Maura Montani

Mesenchymal stem cells share molecular signature with mesenchymal tumor cells and favor early tumor growth in syngeneic mice

Tumor microenvironment in carcinomas recruits mesenchymal cells with an abnormal proangiogenic and invasive phenotype. It is not clear whether mesenchymal tumor cells (MTCs) derive from the activation of mature fibroblasts or from their stem cell precursors. However, stromal cell activation in tumors resembles in several aspects the mesenchymal rearrangement which normally occurs during reparative processes such as wound healing. Mesenchymal stem cells (MSCs) play a crucial role in developmental and reparative processes and have extraordinary proangiogenic potential, on the basis of which they are thought to show great promise for the treatment of ischemic disorders. Here, we show that MTCs have proangiogenic potential and that they share the transcriptional expression of the best-known proangiogenic factors with MSCs. We also found that MTCs and MSCs have the same molecular signature for stemness-related genes, and that when co-implanted with cancer cells in syngeneic animals MSCs determine early tumor appearance, probably by favoring the angiogenic switch. Our data (1) reveal crucial aspects of the proangiogenic phenotype of MTCs, (2) strongly suggest their stem origin and (3) signal the risk of therapeutic use of MSCs in tumor-promoting conditions.

Factors determining the superior performance of lipid/DNA/protammine nanoparticles over lipoplexes.

The utility of using a protammine/DNA complex coated with a lipid envelope made of cationic 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) for transfecting CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), NIH 3T3 (mouse embryonal cells), and A17 (murine cancer cells) cells was examined. The widely used DOTAP/DNA lipoplex was employed as a reference. In all the tested cell lines lipid/protamine/DNA (LPD) nanoparticles were more efficient in transfecting cells than lipoplexes even though the lipid composition of the lipid envelope was the same in both devices. Physical-chemical properties were found to control the ability of nanocarriers to release DNA upon interaction with cellular membranes. LPD complexes easily release their DNA payload, while lipoplexes remain largely intact and accumulate at the cell nucleus. Collectively, these data explain why LPD nanoparticles often exhibit superior performances compared to lipoplexes in trasfecting cells and represent a promising class of nanocarriers for gene delivery.

A better immune reaction to Erbb-2 tumors is elicited in mice by DNA vaccines encoding rat/human chimeric proteins.

The Erbb-2 (neu in rat and Her-2 in humans) tyrosine kinase receptor is an oncoantigen (i.e., a tumor-associated molecule directly involved in cancer progression). Because oncoantigens are self-tolerated molecules, to trigger a response circumventing tolerance, we generated two plasmids (RHuT and HuRT) coding for chimeric neu-Her-2 extracellular and transmembrane proteins that are expressed on the cell membrane of the transfected cells and recognized by monoclonal antibodies reacting against neu and Her-2. RHuT encodes a protein in which the 410 NH(2)-terminal residues are from the neu extracellular domain and the remaining residues from Her-2. Almost symmetrically, HuRT encodes for a protein in which the 390 NH(2)-terminal residues are from Her-2 and the remainder from neu. The ability of RHuT and HuRT to elicit a protective response to neu and Her-2 in wild-type mice and in transgenic mice tolerant to neu and Her-2 proteins was compared with that of plasmids coding for the fully rat or fully human extracellular and transmembrane domains of the Erbb-2 receptor. In most cases, RHuT and HuRT elicited a stronger response, although this chimeric benefit is markedly modulated by the location of the heterologous moiety in the protein coded by the plasmid, the immune tolerance of the responding mouse, and the kind of Erbb-2 orthologue on the targeted tumor.

The impact of early life permethrin exposure on development of neurodegeneration in adulthood.

Early life environmental exposure to pesticides could play a critical role in the onset of age-related diseases. The present study aims to evaluate in brain, plasma and leukocytes of 300 day-old rats, the effect of a low dose of the insecticide permethrin administered during early life (1/50 LD(50), from 6th to 21st day of life). The outcomes show that Nurr1, mRNA and protein expression, as well as calcium and NO levels are decreased in striatum. Moreover, the pesticide induces an imbalance in glutamate, calcium and NO in hippocampus. Low calcium concentrations in leukocytes and in plasma were observed, while increased NO and decreased SOD plasma levels were measured. The results suggest that permethrin intake at a dose close to the NOAEL (25 mg/kg) during the perinatal period can interact with Nurr1 by reducing its expression on striatum nucleus. Consequently, the maintenance of dopaminergic neurons as well as Nurr1 inhibitory effect on the production of proinflammatory mediators fails. The changes in biological markers found in our animal model could represent the basis to study neurodegenerative diseases whose development depends on individual gene signature and life style.

