End-To-End Procedure For IORT in Brain Metastases and Film Dosimetry.

Purpose: The study is intended to perform an end-to-end test of the entire intraoperative process using cadaver heads. A simulation of tumor removal was performed, followed by irradiation of the bed and measurement of absorbed doses with radiochromic films. Materials and Methods: Low-energy X-ray intraoperative radiotherapy (IORT) was used for irradiation. A computed tomography study was performed at each site and the absorbed doses calculated by the treatment planning system, as well as absorbed doses with radiochromic films, were studied. Results: The absorbed doses in the organs at risk (OAR) were evaluated in each case, obtaining maximum doses within the tolerance limits. The absorbed doses in the target were verified and the deviations were <1%. Conclusions: These tests demonstrated that this comprehensive procedure is a reproducible quality assurance tool which allows continuous assessment of the dosimetric and geometric accuracy of clinical brain IORT treatments. Furthermore, the absorbed doses measured in both target and OAR are optimal for these treatments.

Design and Development of Antibody Functionalized Gold Nanoparticles for Biomedical Applications.

Antibody-functionalized gold nanoparticle constitutes a powerful interface biosystem for biomedical applications where the properties of gold nanoparticles and the specificity of antibody-antigen interactions are combined. This study provides insight into the key factors for the development of antibody functionalized gold nanoparticles focusing on the immobilization of the antibody. Here, we address an oriented antibody immobilization procedure on gold nanoparticles. It comprises chelatemodified gold nanoparticles that are designed for oriented immobilization of IgG antibodies (end on spatial orientation) through the metal-chelation to histidine-rich metal binding site in the heavy chain (Fc) of the antibody.

Intrasession Repeatability of the Contact Angle Measured Using the Captive Bubble Method and Agreement Assessed Between Different Analysis Software Programs.

OBJECTIVES: The material biocompatibility of hydrogel and silicone hydrogel (SiHy) contact lens (CL) is of paramount importance in CL wear because a decrease in CL wettability reduces wearer comfort and increases wearer dropout. The aim of this study is to report on the repeatability and agreement between two different software programs that measure the contact angle with the captive bubble method in marketed CLs, which will help to translate this information into clinical practice. METHODS: The contact angle of 23 different CLs was measured with the captive bubble method using 2 software programs: FTÅ200 and ImageJ. Three consecutive measurements were conducted for each CL. Reproducibility, repeatability, and agreement values were calculated according to the British Standards Institute and the International Organization for Standardization. RESULTS: All methods showed good repeatability values in both CL materials (coefficient of variation <1.51%, Sw <2.26°, intraclass correlation coefficient >0.89, and the range of limits of agreement was between 7.22° and 7.57°). Higher concordance was achieved between the spherical and nonspherical options when using FTÅ200 software than when using ImageJ software. Statistically significant differences (P<0.05) between the 2 software programs were found, and they ranged between 5° and 10°. CONCLUSIONS: The captive bubble method showed great repeatability in measuring the contact angle in marketed CLs with both software programs (FTÅ200 and ImageJ) assessed in this study. However, differences in the measured contact angles suggest that these techniques are not interchangeable. Therefore, standardization is recommended for contact angle measurement in hydrogel CL materials to facilitate comparisons, to improve clinical use of this information, and to analyze their impact in CL user comfort.

Treatment of cervical cancer with electronic brachytherapy.

PURPOSE: We report the first cervical cancer cases treated with interstitial electronic brachytherapy (eBT) at our hospital and compare them with plans made with high-dose-rate interstitial brachytherapy based on Ir192 (HDR-BT). MATERIALS AND METHODS: Eight patients with cervical cancer were treated with the Axxent eBT device (Xoft, Inc.). Planning was with magnetic resonance imaging and computed tomography following the recommendations of the EMBRACE protocol. The dosimetry parameters of organs at risk (OAR) were evaluated for the bladder, rectum, and sigmoid colon (D2cc, D1cc, and D0.1cc). In addition, the V150 and V200 of irradiated tissue were compared for both eBT and HDR-BT. All patients received intensity-modulated external beam radiation therapy with a regimen of 23 sessions of 2 Gy followed by four sessions of 7 Gy of eBT performed over 2 weeks (two sessions followed by another two sessions a week later) following the EMBRACE recommendations. Each of the eight patients was followed to assess acute toxicity associated with treatment. RESULTS: The doses reaching OAR for eBT plans were lower than for HDR-BT plans. As for acute toxicity associated with eBT, very few cases of mucositis were detected. No cases of rectal toxicity and one case with grade 1 urinary toxicity were detected. The results at 1 month are equally good, and no relapses have occurred to date. CONCLUSIONS: The first results of treatment with the Axxent eBT device are promising, as no recurrences have been observed and toxicity is very low. eBT is a good alternative for treating cervical cancer in centers without access to conventional HDR.

