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Background The accumulation of liposome encapsulated chemotherapy in solid cancers is dependent on the presence of the enhanced permeability and retention (EPR) effect. Positron emission tomography (PET) imaging with a liposome encapsulated radioisotope, such as liposome encapsulated Cu-64 (64Cu-liposome) may help to identify tumors with high liposome accumulation, and thereby stratify patients based on expected benefit from liposomal chemotherapy. However, intravenous administration of liposomes without a cytotoxic content is complicated by the accelerated blood clearance (ABC) phenomenon for succeeding therapeutic liposome dosing. Alternative markers for assessing the tumor's EPR level are therefore warranted. Materials and Methods To increase our understanding of EPR variations and to ultimately identify an alternative marker for the EPR effect, we investigated the correlation between 64Cu-liposome PET/CT (EPR effect) and 68Ga-RGD PET/CT (neoangiogenesis), 18F-FDG PET/CT (glycolysis), diffusion-weighted MRI (diffusivity) and interstitial fluid pressure in two experimental cancer models (CT26 and COLO 205). Results 64Cu-liposome and 68Ga-RGD SUVmax displayed a significant moderate correlation, however, none of the other parameters evaluated displayed significant correlations. These results indicate that differences in neoangiogenesis may explain some EPR variability, however, as correlations were only moderate and not observed for SUVmean, 68Ga-RGD is probably insufficient to serve as a stand-alone surrogate marker for quantifying the EPR effect and stratifying patients.

Precise methods for quantifying drug accumulation in brain tissue are currently very limited, challenging the development of new therapeutics for brain disorders. Transcardial perfusion is instrumental for removing the intravascular fraction of an injected compound, thereby allowing for ex vivo assessment of extravasation into the brain. However, pathological remodeling of tissue microenvironment can affect the efficiency of transcardial perfusion, which has been largely overlooked. We show that, in contrast to healthy vasculature, transcardial perfusion cannot remove an injected compound from the tumor vasculature to a sufficient extent leading to considerable overestimation of compound extravasation. We demonstrate that 3D deep imaging of optically cleared tumor samples overcomes this limitation. We developed two machine learning-based semi-automated image analysis workflows, which provide detailed quantitative characterization of compound extravasation patterns as well as tumor angioarchitecture in large three-dimensional datasets from optically cleared samples. This methodology provides a precise and comprehensive analysis of extravasation in brain tumors and allows for correlation of extravasation patterns with specific features of the heterogeneous brain tumor vasculature., Kostrikov et al. report a deficiency of transcardial perfusion in brain tumor vasculature, which leads to exaggeration of drug extravasation measurements. They then demonstrate how optical tissue clearing can help to overcome this limitation and provide two machine learning-based image analysis workflows enabling detailed quantitative characterization of compound extravasation patterns as well as tumor angioarchitecture in large three-dimensional datasets.

Local application of radioactive sources as brachytherapy is well established in oncology. This treatment is highly invasive however, due to the insertion of millimeter sized metal seeds. The authors report the development of a new concept for brachytherapy, based on gold-palladium (AuPd) alloy nanoparticles, intrinsically radiolabeled with

Increasing numbers of lung tumors are identified at early disease stages by diagnostic imaging in screening programs, but difficulties in locating these during surgical intervention has prevented an improved treatment outcome. Surgical biomarkers that are visible on diagnostic images, and that provide the surgeon with real-time image guidance during the intervention are thus highly warranted to bridge diagnostic precision into enhanced therapeutic outcome. In this paper, a liquid soft tissue marker for near infrared fluorescence and radio-guidance is presented. The biocompatible marker is based on the carbohydrate ester, sucrose acetate isobutyrate, ethanol, and a multifunctional naphthalocyanine dye, which enable near infrared fluorescence image-guided resection at short, medium and long tissue depths. Naphthalocyanine dyes have high quantum yields and may further act as chelators of radionuclides. Upon injection of the liquid marker, a gel-like depot is formed in situ at the site of injection, wherein the fluorescent dye and radionuclide is retained. The radiolabeled markers were optimized for minimal fluorescence quenching and high retention of the positron emission tomography radionuclide64Cu. The performance of the radiolabeled marker was tested in vivo in mice, where it displayed high photostability over a period of 4 weeks, and high retention of64Cu for 48 hours. The retention and biodistribution of 64Cu was quantified via PET/CT, and the fluorescence emission by an in vivo imaging system. The presented data demonstrate proof-of-concept for naphthalocyanine markers as multimodal imaging agents that can bridge the precision of diagnostic imaging into surgical interventions.

