Marco Colombatti

A comparative analysis of three different techniques for the detection of breast cancer cells in bone marrow

Three different methods, morphologic, immunocytochemic, and fluorescence activated cell sorter (FC) analysis, were compared with respect to their efficiency in detecting breast cancer cells in bone marrow. In the first series of experiments, the three techniques were compared using bone marrow cells artificially mixed with a known amount of breast cancer cells, whereas in a second series bone marrow from breast cancer patients with bone metastases were used. The following results were obtained: When mixtures of the first series were analyzed, FC analysis detected from 1% to 10% of breast cancer cells in bone marrow (0.2% was a border line value), the morphologic method detected from 0.05% to 10%, and the immunocytochemic method, which was clearly superior, detected breast cancer cells in all mixtures (from 0.00025% to 10%), It was noted that, with both the morphologic and immunocytochemic methods, the percentage of breast cancer cells detected was 2 to 360 times higher than the percentage of added cells, and enrichment was inversely proportional to the percentage of added cells. This result could be a result of different separation of cells during centrifugation due to the different density of breast cancer cells. The superiority of the immunocytochemic method was confirmed in the second series of experiments.

The Prostate Specific Membrane Antigen Regulates the Expression of IL-6 and CCL5 in Prostate Tumour Cells by Activating the MAPK Pathways1

The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-κB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis.

Magneto‐Plasmonic Au‐Fe Alloy Nanoparticles Designed for Multimodal SERS‐MRI‐CT Imaging

Diagnostic approaches based on multimodal imaging are needed for accurate selection of the therapeutic regimens in several diseases, although the dose of administered contrast drugs must be reduced to minimize side effects. Therefore, large efforts are deployed in the development of multimodal contrast agents (MCAs) that permit the complementary visualization of the same diseased area with different sensitivity and different spatial resolution by applying multiple diagnostic techniques. Ideally, MCAs should also allow imaging of diseased tissues with high spatial resolution during surgical interventions. Here a new system based on multifunctional Au-Fe alloy nanoparticles designed to satisfy the main requirements of an ideal MCA is reported and their biocompatibility and imaging capability are described. The MCAs show easy and versatile surface conjugation with thiolated molecules, magnetic resonance imaging (MRI) and computed X-ray tomography (CT) signals for anatomical and physiological information (i.e., diagnostic and prognostic imaging), large Raman signals amplified by surface enhanced Raman scattering (SERS) for high sensitivity and high resolution intrasurgical imaging, biocompatibility, exploitability for in vivo use and capability of selective accumulation in tumors by enhanced permeability and retention effect. Taken together, these results show that Au-Fe nanoalloys are excellent candidates as multimodal MRI-CT-SERS imaging agents.

IFN-γ-mediated upmodulation of MHC class I expression activates tumor-specific immune response in a mouse model of prostate cancer

De novo expression of B7-1 impaired tumorigenicity of TRAMP-C2 mouse prostate adenocarcinoma (TRAMP-C2/B7), but it did not elicit a protective response against TRAMP-C2 parental tumor, unless after in vitro treatment with IFN-gamma. TRAMP-C2 cells secrete TGF-beta and show low MHC-I expression. Treatment with IFN-gamma increased MHC-I expression by induction of some APM components and antagonizing the immunosuppressant activity of TGF-beta. Thus, immunization with TRAMP-C2/B7 conferred protection against TRAMP-C2-derived tumors in function of the IFN-gamma-mediated fine-tuned modulation of either APM expression or TGF-beta signaling. To explore possible clinical translation, we delivered IFN-gamma to TRAMP-C2 tumor site by means of genetically engineered MSCs secreting IFN-gamma.

A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin's lymphoma patients treated with an anti-CD25 immunotoxin

Hodgkins lymphoma patients treated with an anti‐CD25 Ricin toxin A‐chain (RTA)‐based Immunotoxin (RFT5.dgA) develop an immune response against the toxic moiety of the immunoconjugate. The anti‐RTA antibody response of 15 patients showing different clinical features and receiving different total amounts of RFT5.dgA was therefore studied in detail, considering antibody titre, IgG and IgM content, average binding efficacy and ability to inhibit in vitro the cytotoxicity of a RTA‐based Immunotoxin. No correlations were found between these parameters and the clinical features of the patients or the total amount of Immunotoxin administered. However, using a peptide scan approach we have identified a continuous epitope recognized by all patients studied, located within the stretch L161‐I175 of the RTA primary sequence, close to a previously identified T‐cell epitope. The ability of anti‐L161‐I175 antibodies to recognize folded RTA and to affect the biological activity of RTA by inhibiting RTA‐IT cytotoxicity in vitro revealed that they may exert an important role in IT neutralization in vivo. Discovery of RTA immunodominant epitopes which are the target of anti‐RTA immune response may lead to the development of immunomodulating strategies and to more successful treatment schedules.

