We previously reported that eribulin mesylate (eribulin), a tubulin‐binding drug (TBD), could remodel tumor vasculature (i.e. increase tumor vessels and perfusion) in human breast cancer xenograft ...models. However, the role of this vascular remodeling in antitumor effects is not fully understood. Here, we investigated the effects of eribulin‐induced vascular remodeling on antitumor activities in multiple human cancer xenograft models. Microvessel densities (MVD) were evaluated by immunohistochemistry (CD31 staining), and antitumor effects were examined in 10 human cancer xenograft models. Eribulin significantly increased MVD compared to the controls in six out of 10 models with a correlation between enhanced MVD levels and antitumor effects (R2 = 0.54). Because of increased MVD, we next used radiolabeled liposomes to examine whether eribulin treatment would result in increased tumoral accumulation levels of these macromolecules and, indeed, we found that eribulin, unlike vinorelbine (another TBD) enhanced them. As eribulin increased accumulation of radiolabeled liposomes, we postulated that this treatment might enhance the antitumor effect of Doxil (a liposomal anticancer agent) and facilitate recruitment of immune cells into the tumor. As expected, eribulin enhanced antitumor activity of Doxil in a post‐erlotinib treatment H1650 (PE‐H1650) xenograft model. Furthermore, infiltrating CD11b‐positive immune cells were significantly increased in multiple eribulin‐treated xenografted tumors, and natural killer (NK) cell depletion reduced the antitumor effects of eribulin. These findings suggest a contribution of the immune cells for antitumor activities of eribulin. Taken together, our results suggest that vascular remodeling induced by eribulin acts as a microenvironment modulator and, consequently, this alteration enhanced the antitumor effects of eribulin.
Eribulin, a nontaxane synthetic microtubule dynamic inhibitor, was approved by FDA and used for cancer treatment. Previously, we reported that eribulin induced vascular remodeling in the tumors by using preclinical mouse and rat xenograft models through increasing microvessel density, which consequently enhanced perfusion in the tumors (Funahashi et al., Cancer Sci., 105, 2014, 1334). Despite these advances, during eribulin treatment, a role of vascular remodeling for anti‐tumor effects is not fully elucidated. In this report, we further expanded our research by investigating pharmacological action of eribulin during the cancer treatment and found eribulin might function as a tumor microenvironment modulator in addition to its classic cytotoxic effect through vascular remodeling and contribute to effective cancer treatment.
Radionuclide imaging and therapy are promising methods for controlling systemic cancers; however, their clinical application has been limited by excessive radionuclide accumulation in healthy ...tissues. To minimize radionuclide accumulation in non-cancerous tissues while ensuring sufficient build up in tumors, we aimed to develop a method that controlled the in vivo dynamics of radionuclides post-administration. To this end, we describe a novel strategy that combines liposomes, a potent carrier system for drug delivery, with unique radionuclide-ligand complexes based on 111In-ethylenedicysteine.
Conventional 111In-ligand-complexes-carrying liposomes delivered substantial amounts of radionuclides to tumors; however, they also accumulated in the liver and spleen. In contrast, 111In-ethylenedicysteine-carrying liposomes greatly reduced non-specific accumulation, while being retained selectively at high doses within tumors. Liposomes were rapidly broken down in the liver, releasing encapsulated 111In-ligand complexes. Among the chelates used, only 111In-ethylenedicysteine could escape from the liver and be excreted in the urine. Instead, most liposomes remained intact in tumors, retaining the radionuclide-ligand complexes within them. Therefore, high tumor accumulation was obtained regardless of the type of 111In-ligand complexes in the liposomes. In vivo single photon emission computed tomography/computed tomography imaging with 111In-ethylenedicysteine-carrying liposomes accurately revealed tumor-selective radionuclide retention with little background. Hence, our new strategy could greatly enhance tumor-to-healthy tissue ratios, improve diagnostic imaging, boost therapeutic efficacy, reduce toxicity to healthy tissues, and facilitate radionuclide imaging and therapy.
