ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC), characterized by hypovascularity, hypoxia, and desmoplastic stroma is one of the deadliest malignancies in humans, with a 5-year survival rate of only 7%. The anatomical location of the pancreas and lack of symptoms in patients with early onset of disease accounts for late diagnosis. Consequently, 85% of patients present with non-resectable, locally advanced, or advanced metastatic disease at diagnosis and rely on alternative therapies such as chemotherapy, immunotherapy, and others. The response to these therapies highly depends on the stage of disease at the start of therapy. It is, therefore, vital to consider the stages of PDAC models in preclinical studies when testing new therapeutics and treatment modalities. We report a standardized induction of cell-based orthotopic pancreatic cancer models in mice and the identification of vital features of their progression by ultrasound imaging and histological analysis of the level of pancreatic stellate cells, mature fibroblasts, and collagen. The results highlight that early-stage primary tumors are secluded in the pancreas and advance towards infiltrating the omentum at week 5-7 post implantation of the BxPC-3 and Panc-1 models investigated. Late stages show extensive growth, the infiltration of the omentum and/or stomach wall, metastases, augmented fibroblasts, and collagen levels. The findings can serve as suggestions for defining growth parameter-based stages of orthotopic pancreatic cancer models for the preclinical testing of drug efficacy in the future.
Subject(s)
Carcinoma, Pancreatic Ductal , Disease Models, Animal , Pancreatic Neoplasms , Animals , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/genetics , Mice , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/metabolism , Humans , Cell Line, TumorABSTRACT
Tumors are a highly heterogeneous mass of tissue showing distinct therapy responses. In particular, the therapeutic outcome of tumor hyperthermia treatments has been inconsistent, presumably due to tumor versus endothelial cell cross-talks related to the treatment temperature and the tumor tissue environment. Here, we investigated the impact of the average or strong hyperthermic treatment (43 °C or 47 °C for 1 h) of the human pancreatic adenocarcinoma cell line (PANC-1 and BxPC-3) on endothelial cells (HUVECs) under post-treatment normoxic or hypoxic conditions. Immediately after the hyperthermia treatment, the distinct repression of secreted pro-angiogenic factors (e.g., VEGF, PDGF-AA, PDGF-BB, M-CSF), intracellular HIF-1α and the enhanced phosphorylation of ERK1/2 in tumor cells were detectable (particularly for strong hyperthermia, 2D cell monolayers). Notably, there was a significant increase in endothelial sprouting when 3D self-organized pancreatic cancer cells were treated with strong hyperthermia and the post-treatment conditions were hypoxic. Interestingly, for the used treatment temperatures, the intracellular HIF-1α accumulation in tumor cells seems to play a role in MAPK/ERK activation and mediator secretion (e.g., VEGF, PDGF-AA, Angiopoietin-2), as shown by inhibition experiments. Taken together, the hyperthermia of pancreatic adenocarcinoma cells in vitro impacts endothelial cells under defined environmental conditions (cell-to-cell contact, oxygen status, treatment temperature), whereby HIF-1α and VEGF secretion play a role in a complex context. Our observations could be exploited for the hyperthermic treatment of pancreatic cancer in the future.