Top-down synthesis of multifunctional iron oxide nanoparticles for macrophage labelling and manipulation

Multifunctional iron oxide (FeOx) magnetic nanoparticles (MNPs) are promising items for biomedical applications. They are studied as theranostic agents for cancer treatment, selective probes for bioanalytical assays, controllable carriers for drug delivery and biocompatible tools for cell sorting or tissue repair. Here we report a new method for the synthesis in water of FeOx–MNPsvia a top-down physical technique consisting in Laser Ablation Synthesis in Solution (LASiS). LASiS is a green method that does not require chemicals or stabilizers, because nanoparticles are directly obtained in water as a stable colloidal system. A gamut of characterization techniques was used for investigating the structure of FeOx–MNPs that have a polycrystalline structure prevalently composed of magnetite (ca. 75%) and hematite (ca. 22%). The FeOx–MNPs exhibit very good magnetic properties if compared to what is usually reported for iron oxide nanoparticles, with saturation magnetization close to the bulk value (ca. 80 emu g−1) and typical signatures of the coexistence of ferrimagnetic and antiferromagnetic phases in the same particle. The functionalization of FeOx–MNPs after the synthesis was possible with a variety of ligands. In particular, we succeeded in the functionalization of FeOx–MNPs with carboxylated phosphonates, fluorescent alkylamines, fluorescent isothiocyanates and bovine serum albumin. Our FeOx–MNPs showed excellent biocompatibility. Multifunctional FeOx–MNPs were exploited for macrophage cell labelling with fluorescent probes as well as for cell sorting and manipulation by external magnetic fields.

Induction of interferon pathways mediates in vivo resistance to oncolytic adenovirus.

Oncolytic adenoviruses are an emerging experimental approach for treatment of tumors refractory to available modalities. Although preclinical results have been promising, and clinical safety has been excellent, it is also apparent that tumors can become virus resistant. The resistance mechanisms acquired by advanced tumors against conventional therapies are increasingly well understood, which has allowed development of countermeasures. To study this in the context of oncolytic adenovirus, we developed two in vivo models of acquired resistance, where initially sensitive tumors eventually gain resistance and relapse. These models were used to investigate the phenomenon on RNA and protein levels using two types of analysis of microarray data, quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. Interferon (IFN) signaling pathways were found upregulated and Myxovirus resistance protein A (MxA) expression was identified as a marker correlating with resistance, while transplantation experiments suggested a role for tumor stroma in maintaining resistance. Furthermore, pathway analysis suggested potential therapeutic targets in oncolytic adenovirus-resistant cells. Improved understanding of the antiviral phenotype causing tumor recurrence is of key importance in order to improve treatment of advanced tumors with oncolytic adenoviruses. Given the similarities between mechanisms of action, this finding might be relevant for other oncolytic viruses as well.

Constitutive Activation of p38 and ERK1/2 MAPKs in Epithelial Cells of Myasthenic Thymus Leads to IL-6 and RANTES Overexpression: Effects on Survival and Migration of Peripheral T and B Cells

Myasthenia gravis (MG) is an autoimmune disease of neuromuscular junctions where thymus plays a pathogenetic role. Thymectomy benefits patients, and thymic hyperplasia, a lymphoid infiltration of perivascular spaces becoming site of autoantibody production, is recurrently observed. Cytokines and chemokines, produced by thymic epithelium and supporting survival and migration of T and B cells, are likely to be of great relevance in pathogenesis of thymic hyperplasia. In thymic epithelial cell (TEC) cultures derived “in vitro” from normal or hyperplastic age-matched MG thymuses, we demonstrate by gene profiling analysis that MG-TEC basally overexpress genes coding for p38 and ERK1/2 MAPKs and for components of their signaling pathways. Immunoblotting experiments confirmed that p38 and ERK1/2 proteins were overexpressed in MG-TEC and, in addition, constitutively activated. Pharmacological blockage with specific inhibitors confirmed their role in the control of IL-6 and RANTES gene expression. According to our results, IL-6 and RANTES levels were abnormally augmented in MG-TEC, either basally or upon induction by adhesion-related stimuli. The finding that IL-6 and RANTES modulate, respectively, survival and migration of peripheral lymphocytes of myasthenic patients point to MAPK transcriptional and posttranscriptional abnormalities of MG-TEC as a key step in the pathological remodelling of myasthenic thymus.

