Stefania Lanzardo

Iopamidol as a responsive MRI-chemical exchange saturation transfer contrast agent for pH mapping of kidneys: In vivo studies in mice at 7 T.

Iopamidol (Isovue®—Bracco Diagnostic Inc.) is a clinically approved X‐Ray contrast agent used in the last 30 years for a wide variety of diagnostic applications with a very good clinical acceptance. Iopamidol contains two types of amide functionalities that can be exploited for the generation of chemical exchange saturation transfer effect. The exchange rate of the two amide proton pools is markedly pH‐dependent. Thus, a ratiometric method for pH assessment has been set‐up based on the comparison of the saturation transfer effects induced by selective irradiation of the two resonances. This ratiometric approach allows to rule out the concentration effect of the contrast agent and provides accurate pH measurements in the 5.5–7.4 range. Upon injection of Iopamidol into healthy mice, it has been possible to acquire pH maps of kidney regions. Furthermore, it has been also shown that the proposed method is able to report about pH‐changes induced in control mice fed with acidified or basified water for a period of a week before image acquisition. Magn Reson Med, 2010.

Magnetic Resonance Visualization of Tumor Angiogenesis by Targeting Neural Cell Adhesion Molecules with the Highly Sensitive Gadolinium-Loaded Apoferritin Probe

Tumor vessel imaging could be useful in identifying angiogenic blood vessels as well as being a potential predictive marker of antiangiogenic treatment response. We recently reported the expression of the neural cell adhesion molecule (NCAM) in the immature and tumor endothelial cell (TEC) lining vessels of human carcinomas. Exploiting an in vivo model of human tumor angiogenesis obtained by implantation of TEC in Matrigel in severe combined immunodeficiency mice, we aimed to image angiogenesis by detecting the expression of NCAM with magnetic resonance imaging. The imaging procedure consisted of (a) targeting NCAMs with a biotinylated derivative of C3d peptide that is known to have high affinity for these epitopes and (b) delivery of a streptavidin/gadolinium (Gd)-loaded apoferritin 1:1 adduct at the biotinylated target sites. The remarkable relaxation enhancement ability of the Gd-loaded apoferritin system allowed the visualization of TEC both in vitro and in vivo when organized in microvessels connected to the mouse vasculature. Gd-loaded apoferritin displayed good in vivo stability and tolerability. The procedure reported herein may be easily extended to the magnetic resonance visualization of other epitopes suitably targeted by proper biotinylated vectors.

WIP Regulates the Stability and Localization of WASP to Podosomes in Migrating Dendritic Cells

Summary The Wiskott-Aldrich Syndrome protein (WASP) is an adaptor protein that is essential for podosome formation in hematopoietic cells [1]. Given that 80% of identified Wiskott-Aldrich Syndrome patients result from mutations in the binding site for WASP-interacting-protein (WIP) [2], we examined the possible role of WIP in the regulation of podosome architecture and cell motility in dendritic cells (DCs). Our results show that WIP is essential both for the formation of actin cores containing WASP and cortactin and for the organization of integrin and integrin-associated proteins in circular arrays, specific characteristics of podosome structure. We also found that WIP is essential for the maintenance of the high turnover of adhesions and polarity in DCs. WIP exerts these functions by regulating calpain-mediated cleavage of WASP and by facilitating the localization of WASP to sites of actin polymerization at podosomes. Taken together, our results indicate that WIP is critical for the regulation of both the stability and localization of WASP in migrating DCs and suggest that WASP and WIP operate as a functional unit to control DC motility in response to changes in the extracellular environment.

Concordant morphologic and gene expression data show that a vaccine halts HER-2/neu preneoplastic lesions

While much experimental data shows that vaccination efficiently inhibits a subsequent challenge by a transplantable tumor, its ability to inhibit the progress of autochthonous preneoplastic lesions is virtually unknown. In this article, we show that a combined DNA and cell vaccine persistently inhibits such lesions in a murine HER-2/neu mammary carcinogenesis model. At 10 weeks of age, all of the ten mammary gland samples from HER-2/neu-transgenic mice displayed foci of hyperplasia that progressed to invasive tumors. Vaccination with plasmids coding for the transmembrane and extracellular domain of rat p185neu followed by a boost with rp185neu+ allogeneic cells secreting IFN-gamma kept 48% of mice tumor free. At 22 weeks, their mammary glands were indistinguishable from those of 10-week-old untreated mice. Furthermore, the transcription patterns of the two sets of glands coincided. Of the 12,000 genes analyzed, 17 were differentially expressed and related to the antibody response. The use of B cell knockout mice as well as the concordance of morphologic and gene expression data demonstrated that the Ab response is the main mechanism facilitating tumor growth arrest. This finding suggests that a new way can be found to secure the immunologic control of the progression of HER-2/neu preneoplastic lesions.

Imaging the pH evolution of an acute kidney injury model by means of iopamidol, a MRI‐CEST pH‐responsive contrast agent

To investigate in vivo possible pH level alterations following an acute renal failure disease using a MRI‐CEST pH responsive contrast agent. The impact of functional evolution in different renal compartments over time was also investigated.