Design of stable magnetic hybrid nanoparticles of Si-entrapped HRP.

Hybrid and composite nanoparticles represent an attractive material for enzyme integration due to possible synergic advantages of the structural builders in the properties of the nanobiocatalyst. In this study, we report the synthesis of a new stable hybrid nanobiocatalyst formed by biomimetic silica (Si) nanoparticles entrapping both Horseradish Peroxidase (HRP) (EC 1.11.1.7) and magnetic nanoparticles (MNPs). We have demonstrated that tailoring of the synthetic reagents and post immobilization treatments greatly impacted physical and biocatalytic properties such as an unprecedented ~280 times increase in the half-life time in thermal stability experiments. The optimized nanohybrid biocatalyst that showed superparamagnetic behaviour, was effective in the batch conversion of indole-3-acetic acid, a prodrug used in Direct Enzyme Prodrug Therapy (DEPT). Our system, that was not cytotoxic per se, showed enhanced cytotoxic activity in the presence of the prodrug towards HCT-116, a colorectal cancer cell line. The strategy developed proved to be effective in obtaining a stabilized nanobiocatalyst combining three different organic/inorganic materials with potential in DEPT and other biotechnological applications.

Orthoxenografts of Testicular Germ Cell Tumors Demonstrate Genomic Changes Associated with Cisplatin Resistance and Identify PDMP as a Resensitizing Agent.

Purpose: To investigate the genetic basis of cisplatin resistance as efficacy of cisplatin-based chemotherapy in the treatment of distinct malignancies is often hampered by intrinsic or acquired drug resistance of tumor cells.Experimental Design: We produced 14 orthoxenograft transplanting human nonseminomatous testicular germ cell tumors (TGCT) in mice, keeping the primary tumor features in terms of genotype, phenotype, and sensitivity to cisplatin. Chromosomal and genetic alterations were evaluated in matched cisplatin-sensitive and their counterpart orthoxenografts that developed resistance to cisplatin in nude mice.Results: Comparative genomic hybridization analyses of four matched orthoxenografts identified recurrent chromosomal rearrangements across cisplatin-resistant tumors in three of them, showing gains at 9q32-q33.1 region. We found a clinical correlation between the presence of 9q32-q33.1 gains in cisplatin-refractory patients and poorer overall survival (OS) in metastatic germ cell tumors. We studied the expression profile of the 60 genes located at that genomic region. POLE3 and AKNA were the only two genes deregulated in resistant tumors harboring the 9q32-q33.1 gain. Moreover, other four genes (GCS, ZNF883, CTR1, and FLJ31713) were deregulated in all five resistant tumors independently of the 9q32-q33.1 amplification. RT-PCRs in tumors and functional analyses in Caenorhabditis elegans (C. elegans) indicate that the influence of 9q32-q33.1 genes in cisplatin resistance can be driven by either up- or downregulation. We focused on glucosylceramide synthase (GCS) to demonstrate that the GCS inhibitor DL-threo-PDMP resensitizes cisplatin-resistant germline-derived orthoxenografts to cisplatin.Conclusions: Orthoxenografts can be used preclinically not only to test the efficiency of drugs but also to identify prognosis markers and gene alterations acting as drivers of the acquired cisplatin resistance. Clin Cancer Res; 24(15); 3755-66. ©2018 AACR.

Assessment of the Monitor Unit Objective tool for VMAT in the Eclipse treatment planning system.