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•Novel strategy: liposomes+111In-ethylenedicysteine for precise radionuclide control.•Significantly reduced healthy tissue accumulation while preserving tumor targeting.•High tumor-to-healthy tissue ratio, precise imaging, and lower toxicity are achieved.•In vivo SPECT/CT imaging demonstrates tumor-selective radionuclide retention.•Potential for improved radionuclide imaging and radionuclide therapy for cancers.
Development of monoclonal antibody is critical for targeted drug delivery because its characteristics determine improved therapeutic efficacy and reduced side-effect. Antibody therapeutics target ...surface molecules; hence, internalization is desired for drug delivery. As an antibody-drug conjugate, a critical parameter is drug-to-antibody ratio wherein the quantity of drugs attached to the antibody influences the antibody structure, stability, and efficacy. Here, we established a cell-based immunotoxin screening system to facilitate the isolation of functional antibodies with internalization capacities, and discovered an anti-human CD71 monoclonal antibody. To overcome the limitation of drug-to-antibody ratio, we employed the encapsulation capacity of liposome, and developed anti-CD71 antibody-conjugated liposome that demonstrated antigen-antibody dependent cellular uptake when its synthesis was optimized. Furthermore, anti-CD71 antibody-conjugated liposome encapsulating doxorubicin demonstrated antigen-antibody dependent cytotoxicity. In summary, this study demonstrates the powerful pipeline to discover novel functional antibodies, and the optimal method to synthesize immunoliposomes. This versatile technology offers a rapid and direct approach to generate antibodies suitable for drug delivery modalities.
Liposomal anticancer agents can effectively deliver drugs to tumor lesions, but their therapeutic effects are enhanced in only limited number of patients. Appropriate biomarkers to identify responder ...patients to these liposomal agents will improve their treatment efficacies. We carried out pharmacological and histopathological analyses of mouse xenograft models bearing human ovarian cancers (Caov‐3, SK‐OV‐3, KURAMOCHI, and TOV‐112D) to correlate the therapeutic effects of doxorubicin‐encapsulated liposome (Doxil®) and histological characteristics linked to the enhanced permeability and retention effect. We next generated 111In‐encapsulated liposomes to examine their capacities to determine indications for Doxil® treatment by single‐photon emission computed tomography (SPECT)/CT imaging. Antitumor activities of Doxil® were drastically enhanced in Caov‐3, moderately in SK‐OV‐3, and minimally in KURAMOCHI and TOV‐112D when compared to doxorubicin. Microvessel density and vascular perfusion were high in Caov‐3 and SK‐OV‐3, indicating a close relation with the enhanced antitumor effects. Next, 111In‐encapsulated liposomes were given i.v. to the animals. Their tumor accumulation and area under the curve values over 72 h were high in Caov‐3, relatively high in SK‐OV‐3, and low in two other tumors. Importantly, as both Doxil® effects and liposomal accumulation varied in the SK‐OV‐3 group, we individually obtained SPECT/CT images of SK‐OV‐3‐bearing mouse (n = 11) before Doxil® treatment. Clear correlation between liposomal tumor accumulation and effects of Doxil® was confirmed (R2 = 0.73). Taken together, our experiments definitely verified that enhanced therapeutic effects through liposomal formulations of anticancer agents depend on tumor accumulation of liposomes. Tumor accumulation of the radiolabeled liposomes evaluated by SPECT/CT imaging is applicable to appropriately determine indications for liposomal antitumor agents.
Advantages of liposomal anti‐cancer agents include increased anti‐tumor activities through EPR effect, and reduced side effects associated with their free drugs; however, despite these clear benefits, we are unequivocally reminded that liposomal agents enhance therapeutic effects to the limited number of cancer patients. Here, we performed pharmacological and histopathological analyses of mouse xenograft models bearing human ovarian cancers to correlate therapeutic effects of Doxil and histological characteristics linked to EPR, and we subsequently generated radiolabeled liposomes to examine their capacities as biomarkers to determine indications for Doxil treatment by SPECT/CT imaging. Our preclinical study clearly indicates the correlations among pharmacological effects of Doxil, histological factors linked to EPR, tumor accumulation of the radiolabeled liposomes, as well as SPECT/CT tests of the radiolabeled liposomes, and demonstrates applicability of SPECT/CT imaging to properly decide the indications for liposomal anti‐cancer agents with significant potential towards the clinical practice.