ABSTRACT
There is a growing interest in exploring the therapeutically mediated modulation of tumor vascularization of pancreatic cancer, which is known for its poorly perfused tumor microenvironment limiting the delivery of therapeutic agents to the tumor site. Here, we assessed how magnetic hyperthermia in combination with chemotherapy selectively affects growth, the vascular compartment of tumors, and the presence of tumor cells expressing key regulators of angiogenesis. To that purpose, a orthotopic PANC-1 (fluorescent human pancreatic adenocarcinoma) mouse tumor model (Rj:Athym-Foxn1nu/nu) was used. Magnetic hyperthermia was applied alone or in combination with systemic chemotherapy (gemcitabine 50 mg per kg body weight, nab-pacitaxel 30 mg/kg body weight) on days 1 and 7 following magnetic nanoparticle application (dose: 1 mg per 100 mm3 of tumor). We used ultrasound imaging, immunohistochemistry, multi-spectral optoacoustic tomography (MSOT), and hematology to assess the biological parameters mentioned above. We found that magnetic hyperthermia in combination with gemcitabine/paclitaxel chemotherapy was able to impact tumor growth (decreased volumes and Ki67 expression) and to trigger neo-angiogenesis (increased small vessel diameter) as a result of the therapeutically mediated cell damages/stress in tumors. The applied stressors activated specific pro-angiogenic mechanisms, which differed from those seen in hypoxic conditions involving HIF-1α, since (a) treated tumors showed a significant decrease of cells expressing VEGF, CD31, HIF-1α, and neuropilin-1; and (b) the relative tumor blood volume and oxygen level remained unchanged. Neo-angiogenesis seems to be the result of the activation of cell stress pathways, like MAPK pathways (high number of pERK-expressing tumor cells). In the long term, the combination of magnetic hyperthermia and chemotherapy could potentially be applied to transiently modulate tumor angiogenesis and to improve drug accessibility during oncologic therapies of pancreatic cancer.
ABSTRACT
OBJECTIVE: Deep-tissue localization of thermal doses is a long-standing challenge in magnetic field hyperthermia (MFH), and remains a limitation of the clinical application of MFH to date. Here, we show that pulse sequencing of MFH leads to a more persistent inhibition of tumor growth and less systemic impact than continuous MFH, even when delivering the same thermal dose. METHODS: We used an in vivo orthotopic murine model of pancreatic PANC-1 cancer, which was designed with a view to the forthcoming 'NoCanTher' clinical study, and featured MFH alongside systemic chemotherapy (SyC: gemcitabine and nab-paclitaxel). In parallel, in silico thermal modelling was implemented. RESULTS: Tumor volumes 27 days after the start of MFH/SyC treatment were 53% (of the initial volume) in the pulse MFH group, compared to 136% in the continuous MFH group, and 337% in the non-treated controls. Systemically, pulse MFH led to ca. 50% less core-temperature increase in the mice for a given injected dose of magnetic heating agent, and inflicted lower levels of the stress marker, as seen in the blood-borne neutrophil-to-lymphocyte ratio (1.7, compared to 3.2 for continuous MFH + SyC, and 1.2 for controls). CONCLUSION: Our data provided insights into the influence of pulse sequencing on the observed biological outcomes, and validated the nature of the improved thermal dose localization, alongside significant lowering of the overall energy expenditure entailed in the treatment.
Subject(s)
Hyperthermia, Induced , Pancreatic Neoplasms , Animals , Hyperthermia , Magnetic Fields , Magnetics , Mice , Pancreatic Neoplasms/therapyABSTRACT
Magnetic hyperthermia can cause localized thermal eradication of several solid cancers. However, a localized and homogenous deposition of high concentrations of magnetic nanomaterials into the tumor stroma and tumor cells is mostly required. Poorly responsive cancers such as the pancreatic adenocarcinomas are hallmarked by a rigid stroma and poor perfusion to therapeutics and nanomaterials. Hence, approaches that enhance the infiltration of magnetic nanofluids into the tumor stroma convey potentials to improve thermal tumor therapy. We studied the influence of the matrix-modulating enzymes hyaluronidase and collagenase on the uptake of magnetic nanoparticles by pancreatic cancer cells and 3D spheroids thereof, and the overall impact on magnetic heating and cell death. Furthermore, we validated the effect of hyaluronidase on magnetic hyperthermia treatment of heterotopic pancreatic cancer models in mice. Treatment of cultured cells with the enzymes caused higher uptake of magnetic nanoparticles (MNP) as compared to nontreated cells. For example, hyaluronidase caused a 28% increase in iron deposits per cell. Consequently, the thermal doses (cumulative equivalent minutes at 43 °C, CEM43) increased by 15-23% as compared to heat dose achieved for cells treated with magnetic hyperthermia without using enzymes. Likewise, heat-induced cell death increased. In in vivo studies, hyaluronidase-enhanced infiltration and distribution of the nanoparticles in the tumors resulted in moderate heating levels (CEM43 of 128 min as compared to 479 min) and a slower, but persistent decrease in tumor volumes over time after treatment, as compared to comparable treatment without hyaluronidase. The results indicate that hyaluronidase, in particular, improves the infiltration of magnetic nanoparticles into pancreatic cancer models, impacts their thermal treatment and cell depletion, and hence, will contribute immensely in the fight against pancreatic and many other adenocarcinomas.
