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Purpose: This study was aimed at investigating whether the PPARγ agonist pioglitazone—given in combination with trabectedin—is able to reactivate adipocytic differentiation in myxoid liposarcoma (MLS) patient-derived xenografts, overcoming resistance to trabectedin. Experimental Design: The antitumor and biological effects of trabectedin, pioglitazone, and the combination of the two drugs were investigated in nude mice bearing well-characterized MLS xenografts representative of innate or acquired resistance against trabectedin. Pioglitazone and trabectedin were given by daily oral and weekly i.v. administrations, respectively. Molecular studies were performed by using microarrays approach, real-time PCR, and Western blotting. Results: We found that the resistance of MLS against trabectedin is associated with the lack of activation of adipogenesis. The PPARγ agonist pioglitazone reactivated adipogenesis, assessed by histologic and gene pathway analyses. Pioglitazone was well tolerated and did not increase the toxicity of trabectedin. The ability of pioglitazone to reactivate adipocytic differentiation was observed by morphologic examination, and it is consistent with the increased expression of genes such as ADIPOQ implicated in the adipogenesis process. The determination of adiponectin by Western blotting constitutes a good and reliable biomarker related to MLS adipocytic differentiation. Conclusions: The finding that the combination of pioglitazone and trabectedin induces terminal adipocytic differentiation of some MLSs with the complete pathologic response and cure of tumor-bearing mice provides a strong rationale to test the combination of trabectedin and pioglitazone in patients with MLS.

Background Cancer stem cells (CSC) have been implicated in tumor progression. In ovarian carcinoma (OC), CSC drive tumor formation, dissemination and recurrence, as well as drug resistance, thus contributing to the high death-to-incidence ratio of this disease. However, the molecular basis of such a pathogenic role of ovarian CSC (OCSC) has been elucidated only to a limited extent. In this context, the functional contribution of the L1 cell adhesion molecule (L1CAM) to OC stemness remains elusive. Methods The expression of L1CAM was investigated in patient-derived OCSC. The genetic manipulation of L1CAM in OC cells provided gain and loss-of-function models that were then employed in cell biological assays as well as in vivo tumorigenesis experiments to assess the role of L1CAM in OC cell stemness and in OCSC-driven tumor initiation. We applied antibody-mediated neutralization to investigate L1CAM druggability. Biochemical approaches were then combined with functional in vitro assays to study the molecular mechanisms underlying the functional role of L1CAM in OCSC. Results We report that L1CAM is upregulated in patient-derived OCSC. Functional studies showed that L1CAM promotes several stemness-related properties in OC cells, including sphere formation, tumor initiation and chemoresistance. These activities were repressed by an L1CAM-neutralizing antibody, pointing to L1CAM as a druggable target. Mechanistically, L1CAM interacted with and activated fibroblast growth factor receptor-1 (FGFR1), which in turn induced the SRC-mediated activation of STAT3. The inhibition of STAT3 prevented L1CAM-dependent OC stemness and tumor initiation. Conclusions Our study implicate L1CAM in the tumorigenic function of OCSC and point to the L1CAM/FGFR1/SRC/STAT3 signaling pathway as a novel driver of OC stemness. We also provide evidence that targeting this pathway can contribute to OC eradication.

Rationale: Optimal intratumor distribution of an anticancer drug is fundamental to reach an active concentration in neoplastic cells, ensuring the therapeutic effect. Determination of drug concentration in tumor homogenates by LC-MS/MS gives important information about this issue but the spatial information gets lost. Targeted mass spectrometry imaging (MSI) has great potential to visualize drug distribution in the different areas of tumor sections, with good spatial resolution and superior specificity. MSI is rapidly evolving as a quantitative technique to measure the absolute drug concentration in each single pixel. Methods: Different inorganic nanoparticles were tested as matrices to visualize the PARP inhibitors (PARPi) niraparib and olaparib. Normalization by deuterated internal standard and a custom preprocessing pipeline were applied to achieve a reliable single pixel quantification of the two drugs in human ovarian tumors from treated mice. Results: A quantitative method to visualize niraparib and olaparib in tumor tissue of treated mice was set up and validated regarding precision, accuracy, linearity, repeatability and limit of detection. The different tumor penetration of the two drugs was visualized by MSI and confirmed by LC-MS/MS, indicating the homogeneous distribution and higher tumor exposure reached by niraparib compared to olaparib. On the other hand, niraparib distribution was heterogeneous in an ovarian tumor model overexpressing the multidrug resistance protein P-gp, a possible cause of resistance to PARPi. Conclusions: The current work highlights for the first time quantitative distribution of PAPRi in tumor tissue. The different tumor distribution of niraparib and olaparib could have important clinical implications. These data confirm the validity of MSI for spatial quantitative measurement of drug distribution providing fundamental information for pharmacokinetic studies, drug discovery and the study of resistance mechanisms.

