Liposomes and Immunoliposomes as Carriers for Cytostatic Drugs, Magnetic Resonance Contrast Agents, and Fluorescent Chelates

Biological and medical applications of liposomes as carriers for cytostatic drugs, magnetic-resonance contrast agents, and fluorescent markers are presented. The cytostatic effects of liposomal preparations of lipophilic derivatives of cytosine arabinoside (ara-C), namely N4-oleyl-ara-C (NOAC) and N4-alkyl-ara-C are demonstrated in the mouse Ll210 tumor model. Liposomal drugs were more effective by factors of 2–10 as compared to ara-C administered in aqueous preparations. Synergistic effects of the combination of ara-C and mitoxantrone could be shown, again with significantly increased effects, when the drugs were administered in liposomes. A practical detergent-dialysis method for the preparation of large volumes of sterile liposomes for clinical use is presented. Clinical phase I/II studies of Iiposoma I NOAC and mitoxantrone are currently in progress. Methods of preparation of immunoliposomes, i.e. the coupling of antibodies or antibody subunits to liposome membranes, are discussed' and results of specific immunoliposome – cell binding are presented. Possibilities of increasing the blood circulation times of liposomes by incorporation of lipophilic derivatives of poly(ethylene glycol) into the liposome membranes are shown. The successful application of liposomes as carriers of paramagnetic metal complexes as contrast agents in magnetic-resonance imaging for liver and spleen is documented. Finally, the concept of liposomes as carriers for fluorescent Europium chelates as markers for time-resolved flow cytometric applications are presented

Modulation of tumour angiogenesis by targeting p38 MAPK signalling in tumour-associated macrophages

Cellular crosstalk between cancer cells and tumour-associated macrophages (TAMs) plays an important role in tumour growth and metastasis by promoting tumour angiogenesis. Western blot analyses of cell lysates from in vitro studies revealed that RAW macrophages co-cultured with Murine Lewis lung carcinoma (LLC) cells display elevated levels of p38 MAPK (p38) activation. To further investigate this signalling pathway we used the p38 inhibitor BIRB796. Exposure of cancer cells to macrophage-conditioned medium pre-incubated with BIRB796 showed a clear dose-dependent increase in apoptosis. By preparing liposomal formulations of BIRB796 (Lip-BIRB796) we can spare normal cells from the drug’s cytotoxic effects and specifically target p38 signalling in phagocytic macrophages. In vitro cytotoxicity studies revealed an increased chemosensitivity to doxorubicin treatment when macrophage/cancer cell co-cultures were treated with Lip-BIRB796 prior to doxorubicin exposure. Furthermore, in vivo CAM assays were able to demonstrate both a marked increase in angiogenesis in CAMs inoculated with RAW and LLC cell co-cultures as compared to the respective monocultures and a significant decrease where these co-cultures were treated with Lip-BIRB796. To conclude, our results suggest that p38 is a promising target in TAMs for cancer adjuvant therapy

Soluble vascular endothelial growth factor receptor-3 suppresses lymphangiogenesis and lymphatic metastasis in bladder cancer

Peer reviewe

Cellular pharmacology of multi-and duplex drugs consisting of ethynylcytidine and 5-fluoro-2′-deoxyuridine

In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation

Cellular pharmacology of multi- and duplex drugsconsisting of ethynylcytidine and 5-fluoro-2′-deoxyuridine

Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2′deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK−. ETC-FdUrd was active (IC50 of 2.2 and 79 nM) in FM3A/0 and TK− cells, respectively. ETC-L-FdUrd was less active (IC50: 7 nM in FM3A/0 vs 4500 nM in FM3A/TK−). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC50:19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80–90%), but to a lower extent in FM3A/TK− (10–50%). FdUMP was hardly detected in FM3A/TK− cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation

Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors

<p>Abstract</p> <p>Background</p> <p>Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-β signaling and activation of the β-catenin pathway can induce EMT in intratumoral cancer cells.</p> <p>Methods</p> <p>We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized <it>in vitro </it>in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC).</p> <p>Results</p> <p>Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. <it>In vitro</it>, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating β-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-β was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-β levels and tumor grade.</p> <p>Conclusions</p> <p>Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.</p

Myeloid Cells Contribute to Tumor Lymphangiogenesis

The formation of new blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis) promotes tumor outgrowth and metastasis. Previously, it has been demonstrated that bone marrow-derived cells (BMDC) can contribute to tumor angiogenesis. However, the role of BMDC in lymphangiogenesis has largely remained elusive. Here, we demonstrate by bone marrow transplantation/reconstitution and genetic lineage-tracing experiments that BMDC integrate into tumor-associated lymphatic vessels in the Rip1Tag2 mouse model of insulinoma and in the TRAMP-C1 prostate cancer transplantation model, and that the integrated BMDC originate from the myelomonocytic lineage. Conversely, pharmacological depletion of tumor-associated macrophages reduces lymphangiogenesis. No cell fusion events are detected by genetic tracing experiments. Rather, the phenotypical conversion of myeloid cells into lymphatic endothelial cells and their integration into lymphatic structures is recapitulated in two in vitro tube formation assays and is dependent on fibroblast growth factor-mediated signaling. Together, the results reveal that myeloid cells can contribute to tumor-associated lymphatic vessels, thus extending the findings on the previously reported role of hematopoietic cells in lymphatic vessel formation