Structural stability and increase in size rationalize the efficiency of lipoplexes in serum.

We have investigated the effect of serum on nanometric structure, size, surface potential, DNA-binding capacity, and transfection efficiency of DDAB-DOPE/DNA and DC-Chol-DOPE/DNA lipoplexes as a function of membrane charge density and cationic lipid/DNA charge ratio. In the absence of serum, the nanometric structure and DNA binding capacity of lipoplexes determined the transfection efficiency. When serum was added, the transfection efficiency of all lipoplex formulations was found to increase. We identified structural stability and an increase in size in serum as major parameters regulating the efficiency of lipofection. By extrapolation, we propose that serum, regulating the size of resistant lipid-DNA complexes, can control the mechanism of internalization of lipoplexes and, in turn, their efficiency.

Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: interplay between nanostructure and composition.

Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

p130Cas is an essential transducer element in ErbB2 transformation

The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2‐dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202‐1A cells), and by its reexpression in ErbB2‐transformed p130Cas‐null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2‐dependent foci formation, anchorage‐independent growth, and in vivo growth of orthotopic N202‐1A tumors. Moreover, intranipple injection of p130Cas‐stabilized siRNAs in the mammary gland of Balbc‐NeuT mice decreases the growth of spontaneous tumors. In ErbB2‐transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c‐Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202‐1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.—Cabodi, S., Tinnirello, A., Bisaro, B., Tornillo, G., Camacho‐Leal, M. P., Forni, G., Cojoca, R., Iezzi, M., Amici, A., Montani, M., Eva, A., Di Stefano, P., Muthuswamy, S. K., Tarone, G., Turco, E., Defilippi, P. p130Cas is an essential transducer element in ErbB2 transformation. FASEB J. 24, 3796–3808 (2010). www.fasebj.org

Tailoring Lipoplex Composition to the Lipid Composition of Plasma Membrane: A Trojan Horse for Cell Entry?

The first interaction between lipoplexes and cells is charge-mediated and not specific. Endocytosis is considered to be the main pathway for lipoplex entry. Upon interaction between lipoplexes and the plasma membrane, intermixing between lipoplex and membrane lipids is necessary for efficient endocytosis. Here we study the mechanism of the different endocytic pathways in lipid-mediated gene delivery. We show that DC-Chol-DOPE/DNA lipoplexes preferentially use a raft-mediated endocytosis, while DOTAP-DOPC/DNA systems are mainly internalized by not specific fluid phase macropinocitosys. On the other hand, most efficient multicomponent lipoplexes, incorporating different lipid species in their lipid bilayer, can use multiple endocytic pathways to enter cells. Our data demonstrate that efficiency of endocytosis is regulated by shape coupling between lipoplex and membrane lipids. We suggest that such a shape-dependent coupling regulates efficient formation of endocytic vesicles thus determining the success of internalization. Our results suggest that tailoring the lipoplex lipid composition to the patchwork-like plasma membrane profile could be a successful machinery of coordinating the endocytic pathway activities and the subsequent intracellular processing.

Mechanistic Understanding of Gene Delivery Mediated by Highly Efficient Multicomponent Envelope-Type Nanoparticle Systems

We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Toward the rational design of lipid gene vectors: Shape coupling between lipoplex and anionic cellular lipids controls the phase evolution of lipoplexes and the efficiency of DNA release

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interaction with anionic cellular lipids, resulting in DNA release. At the early stages of interaction, we found a universal behavior of lipoplex/anionic lipid (AL) mixtures: the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely diluted by ALs. This finding is in excellent agreement with previous suggestions on the mechanism of DNA unbinding from lipoplexes by ALs. Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs. Furthermore, we investigated the effect of the membrane charge density and a general correlation resulted: the higher the membrane charge density of anionic membranes, the higher their ability to solubilize the structure of lipoplexes and to promote DNA release. Lastly, the formation of nonlamellar phases in lipoplex/AL mixtures is regulated by the propensity of anionic cellular lipids to adopt nonlamellar phases. Remarkably, also phase transition rates and DNA release were found to be strongly affected by the shape coupling between lipoplex and ALs. It thus seems likely that the structural and phase evolution of lipoplexes may only be meaningful in the context of specific anionic cellular membranes. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for optimal DNA release and suggest a potential strategy for the rational design of efficient cationic lipid carriers.