ABSTRACT
Liposomes represent suitable tools for the diagnosis and treatment of a variety of diseases, including cancers. To study the role of the human epidermal growth factor receptor 2 (HER2) as target in cancer imaging and image-guided deliveries, liposomes were encapsulated with an intrinsically quenched concentration of a near-infrared fluorescent dye in their aqueous interior. This resulted in quenched liposomes (termed LipQ), that were fluorescent exclusively upon degradation, dye release, and activation. The liposomes carried an always-on green fluorescent phospholipid in the lipid layer to enable tracking of intact liposomes. Additionally, they were functionalized with single-chain antibody fragments directed to fibroblast activation protein (FAP), a marker of stromal fibroblasts of most epithelial cancers, and to HER2, whose overexpression in 20-30% of all breast cancers and many other cancer types is associated with a poor treatment outcome and relapse. We show that both monospecific (HER2-IL) and bispecific (Bi-FAP/HER2-IL) formulations are quenched and undergo HER2-dependent rapid uptake and cargo release in cultured target cells and tumor models in mice. Thereby, tumor fluorescence was retained in whole-body NIRF imaging for 32-48 h post-injection. Opposed to cell culture studies, Bi-FAP/HER2-IL-based live confocal microscopy of a high HER2-expressing tumor revealed nuclear delivery of the encapsulated dye. Thus, the liposomes have potentials for image-guided nuclear delivery of therapeutics, and also for intraoperative delineation of tumors, metastasis, and tumor margins.
ABSTRACT
Liposomes are biocompatible nanocarriers with promising features for targeted delivery of contrast agents and drugs into the tumor microenvironment, for imaging and therapy purposes. Liposome-based simultaneous targeting of tumor associated fibroblast and the vasculature is promising, but the heterogeneity of tumors entails a thorough validation of suitable markers for targeted delivery. Thus, we elucidated the potential of bispecific liposomes targeting the fibroblast activation protein (FAP) on tumor stromal fibroblasts, together with endoglin which is overexpressed on tumor neovascular cells and some neoplastic cells. Fluorescence-quenched liposomes were prepared by hydrating a lipid film with a high concentration of the self-quenching near-infrared fluorescent dye, DY-676-COOH, to enable fluorescence detection exclusively upon liposomal degradation and subsequent activation. A non-quenched green fluorescent phospholipid was embedded in the liposomal surface to fluorescence-track intact liposomes. FAP- and murine endoglin-specific single chain antibody fragments were coupled to the liposomal surface, and the liposomal potentials validated in tumor cells and mice models. The bispecific liposomes revealed strong fluorescence quenching, activatability, and selectivity for target cells and delivered the encapsulated dye selectively into tumor vessels and tumor associated fibroblasts in xenografted mice models and enabled their fluorescence imaging. Furthermore, detection of swollen lymph nodes during intra-operative simulations was possible. Thus, the bispecific liposomes have potentials for targeted delivery into the tumor microenvironment and for image-guided surgery.