Mass spectrometry imaging (MSI) allows visualization of endogenous and exogenous compound in tissue sections based on its molecular mass. The 3D reconstruction by MSI provides a more informative description of the tumor drug distribution compared to the high-performance liquid chromatography method, highlighting the heterogeneity of intratumor drug concentration. This additional information can be important in understanding chemoresistance to target agents. Here, we present the 3D visualization of the tyrosine kinase inhibitor (TKI), imatinib, in a xenograft model of resistant malignant pleural mesothelioma.

Polymer nanoparticles (NPs) represent a promising way to deliver poorly water-soluble anticancer drugs without the use of unwanted excipients, whose presence can be the cause of severe side effects. In this work, a Cremophor-free formulation for paclitaxel (PTX) has been developed by employing PEGylated polymer nanoparticles (NPs) as drug delivery carriers based on modified poly(Ε-caprolactone) macromonomers and synthesized through free radical emulsion polymerization. Paclitaxel was loaded in the NPs in a postsynthesis process which allowed to obtain a drug concentration suitable for in vivo use. In vivo experiments on drug biodistribution and therapeutic efficacy show comparable behavior between the NPs and the Cremophor formulation, also showing good tolerability of the new formulation proposed.

The advent of nanotechnology in medicine has allowed to eliminate the toxic excipients that are often necessary to formulate lipophilic drugs in clinics. An example is paclitaxel, one of the most important chemotherapeutic drugs developed so far, where the Cremophor EL has been eliminated in the Genexol and Abraxane formulations. However, the complex procedures to synthesize these formulations hamper their cost-effective use and, in turn, their distribution among the patient population. For this reason, a simplified method to formulate this drug directly at the bed of the patient has been adopted. It requires only the use of a syringe and it starts from a native dry amphiphilic biodegradable and biocompatible block-copolymer obtained via the combination of the reversible addition–fragmentation chain transfer polymerization and ring-opening polymerization. In this way, a novel paclitaxel formulation with the same drug pharmacological properties, but without the use of the Cremophor EL, can be produced. In addition, as long as these nanoparticles are engineered to act as solubility enhancers, a lower burden for its approval from the pharmaceutical regulatory agencies is also expected.

One of the major drawbacks that limits the clinical application of nanoparticles is the lack of preliminary investigations related to their biocompatibility, biodegradability and biodistribution. In this work, biodegradable PEGylated polymer nanoparticles (NPs) have been synthesized by using macromonomers based on poly(ε-caprolaconte) oligomers. More in detail, NPs have been produced by adopting a surfactant-free semibatch emulsion polymerization process using PEG chains as a stabilizing agent. The NPs were also labeled with rhodamine B covalently bound to the NPs to quantitatively study their biodistribution in vivo. NPs were investigated in both in vitro and in vivo preclinical systems to study their biodistribution in mice bearing B16/F10 melanoma, as well as their biocompatibility and biodegradability. The NP concentration was evaluated in different tissues at several times after intravenous injection. The disappearance of the NPs from the plasma was biphasic, with distribution and elimination half-lives of 30 min and 15 h, respectively. NPs were retained in tumors and in filter organs for a long time, were still detectable after 7 d and maintained a steady concentration in the tumor for 120 h. 48 h after injection, 70± 15% of the inoculated NPs were excreted in the feces. The favorable tumor uptake, fast excretion and absence of cytotoxicity foster the further development of produced NPs as drug delivery carriers. © 2014 IOP Publishing Ltd.

