Assessment of a liposomal CT/optical contrast agent for image-guided head and neck surgery.

This study evaluates a long-acting liposomal fluorescence / CT dual-modality contrast agent (CF800) in head and neck cancer to enhance intraoperative tumor demarcation with fluorescence imaging and cone-beam computed tomography (CBCT). CF800 was administered to 12 buccal cancer-bearing rabbits. Imaging was acquired at regular time points to quantify time-dependent contrast enhancement. Surgery was performed 5-7 days after, with intraoperative near-infrared fluorescence endoscopy and CBCT, followed by histological and ex-vivo fluorescence assessment. Tumor enhancement on CT was significant at 24, 96 and 120 hours. Volumetric analysis of tumor segmentation showed high correlation between CBCT and micro-CT. Fluorescence signal was apparent in both ex-vivo and in-vivo imaging. Histological correlation showed [100%] specificity for primary tumor. Sensitivity and specificity of CF800 in detecting nodal involvement require further investigation.CF800 is long acting and has dual function for CT and fluorescence contrast, making it an excellent candidate for image-guided surgery.

Enteric Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation in Mice With Colitis.

BACKGROUND & AIMS: Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. METHODS: We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2',7'-dichlorofluorescein diacetate and flow cytometry. RESULTS: The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A-TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A-TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A-TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria (Faecalibacterium prausnitzii and Roseburia intestinalis). CONCLUSIONS: Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A-TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation.

Liposomal Irinotecan Achieves Significant Survival and Tumor Burden Control in a Triple Negative Breast Cancer Model of Spontaneous Metastasis.

Triple negative breast cancer (TNBC) represents a significant therapeutic challenge due to its highly aggressive nature and lack of effective treatment options. Liposomal irinotecan (nal-IRI, ONIVYDE) was approved in 2015 (by the Food and Drug Administration, European Medicines Agency, and Therapeutic Goods Administration) and is a topoisomerase inhibitor indicated, in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. This study investigates the potential therapeutic benefit of nal-IRI for the treatment of advanced TNBC in a clinically relevant mouse model of spontaneous metastasis (LM2-4). Female SCID mice were orthotopically inoculated with TNBC LM2-4-luc cells in the lower mammary fat pad. Following primary tumor resection, bioluminescence imaging (BLI) was used to monitor both metastasis formation and spread as well as response to treatment with nal-IRI. Weekly treatment with 10 mg/kg of nal-IRI provided a 4.9-times longer median survival compared to both 50 mg/kg irinotecan treated and untreated animals. The survival benefit was supported by a significant delay in the regrowth of the primary tumor, effective control, and eventual regression of metastases assessed using longitudinal BLI, which was confirmed at the study end point with magnetic resonance (MR) imaging and post-mortem observation. This preclinical investigation demonstrates that, at a five-times lower dose compared to the free drug, liposomal irinotecan provides significant survival benefit and effective management of metastatic disease burden in a clinically relevant model of spontaneous TNBC metastases. These findings support the evaluation of nal-IRI in patients with advanced and metastatic TNBC.

Contrast Agent Mass Spectrometry Imaging Reveals Tumor Heterogeneity.

Mapping intratumoral heterogeneity such as vasculature and margins is important during intraoperative applications. Desorption electrospray ionization mass spectrometry (DESI-MS) has demonstrated potential for intraoperative tumor imaging using validated MS profiles. The clinical translation of DESI-MS into a universal label-free imaging technique thus requires access to MS profiles characteristic to tumors and healthy tissues. Here, we developed contrast agent mass spectrometry imaging (CA-MSI) that utilizes a magnetic resonance imaging (MRI) contrast agent targeted to disease sites, as a label, to reveal tumor heterogeneity in the absence of known MS profiles. Human breast cancer tumors grown in mice were subjected to CA-MSI using Gadoteridol revealing tumor margins and vasculature from the localization of [Gadoteridol+K](+) and [Gadoteridol+Na](+) adducts, respectively. The localization of the [Gadoteridol+K](+) adduct as revealed through DESI-MS complements the in vivo MRI results. DESI-MS imaging is therefore possible for tumors for which no characteristic MS profiles are established. Further DESI-MS imaging of the flux of the contrast agent through mouse kidneys was performed indicating secretion of the intact label.