ABSTRACT
Desmoplasia, an aberrant production of extracellular matrix (ECM), is considered as one predictive marker of malignancy of pancreatic cancer. In this paper, we study the effect of mild hyperthermia on fibrillary collagen architecture in murine Achilles tendons and in a pancreatic cancer model, in vitro, i.e. 3D hetero-type tumor spheroids, consisting of pancreatic cancer (Panc-1) cells and fibroblasts (WI-38), producing collagen fibers. We clearly demonstrate that i) mild hyperthermia (40 °C, 42 °C) damages the collagen architecture in murine Achilles tendons. ii) Mild extrinsic (hot air) and iron oxide nanoparticle based magnetic hyperthermia reduce the level of collagen fiber architecture in the generated hetero-type tumor spheroids. iii) Mild magnetic hyperthermia reduces cell vitality mainly through apoptotic and necrotic processes in the generated tumor spheroids. In conclusion, hetero-type 3D tumor spheroids are suitable for studying the effect of hyperthermia on collagen fibers, in vitro.
Subject(s)
Collagen/metabolism , Hyperthermia/metabolism , Pancreatic Neoplasms/metabolism , Spheroids, Cellular/metabolism , Spheroids, Cellular/pathology , Animals , Apoptosis/physiology , Cell Line, Tumor , Cell Survival/physiology , Humans , Magnetic Iron Oxide Nanoparticles/chemistry , Mice , Pancreatic Neoplasms/pathology , Pancreatic NeoplasmsABSTRACT
In the last three decades, many new cell-penetrating peptides (CPPs) were developed that exhibited enhanced cell selectivity. Thus, we aimed to validate the tumor cell selectivity of peptides from this new generation, namely fragments mini-crotamine and mini-maurocalcine. Both of these peptides are derived from venoms. Furthermore, we studied an analog of the classical CPP HIV-TAT(47-57) with alternating chirality of Arg residues. To allow covalent coupling of cargoes or fluorophores, a cysteine residue was introduced to the N-terminus of the synthesized peptides. The therapeutic antibody trastuzumab conjugated to different fluorescent dyes was used for internalization studies. Comparison of uptake efficiencies revealed that CPPs of the new generation are in contrast to MPG-peptides, nearly unable to internalize the noncovalently formed complexes with trastuzumab. Interestingly, the fluorescent derivative of the crotamine fragment was mainly observed in a subpopulation of breast cancer cells, whereas it was homogenously distributed in fibrosarcoma, colon cancer, and noncancerous endothelia cells. Thus, the fluorescent crotamine fragment reported herein is a potent theranostic tool for image-guided applications. This peptide can be used to pinpoint the level of heterogeneity present within tumors and aid in the generation of therapeutics that target heterogenic subpopulations.
Subject(s)
Antineoplastic Agents/pharmacology , Cell-Penetrating Peptides/pharmacology , Crotalid Venoms/chemistry , Endothelial Cells/drug effects , Neoplasms/drug therapy , Scorpion Venoms/chemistry , tat Gene Products, Human Immunodeficiency Virus/chemistry , Antineoplastic Agents/chemistry , Cell-Penetrating Peptides/chemistry , Cells, Cultured , Endothelial Cells/cytology , Humans , Neoplasms/pathology , Protein TransportABSTRACT
The underlying data demonstrates that the expression of endoglin in murine melanoma cells influences melanin production in the cells. Also, the data shows that melanin production is further increased when the cells are subcutaneously implanted in mice models and that the high melanin production prevents detection of the cells by fluorescence imaging. The processed data presented herein is related to a research article by Tansi et al. (2018) entitled "Endoglin based in vivo near-infrared fluorescence imaging of tumor models in mice using activatable liposomes".