Liposomal oxaliplatin remodels the tumor microenvironment and potentiate TLR-agonist–mediated immune responses in cancer., Immunosuppressive cells in the tumor microenvironment allow cancer cells to escape immune recognition and support cancer progression and dissemination. To improve therapeutic efficacy, we designed a liposomal oxaliplatin formulation (PCL8-U75) that elicits cytotoxic effects toward both cancer and immunosuppressive cells via protease-mediated, intratumoral liposome activation. The PCL8-U75 liposomes displayed superior therapeutic efficacy across all syngeneic cancer models in comparison to free-drug and liposomal controls. The PCL8-U75 depleted myeloid-derived suppressor cells and tumor-associated macrophages in the tumor microenvironment. The combination of improved cancer cell cytotoxicity and depletion of immunosuppressive populations of immune cells is attractive for combination with immune-activating therapy. Combining the PCL8-U75 liposomes with a TLR7 agonist induced immunological rejection of established tumors. This combination therapy increased intratumoral numbers of cancer antigen–specific cytotoxic T cells and Foxp3− T helper cells. These results are encouraging toward advancing liposomal drug delivery systems with anticancer and immune-modulating properties into clinical cancer therapy.

The liquid-based Carbo-gel technology enables real-time multimodal image guidance for improved therapeutic precision., Diagnostic imaging often outperforms the surgeon’s ability to identify small structures during therapeutic procedures. Smart soft tissue markers that translate the sensitivity of diagnostic imaging into optimal therapeutic intervention are therefore highly warranted. This paper presents a unique adaptable liquid soft tissue marker system based on functionalized carbohydrates (Carbo-gel). The liquid state of these markers allows for high-precision placement under image guidance using thin needles. Based on step-by-step modifications, the image features and mechanical properties of markers can be optimized to bridge diagnostic imaging and specific therapeutic interventions. The performance of Carbo-gel is demonstrated for markers that (i) have radiographic, magnetic resonance, and ultrasound visibility; (ii) are palpable and visible; and (iii) are localizable by near-infrared fluorescence and radio guidance. The study demonstrates encouraging proof of concept for the liquid marker system as a well-tolerated multimodal imaging marker that can improve image-guided radiotherapy and surgical interventions, including robotic surgery.

The first developed secretory phospholipase A2 (sPLA2) sensitive liposomal cisplatin formulation (LiPlaCis®) is currently undergoing clinical evaluation. In the present study we revisit and evaluate critical preclinical parameters important for the therapeutic potential and safety of platinum drugs, here oxaliplatin (L-OHP), formulated in sPLA2 sensitive liposomes. We show the mole percentage of negatively charged phospholipid needed to obtain enzyme-sensitivity for saturated systems is ≥25% for 16-carbon chain lipid membranes, and >40% for 18-chain lipid membranes, which was surprising as 25% is used clinically in LiPlaCis®. Efficient sPLA2-dependent growth inhibition of colorectal cancer cells was demonstrated in vitro, where cell membrane degradation and cytolysis depends on the sensitivity of the formulation towards the enzyme and is governed by the amount of lysolipids generated and the presence of serum proteins. We found that serum proteins did not affect the lipase activity of the enzyme towards the membranes but instead sequester the lysolipid byproducts consequently inhibiting their detergent-like cytotoxic properties. In vivo therapeutic potential and safety of the liposomes was investigated in nude mice bearing sPLA2-deficient FaDu squamous carcinoma and sPLA2-expressing Colo205 colorectal adenocarcinoma. After intravenous injections, the tumor growth was suppressed for liposomal L-OHP relative to free drug, but only a weak response was observed for both slow- and fast-releasing sPLA2-sensitive formulations compared to non-sensitive liposomes. Also, the mice did not show longer survival. In turn, for the highly sPLA2-sensitive liposomes, multiple high doses caused petechial cutaneous hemorrhages, along with multifocal hepatonecrotic lesions, suggestive of premature activation in skin and liver irrespective of sPLA2-status of the tumor engraft. These results indicate that although liposomal carriers can improve the antitumor efficacy of platinum drugs, sPLA2-triggered release suffers from a narrow therapeutic index and has safety concerns.