Liposomes as vaccine delivery systems: A review of the recent advances

Liposomes and liposome-derived nanovesicles such as archaeosomes and virosomes have become important carrier systems in vaccine development and the interest for liposome-based vaccines has markedly increased. A key advantage of liposomes, archaeosomes and virosomes in general, and liposome-based vaccine delivery systems in particular, is their versatility and plasticity. Liposome composition and preparation can be chosen to achieve desired features such as selection of lipid, charge, size, size distribution, entrapment and location of antigens or adjuvants. Depending on the chemical properties, water-soluble antigens (proteins, peptides, nucleic acids, carbohydrates, haptens) are entrapped within the aqueous inner space of liposomes, whereas lipophilic compounds (lipopeptides, antigens, adjuvants, linker molecules) are intercalated into the lipid bilayer and antigens or adjuvants can be attached to the liposome surface either by adsorption or stable chemical linking. Coformulations containing different types of antigens or adjuvants can be combined with the parameters mentioned to tailor liposomal vaccines for individual applications. Special emphasis is given in this review to cationic adjuvant liposome vaccine formulations. Examples of vaccines made with CAF01, an adjuvant composed of the synthetic immune-stimulating mycobacterial cordfactor glycolipid trehalose dibehenate as immunomodulator and the cationic membrane forming molecule dimethyl dioctadecylammonium are presented. Other vaccines such as cationic liposome-DNA complexes (CLDCs) and other adjuvants like muramyl dipeptide, monophosphoryl lipid A and listeriolysin O are mentioned as well. The field of liposomes and liposome-based vaccines is vast. Therefore, this review concentrates on recent and relevant studies emphasizing current reports dealing with the most studied antigens and adjuvants, and pertinent examples of vaccines. Studies on liposome-based veterinary vaccines and experimental therapeutic cancer vaccines are also summarized

A New Approach to Cancer Therapy: The Tumor Microenvironment as Target

Solid tumors grow within a complex microenvironment composed of diverse cell types such as fibroblasts, endothelial cells, mast cells, macrophages and immune cells that are attracted by tumor cell derived factors and embedded in an extracellular matrix. Molecular and cellular interactions between epithelial cells and cells surrounding the tumor stroma promote growth, invasion and spread of tumors. To delay or impede tumor growth, the tumor microenvironment (TME) is increasingly being explored as a potential therapeutic target for which novel strategies are developed. &lt;/p&gt;&lt;p&gt; This article reviews how key interactions between tumor cells and surrounding mesenchymal and immune cells in the TME can promote tumor progression and it highlights cellular and molecular elements that might represent novel therapeutic targets. Special emphasis is given on therapies targeted towards tumor-associated macrophages. As main class of drugs the bisphosphonates are covered with their properties to repolarize a pro-tumorigenic, immunosuppressive environment to a tumor growth inhibiting and immunocompetent microenvironment. Properties and advantages of liposome-encapsulated bisphosphonates as macrophage depleting or modulating agents as well as the latest developments towards clinical applications of compounds targeting cellular and molecular components of the TME are described and reviewed

Cytotoxic tumor targeting with scFv antibody-modified liposomes

Specific targeting of liposome-formulated cytotoxic drugs or antigens to receptors expressed selectively on target cells represents an effective strategy for increasing the pharmacological efficacy of the delivered molecules. We have developed a feasible technique to selectively attach antibodies and fragments thereof, but also small-mol-wt ligands such as peptides, carbohydrates, or any molecules that recognize and bind target antigens or receptors to the surface of small unilamellar liposomes. Our concept is based on the site-specific functionalization of the ligands to be attached to the liposomes by thiol groups. These thiol groups can easily be introduced to antibodies or peptides by addition of cysteines, preferably at sites that do not interfere with the receptor binding domains. Optimally, the site-specific modification is introduced at the C-terminal end of the ligand, separated by an inert spacer sequence located between the thiols and the specific part of the ligand. The thiol-reactive molecules on the liposome surface are maleimides that are linked to phospholipids composing the liposome bilayer membrane. We illustrate the coupling method of a functionalized single-chain antibody fragment with binding specificity to ED-B fibronectin, an isoform of fibronectin exclusively expressed in tumor tissues, to long circulating small unilamellar poly(ethylene glycol) liposomes