Unravelling Heterogeneities in Complement and Antibody Opsonization of Individual Liposomes as a Function of Surface Architecture.

Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long-circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti-PEG side-effects show that PEGylated nanoparticles are not truly "stealth" to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy-based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibodies and complement C3b is highly dependent on the surface chemistry. It is further demonstrated that this opsonization is heterogeneous, with opsonized and non-opsonized liposomes co-existing in the same ensemble. Surface coatings modify the percentage of opsonized liposomes and/or opsonin surface density on the liposomes, with strikingly different patterns for antibodies and complement. Thus, this assay provides mechanistic details about opsonization at the single nanoparticle level previously inaccessible to established bulk assays.

Accelerated blood clearance and hypersensitivity by PEGylated liposomes containing TLR agonists.

Systemic administration of toll-like receptor (TLR) agonists have demonstrated impressive preclinical results as an anti-cancer therapy due to their potent innate immune-stimulatory properties. The clinical advancement has, however, been hindered by severe adverse effects due to systemic activation of the immune system. Liposomal drug delivery systems may modify biodistribution, cellular uptake, and extend blood circulation, and thus, potentially enable systemic administration of TLR agonists at therapeutic doses. In this study, we investigated potential barriers for the administration of TLR agonists formulated in polyethylene glycosylated (PEGylated) liposomes with regards to liposome formulation, TLR agonist, administration route, administration schedule, biodistribution, blood clearance, and anti-PEG antibodies. We found that administration of TLR agonists formulated in PEGylated liposomes led to high anti-PEG antibody titers, which upon multiple intravenous administrations, resulted in accelerated blood clearance and acute hypersensitivity reactions. The latter was found to be associated with anti-PEG IgG antibody and not anti-PEG IgM antibody opsonization. This study highlights the need to carefully design and evaluate nanoparticle delivery systems for immunotherapy as anti-nanoparticle immune responses may challenge the therapeutic application.

Enhancing adoptive CD8 T cell therapy by systemic delivery of tumor associated antigens.

Adoptive T-cell transfer (ACT) offers a curative therapeutic option for subsets of melanoma and hematological cancer patients. To increase response rates and broaden the applicability of ACT, it is necessary to improve the post-infusion performance of the transferred T cells. The design of improved treatment strategies includes transfer of cells with a less differentiated phenotype. Such T cell subsets have high proliferative potential but require stimulatory signals in vivo to differentiate into tumor-reactive effector T cells. Thus, combination strategies are needed to support the therapeutic implementation of less differentiated T cells. Here we show that systemic delivery of tumor-associated antigens (TAAs) facilitates in vivo priming and expansion of previously non-activated T cells and enhance the cytotoxicity of activated T cells. To achieve this in vivo priming, we use flexible delivery vehicles of TAAs and a TLR7/8 agonist. Contrasting subcutaneous delivery systems, these vehicles accumulate TAAs in the spleen, thereby achieving close proximity to both cross-presenting dendritic cells and transferred T cells, resulting in robust T-cell expansion and anti-tumor reactivity. This TAA delivery platform offers a strategy to safely potentiate the post-infusion performance of T cells using low doses of antigen and TLR7/8 agonist, and thereby enhance the effect of ACT.

Optical tissue clearing and machine learning can precisely characterize extravasation and blood vessel architecture in brain tumors.

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.

Carbohydrate based biomarkers enable hybrid near infrared fluorescence and 64Cu based radio-guidance for improved surgical precision.

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 radionuclide 64Cu. 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 of 64Cu 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.

Establishment and Initial Experience of Clinical FLASH Radiotherapy in Canine Cancer Patients.

