the first time that disruption of a key apoptotic gene has a significant impact on aging.

Use of avidin/biotin-liposome system for enhanced peritoneal drug delivery in an ovarian cancer model.

The goal of this study was to determine the distribution of the avidin/biotin-liposome system in an ovarian cancer xenograft model. Optimal avidin/biotin-liposome injection sequence with enhanced liposome accumulation to the peritoneum was determined. Two weeks after NIH:OVCAR-3 cell inoculation, rats were divided into three groups. Group 1 (B-A) (n=4), received an intraperitoneal injection of (99m)Tc-blue-biotin-liposomes 30 min before an intraperitoneal injection of avidin. Group 2 (A-B) (n=4), received an intraperitoneal injection of avidin 30 min before an intraperitoneal injection of (99m)Tc-blue-biotin-liposomes. Group 3 (A-B 2h) (n=5), received an intraperitoneal injection of avidin 2h before an intraperitoneal injection of (99m)Tc-blue-biotin-liposomes. Three additional non-tumor nude rats served as controls in each group, and were subjected to the same injection sequences. Scintigraphic imaging commenced at various times post (99m)Tc-blue-biotin-liposome injection. After imaging, rats were euthanized at 23 h post-liposome injection for tissue biodistribution. Images showed no apparent difference in liposome distribution between control and tumor animals. Regional uptake analysis at 4h for tumor rats showed significantly higher lymphatic channel uptake in the A-B 2h group (p<0.05) and a trend of increased peritoneal uptake in A-B group. By 22 h, peritoneal and lymphatic channel uptake was similar for all groups. At necropsy, most activity was found in blue-stained omentum, diaphragm, mediastinal and abdominal nodes. Bowel activity was minimal. These results correlate with previous normal rat studies, and demonstrate potential use of this avidin/biotin-liposome system for prolonging drug delivery to the peritoneal cavity and associating lymph nodes in this ovarian cancer xenograft model.

Image-guided interventional therapy for cancer with radiotherapeutic nanoparticles.

One of the major limitations of current cancer therapy is the inability to deliver tumoricidal agents throughout the entire tumor mass using traditional intravenous administration. Nanoparticles carrying beta-emitting therapeutic radionuclides that are delivered using advanced image-guidance have significant potential to improve solid tumor therapy. The use of image-guidance in combination with nanoparticle carriers can improve the delivery of localized radiation to tumors. Nanoparticles labeled with certain beta-emitting radionuclides are intrinsically theranostic agents that can provide information regarding distribution and regional dosimetry within the tumor and the body. Image-guided thermal therapy results in increased uptake of intravenous nanoparticles within tumors, improving therapy. In addition, nanoparticles are ideal carriers for direct intratumoral infusion of beta-emitting radionuclides by convection enhanced delivery, permitting the delivery of localized therapeutic radiation without the requirement of the radionuclide exiting from the nanoparticle. With this approach, very high doses of radiation can be delivered to solid tumors while sparing normal organs. Recent technological developments in image-guidance, convection enhanced delivery and newly developed nanoparticles carrying beta-emitting radionuclides will be reviewed. Examples will be shown describing how this new approach has promise for the treatment of brain, head and neck, and other types of solid tumors.

Biodistribution and lymph node retention of polysaccharide-based immunostimulating nanocapsules.

The adjuvant properties of polyglucosamine/squalene-based nanocapsules (PG-nanocapsules) associated with different subunit antigens has been previously reported. Thus, the aim of the present study was to monitor the biodistribution of PG-nanocapsules and their affinity for the draining lymph nodes after subcutaneous (s.c.) injection. The nanocapsules were efficiently radiolabeled with indium-111 ((111)In) (labeling efficiency of 98%). The diameter and zeta potential values of the unlabeled nanocapsules was preserved after the radiolabeling process and only 20% of the (111)In dissociated from the nanocapsules after 48h of incubation in serum. The radiolabeled nanocapsules and the control (111)InCl3 in saline solution (18.5MBq (500µCi) in 100µL) were injected s.c. in New Zealand White rabbits. The γ-scintigraphy imaging analysis revealed a slow clearance of the nanocapsules from the injection site and their progressive accumulation in the popliteal lymph node over time (3.8%±1.2 of the injected dose at 48h). Indeed, the clearance rate of the nanocapsules from the injection site was significantly slower than that of the control (free (111)InCl3), which rapidly drained into systemic circulation and accumulated mainly in excretion organs (i.e. kidneys and liver). In contrast, the biodistribution of nanocapsules was preferably limited to the lymphatic circulation. These results suggest that the immune potentiating effect previously observed for PG-nanocapsules is mainly due to the formation of a depot at the injection site, which was followed by a slow drainage into the lymphatic system and a prolonged retention in the lymph nodes.