Ambient Mass Spectrometry Imaging with Picosecond Infrared Laser Ablation Electrospray Ionization (PIR-LAESI).

A picosecond infrared laser (PIRL) is capable of cutting through biological tissues in the absence of significant thermal damage. As such, PIRL is a standalone surgical scalpel with the added bonus of minimal postoperative scar tissue formation. In this work, a tandem of PIRL ablation with electrospray ionization (PIR-LAESI) mass spectrometry is demonstrated and characterized for tissue molecular imaging, with a limit of detection in the range of 100 nM for reserpine or better than 5 nM for verapamil in aqueous solution. We characterized PIRL crater size using agar films containing Rhodamine. PIR-LAESI offers a 20-30 µm vertical resolution (∼3 µm removal per pulse) and a lateral resolution of ∼100 µm. We were able to detect 25 fmol of Rhodamine in agar ablation experiments. PIR-LAESI was used to map the distribution of endogenous methoxykaempferol glucoronide in zebra plant (Aphelandra squarrosa) leaves producing a localization map that is corroborated by the literature. PIR-LAESI was further used to image the distribution inside mouse kidneys of gadoteridol, an exogenous magnetic resonance contrast agent intravenously injected. Parallel mass spectrometry imaging (MSI) using desorption electrospray ionization (DESI) and matrix assisted laser desorption ionization (MALDI) were performed to corroborate PIR-LAESI images of the exogenous agent. We further show that PIR-LAESI is capable of desorption ionization of proteins as well as phospholipids. This comparative study illustrates that PIR-LAESI is an ion source for ambient mass spectrometry applications. As such, a future PIRL scalpel combined with secondary ionization such as ESI and mass spectrometry has the potential to provide molecular feedback to guide PIRL surgery.

A gradient-loadable (64)Cu-chelator for quantifying tumor deposition kinetics of nanoliposomal therapeutics by positron emission tomography.

Effective drug delivery to tumors is a barrier to treatment with nanomedicines. Non-invasively tracking liposome biodistribution and tumor deposition in patients may provide insight into identifying patients that are well-suited for liposomal therapies. We describe a novel gradient-loadable chelator, 4-DEAP-ATSC, for incorporating (64)Cu into liposomal therapeutics for positron emission tomographic (PET). (64)Cu chelated to 4-DEAP-ATSC (>94%) was loaded into PEGylated liposomal doxorubicin (PLD) and HER2-targeted PLD (MM-302) with efficiencies >90%. (64)Cu-MM-302 was stable in human plasma for at least 48h. PET/CT imaging of xenografts injected with (64)Cu-MM-302 revealed biodistribution profiles that were quantitatively consistent with tissue-based analysis, and tumor (64)Cu positively correlated with liposomal drug deposition. This loading technique transforms liposomal therapeutics into theranostics and is currently being applied in a clinical trial (NCT01304797) to non-invasively quantify MM-302 tumor deposition, and evaluate its potential as a prognostic tool for predicting treatment outcome of nanomedicines.

The translocator protein ligand [¹8F]DPA-714 images glioma and activated microglia in vivo.

PURPOSE: In recent years there has been an increase in the development of radioligands targeting the 18-kDa translocator protein (TSPO). TSPO expression is well documented in activated microglia and serves as a biomarker for imaging neuroinflammation. In addition, TSPO has also been reported to be overexpressed in a number of cancer cell lines and human tumours including glioma. Here we investigated the use of [(18)F]DPA-714, a new TSPO positron emission tomography (PET) radioligand to image glioma in vivo. METHODS: We studied the uptake of [(18)F]DPA-714 in three different rat strains implanted with 9L rat glioma cells: Fischer (F), Wistar (W) and Sprague Dawley (SD) rats. Dynamic [(18)F]DPA-714 PET imaging, kinetic modelling of PET data and in vivo displacement studies using unlabelled DPA-714 and PK11195 were performed. Validation of TSPO expression in 9L glioma cell lines and intracranial 9L gliomas were investigated using Western blotting and immunohistochemistry of brain tissue sections. RESULTS: All rats showed significant [(18)F]DPA-714 PET accumulation at the site of 9L tumour implantation compared to the contralateral brain hemisphere with a difference in uptake among the three strains (F > W > SD). The radiotracer showed high specificity for TSPO as demonstrated by the significant reduction of [(18)F]DPA-714 binding in the tumour after administration of unlabelled DPA-714 or PK11195. TSPO expression was confirmed by Western blotting in 9L cells in vitro and by immunohistochemistry ex vivo. CONCLUSION: The TSPO radioligand [(18)F]DPA-714 can be used for PET imaging of intracranial 9L glioma in different rat strains. This preclinical study demonstrates the feasibility of employing [(18)F]DPA-714 as an alternative radiotracer to image human glioma.