Liquid brachytherapy is an emerging technology for internal radiation therapy where liquids containing radionuclides are administered directly into solid tumors. These technologies are less invasive than conventional brachytherapy, and can potentially improve the dose coverage and homogeneity of the radioactivity distribution within the tumor. For this purpose, we have developed a novel cationic micelle system for delivery of a range of radionuclides. The system is applicable for emitters of alpha, beta or photon radiation, and enables dose-mapping via theranostic nuclear imaging. Methods: The cationic micelles were developed as linear surfactants comprising the chelator DOTA, a triarginine sequence and a palmitoyl or stearoyl fatty acid chain. The critical micelle concentration of the surfactants was determined, and the micelles were radiolabelled with 64Cu or 177Lu in high radiochemical purity (>95%). The tumor retention and biodistribution of the 64Cu-radiolabeled surfactants, administered as micelles or formulated in liposomes, were investigated in vivo by PET/CT in a tumor bearing mouse model. Results: The interaction of the micelles with anionic lipid membranes was demonstrated to be favourable, using a liposome partition assay. In vivo, the surfactants formulated both as cationic micelles and liposomes displayed the best intratumoral retention, with micelles providing more homogeneous activity distribution. Conclusion: A cationic, surfactant-based drug delivery system was developed and demonstrated promise as a vehicle for liquid brachytherapy when formulated as micelles or in liposomes. The system enables accurate dosimetry due to the flexible radiochemistry of DOTA.

Background Initiating mechanisms of migraine headache remain poorly understood and a biomarker of migraine does not exist. Inflammation pertaining to the wall of cerebral arteries and brain parenchyma has been suggested to play a role in migraine pathophysiology. Objective We conducted the first experimental human study to investigate macrophage-mediated inflammation as a possible biomarker of migraine. Methods Using ultrasmall superparamagnetic iron oxide (USPIO)-enhanced 3T magnetic resonance imaging (MRI), we investigated the presence of macrophages in cerebral artery walls and in brain parenchyma of patients with migraine without aura. We used the phosphodiesterase-3-inhibitor cilostazol as an experimental migraine trigger, and investigated both patients who received sumatriptan treatment, and patients who did not. To validate our use of USPIO-enhanced MRI, we included a preclinical mouse model with subcutaneous capsaicin injection in the trigeminal V1 area. The study is registered at ClinicalTrials.gov with the identifier NCT02549898. Results A total of 28 female patients with migraine without aura underwent a baseline MRI scan, ingested cilostazol, developed a migraine-like attack, and underwent an USPIO-enhanced MRI scan > 24 hours after intravenous administration of USPIO. Twelve patients treated their attack with 6 mg s.c. sumatriptan, while the remaining 16 patients received no migraine-specific rescue medication. The preclinical model confirmed that USPIO-enhanced MRI detects macrophage-mediated inflammation. In patients, however, migraine attacks were not associated with increased USPIO signal on the pain side of the head compared to the non-pain side. Conclusion Our findings suggest that migraine without aura is not associated with macrophage-mediated inflammation specific to the head pain side.

Background Diffusion weighted magnetic resonance imaging (DW-MRI) holds great potential for monitoring treatment response in cancer patients shortly after initiation of radiotherapy. It is hypothesized that a decrease in cellular density of irradiated cancerous tissue will lead to an increase in quantitative apparent diffusion coefficient (ADC) values. DW-MRI can therefore serve as a non-invasive marker of cell death and apoptosis in response to treatment. In the present study, we aimed to investigate the applicability of DW-MRI in preclinical models to monitor radiation-induced treatment response. In addition, we compared DW-MRI with ex vivo measures of cell density, cell death and apoptosis. Methods DW-MRI was tested in two different syngeneic mouse models, a colorectal cancer (CT26) and a breast cancer (4 T1). ADC values were compared with quantitative determinations of apoptosis and cell death by flow cytometry. Furthermore, ADC-values were also compared to histological measurement of cell density on tumor sections. Results We found a significant correlation between ADC-values and apoptotic state in the CT26 model (P = 0.0031). A strong correlation between the two measurements of ADC-value and apoptotic state was found in both models, which were also present when comparing ADC-values to cell densities. Conclusions Our findings demonstrate that DW-MRI can be used for non-invasive monitoring of radiation-induced changes in cell state during cancer therapy. ADC values reflect ex vivo cell density and correlates well with apoptotic state, and can hereby be described as a marker for the cell state after therapy and used as a non-invasive response marker.