Construction of a preclinical multimodality phantom using tissue-mimicking materials for quality assurance in tumor size measurement.

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible (1, 2, 3,4,5). These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) (6). In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed. To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom (7). The Appendix in Lee et al. demonstrates the procedures of phantom fabrication (7). In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. (8).

Chemoradionuclide therapy with 186re-labeled liposomal doxorubicin: toxicity, dosimetry, and therapeutic response.

This study was performed to determine the maximum tolerated dose (MTD) and therapeutic effects of rhenium-186 ((186)Re)-labeled liposomal doxorubicin (Doxil), investigate associated toxicities, and calculate radiation absorbed dose in head and neck tumor xenografts and normal organs. Doxil and control polyethylene glycol (PEG)-liposomes were labeled using (186)Re-N,N-bis(2-mercaptoethyl)-N',N'-diethylethylenediamine (BMEDA) method. Tumor-bearing rats received either no therapy (n=6), intravenous Doxil (n=4), or escalating radioactivity of (186)Re-Doxil (185-925 MBq/kg) or (186)Re-PEG-liposomes (1110-1665 MBq/kg) and were monitored for 28 days. Based on body weight loss and systemic toxicity, MTD for (186)Re-Doxil and (186)Re-PEG-liposomes were established at injected radioactivity/body weight of 740 and 1480 MBq/kg, respectively. (186)Re-injected radioactivity/body weight for therapy studies was determined to be 555 MBq/kg for (186)Re-Doxil and 1295 MBq/kg for (186)Re-PEG-liposomes. All groups recovered from their body weight loss, leucopenia, and thrombocytopenia by 28 days postinjection. Normalized radiation absorbed dose to tumor was significantly higher for (186)Re-Doxil (0.299±0.109 Gy/MBq) compared with (186)Re-PEG-liposomes (0.096±0.120 Gy/MBq) (p<0.05). In a separate therapy study, tumor volumes were significantly smaller for (186)Re-Doxil (555 MBq/kg) compared with (186)Re-PEG-liposomes (1295 MBq/kg) (p<0.01) at 42 days postinjection. In conclusion, combination chemoradionuclide therapy with (186)Re-Doxil has promising potential, because good tumor control was achieved with limited associated toxicity.

Ultrasound-guided intratumoral administration of collagenase-2 improved liposome drug accumulation in solid tumor xenografts.

PURPOSE: To investigate the effect of intratumoral administration of collagenase-2 on liposomal drug accumulation and diffusion in solid tumor xenografts. METHODS: Correlation between tumor interstitial fluid pressure (IFP) and tumor physiological properties (size and vessel fraction by B-mode and Doppler ultrasound, respectively) was determined. IFP response to intravenous or intratumoral collagenase-2 (0.1%) treatment was compared with intratumoral deactivated collagenase-2. To evaluate drug accumulation and diffusion, technetium-99 m-((99m)Tc)-liposomal doxorubicin (Doxil) was intravenously injected after collagenase-2 (0.1 and 0.5%, respectively) treatment, and planar scintigraphic images acquired and percentage of the injected dose per gram tissue calculated. Subsequently, tumors were subjected to autoradiography and histopathology. RESULTS: IFP in two-week-old head and neck squamous cell carcinoma xenografts was 18 ± 3.7 mmHg and not correlated to the tumor size but had reverse correlation with the vessel fraction (r = -0.91, P < 0.01). Intravenous and intratumoral collagenase-2 use reduced IFP by a maximum of 35-40%. Compared to the control, the low IFP level achieved through intratumoral route remained for a long period (24 vs. 2 h, P < 0.05). SPECT images and autoradiography showed significantly higher (99m)Tc-Doxil accumulation in tumors with intratumoral collagenase-2 treatment, confirmed by %ID/g in tumors (P < 0.05), and pathological findings showed extensive distribution of Doxil in tumors. CONCLUSIONS: Intratumoral injection of collagenase-2 could effectively reduce IFP in HNSCC xenografts for a longer period than using intravenous approach, which allowed for more efficient accumulation and homogeneous diffusion of the Doxil within the tumor interstitium.