Differential expression of the 18 kDa translocator protein (TSPO) by neoplastic and inflammatory cells in mouse tumors of breast cancer.

Tumor-associated inflammation has been linked to angiogenesis, metastasis and poor prognosis. The 18 kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor (PBR), is expressed in activated immune cells such as macrophages, but also in a number of cancer cell lines such as those of breast cancer. There is an increasing clinical interest in TSPO expression as it has been proposed as a poor prognostic factor for survival in lymph-node negative breast cancer patients. This study aims to assess of the presence of neoplastic cell-associated TSPO and tumor macrophage-associated TSPO in mouse xenografts generated from the MDA-MB-231 and the MCF-7 breast cancer cell lines, as well as 25 different breast tumors originally derived from patient-tissue but propagated in mice using two antibodies, each specific to either the human or the murine form of TSPO. Autoradiography with the TSPO ligand [¹8F]DPA-714 and immunohistochemistry were also performed on the excised tumor tissues from the MDA-MB-231, MCF-7 and one of the patient-derived xenografts (HBCx-12B). High TSPO expression (either cancer or stromal cell-associated, or both) was measured in 20/25 (80%) of the patient-derived breast cancer xenografts. [¹8F]DPA-714 showed displaceable binding to both the human and murine TSPO on tumor tissue sections. Immunohistochemistry demonstrated that a significant portion of the tumor stromal TSPO expression colocalized with F4/80 positive macrophages cells. This study constitutes a first report of the tumor TSPO expression by mixed cell populations, and it may have important implications for cancer biology as well as for the development of imaging and therapeutic ligands targeted to TSPO.

An integrated augmented reality surgical navigation platform using multi-modality imaging for guidance.

An integrated augmented reality (AR) surgical navigation system that potentially improves intra-operative visualization of concealed anatomical structures. Integration of real-time tracking technology with a laser pico-projector allows the surgical surface to be augmented by projecting virtual images of lesions and critical structures created by multimodality imaging. We aim to quantitatively and qualitatively evaluate the performance of a prototype interactive AR surgical navigation system through a series of pre-clinical studies. Four pre-clinical animal studies using xenograft mouse models were conducted to investigate system performance. A combination of CT, PET, SPECT, and MRI images were used to augment the mouse body during image-guided procedures to assess feasibility. A phantom with machined features was employed to quantitatively estimate the system accuracy. All the image-guided procedures were successfully performed. The tracked pico-projector correctly and reliably depicted virtual images on the animal body, highlighting the location of tumour and anatomical structures. The phantom study demonstrates the system was accurate to 0.55 ± 0.33mm. This paper presents a prototype real-time tracking AR surgical navigation system that improves visualization of underlying critical structures by overlaying virtual images onto the surgical site. This proof-of-concept pre-clinical study demonstrated both the clinical applicability and high precision of the system which was noted to be accurate to <1mm.

Longitudinal PET Imaging to Monitor Treatment Efficacy by Liposomal Irinotecan in Orthotopic Patient-Derived Pancreatic Tumor Models of High and Low Hypoxia.