FLASH radiotherapy has emerged as a treatment technique with great potential to increase the differential effect between normal tissue toxicity and tumor response compared to conventional radiotherapy. To evaluate the feasibility of FLASH radiotherapy in a relevant clinical setting, we have commenced a feasibility and safety study of FLASH radiotherapy in canine cancer patients with spontaneous superficial solid tumors or microscopic residual disease, using the electron beam of our modified clinical linear accelerator. The setup for FLASH radiotherapy was established using a short electron applicator with a nominal source-to-surface distance of 70 cm and custom-made Cerrobend blocks for collimation. The beam was characterized by measuring dose profiles and depth dose curves for various field sizes. Ten canine cancer patients were included in this initial study; seven patients with nine solid superficial tumors and three patients with microscopic disease. The administered dose ranged from 15 to 35 Gy. To ensure correct delivery of the prescribed dose, film measurements were performed prior to and during treatment, and a Farmer-type ion-chamber was used for monitoring. Treatments were found to be feasible, with partial response, complete response or stable disease recorded in 11/13 irradiated tumors. Adverse events observed at follow-up ranging from 3-6 months were mild and consisted of local alopecia, leukotricia, dry desquamation, mild erythema or swelling. One patient receiving a 35 Gy dose to the nasal planum, had a grade 3 skin adverse event. Dosimetric procedures, safety and an efficient clincal workflow for FLASH radiotherapy was established. The experience from this initial study will be used as a basis for a veterinary phase I/II clinical trial with more specific patient inclusion selection, and subsequently for human trials.

Effective Intratumoral Retention of [103 Pd]AuPd Alloy Nanoparticles Embedded in Gel-Forming Liquids Paves the Way for New Nanobrachytherapy.

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.

Effect of apoA-I PEGylation on the Biological Fate of Biomimetic High-Density Lipoproteins.

Biomimetic high-density lipoproteins (b-HDL) have in the past two decades been applied for various drug delivery applications. As b-HDL inherently have relatively long circulation half-life and high tumor accumulation, this has inspired researchers to use b-HDL to selectively deliver drugs to tumors. PEGylation of the b-HDL has been pursued to increase the circulation half-life and therapeutic efficacy even further. The b-HDL consist of lipids stabilized by a protein/peptide scaffold, and while PEGylation of the scaffold has been shown to greatly increase the circulation half-life of the scaffold, the effect of PEGylation of the lipids is much less significant. Still, it remains to be evaluated how the biological fate, including cellular uptake, biodistribution, and circulation half-life, of the b-HDL lipids is affected by PEGylation of the b-HDL scaffold. We studied this with apolipoprotein A-I (apoA-I)-based b-HDL and mono-PEGylated b-HDL (PEG b-HDL) both in vitro and in vivo. We found that PEGylation of the b-HDL scaffold only seemed to have minimal effect on the biological fate of the lipids. Both b-HDL and PEG b-HDL overall shared similar biological fates, which includes cellular uptake through the scavenger receptor class B type 1 (SR-BI) and relatively high tumor accumulation. This highlights that b-HDL are dynamic particles, and the biological fates of the b-HDL components (lipids and scaffold) can differ. A phenomenon that may also apply for other multicomponent nanoparticles.

Multimodal soft tissue markers for bridging high-resolution diagnostic imaging with therapeutic intervention.

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.

Preclinical evaluation of cationic DOTA-triarginine-lipid conjugates for theranostic liquid brachytherapy.

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.

Folate receptor targeting of radiolabeled liposomes reduces intratumoral liposome accumulation in human KB carcinoma xenografts.

BACKGROUND: Active, ligand-mediated, targeting of functionalized liposomes to folate receptors (FRs) overexpressed on cancer cells could potentially improve drug delivery and specificity. Studies on folate-targeting liposomes (FTLs) have, however, yielded varying results and generally fail to display a clear benefit of FR targeting. METHOD: Tumor accumulating potential of FTLs and NTLs were investigated in a FR overex-pressing xenograft model by positron emission tomography/computed tomography imaging. RESULTS: Tumors displayed significantly lower activity of FTLs than NTLs. Furthermore, FTLs displayed worse circulating properties and increased liver-accumulation than NTLs. CONCLUSION: This study underlines that long-circulating properties of liposomes must be achieved to take advantage of EPR-dependent tumor accumulation which may be lost by functionalization. FR-functionalization negatively affected both tumor accumulation and circulation properties.

Theranostic Imaging May Vaccinate against the Therapeutic Benefit of Long Circulating PEGylated Liposomes and Change Cargo Pharmacokinetics.