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 noninflammatory role of high mobility group box 1/toll-like receptor 2 axis in the self-renewal of mammary cancer stem cells

Cancer stem cells (CSCs) are responsible for tumor progression, metastases, resistance to therapy, and tumor recurrence. Therefore, the identification of molecules involved in CSC self‐renewal is a necessary step toward more effective therapies. To this aim, through the transcription profiling of the murine ErbB2+ tumor cell line TUBO vs. derived CSC‐enriched mammospheres, Toll‐like receptor 2 (TLR2) was identified as 2‐fold overexpressed in CSCs, as confirmed by qPCR and cytofluorimetric analysis. TLR2 signaling inhibition impaired in vitro mammosphere generation in murine TUBO (60%) and 4T1 (30%) and human MDA‐MB‐231 (50%), HCC1806 (60%), and MCF7 (50%) cells. In CSC, TLR2 was activated by endogenous high‐mobility‐group box 1 (HMGB1), inducing IκBα phosphorylation, IL‐6 and TGFβ secretion, and, consequently, STAT3 and Smad3 activation. In vivo TLR2 inhibition blocked TUBO tumor takes in 9/14 mice and induced a 2‐fold reduction in lung metastases development by decreasing cell proliferation and vascularization and increasing apoptosis. Collectively, these results demonstrate that murine and human mammary CSCs express TLR2 and its ligand HMGB1; this autocrine loop plays a pivotal role in CSC self‐renewal, tumorigenesis, and metastatic ability. These findings, while providing evidence against the controversial use of TLR2 agonists in antitumor therapy, lay out new paths toward the design of anticancer treatments.—Conti, L., Lanzardo, S., Arigoni, M., Antonazzo, R., Radaelli, E., Cantarella, D., Calogero, R. A., Cavallo, F., The noninflammatory role of high mobility group box 1/toll‐like receptor 2 axis in the self‐renewal of mammary cancer stem cells. FASEB J. 27, 4731–4744 (2013). www.fasebj.org

A vaccine targeting angiomotin induces an antibody response which alters tumor vessel permeability and hampers the growth of established tumors

Angiomotin (Amot) is one of several identified angiostatin receptors expressed by the endothelia of angiogenic tissues. We have shown that a DNA vaccine targeting Amot overcome immune tolerance and induce an antibody response that hampers the progression of incipient tumors. Following our observation of increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, we evaluated the effect of anti-Amot vaccination on clinically evident tumors. Electroporation of plasmid coding for the human Amot (pAmot) significantly delayed the progression both of autochthonous tumors in cancer prone BALB-neuT and PyMT genetically engineered mice and transplantable TUBO tumor in wild-type BALB/c mice. The intensity of the inhibition directly correlated with the titer of anti-Amot antibodies induced by the vaccine. Tumor inhibition was associated with an increase of vessels diameter with the formation of lacunar spaces, increase in vessel permeability, massive tumor perivascular necrosis and an effective epitope spreading that induces an immune response against other tumor associated antigens. Greater tumor vessel permeability also markedly enhances the antitumor effect of doxorubicin. These data provide a rationale for the development of novel anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy regimens.

WIP null mice display a progressive immunological disorder that resembles Wiskott–Aldrich syndrome

The Wiskott–Aldrich syndrome (WAS) is an X‐linked immunodeficiency syndrome caused by mutations in the WAS protein (WASP). This participates in signalling and cytoskeletal homoeostasis, and some of its activities are regulated by its binding to the WASP interacting protein (WIP). WIP deficiency, however, has not yet been shown to be of pathological significance in humans. Here we show that, in WIP null (WIP−/−) mice, it produces haematological alterations and anatomical abnormalities in several organs, most probably as a consequence of autoimmune attacks. Granulocytosis and severe lymphopenia are associated with a proportional increase in segmented cells and fewer bone marrow erythrocytes and lymphocytes. Splenomegaly is accompanied by an increase of haematopoietic tissue and red pulp, reduction of the white pulp, and fewer B (B220+) lymphocytes (also apparent in the lymph nodes and Peyers patches). Ulcerative colitis, interstitial pneumonitis, glomerular nephropathy with IgA deposits, autoantibodies, and joint inflammation are also evident. These progressive immunological disorders closely mimic those seen in WAS. WIP deficiency may thus be implicated in some cases in which mutations in the gene encoding WASP are not detected. Copyright

CSPG4-Specific Immunity and Survival Prolongation in Dogs with Oral Malignant Melanoma Immunized with Human CSPG4 DNA

Purpose: Due to the many similarities with its human counterpart, canine malignant melanoma (cMM) is a valuable model in which to assess the efficacy of novel therapeutic strategies. The model is herein used to evaluate the immunogenicity, safety, and therapeutic efficacy of a human chondroitin sulfate proteoglycan-4 (hCSPG4) DNA-based vaccine. The fact that homology between hCSPG4 and cCSPG4 amino-acidic sequences stands at more than 80% provides the rationale for using an hCSPG4 DNA vaccine in the cMM model. Experimental Design: Dogs with stage II–III surgically resected CSPG4-positive oral MM were subjected to monthly intramuscular plasmid administration, which was followed immediately by electroporation (electrovaccination) for at least 6, and up to 20, months. The immunogenicity, safety, and therapeutic efficacy of the vaccine have been evaluated. Results: hCSPG4 electrovaccination caused no clinically relevant local or systemic side effects and resulted in significantly longer overall and disease-free survival times in 14 vaccinated dogs as compared with 13 nonvaccinated controls. All vaccinated dogs developed antibodies against both hCSPG4 and cCSPG4. Seven vaccinated dogs were also tested for a cCSPG4-specific T-cell response and only two gave a detectable interferon (IFN)γ response. Conclusion: Xenogeneic electrovaccination against CSPG4 is able to overcome host unresponsiveness to the “self” antigen and seems to be effective in treating cMM, laying the foundation for its translation to a human clinical setting. Clin Cancer Res; 20(14); 3753–62. ©2014 AACR.