PURPOSE: Hypoxia is linked to aggressiveness, resistance to therapy, and poor prognosis of pancreatic tumors. Liposomal irinotecan (nal-IRI, ONIVYDE®) has shown potential in reducing hypoxia in the HT29 colorectal cancer model, and here, we investigate its therapeutic activity and ability to modulate hypoxia in patient-derived orthotopic tumor models of pancreatic cancer. PROCEDURES: Mice were randomized into nal-IRI treated and untreated controls. Magnetic resonance imaging was used for monitoring treatment efficacy, positron emission tomography (PET) imaging with F-18-labelled fluoroazomycinarabinoside ([18F]FAZA) for tumor hypoxia quantification, and F-18-labelled fluorothymidine ([18F]FLT) for tumor cell proliferation. RESULTS: The highly hypoxic OCIP51 tumors showed significant response following nal-IRI treatment compared with the less hypoxic OCIP19 tumors. [18F]FAZA-PET detected significant hypoxia reduction in treated OCIP51 tumors, 8 days before significant changes in tumor volume. OCIP19 tumors also responded to therapy, although tumor volume control was not accompanied by any reduction in [18F]FAZA uptake. In both models, no differences were observable in [18F]FLT uptake in treated tumors compared with control mice. CONCLUSIONS: Hypoxia modulation may play a role in nal-IRI's mechanism of action. Nal-IRI demonstrated greater anti-tumor activity in the more aggressive and hypoxic tumor model. Furthermore, hypoxia imaging provided early prediction of treatment response.

Perfluorocarbon nanodroplets can reoxygenate hypoxic tumors in vivo without carbogen breathing.

Nanoscale perfluorocarbon (PFC) droplets have enormous potential as clinical theranostic agents. They are biocompatible and are currently used in vivo as contrast agents for a variety of medical imaging modalities, including ultrasound, computed tomography, photoacoustic and 19F-magnetic resonance imaging. PFC nanodroplets can also carry molecular and nanoparticulate drugs and be activated in situ by ultrasound or light for targeted therapy. Recently, there has been renewed interest in using PFC nanodroplets for hypoxic tumor reoxygenation towards radiosensitization based on the high oxygen solubility of PFCs. Previous studies showed that tumor oxygenation using PFC agents only occurs in combination with enhanced oxygen breathing. However, recent studies suggest that PFC agents that accumulate in solid tumors can contribute to radiosensitization, presumably due to tumor reoxygenation without enhanced oxygen breathing. In this study, we quantify the impact of oxygenation due to PFC nanodroplet accumulation in tumors alone in comparison with other reoxygenation methodologies, in particular, carbogen breathing. Methods: Lipid-stabilized, PFC (i.e., perfluorooctyl bromide, CF3(CF2)7Br, PFOB) nanoscale droplets were synthesized and evaluated in xenograft prostate (DU145) tumors in male mice. Biodistribution assessment of the nanodroplets was achieved using a fluorescent lipophilic indocarbocyanine dye label (i.e., DiI dye) on the lipid shell in combination with fluorescence imaging in mice (n≥3 per group). Hypoxia reduction in tumors was measured using PET imaging and a known hypoxia radiotracer, [18F]FAZA (n≥ 3 per group). Results: Lipid-stabilized nanoscale PFOB emulsions (mean diameter of ~250 nm), accumulated in the xenograft prostate tumors in mice 24 hours post-injection. In vivo PET imaging with [18F]FAZA showed that the accumulation of the PFOB nanodroplets in the tumor tissues alone significantly reduced tumor hypoxia, without enhanced oxygen (i.e., carbogen) breathing. This reoxygenation effect was found to be comparable with carbogen breathing alone. Conclusion: Accumulation of nanoscale PFOB agents in solid tumors alone successfully reoxygenated hypoxic tumors to levels comparable with carbogen breathing alone, an established tumor oxygenation method. This study confirms that PFC agents can be used to reoxygenate hypoxic tumors in addition to their current applications as multifunctional theranostic agents.

Nanoparticle-based CT visualization of pulmonary vasculature for minimally-invasive thoracic surgery planning.