The enhanced permeability and retention (EPR) effect increases tumor accumulation of liposomal chemotherapy and should, in theory, increase anticancer effects and lower toxicity. Unfortunately, liposomal chemotherapy has generally not met the expected potential, perhaps because the EPR effect is not ubiquitous. PET imaging using radiolabeled liposomes can identify cancers positive for the EPR effect. In the current study, we show in clinical canine cancer patients that repeated imaging with radiolabeled liposomes (64Cu-liposome) induces the accelerated blood clearance (ABC) phenomenon. This was observed even with very long intervals between PEGylated liposome injections, which contradict previous reporting in experimental animal models. The induction of ABC may be devastating for the theranostic use of liposomal imaging, as this could vaccinate patients against therapeutic efficacy. To investigate and solve this important problem, an additional study part was designed in which rats were subjected to repeated liposomal administrations, including stealth 64Cu-liposome PET imaging and Caelyx chemotherapy. Most importantly, it was found that, by increasing the lipid dose at the first injection or by supplying a small predose before the second 64Cu-liposome injection, ABC could be prevented. Importantly, signs of liposome tracer breakdown with subsequent renal excretion were observed. These findings highlight the importance of the ABC phenomenon for liposomal predictive imaging in a clinically relevant setting and show that carefully planned application is central to avoid potential detrimental effects on patient benefit.

Methotrexate prodrugs sensitive to reactive oxygen species for the improved treatment of rheumatoid arthritis.

Methotrexate (MTX) is the standard of care in the treatment of rheumatoid arthritis (RA), a common autoimmune disease that is characterized by chronic inflammation in the synovial membrane of joints. Unfortunately, MTX suffers from high discontinuation rates due to a large variability in efficacy and, in particular, adverse effects. As inflammation is associated with elevated levels of reactive oxygen species (ROS) like H2O2, we propose to improve treatment through site-selective delivery of MTX to inflammatory tissue by use of a H2O2 sensitive MTX prodrug. To establish proof proof-of-concept, two novel H2O2 sensitive, thiazolidinone-based MTX prodrugs were synthesized and evaluated for this purpose. MTX-γ-thiazolidinone (MTX-γ-TZ) exhibited the most promising properties - good to high chemical and metabolic stability, excellent aqueous solubility, while being activated when subjected to patho-physiological concentrations of H2O2. In vivo, MTX-γ-TZ exhibited comparable efficacy to MTX in a murine collagen type II-induced arthritis (CIA) model while treated mice showed indications of reduced toxicity as their body weight decreased less towards the end of the study, compared to the MTX-treated group.

Synthesis and Evaluation of Hydrogen Peroxide Sensitive Prodrugs of Methotrexate and Aminopterin for the Treatment of Rheumatoid Arthritis.

A series of novel hydrogen peroxide sensitive prodrugs of methotrexate (MTX) and aminopterin (AMT) were synthesized and evaluated for therapeutic efficacy in mice with collagen induced arthritis (CIA) as a model of chronic rheumatoid arthritis (RA). The prodrug strategy selected is based on ROS-labile 4-methylphenylboronic acid promoieties linked to the drugs via a carbamate linkage or a direct C-N bond. Activation under pathophysiological concentrations of H2O2 proved to be effective, and prodrug candidates were selected in agreement with relevant in vitro physicochemical and pharmacokinetic assays. Selected candidates showed moderate to good solubility, high chemical and enzymatic stability, and therapeutic efficacy comparable to the parent drugs in the CIA model. Importantly, the prodrugs displayed the expected safer toxicity profile and increased therapeutic window compared to MTX and AMT while maintaining a comparable therapeutic efficacy, which is highly encouraging for future use in RA patients.

Remote loading of liposomes with a 124I-radioiodinated compound and their in vivo evaluation by PET/CT in a murine tumor model.

Long circulating liposomes entrapping iodinated and radioiodinated compounds offer a highly versatile theranostic platform. Here we report a new methodology for efficient and high-yield 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.

Remote-loading of liposomes with manganese-52 and in vivo evaluation of the stabilities of 52Mn-DOTA and 64Cu-DOTA using radiolabelled liposomes and PET 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 (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.

Revisiting the use of sPLA2-sensitive liposomes in cancer therapy.

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.

Mouse Positron Emission Tomography Study of the Biodistribution of Gold Nanoparticles with Different Surface Coatings Using Embedded Copper-64.