ABSTRACT
BACKGROUND: Endoglin (CD105) is overexpressed on tumor cells and tumor vasculatures, making it a potential target for diagnostic imaging and therapy of different neoplasms. Therefore, studies on nanocarrier systems designed for endoglin-directed diagnostic and drug delivery purposes would expose the feasibility of targeting endoglin with therapeutics. METHODS: Liposomes carrying high concentrations of a near-infrared fluorescent dye in the aqueous interior were prepared by the lipid film hydration and extrusion procedure, then conjugated to single chain antibody fragments either selective for murine endoglin (termed mEnd-IL) or directed towards human endoglin (termed hEnd-IL). A combination of Dynamic Light Scattering, electron microscopy, cell binding and uptake assays, confocal microscopy and in vivo fluorescence imaging of mice bearing xenografted human breast cancer and human fibrosarcoma models were implemented to elucidate the potentials of the liposomes. RESULTS: The mEnd-IL and hEnd-IL were highly selective for the respective murine- and human endoglin expressing cells in vitro and in vivo. Hence, the hEnd-IL bound distinctly to the tumor cells and enabled suitable fluorescence imaging of the tumors, whereas the mEnd-IL bound the tumor vasculature, but also to the liver, kidney and lung vasculature of mice. CONCLUSIONS: The work highlights key differences between targeting vascular (murine) and neoplastic (human) endoglin in animal studies, and suggests that the hEnd-IL can serve as a delivery system that targets human endoglin overexpressed in pathological conditions. GENERAL SIGNIFICANCE: The endoglin-targeting liposomes presented herewith represent strategic tools for the future implementation of endoglin-directed neoplastic and anti-angiogenic therapies.
Subject(s)
Breast Neoplasms/metabolism , Endoglin/metabolism , Fibrosarcoma/metabolism , Fluorescence , Liposomes , Single-Chain Antibodies/immunology , Spectroscopy, Near-Infrared/methods , Animals , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Endoglin/immunology , Female , Fibrosarcoma/immunology , Fibrosarcoma/pathology , Fluorescent Dyes , Humans , Mice , Optical Imaging/methods , Single-Chain Antibodies/administration & dosage , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Molecular targeting plays a significant role in cancer diagnosis and therapy. However, the heterogeneity of tumors is a limiting obstacle for molecular targeting. Consequently, clinically approved drug delivery systems such as liposomes still rely on passive targeting to tumors, which does not address tumor heterogeneity. In this work, we therefore designed and elucidated the potentials of activatable bispecific targeted liposomes for simultaneous detection of fibroblast activation protein (FAP) and the human epidermal growth factor receptor 2 (HER2). The bispecific liposomes were encapsulated with fluorescence-quenched concentrations of the near-infrared fluorescent dye, DY-676-COOH, making them detectable solely post processing within target cells. The liposomes were endowed with a combination of single chain antibody fragments specific for FAP and HER2 respectively, or with the FAP single chain antibody fragment in combination with Trastuzumab, which is specific for HER2. The Trastuzumab based bispecific formulation, termed Bi-FAP/Tras-IL revealed delivery of the encapsulated dye into the nuclei of HER2 expressing cancer cells and caused cell death at significantly higher rates than the free Trastuzumab. Furthermore, fluorescence imaging and live microscopy of tumor models in mice substantiated the delivery of the encapsulated cargo into the nuclei of target tumor cells and tumor stromal fibroblasts. Hence, they convey potentials to address tumor plasticity, to improve targeted cancer therapy and reduce Trastuzumab resistance in the future. STATEMENT OF SIGNIFICANCE: This work demonstrates the design of activatable bispecific liposomes aimed to target HER2, a poor prognosis tumor marker in many tumor types, and fibroblast activation protein (FAP), a universal tumor marker overexpressed on tumor fibroblasts and pericytes of almost all solid tumors. Encapsulating liposomes with a quenched concentration of a NIRF dye which only fluoresced after cellular degradation and activation enabled reliable visualization of the destination of the cargo in cells and animal studies. Conjugating single chain antibody fragments directed to FAP, together with Trastuzumab, a humanized monoclonal antibody for HER2 resulted in the activatable bispecific liposomes. In animal models of xenografted human breast tumors, the remarkable ability of the bispecific probes to simultaneously deliver the encapsulated dye into the nuclei of target tumor cells and tumor fibroblasts could be demonstrated. Hence, the bispecific probes represent model tools with high significance to address tumor heterogeneity and manage Trastuzumab resistance in the future.