PURPOSE: To evaluate CF800, a novel lipid-based liposomal nanoparticle that co-encapsulates indocyanine green (ICG) and iohexol, for CT imaging of pulmonary vasculature in minimally-invasive thoracic surgery planning. METHODS: CF800 was intravenously administered to 7 healthy rabbits. In vivo CT imaging was performed 15 min post-injection, with a subset of animals imaged at 24h, 48h, and 72h post injection. Signal-to-background ratios (SBR) were calculated at the inferior vena cava and compared across time-points. A similar protocol was applied to 2 healthy pigs to evaluate the feasibility and efficacy in a large animal model. To evaluate the feasibility of clinical application, a survey was completed by 7 surgical trainees to assess pre- and post-injection CT images of rabbits and pigs. Responses on the discernibility of pulmonary vasculature sub-branches and comfort level to use the images for pre-operative planning were collected and analyzed. RESULTS: CF800 injection improved visualization of pulmonary vessels in both rabbit and pig models. The SBR of rabbit pulmonary vasculature was significantly higher after CF800 injection (range 3.7-4.4) compared to pre-injection (range 3.3-3.8, n = 7; p<0.05). SBR remained significantly different up to 24 hours after injection (range 3.7-4.3, n = 4; p<0.05). Trainees' evaluation found the post-injection CT images had significantly higher discernibility at the second vessel branch generation in both rabbit and pig models. Trainees identified smaller vasculature branch generations in the post-injection images compared to the pre-treatment images in both rabbit (mean 6.7±1.8 vs 5.4±2.1; p<0.05) and pig (mean 6.7±1.8 vs 5.4±2.1; p<0.05). Trainees were significantly more comfortable using post-injection images for surgical planning compared to the pre-injection images (rabbit: 8.1±1.1 vs. 4.7±2.1; pig: 7.6±2.1 vs. 4.9±2.2; p<0.05). CONCLUSION: CF800 provides SBR and contrast enhancement of pulmonary vasculature which may assist in pre-surgical CT planning of minimally invasive thoracic surgery.

Intraoperative Near-Infrared Fluorescence-Guided Peripheral Lung Tumor Localization in Rabbit Models.

BACKGROUND: A novel liposomal nanoparticle, CF800, that co-encapsulates indocyanine green for near-infrared (NIR) imaging and iohexol for computed tomography (CT) imaging has shown preferential tumor accumulation after intravenous injection by the enhanced permeability and retention effect. We hypothesized that CF800-enhanced NIR imaging would facilitate intraoperative localization of small lung nodules. METHODS: A rabbit VX2 lung tumor model was implemented. CF800 was injected intravenously, followed by sequential CT acquisitions to track the biodistribution of CF800. Eleven rabbits were used for NIR fluorescence evaluation after thoracotomy at time points until 7 days after injection by using a NIR fluorescence thoracoscope in vivo. Organs of interests were removed for ex vivo analysis by using NIR imaging. Tumor-to-background (inflated lung) ratio was calculated and compared among the time points. RESULTS: Both CT and NIR imaging indicated enhanced accumulation of CF800 within the VX2 tumor. NIR image analysis revealed the highest tumor-to-background ratio on days 4 and 5. High background at day 2 and low tumor signal at day 7 prevented distinct demarcation. Metastatic pulmonary small nodules (less than 2 mm in diameter) were successfully visualized by NIR imaging on day 4. However, NIR signal penetration was limited, resulting in localization failure for the few tumors deep (>0 mm) to the lung surface. CONCLUSIONS: NIR image-guided localization of small lung nodules appears to be feasible under certain conditions. However, further refinement will be required to increase tumor signal intensity and to reduce background signal from normal lung parenchyma, which is at least in part a consequence of persistent CF800 in the vasculature.

Spatiotemporal assessment of spontaneous metastasis formation using multimodal in vivo imaging in HER2+ and triple negative metastatic breast cancer xenograft models in mice.