By taking advantage of the ability of 64Cu to bind nonspecifically to gold surfaces, we have developed a methodology to embed this radionuclide inside gold nanoparticles (AuNPs). 64Cu enables the in vivo imaging of AuNPs by positron emission tomography (PET). AuNPs have a multitude of uses within health technology and are useful tools for general nanoparticle research. 64Cu-AuNPs were prepared by incubating AuNP seeds with 64Cu2+, followed by the entrapment of the radionuclide by grafting on a second layer of gold. This resulted in radiolabeling efficiencies of 53 ± 6%. The radiolabel showed excellent stability when incubated with EDTA for 2 days (95% radioactivity retention) and showed no loss of 64Cu when incubated with 50% mouse serum for 2 days. The methodology was chelator-free, removing traditional concerns over chelator instability and altered AuNP properties due to surface modification. Radiolabeled 64Cu-AuNP cores were prepared in biomedically relevant sizes of 20-30 nm and used to investigate the in vivo stability of three different AuNP coatings by PET imaging in a murine xenograft tumor model. We found the longest plasma half-life (T1/2 about 9 h) and tumor accumulation (3.9%ID/g) to result from a polyethylene glycol coating, while faster elimination from the bloodstream was observed with both a Tween 20-stabilized coating and a zwitterionic coating based on a mixture of sulfonic acids and quaternary amines. In the in vivo model, the 64Cu was observed to closely follow the AuNPs for each coating, again attributed to the excellent stability of the radiolabel.

The use of matrigel has no influence on tumor development or PET imaging in FaDu human head and neck cancer xenografts.

BACKGROUND: In preclinical research Matrixgel(TM) 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 (18)F- fluorodeoxyglucose ((18)F-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 mm(3), 500-700 mm(3), 800-1100 mm(3)). 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 mm(3) and 800-1100 mm(3) and average vessel area for tumor size 500-700 mm(3) 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 (18)F-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.

64Cu-ATSM Reflects pO2 Levels in Human Head and Neck Cancer Xenografts but Not in Colorectal Cancer Xenografts: Comparison with 64CuCl2.

UNLABELLED: The hypoxia PET tracer (64)Cu-diacetyl-bis(N(4)-methylthiosemicarbazonate) ((64)Cu-ATSM) has shown promising results in clinical studies. However, concerns have been raised with regard to the possible effect of copper metabolism and free copper on tumor uptake and thereby the robustness of (64)Cu-ATSM as a hypoxia marker. In this study, accumulation and distribution of (64)Cu-ATSM and (64)CuCl2 in tumor tissue were compared with partial pressure of oxygen (pO2) probe measurements. METHODS: One-hour dynamic PET scans were performed on nude mice bearing subcutaneous human head and neck tumors (FaDu) and human colorectal tumors (HT29) after administration of either (64)Cu-ATSM or (64)CuCl2. Subsequently, tracks were generated and track markers were positioned in tumors to allow for registration of their exact location on the high-resolution CT scan. After completion of the CT scan, pO2 probe measurements were performed along each track. PET and CT images were coregistered and ROIs drawn on the basis of the location of track markers and pO2 probe measurement depth. A linear mixed model for repeated measures was applied for the comparison of PET tracer uptake to corresponding pO2 values. RESULTS: Comparable uptake of (64)Cu-ATSM and (64)CuCl2 was found in the kidney, muscle, and liver of all animals, but (64)CuCl2 showed a higher uptake 10-60 min after injection in both tumor models. Significant differences were also found for both tumor-to-muscle and tumor-to-liver ratios. The intratumoral distribution of (64)Cu-ATSM, but not (64)CuCl2, showed a significant negative relationship with pO2 measurements in FaDu tumors. However, this relationship was not found in HT29 tumors. CONCLUSION: (64)Cu-ATSM and (64)CuCl2 displayed different uptake in tumors. In human head and neck xenografts, (64)Cu-ATSM but not (64)CuCl2 reflected pO2 measurements, indicating that (64)Cu-ATSM is a hypoxia-specific marker in this tumor type. However, data from colorectal cancer xenografts indicated that (64)Cu-ATSM may not be a hypoxia marker in all tumor types.