Local application of radioactive sources as brachytherapy is well established in oncology. This treatment is highly invasive however, due to the insertion of millimeter sized metal seeds. The authors report the development of a new concept for brachytherapy, based on gold-palladium (AuPd) alloy nanoparticles, intrinsically radiolabeled with 103 Pd. These are formulated in a carbohydrate-ester based liquid, capable of forming biodegradable gel-like implants upon injection. This allows for less invasive administration through small-gauge needles. [103 Pd]AuPd nanoparticles with sizes around 20 nm are prepared with radiolabeling efficiencies ranging from 79% to >99%. Coating with the hydrophobic polymer poly(N-isopropylacrylamide) leads to nanoparticle diameters below 40 nm. Dispersing the nanoparticles in ethanol with water insoluble carbohydrate esters gives "nanogels", a low viscosity liquid capable of solidifying upon injection into aqueous environments. Both nanoparticles and radioactivity are stably retained in the nanogel over 25 days (>99%) after formation in aqueous buffers. Animals bearing CT26 murine tumors are injected intratumorally with 25 MBq of the 103 Pd-nanogel, and display tumor growth delay and significantly increase median survival times compared with control groups. Excellent retention in the tumor of both the 103 Pd and the nanoparticle matrix itself is observed, demonstrating a potential for replacing currently used brachytherapy seeds.

Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumor and is characterized by its sudden onset and invasive growth into the brain parenchyma. The invasive tumor cells evade conventional treatments and are thought to be responsible for the ubiquitous tumor regrowth. Understanding the behavior of these invasive tumor cells and their response to therapeutic agents could help improve patient outcome. In this study, we present a GBM tumorsphere migration model with high biological complexity to study migrating GBM cells in a quantitative and qualitative manner. We demonstrated that the in vitro migration model could be used to investigate both inhibition and stimulation of cell migration with oxaliplatin and GBM-derived extracellular vesicles, respectively. The intercellular heterogeneity within the GBM tumorspheres was examined by immunofluorescent staining of nestin/vimentin and GFAP, which showed nestin and vimentin being highly expressed in the periphery of tumorspheres and GFAP mostly in cells in the tumorsphere core. We further showed that this phenotypic gradient was present in vivo after implanting dissociated GBM tumorspheres, with the cells migrating away from the tumor being nestin-positive and GFAP-negative. These results indicate that GBM tumorsphere migration models, such as the one presented here, could provide a more detailed insight into GBM cell biology and prove highly relevant as a pre-clinical platform for drug screening and assessing drug response in the treatment of GBM.

Long circulating liposomes entrapping iodinated and radioiodinated compounds offer a highly versatile theranostic platform. Here we report a new methodology for efficient and high-yielding loading of such compounds into liposomes, enabling CT/SPECT/PET imaging and 131I-radiotherapy. Methods: The CT contrast agent diatrizoate was synthetically functionalized with a primary amine, which enabled its remote loading into PEGylated liposomes by either an ammonium sulfate or a citrate based pH transmembrane gradient. Further, the amino-diatrizoate was radiolabeled with either 124I (t1/2 = 4.18 days) for PET or 125I (t1/2 = 59.5 days) for SPECT, through an aromatic Finkelstein reaction. Results: Quantitative loading efficiencies (>99%) were achieved at optimized conditions. The 124I-labeled compound was remote-loaded into liposomes, with an overall radiolabeling efficiency of 77 ± 1%, and imaged in vivo in a CT26 murine colon cancer tumor model by PET/CT. A prolonged blood circulation half-life of 19.5 h was observed for the radiolabeled liposomes, whereas injections of the free compound were rapidly cleared. Lower accumulation was observed in the spleen, liver, kidney and tumor than what is usually seen for long-circulating liposomes. Conclusion: The lower accumulation was interpreted as release of the tracer from the liposomes within these organs after accumulation. These results may guide the design of systems for controlled release of remote loadable drugs from liposomes.

Radiation therapy may affect several important parameters in the tumor microenvironment and thereby influence the accumulation of liposomes by the enhanced permeability and retention (EPR)-effect. Here we investigate the effect of single dose radiation therapy on liposome tumor accumulation by PET/CT imaging using radiolabeled liposomes. Head and neck cancer xenografts (FaDu) and syngenic colorectal (CT26) cancer models were investigated. Radiotherapy displayed opposite effects in the two models. FaDu tumors displayed increased mean accumulation of liposomes for radiation doses up to 10 Gy, whereas CT26 tumors displayed a tendency for decreased accumulation. Tumor hypoxia was found negatively correlated to microregional distribution of liposomes. However, liposome distribution in relation to hypoxia was improved at lower radiation doses. The study reveals that the heterogeneity in liposome tumor accumulation between tumors and different radiation protocols are important factors that need to be taken into consideration to achieve optimal effect of liposome based radio-sensitizer therapy.