BACKGROUND: Preclinical breast cancer models recapitulating the clinical course of metastatic disease are crucial for drug development. Highly metastatic cell lines forming spontaneous metastasis following orthotopic implantation were previously developed and characterized regarding their biological and histological characteristics. This study aimed to non-invasively and longitudinally characterize the spatiotemporal pattern of metastasis formation and progression in the MDA-MB-231-derived triple negative LM2-4 and HER2+ LM2-4H2N cell lines, using bioluminescence imaging (BLI), contrast enhanced computed tomography (CT), fluorescence imaging, and 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography ([18F]FDG-PET). MATERIAL AND METHODS: LM2-4, LM2-4H2N, and MDA-MB-231 tumors were established in the right inguinal mammary fat pad (MFP) of female SCID mice and resected 14-16 days later. Metastasis formation was monitored using BLI. Metabolic activity of primary and metastatic lesions in mice bearing LM2-4 or LM2-4H2N was assessed by [18F]FDG-PET. Metastatic burden at study endpoint was assessed by CT and fluorescence imaging following intravenous dual-modality liposome agent administration. RESULTS: Comparable temporal metastasis patterns were observed using BLI for the highly metastatic cell lines LM2-4 and LM2-4H2N, while metastasis formed about 10 days later for MDA-MB-231. 21 days post primary tumor resection, metastases were detected in 86% of LM2-4, 69% of LM2-4H2N, and 60% of MDA-MB-231 inoculated mice, predominantly in the axillary region, contralateral MFP, and liver/lung. LM2-4 and LM2-4H2N tumors displayed high metabolism based on [18F]FDG-PET uptake. Lung metastases were detected as the [18F]FDG-PET uptake increased significantly between pre- and post-metastasis scan. Using a liposomal dual-modality agent, CT and fluorescence confirmed BLI detected lesions and identified additional metastatic nodules in the intraperitoneal cavity and lung. CONCLUSIONS: The combination of complementary anatomical and functional imaging techniques can provide high sensitivity characterization of metastatic disease spread, progression and overall disease burden. The described models and imaging toolset can be implemented as an effective means for quantitative treatment response evaluation in metastatic breast cancer.

Companion Diagnostic 64Cu-Liposome Positron Emission Tomography Enables Characterization of Drug Delivery to Tumors and Predicts Response to Cancer Nanomedicines.

Deposition of liposomal drugs into solid tumors is a potentially rate-limiting step for drug delivery and has substantial variability that may influence probability of response. Tumor deposition is a shared mechanism for liposomal therapeutics such that a single companion diagnostic agent may have utility in predicting response to multiple nanomedicines. Methods: We describe the development, characterization and preclinical proof-of-concept of the positron emission tomography (PET) agent, MM-DX-929, a drug-free untargeted 100 nm PEGylated liposome stably entrapping a chelated complex of 4-DEAP-ATSC and 64Cu (copper-64). MM-DX-929 is designed to mimic the biodistribution of similarly sized therapeutic agents and enable quantification of deposition in solid tumors. Results: MM-DX-929 demonstrated sufficient in vitro and in vivo stability with PET images accurately reflecting the disposition of liposome nanoparticles over the time scale of imaging. MM-DX-929 is also representative of the tumor deposition and intratumoral distribution of three different liposomal drugs, including targeted liposomes and those with different degrees of PEGylation. Furthermore, stratification using a single pre-treatment MM-DX-929 PET assessment of tumor deposition demonstrated that tumors with high MM-DX-929 deposition predicted significantly greater anti-tumor activity after multi-cycle treatments with different liposomal drugs. In contrast, MM-DX-929 tumor deposition was not prognostic in untreated tumor-bearing xenografts, nor predictive in animals treated with small molecule chemotherapeutics. Conclusions: These data illustrate the potential of MM-DX-929 PET as a companion diagnostic strategy to prospectively select patients likely to respond to liposomal drugs or nanomedicines of similar molecular size.

Multimodal contrast agent for combined computed tomography and magnetic resonance imaging applications.