Tumor angiogenesis induced by vascular endothelial growth factor (VEGF) and/or fibroblast growth factor (FGF) plays an important role in tumor growth, metastasis, and drug resistance. However, the ...characteristics of tumor vessels derived from these angiogenic factors have not been fully explored.
To functionally examine tumor vessels, we developed in vivo VEGF- and FGF-induced tumor blood vessel models. We performed immunohistochemistry and Hoechst perfusion assay to elucidate histopathological differences between the derived tumor vessels. To kinetically understand tumor perfusion, we employed radiolabeled PEGylated liposomes.
While tumor vessel density was substantially increased by enhanced expression levels of VEGF and FGF, permeability of VEGF-driven tumor vessels was significantly higher than that of FGF-driven ones, the latter demonstrating an increased number of pericyte-covered vessels. Accordingly, we observed an increased tumor retention of the PEGylated liposomes in the VEGF-driven tumor.
Our in vivo models of tumor vessel demonstrate the frequency of pericyte coverage and tumor perfusion levels as major functional differences between VEGF- and FGF-driven tumor vessels.
In this study, we aimed to develop a dual-modal in vivo imaging based on over-1000 nm near-infrared (OTN-NIR) fluorescence and single photon emission computed tomography (SPECT)/computed tomography ...(CT). As an OTN-NIR nanophosphor material, Ytterbium and erbium ion co-doped yttrium phosphate nanoparticles (YPO4 NPs) was synthesized by a hydrothermal synthesis method. Biocompatible poly(ethylene glycol) (PEG)/polycation block copolymer and radioactive 111In were introduced on the YPO4 NPs surface (PEG-111In-YPO4 NPs). The PEG-111In-YPO4 NPs showed high dispersion stability in physiological saline and excellent radioactivity in any of PBS and FBS solutions. Furthermore, in vivo OTN-NIR fluorescence and SPECT /CT imaging of live mice was performed. Strong OTN-NIR emission from the blood vessel and organs of live mice were observed. Moreover, SPECT/CT images clearly showed the three dimensional images of the live mice. Therefore, PEG-111In-YPO4 NPs is an attractive candidate for the OTN-NIR fluorescence and SPECT/CT dual-modal imaging probe, and the concept can be served as a novel platform for real-time imaging and three dimensional quantitative determinations.
Objectives
Tumor interiors are never homogeneous and in vivo visualization of intratumoral heterogeneity would be an innovation that contributes to improved cancer therapy. But, conventional nuclear ...medicine tests have failed to visualize heterogeneity in vivo because of limited spatial resolution. Recently developed single photon emission computed tomographic (SPECT) scanners dedicated for small animal imaging are of interest due to their excellent spatial resolution of <1 mm, but few studies have focused on the evaluation of intratumoral heterogeneity. We investigated the optimal conditions related to high resolution imaging of heterogeneous tumor interiors using a small animal SPECT scanner.
Methods
The conditions related to SPECT/CT visualization of heterogeneous tumor interiors were investigated using phantoms with
111
In and simulations of actual small animal imaging. The optimal conditions obtained were validated by in vivo imaging of sarcoma 180-bearing mice.
Results
Larger number of counts must be obtained within limited acquisition time to visualize tumor heterogeneity in vivo in animal imaging, compared to cases that simply detect tumors. At an acquisition time of 30 min, better image quality was obtained with pinhole apertures diameter of 1.4 mm than of 1.0 mm. The obtained best spatial resolution was 1.3 mm, it was acceptable for our purpose, though a little worse than the best possible performance of the scanner (1.0 mm). Additionally, the reconstruction parameters, such as noise suppression, voxel size, and iteration/subset number, needed to be optimized under the limited conditions and were different from those found under the ideal condition. The minimal radioactivity concentration for visualization of heterogeneous tumor interiors was estimated to be as high as 0.2–0.5 MBq/mL. Liposomes containing
111
In met this requirement and were administered to tumor-bearing mice. SPECT imaging successfully showed heterogeneous
111
In distribution within the tumors in vivo with good spatial resolution. A threshold of 0.2 MBq/g for clear visualization of tumor heterogeneity was validated. Autoradiograms obtained ex vivo of excised tumors confirmed that the in vivo SPECT images accurately depicted the heterogeneous intratumoral accumulation of liposomes.