Subject(s)
Antineoplastic Agents, Immunological , Gelatinases/antagonists & inhibitors , Membrane Proteins/antagonists & inhibitors , Neoplasms, Experimental/drug therapy , Single-Chain Antibodies , Trastuzumab , Animals , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/pharmacology , Endopeptidases , Female , Gelatinases/metabolism , Humans , Liposomes , MCF-7 Cells , Membrane Proteins/metabolism , Mice , Mice, Nude , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Serine Endopeptidases/metabolism , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/pharmacology , Trastuzumab/chemistry , Trastuzumab/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Preclinical research implementing fluorescence-based approaches is inevitable for drug discovery and technology. For example, a variety of contrast agents developed for biomedical imaging are usually evaluated in cell systems and animal models based on their conjugation to fluorescent dyes. Biodistribution studies of excised organs are often performed by macroscopic imaging, whereas the subcellular localization though vital, is often neglected or further validated by histological procedures. Available systems used to define the subcellular biodistribution of contrast agents such as intravital microscopes or ex vivo histological analysis are expensive and not affordable by the majority of researchers, or encompass tedious and time consuming steps that may modify the contrast agents and falsify the results. Thus, affordable and more reliable approaches to study the biodistribution of contrast agents are required. We developed fluorescent immunoliposomes specific for human fibroblast activation protein and murine endoglin, and used macroscopic fluorescence imaging and confocal microscopy to determine their biodistribution and subcellular localization in freshly excised mice organs at different time points post intravenous injection. RESULTS: Near infrared fluorescence macroscopic imaging revealed key differences in the biodistribution of the respective immunoliposomes at different time points post injection, which correlated to the first-pass effect as well as the binding of the probes to molecular targets within the mice organs. Thus, a higher accumulation and longer retention of the murine endoglin immunoliposomes was seen in the lungs, liver and kidneys than the FAP specific immunoliposomes. Confocal microscopy showed that tissue autofluorescence enables detection of organ morphology and cellular components within freshly excised, non-processed organs, and that fluorescent probes with absorption and emission maxima beyond the tissue autofluorescence range can be easily distinguished. Hence, the endoglin targeting immunoliposomes retained in some organs could be detected in the vascular endothelia cells of the organs. CONCLUSIONS: The underlying work represents a quick, effective and more reliable setup to validate the macroscopic and subcellular biodistribution of contrast agents in freshly excised animal organs. The approach will be highly beneficial to many researchers involved in nanodrug design or in fluorescence-based studies on disease pathogenesis.
Subject(s)
Antibodies, Monoclonal/immunology , Liposomes/immunology , Microscopy, Fluorescence/methods , Molecular Imaging/methods , Subcellular Fractions/immunology , Viscera/immunology , Animals , Female , In Vitro Techniques , Metabolic Clearance Rate/immunology , Mice , Mice, Nude , Microscopy, Confocal/methods , Organ Specificity/immunology , Tissue Distribution/immunologyABSTRACT
The underlying data demonstrates that fibroblast activation protein (FAP) paves the way for fibrosarcoma cells, which require the proteolysis of the extracellular matrix (ECM) and basement membranes to intravasate from implanted subcutaneous primary tumors into blood vessels, be transported to distant organs where they extravasate from the blood vessels, reattach and proliferate to metastases. The data additionally shows that FAP, when overexpressed on fibrosarcoma cells induces their invasion and formation of spontaneous metastases in multiple organs, particularly after subcutaneous co-implantation of the FAP-expressing and wildtype fibrosarcoma. The raw and processed data presented herein is related to a research article entitled "Potential of activatable FAP-targeting immunoliposomes in intraoperative imaging of spontaneous metastases" (F.L. Tansi, R. Rüger, C. Böhm, R.E. Kontermann, U.K. Teichgraeber, A. Fahr, I. Hilger, 2016) [1]. Furthermore, evidence for the detection of FAP-expressing tumor cells and cells of the tumor stroma by activatable FAP-targeting liposomes is presented in this dataset.