Pichia pastoris as a host for secretion of toxic saporin chimeras

Most of the targeting moieties, such as antibody fragments or growth factor domains, used to construct targeted toxins for anticancer therapy derive from secretory proteins. These normally fold in the oxidative environment of the endoplasmic reticulum, and hence their folding in bacterial cells can be quite inefficient. For instance, only low amounts of properly folded antimetastatic chimera constituted by the amino‐terminal fragment of human urokinase (ATF) fused to the plant ribosome‐inactivating protein saporin could be recovered. ATF‐saporin was instead secreted efficiently when expressed in eukaryotic cells protected from autointoxication with neutralizing anti‐saporin antibodies. Pichia pastoris is a microbial eukaryotic host where these domains can fold into a transport‐competent conformation and reach the extracellular medium. We show here that despite some host toxicity codon‐usage optimization greatly increased the expression levels of active saporin but not those of an active‐site mutant SAP‐KQ in GS115 (his4) strain. The lack of any toxicity associated with expression of the latter confirmed that toxicity is due to saporin catalytic activity. Nevertheless, GS115 (his4) cells in flask culture secreted 3.5 mg/L of a histidine‐tagged ATF‐saporin chimera showing an IC50 of 6 X 10−11 M against U937 cells, thus demonstrating the suitability of this expression platform for secretion of toxic saporin‐based chimeras.—Lombardi, A., Bursomanno, S., Lopardo, T., Traini, R., Colombatti, M., Ippoliti, R., Flavell, D. J., Flavell, S. U., Ceriotti, A., Fabbrini, M. S. Pichia pastoris as a host for secretion of toxic saporin chimeras. FASEB J. 24, 253–265 (2010). www.fasebj.org

Dual-Modality Image-Guided Surgery of Prostate Cancer with a Radiolabeled Fluorescent Anti-PSMA Monoclonal Antibody

Both radionuclide imaging and near-infrared fluorescent (NIRF) imaging have a high sensitivity to detect tumors in vivo. The combination of these modalities using dual-labeled antibodies may allow both preoperative and intraoperative tumor localization and may be used in image-guided surgery to ensure complete resection of tumor tissue. Here, we evaluated the potential of dual-modality imaging of prostate cancer with the monoclonal antibody D2B, directed against an extracellular domain of prostate-specific membrane antigen (PSMA). For these studies, D2B was labeled both with 111In and with the NIRF dye IRDye800CW. Methods: D2B was conjugated with N-hydroxysuccinimide-IRDye800CW and p-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (ITC-DTPA) and subsequently radiolabeled with 111In. For biodistribution and NIRF imaging, 111In-DTPA-D2B-IRDye800CW (2 μg, 0.55 MBq/mouse) was injected intravenously into BALB/c nude mice with subcutaneous PSMA-expressing LNCaP tumors (right flank) and PSMA-negative PC3 tumors (left flank). The biodistribution was determined at 1, 2, 3, and 7 d after injection. In addition, micro-SPECT/CT and NIRF imaging with 111In-DTPA-D2B-IRDye800CW (3 μg, 8.5 MBq/mouse) was performed on mice with intraperitoneally growing LS174T-PSMA tumors. Results: 111In-DTPA-D2B-IRDye800CW specifically accumulated in subcutaneous PSMA-positive LNCaP tumors (45.8 ± 8.0 percentage injected dose per gram at 168 h after injection), whereas uptake in subcutaneous PSMA-negative PC3 tumors was significantly lower (6.6 ± 1.3 percentage injected dose per gram at 168 h after injection). Intraperitoneal LS174T-PSMA tumors could be visualized specifically with both micro-SPECT/CT and NIRF imaging at 2 d after injection, and the feasibility of image-guided resection of intraperitoneal tumors was demonstrated in this model. Conclusion: Dual-labeled 111In-DTPA-D2B-IRDye800CW enables specific and sensitive detection of prostate cancer lesions in vivo with micro-SPECT/CT and NIRF imaging. In addition to preoperative micro-SPECT/CT imaging to detect tumors, NIRF imaging enables image-guided surgical resection. These preclinical findings warrant clinical studies with 111In-DTPA-D2B-IRDye800CW to improve tumor detection and resection in prostate cancer patients.