OBJECTIVE: The objective of this study was to examine the feasibility of a multimodal system to effectively induce and maintain contrast enhancement in both computed tomography (CT) and magnetic resonance (MR) for radiation therapy applications. MATERIALS AND METHODS: The physicochemical characteristics of a liposome-encapsulated iohexol and gadoteridol formulation were assessed in terms of agent loading efficiencies, size and morphology, in vitro stability, and release kinetics. The imaging properties of the liposome formulation were assessed based on T1 and T2 relaxivity measurements and in vitro CT and MR imaging in a phantom. A preliminary imaging-based evaluation of the in vivo stability of this multimodal contrast agent was also performed in a lupine model. RESULTS: The average agent loading levels achieved were 26.5+/-3.8 mg/mL for iodine and 6.6+/- 1.5 mg/mL for gadolinium. These concentrations correspond to approximately 10% of that found in the commercially available preparations of each of these agents. However, this liposome-based formulation is expected to have a smaller volume of distribution and prolonged circulation lifetime in vivo. This multimodal system was found to have high agent retention in vitro, which translated into maintained contrast enhancement (up to 3 days) and stability in vivo. CONCLUSIONS: This study demonstrated the feasibility of engineering a multimodal contrast agent with prolonged contrast enhancement in vivo for use in CT and MR. This contrast agent may serve as a valuable tool for cardiovascular imaging as well as image registration and guidance applications in radiation therapy.

Evaluation of PET Imaging Performance of the TSPO Radioligand [18F]DPA-714 in Mouse and Rat Models of Cancer and Inflammation.

PURPOSE: Many radioligands have been explored for imaging the 18-kDa translocator protein (TSPO), a diagnostic and therapeutic target for inflammation and cancer. Here, we investigated the TSPO radioligand [(18)F]DPA-714 for positron emission tomography (PET) imaging of cancer and inflammation. PROCEDURES: [(18)F]DPA-714 PET imaging was performed in 8 mouse and rat models of breast and brain cancer and 4 mouse and rat models of muscular and bowel inflammation. RESULTS: [(18)F]DPA-714 showed different uptake levels in healthy organs and malignant tissues of mice and rats. Although high and displaceable [(18)F]DPA-714 binding is observed ex vivo, TSPO-positive PET imaging of peripheral lesions of cancer and inflammation in mice did not show significant lesion-to-background signal ratios. Slower [(18)F]DPA-714 metabolism and muscle clearance in mice compared to rats may explain the elevated background signal in peripheral organs in this species. CONCLUSION: Although TSPO is an evolutionary conserved protein, inter- and intra-species differences call for further exploration of the pharmacological parameters of TSPO radioligands.

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors.

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and CT-liposome accumulation in an individual subcutaneous xenograft mouse model of breast cancer. Measurements led to the discovery that perfusion and plasma volume fraction are strong mediators of the intra-tumoral distribution of liposomes. Furthermore, the results suggest that IFP plays an indirect role in mediating liposome distribution through modulating blood flow.

Multi-Modal Imaging in a Mouse Model of Orthotopic Lung Cancer.

BACKGROUND: Investigation of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine green (ICG) and iohexol, for real-time near infrared (NIR) fluorescence and computed tomography (CT) image-guided surgery in an orthotopic lung cancer model in nude mice. METHODS: CF800 was intravenously administered into 13 mice bearing the H460 orthotopic human lung cancer. At 48 h post-injection (peak imaging agent accumulation time point), ex vivo NIR and CT imaging was performed. A clinical NIR imaging system (SPY®, Novadaq) was used to measure fluorescence intensity of tumor and lung. Tumor-to-background-ratios (TBR) were calculated in inflated and deflated states. The mean Hounsfield unit (HU) of lung tumor was quantified using the CT data set and a semi-automated threshold-based method. Histological evaluation using H&E, the macrophage marker F4/80 and the endothelial cell marker CD31, was performed, and compared to the liposomal fluorescence signal obtained from adjacent tissue sections. RESULTS: The fluorescence TBR measured when the lung is in the inflated state (2.0 ± 0.58) was significantly greater than in the deflated state (1.42 ± 0.380 (n = 7, p<0.003). Mean fluorescent signal in tumor was highly variable across samples, (49.0 ± 18.8 AU). CT image analysis revealed greater contrast enhancement in lung tumors (a mean increase of 110 ± 57 HU) when CF800 is administered compared to the no contrast enhanced tumors (p = 0.0002). CONCLUSION: Preliminary data suggests that the high fluorescence TBR and CT tumor contrast enhancement provided by CF800 may have clinical utility in localization of lung cancer during CT and NIR image-guided surgery.