ABSTRACT
Despite intensive research and medical advances met, metastatic disease remains the most common cause of death in cancer patients. This results from late diagnosis, poor therapeutic response and undetected micrometastases and tumor margins during surgery. One approach to overcome these challenges involves fluorescence imaging, which exploits the properties of fluorescent probes for diagnostic detection of molecular structures at the onset of transformation and for intraoperative detection of metastases and tumor margins in real time. Considering these benefits, many contrast agents suitable for fluorescence imaging have been reported. However, most reports only demonstrate the detection of primary tumors and not the detection of metastases or their application in models of image-guided surgery. In this work, we demonstrate the influence of fibroblast activation protein (FAP) on the metastatic potential of fibrosarcoma cells and elucidate the efficacy of activatable FAP-targeting immunoliposomes (FAP-IL) for image-guided detection of the spontaneous metastases in mice models. Furthermore, we characterized the biodistribution and cellular localization of the liposomal fluorescent components in mice organs and traced their excretion over time in urine and feces. Taken together, activatable FAP-IL enhances intraoperative imaging of metastases. Their high accumulation in metastases, subsequent localization in the bile canaliculi and liver kupffer cells and suitable excretion in feces substantiates their potency as contrast agents for intraoperative imaging.
Subject(s)
Fibrosarcoma/pathology , Gelatinases/metabolism , Liposomes/metabolism , Liposomes/pharmacokinetics , Membrane Proteins/metabolism , Molecular Imaging/methods , Neoplasm Metastasis/diagnosis , Serine Endopeptidases/metabolism , Animals , Cell Line, Tumor , Cell Movement , Endopeptidases , Female , Fibrosarcoma/metabolism , Humans , Mice , Mice, Nude , Neoplasm Metastasis/pathologyABSTRACT
Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.
Subject(s)
Fluorescent Dyes/chemistry , Liposomes/chemistry , Optical Imaging/methods , Spectroscopy, Near-Infrared/methods , Animals , Fluorescence , MiceABSTRACT
Molecular and cellular changes that precede the invasive growth of solid tumors include the release of proteolytic enzymes and peptides in the tumor stroma, the recruitment of phagocytic and lymphoid infiltrates and alteration of the extracellular matrix. The reactive tumor stroma consists of a large number of myofibroblasts, characterized by high expression of fibroblast activation protein alpha (FAP). FAP, a type-II transmembrane sialoglycoprotein is an attractive target in diagnosis and therapy of several pathologic disorders especially cancer. In the underlying work, a fluorescence-activatable liposome (fluorescence-quenched during circulation and fluorescence activation upon cellular uptake), bearing specific single-chain Fv fragments directed against FAP (scFv'FAP) was developed, and its potential for use in fluorescence diagnostic imaging of FAP-expressing tumor cells was evaluated by whole body fluorescence imaging. The liposomes termed anti-FAP-IL were prepared via post-insertion of ligand-phospholipid-conjugates into preformed DY-676-COOH-containing liposomes. The anti-FAP-IL revealed a homogeneous size distribution and showed specific interaction and binding with FAP-expressing cells in vitro. The high level of fluorescence quenching of the near-infrared fluorescent dye sequestered in the aqueous interior of the liposomes enables fluorescence imaging exclusively upon uptake and degradation by cells, which results in fluorescence activation. Only FAP-expressing cells were able to take up and activate fluorescence of anti-FAP-IL in vitro. Furthermore, anti-FAP-IL accumulated selectively in FAP-expressing xenograft models in vivo, as demonstrated by blocking experiments using free scFv'FAP. The local tumor fluorescence intensities were in agreement with the intrinsic degree of FAP-expression in different xenograft models. Thus, anti-FAP-IL can serve as a suitable in vivo diagnostic tool for pathological disorders accompanied by high FAP-expression.