Liposomes are nanoparticles used in drug delivery that distribute over several days in humans and larger animals. Radiolabeling with long-lived positron emission tomography (PET) radionuclides, such as manganese-52 (52Mn, T½=5.6days), allow the imaging of this biodistribution. We report optimized protocols for radiolabeling liposomes with 52Mn, through both remote-loading and surface labeling. For comparison, liposomes were also remote-loaded and surface labeled with copper-64 (64Cu, T½=12.7h) through conventional means. The chelator DOTA was used in all cases. The in vivo stability of radiometal chelates is widely debated but studies that mimic a realistic in vivo setting are lacking. Therefore, we employed these four radiolabeled liposome types as platforms to demonstrate a new concept for such in vivo evaluation, here of the chelates 52Mn-DOTA and 64Cu-DOTA. This was done by comparing "shielded" remote-loaded with "exposed" surface labeled variants in a CT26 tumor-bearing mouse model. Remote loading (90min at 55°C) and surface labeling (55°C for 2h) of 52Mn gave excellent radiolabeling efficiencies of 97-100% and 98-100% respectively, and the liposome biodistribution was imaged by PET for up to 8days. Liposomes with surface-conjugated 52Mn-DOTA exhibited a significantly shorter plasma half-life (T½=14.4h) when compared to the remote-loaded counterpart (T½=21.3h), whereas surface-conjugated 64Cu-DOTA cleared only slightly faster and non-significantly, when compared to remote-loaded (17.2±2.9h versus 20.3±1.2h). From our data, we conclude the successful remote-loading of liposomes with 52Mn, and furthermore that 52Mn-DOTA may be unstable in vivo whereas 64Cu-DOTA appears suitable for quantitative imaging.

Liposomes are nanoparticles used in drug delivery that distribute over several days in humans and larger animals. Radiolabeling with long-lived positron emission tomography (PET) radionuclides, such as manganese-52 (

The first developed secretory phospholipase A

Background: In preclinical research MatrixgelTM Basement Membrane Matrix (MG) is used frequently for the establishment of syngeneic and xenograft cancer models. Limited information on its influence on parameters including; tumor growth, vascularization, hypoxia and imaging characteristics is currently available. This study evaluates the potential effect of matrigel use in a human head and neck cancer xenograft model (FaDu; hypopharyngeal carcinoma) in NMRI nude mice. The FaDu cell line was chosen based on its frequent use in studies of cancer imaging and tumor microenvironment. Methods: NMRI nude mice (n = 34) were divided into two groups and subcutaneously injected with FaDu cells in medium either including (+MG) or excluding matrigel (-MG). In sub study I seven mice from each group (+MG, n = 7; -MG, n = 7) were 18F- fluorodeoxyglucose (18F-FDG) PET/CT scanned on Day 5, 8, 12, 15, and 19. In sub study II ten mice from each group (+MG, n = 10; -MG, n = 10) were included and tumors collected for immunohistochemistry (IHC) analysis of tumor microenvironment including; proliferation ratio, micro vessel density, average vessel area, hypoxia, nuclear density, and necrosis. Tumors for IHC were collected according to size (200-400 mm3, 500-700 mm3, 800-1100 mm3). Results: FDG uptake and tumor growth was statistically compatible for the tumors established with or without MG. The IHC analysis on all parameters only identified a significantly higher micro vessel density for tumor size 500-700 mm3 and 800-1100 mm3 and average vessel area for tumor size 500-700 mm3 in the -MG group. Comparable variations were observed for tumors of both the +MG and -MG groups. No difference in tumor take rate was observed between groups in study. Conclusions: Matrigel did not affect tumor growth or tumor take for the FaDu xenograft model evaluated. Tumors in the -MG group displayed increased angiogenesis compared to the +MG tumors. No difference in 18F-FDG PET uptake for tumors of different groups was found. Based on these observations the influence of matrigel on tumor imaging and tumor microenvironment seems minor for this particular xenograft model.