Preclinical multimodality phantom design for quality assurance of tumor size measurement.

BACKGROUND: Evaluation of changes in tumor size from images acquired by ultrasound (US), computed tomography (CT) or magnetic resonance imaging (MRI) is a common measure of cancer chemotherapy efficacy. Tumor size measurement based on either the World Health Organization (WHO) criteria or the Response Evaluation Criteria in Solid Tumors (RECIST) is the only imaging biomarker for anti-cancer drug testing presently approved by the United States Food and Drug Administration (FDA). The aim of this paper was to design and test a quality assurance phantom with the capability of monitoring tumor size changes with multiple preclinical imaging scanners (US, CT and MRI) in order to facilitate preclinical anti-cancer drug testing. METHODS: Three phantoms (Gammex/UTHSCSA Mark 1, Gammex/UTHSCSA Mark 2 and UTHSCSA multimodality tumor measurement phantom) containing tumor-simulating test objects were designed and constructed. All three phantoms were scanned in US, CT and MRI devices. The size of test objects in the phantoms was measured from the US, CT and MRI images. RECIST, WHO and volume analyses were performed. RESULTS: The smaller phantom size, simplified design and better test object CT contrast of the UTHSCSA multimodality tumor measurement phantom allowed scanning of the phantom in preclinical US, CT and MRI scanners compared with only limited preclinical scanning capability of Mark 1 and Mark 2 phantoms. For all imaging modalities, RECIST and WHO errors were reduced for UTHSCSA multimodality tumor measurement phantom (≤1.69 ± 0.33%) compared with both Mark 1 (≤ -7.56 ± 6.52%) and Mark 2 (≤ 5.66 ± 1.41%) phantoms. For the UTHSCSA multimodality tumor measurement phantom, measured tumor volumes were highly correlated with NIST traceable design volumes for US (R2 = 1.000, p < 0.0001), CT (R2 = 0.9999, p < 0.0001) and MRI (R2 = 0.9998, p < 0.0001). CONCLUSIONS: The UTHSCSA multimodality tumor measurement phantom described in this study can potentially be a useful quality assurance tool for verifying radiologic assessment of tumor size change during preclinical anti-cancer therapy testing with multiple imaging modalities.

Isolation and characterization of a metastatic hybrid cell line generated by ER negative and ER positive breast cancer cells in mouse bone marrow.

BACKGROUND: The origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other's metastatic behavior. METHODS: ER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC. RESULTS: The presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-ß1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines. CONCLUSIONS: Spontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity.

[(186)Re]Liposomal doxorubicin (Doxil): in vitro stability, pharmacokinetics, imaging and biodistribution in a head and neck squamous cell carcinoma xenograft model.