Subject(s)
Gelatinases/metabolism , Membrane Proteins/metabolism , Neoplasms/metabolism , Serine Endopeptidases/metabolism , Single-Chain Antibodies/administration & dosage , Animals , Carbocyanines/administration & dosage , Carbocyanines/chemistry , Cell Line, Tumor , Cells, Cultured , Endopeptidases , Female , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/chemistry , Gelatinases/immunology , Human Umbilical Vein Endothelial Cells , Humans , Indoles/administration & dosage , Indoles/chemistry , Liposomes , Maleimides/chemistry , Membrane Proteins/immunology , Mice, Nude , Optical Imaging , Phosphatidylethanolamines/chemistry , Polyethylene Glycols/chemistry , Serine Endopeptidases/immunology , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/immunology , Spectroscopy, Near-InfraredABSTRACT
In the past decade, there has been significant progress in the development of water soluble near-infrared fluorochromes for use in a wide range of imaging applications. Fluorochromes with high photo and thermal stability, sensitivity, adequate pharmacological properties and absorption/emission maxima within the near infrared window (650-900 nm) are highly desired for in vivo imaging, since biological tissues show very low absorption and auto-fluorescence at this spectrum window. Taking these properties into consideration, a myriad of promising near infrared fluorescent probes has been developed recently. However, a hallmark of most of these probes is a rapid clearance in vivo, which hampers their application. It is hypothesized that encapsulation of the near infrared fluorescent dye DY-676-COOH, which undergoes fluorescence quenching at high concentrations, in the aqueous interior of liposomes will result in protection and fluorescence quenching, which upon degradation by phagocytes in vivo will lead to fluorescence activation and enable imaging of inflammation. Liposomes prepared with high concentrations of DY-676-COOH reveal strong fluorescence quenching. It is demonstrated that the non-targeted PEGylated fluorescence-activatable liposomes are taken up predominantly by phagocytosis and degraded in lysosomes. Furthermore, in zymosan-induced edema models in mice, the liposomes are taken up by monocytes and macrophages which migrate to the sites of inflammation. Opposed to free DY-676-COOH, prolonged stability and retention of liposomal-DY-676-COOH is reflected in a significant increase in fluorescence intensity of edema. Thus, protected delivery and fluorescence quenching make the DY-676-COOH-loaded liposomes a highly promising contrast agent for in vivo optical imaging of inflammatory diseases.
Subject(s)
Fluorescent Dyes/chemistry , Liposomes , Macrophages/chemistry , Optical Imaging , Animals , Cell Line , Fluorescence , Mice , Spectroscopy, Near-InfraredABSTRACT
BACKGROUND: Dipeptidyl peptidase IV (DPPIV) also known as the T cell activation marker CD26 is a multifunctional protein which is involved in various biological processes. The association of human-DPPIV with components of the human immunodeficiency virus type-1 (HIV1) is well documented and raised some discussions. Several reports implicated the interaction of human-DPPIV with the HIV1 transcription transactivator protein (HIV1-Tat) and the inhibition of the dipeptidyl peptidase activity of DPPIV by the HIV1-Tat protein. Furthermore, enzyme kinetic data implied another binding site for the HIV1-Tat other than the active centre of DPPIV. However, the biological significance of this interaction of the HIV1-Tat protein and human-DPPIV has not been studied, yet. Therefore, we focused on the interaction of HIV1-Tat protein with DPPIV and investigated the subsequent biological consequences of this interaction in Spodoptera frugiperda cells, using the BAC-TO-BAC baculovirus system. RESULTS: The HIV1-Tat protein (Tat-BRU) co-localized and co-immunoprecipitated with human-DPPIV protein, following co-expression in the baculovirus-driven Sf9 cell expression system. Furthermore, tyrosine phosphorylation of DPPIV protein was up-regulated in Tat/DPPIV-co-expressing cells after 72 h culturing and also in DPPIV-expressing Sf9 cells after application of purified recombinant Tat protein. As opposed to the expression of Tat alone, serine phosphorylation of the Tat protein was decreased when co-expressed with human-DPPIV protein. CONCLUSIONS: We show for the first time that human-DPPIV and HIV1-Tat co-immunoprecipitate. Furthermore, our findings indicate that the interaction of HIV1-Tat and human-DPPIV may be involved in signalling platforms that regulate the biological function of both human-DPPIV and HIV1-Tat.