Conclusion
Intratumoral heterogeneity was successfully visualized under the optimized conditions using a SPECT/CT scanner.
Sentinel node navigation surgery using real-time, near-infrared imaging with indocyanine green is becoming popular by allowing head and neck surgeons to avoid unnecessary neck dissection. The major ...drawback of this method is its quick migration through the lymphatics, limiting the diagnostic time window and undesirable detection of downstream nodes. We resolved this problem by mixing indocyanine green (ICG) with phytate colloid to retard its migration and demonstrated its feasibility in a nude mouse study.
Experimental prospective animal study.
Animal laboratory.
Indocyanine green at 3 concentrations was tested to determine the optimal concentration for sentinel lymph node detection in a mouse model. Effect of indocyanine green with phytate colloid mixture solutions was also analyzed. Indocyanine green or mixture solution at different mixing ratios were injected into the tongue of nude mice and near-infrared fluorescence images were captured sequentially for up to 48 hours. The brightness of fluorescence in the sentinel lymph node and lymph nodes further downstream were assessed.
Indocyanine green concentration >50 μg/mL did not improve sentinel lymph node detection. The addition of phytate colloid to indocyanine green extended the period when sentinel lymph node was detectable. Second echelon lymph nodes were not imaged in mice injected with the mixture, while these were visualized in mice injected with indocyanine green alone.
This novel technique of ICG-phytate colloid mixture allows prolonged diagnostic time window, prevention of downstream subsequent nodes detection, and improved accuracy for the detection of true sentinel lymph nodes.
The insufficient energy and spatial resolutions of radionuclide imaging with conventional scintillation detectors restrict the visualization of multiple radionuclides and of microstructures in ...tissue. Here we report the development and performance of an imaging system equipped with a cadmium telluride diode detector that achieves an energy resolution of 1.7% at 140 keV and a spatial resolution of 250 μm. The combination of high-resolution spectra fitted to an X-ray analysis model of the emission lines of the radionuclides in a chosen energy band allowed us to accurately determine individual radiation activities from three radionuclides to simultaneously visualize thyroid tissue (via intravenously administered iodine-125), mandibular lymph nodes (via the intramuscular injection of indium-111) and parotid lymph nodes (via a subcutaneous injection of technetium-99m) in mice. Multi-radionuclide imaging may find advantageous applications in biomedical imaging.
Tumor hypoxia is closely associated with the malignant progression and/or the high metastatic ability of tumors and often induces resistance to chemo- and/or radiotherapy. Thus, the detection and ...evaluation of hypoxia is important for the optimization of cancer therapy. We designed a novel
99mTc-labeled probe for tumor hypoxia imaging that utilizes bioreductive reactions in hypoxic cells. This probe, which contains a 4-nitrobenzyl ester group, is reduced in hypoxic cells to produce a corresponding carboxylate anion that cannot penetrate cell membranes because of its hydrophilicity and negative charge; therefore, it is expected to be trapped inside hypoxic cells. Based on this unique strategy, we synthesized the Technetium-99m (
99mTc)-labeled probe
99mTc-SD32. The uptake of
99mTc-SD32 in tumor cells was investigated under normoxic and hypoxic conditions.
99mTc-SD32 showed sufficient accumulation and good retention in hypoxic cells. In addition, we demonstrated that
99mTc-SD32 was subjected to bioreduction in hypoxic cells and was trapped as the corresponding carboxylate anion. These results indicated that
99mTc-SD32 would be a promising agent for in vivo hypoxia imaging.
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