The purpose of this study was to determine the feasibility of radiolabeling liposomal doxorubicin (Doxil) for cancer chemoradionuclide therapy by directly loading the therapeutic radionuclide rhenium-186 ((186)Re) into the liposome interior. The pharmacokinetics, imaging and biodistribution of [(186)Re]Doxil (555 MBq/kg) and control [(186)Re]polyethylene glycol (PEG) liposomes (555 MBq/kg) were determined after intravenous administration in a head and neck cancer xenograft model in nude rats. [(186)Re]Doxil and [(186)Re]PEG liposomes were radiolabeled using [(186)Re]N,N-bis(2-mercaptoethyl)-N',N'-diethylethylenediamine. (186)Re labeling efficiency was 76.1+/-8.3% with Doxil. The in vitro serum stability of [(186)Re]Doxil at 37 degrees C was 38.06+/-12.13% at 24 h. Pharmacokinetic studies revealed that [(186)Re]Doxil had a two-phase blood clearance with half clearance times of 0.8 and 28.2 h. Images acquired over 120 h showed that [(186)Re]Doxil had slow blood clearance, low liver accumulation and increasing spleen accumulation. The biodistribution study at 120 h indicated that the percentage of injected dose (%ID) in the blood and tumor for [(186)Re]Doxil was 20-fold higher than that of [(186)Re]PEG liposomes. The %ID values in the kidney and liver were not significantly different between [(186)Re]Doxil and [(186)Re]PEG liposomes. These results suggest that the long circulation and prolonged bioavailability of [(186)Re]Doxil could potentially deliver high concentrations of both doxorubicin and (186)Re to tumor when encapsulated in the same liposome vehicle.

Radiolabeled lipid nanoparticles for diagnostic imaging.

BACKGROUND: Nanoparticles are increasingly being incorporated into the design of diagnostic imaging agents. Significant research efforts have been conducted with one class of lipid nanoparticle (liposomes) radiolabeled with gamma-emitting radionuclides as radiopharmaceuticals for scintigraphic imaging of cancer, inflammation/infection and sentinel lymph node detection. OBJECTIVE: This article reviews the current literature with special emphasis on the clinical studies performed with liposome radiopharmaceuticals for detection of tumors, infectious/inflammatory sites or metastatic lymph nodes. Future uses of liposome radiopharmaceuticals are also described. METHODS: Characteristics required of the radionuclide, liposome formulation and radiolabeling method for an effective radiopharmaceutical are discussed. A description of the procedures and instrumentation for conducting an imaging study with liposome radiopharmaceutical is included. Clinical studies using liposome radiopharmaceuticals are summarized. Future imaging applications of first- and second-generation radiolabeled liposomes for chemodosimetry and the specific targeting of a disease process are also described. RESULTS/CONCLUSION: The choice of radionuclide, liposome formulation and radiolabeling method must be carefully considered during the design of a liposome radiopharmaceutical for a given application. After-loading and surface chelation methods are the most efficient and practical. Clinical studies with liposome radiopharmaceuticals demonstrated that a wide variety of tumors could be detected with good sensitivity and specificity. Liposome radiopharmaceuticals could also clearly detect various soft tissue and bone inflammatory/infectious lesions, and performed equal to or better than infection imaging agents that are approved at present. Yet, despite these favorable results, no liposome radiopharmaceutical has been approved for any indication. Some of the reasons for this can be attributed to reports of an unexpected infusion-related adverse reaction in two studies, the requirement of more complex liposome manufacturing procedures, and the adoption of other competing imaging procedures. Continued research of liposome radiopharmaceutical design based on a better understanding of liposome biology, improved radiolabeling methodologies and advances in gamma camera technology is warranted.

Imaging of 186Re-liposome therapy in ovarian cancer xenograft model of peritoneal carcinomatosis.

This study determined the biodistribution of rhenium-186 ((186)Re) encapsulated in biotin-liposomes containing patent blue dye, injected intraperitoneally (IP) with avidin in an OVCAR-3 ovarian cancer xenograft model and evaluated tumor response of this therapy with fluorine-18-fluorodeoxyglucose ((18)F-FDG) microPET imaging. Treated rats (n = 8) received an IP injection of (186)Re-blue-biotin-liposomes (1000 MBq/kg) 30 min before an IP injection of avidin (5 mg), whereas control rats (n = 4) received a sham IP injection of saline. Scintigraphic images showed that (186)Re-blue-biotin liposomes/avidin were retained in the peritoneal cavity with 18% of the original activity remaining after 5 days. From 1 to 4 weeks post-treatment, peritoneal (18)F-FDG standard uptake values decreased 30% in treatment group, yet increased 44% in control group. Total number of cells in ascites was significantly higher in control versus treatment group. Omental fat in control rats had numerous tumor cells compared with treated rats. Results show the potential for (186)Re-blue-biotin-liposome/avidin system in treating advanced ovarian cancer involving peritoneal carcinomatosis.