Prion protein (PrP) amyloid formation is a central feature of genetic and acquired prion diseases such as Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob disease. Themajor component of GSS amyloid is a PrP fragment spanning residues ∼82-146, which when synthesized as a peptide, readily forms fibrils featuring GSS amyloid. The present study employed surface plasmon resonance (SPR) to characterize the binding events underlying PrP82-146 oligomerization at the first stages of fibrillization, according to evidence suggesting a pathogenic role of prefibrillar oligomers rather than mature amyloid fibrils. We followed in real time the binding reactions occurring during short term (seconds) addition of PrP82-146 small oligomers (1-5-mers, flowing species) onto soluble prefibrillar PrP82-146 aggregates immobilized on the sensor surface. SPR data confirmed very efficient aggregation/elongation, consistent with the hypothesis of nucleation-dependent polymerization process. Much lower binding was observed when PrP82-146 flowed onto the scrambled sequence of PrP82-146 or onto prefibrillar Aβ42 aggregates. As previously found with Aβ40, SPR data could be adequately fitted by equations modeling the "dock-and-lock" mechanism, in which the "locking" step is due to sequential conformational changes, each increasing the affinity of the monomerfor the fibril until a condition of irreversible binding is reached. However, these conformational changes (i.e. the locking steps) appear to be faster and easier with PrP82-146 than with Aβ40. Such differences suggest that PrP82-146 has a greater propensity to polymerize and greater stability of the aggregates. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, or dFdC) is a promising anticancer agent with demonstrated clinical activity in solid tumours currently undergoing clinical trials. Despite extensive studies on the biochemical mechanism of action, cell cycle perturbations induced by dFdC have not yet been thoroughly investigated, apart from the expected inhibition of DNA synthesis. The aim of our study was to clarify whether cell population kinetics is a vital factor in the cytotoxicity of dFdC in single or repeated treatments and in the dFdC-cisplatin combination. Ovarian cancer cells growing in vitro were treated with dFdC for 1 hr in a range of concentrations from 10 nM to 10 microM. Cell kinetics was investigated by DNA-bromodeoxyuridine flow cytometry, using different experimental protocols to measure either the time course of DNA-synthesis inhibition or the fate of cells in G(1), S or G(2)M at the time of dFdC treatment or 24 hr later. A modified sulforhodamine B test was used to assess the growth inhibition caused by dFdC given alone or with cisplatin. Although dFdC promptly inhibited DNA synthesis, cytotoxicity on proliferating cells was not specific for cells initially in the S phase. DNA synthesis was restored after a G(1) block of variable, dose-dependent length, but recycling cells were intercepted at the subsequent checkpoints, resulting in delays in the G(2)M and G(1) phases. The activity of repeated treatment with dFdC + dFdC or dFdC + cisplatin was highly dependent on the interval length between them. These results suggest that the kinetics of cell recycling from a first dFdC treatment strongly affects the outcome of a second treatment with either dFdC itself or cisplatin.

Background: Cell kinetics could have prognostic significance in human ovarian cancer and might also help in designing optimal therapy. No data are available on the in vivo kinetics of this tumor using bromodeoxyuridine (BrdU) infusion before surgery.Patients and methods: The kinetic parameters of human ovarian carcinoma were investigated in vivo using BrdU incorporation and bivariate BrdU/DNA flow cytometric (FCM) analysis. Fifty-five previously untreated patients with ovarian cancer (F.I.G.O. clinical stage III and IV) were studied. BrdU (250 mg/sqm) was given i.v. 6 h before surgery and samples of primary tumor and metastasis biopsies were fixed in 70% ethanol. By coupling the BrdU immunoreaction with biparametric FCM analysis, the nuclear DNA content (i.e., ploidy status), the tumor labelling index (LI), the synthesis time (TS) and the potential doubling time of the tumor mass (Tpot) were obtained. BrdU immunodetection was done on histological sections of the same tumors.Results: The majority of the tumors had a DNA aneuploid content (71.5%). The amount of BrdU-positive S-phase cells varied in different tumor samples and when several samples were taken from the same tumor. The proportion of BrdU-negative S-phase cells was large (50% of the total S phase cells) in almost all cases. The mean LI was 6.1% using FCM and 7.2% on visual count of the slide. The mean TS and Tpot were 14.7 h and 12.5 days, respectively. LI and TS were not correlated with clinical tumor stage, histological grading, residual tumor size or DNA ploidy, but Tpot was significantly higher (p < 0.05) in patients with residual tumor size

A total of 155 samples from 101 patients with ovarian cancer were investigated using flow cytometry to evaluate the DNA index and the percentage of cells in the various cell cycle phases. Thirty-four samples were DNA diploid tumours, while the other 121 were DNA aneuploid tumours. The DNA index was very stable in different sites and over time in the same patient. Tumour stage and ploidy were significantly associated: stages III and IV tumour stage were more likely to be DNA aneuploid. Patients with residual tumour size at first surgery greater than 2 cm had a significantly larger number of DNA aneuploid than DNA diploid tumours. The DNA index was also related to the degree of differentiation of the tumours. The percentage of cells in the S phase of the cell cycle was significantly higher in DNA aneuploid and in poorly differentiated tumours than DNA diploid and well differentiated tumours. Multivariate analysis using the Cox model showed that the DNA index and the percentage of cells in S phase were not independent prognostic variables in this study. Prospectively collected data should be accumulated before assigning the DNA index an important role as a biological prognostic factor in ovarian cancer.