AIM: This work aims to achieve the highest possible monitor units (MU) reduction using the MU Objective tool included in the Eclipse treatment planning system, while preserving the plan quality. BACKGROUND: The treatment planning system Eclipse (Varian Medical Systems, Palo Alto, CA) includes a control mechanism for the number of monitor units of volumetric modulated arc therapy (VMAT) plans, named the MU Objective tool. MATERIAL AND METHODS: Forty prostate plans, 20 gynecological plans and 20 head and neck plans designed with VMAT were retrospectively studied. Each plan (base plan) was optimized without using the MU Objective tool, and it was re-optimized with different values of the Maximum MU (MaxMU) parameter of the MU Objective tool. MU differences were analyzed with a paired samples t-test and changes in plan quality were assessed with a set of parameters for OARs and PTVs. RESULTS: The average relative MU difference [Formula: see text] considering all treatment sites, was the highest when MaxMU = 400 (-4.2%, p < 0.001). For prostate plans, the lowest [Formula: see text] was obtained (-3.7%, p < 0.001). For head and neck plans [Formula: see text] was -7.3% (p < 0.001) and for gynecological plans [Formula: see text] was 7.0% (p = 0.002). Although similar MU reductions were observed for both sites, for some gynecological plans maximum differences were greater than 10%. All the assessed parameters for PTVs and OARs sparing showed average differences below 2%. CONCLUSION: For the three studied clinical sites, establishing MaxMU = 400 led to the optimum MU reduction, maintaining the original dose distribution and dosimetric parameters practically unaltered.

Modeling lung cancer evolution and preclinical response by orthotopic mouse allografts.

Cancer evolution is a process that is still poorly understood because of the lack of versatile in vivo longitudinal studies. By generating murine non-small cell lung cancer (NSCLC) orthoallobanks and paired primary cell lines, we provide a detailed description of an in vivo, time-dependent cancer malignization process. We identify the acquisition of metastatic dissemination potential, the selection of co-driver mutations, and the appearance of naturally occurring intratumor heterogeneity, thus recapitulating the stochastic nature of human cancer development. This approach combines the robustness of genetically engineered cancer models with the flexibility of allograft methodology. We have applied this tool for the preclinical evaluation of therapeutic approaches. This system can be implemented to improve the design of future treatments for patients with NSCLC.

A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect.

Cancer cells possess aberrant proteomes that can arise by the disruption of genes involved in physiological protein degradation. Here we demonstrate the presence of promoter CpG island hypermethylation-linked inactivation of DERL3 (Derlin-3), a key gene in the endoplasmic reticulum-associated protein degradation pathway, in human tumours. The restoration of in vitro and in vivo DERL3 activity highlights the tumour suppressor features of the gene. Using the stable isotopic labelling of amino acids in cell culture workflow for differential proteome analysis, we identify SLC2A1 (glucose transporter 1, GLUT1) as a downstream target of DERL3. Most importantly, SLC2A1 overexpression mediated by DERL3 epigenetic loss contributes to the Warburg effect in the studied cells and pinpoints a subset of human tumours with greater vulnerability to drugs targeting glycolysis.

Tips for the functionalization of nanoparticles with antibodies.

Multiple antibody immobilization methodologies have been developed for several applications including affinity chromatography, immunosensing, and drug delivery. Most of them have been carried out without considering the orientation of the antigen binding site of the antibody, or after the chemical modification of the antibody. An efficient immobilization to improve the biological activity of the antibody is one of the key fundamental issues to pursue. A simple and effective methodology for well-oriented covalently immobilization of antibodies on nanoparticles is reported in this chapter.

Improving immunosensor performance through oriented immobilization of antibodies on carbon nanotube composite surfaces.

We report the straightforward oriented covalent attachment of antibodies (Abs) on the surface of carboxylated multiwalled carbon nanotube-polystyrene (MWCNT-PS) materials. The combination of this composite material, applied as a robust electrochemical transducer platform, and its covalent functionalization with Abs in a controlled way by means of a two-step process, could contribute to the development of highly sensitive immunosensor devices. Using the simple and versatile carbodiimide chemistry, Abs were attached to the carboxylic groups of the MWCNT-PS composite surfaces via their superficial amine groups. By taking into account the Ab isoelectric point and the net charge of the composite surface, we engineered an immobilization process to achieve the oriented binding of the Ab molecules by favoring an ionic pre-adsorption step before covalent binding occurred. Thus, the antigen binding capacity of the attached Abs was enhanced by up to 10 times with respect to the capacity estimated for a random spatial distribution of these molecules. The proposed strategy would also serve as a model for the efficient biofunctionalization of other carboxylated carbon-based polymer composite materials with potential applications in the biosensor field.

Tackling reproducibility in microcantilever biosensors: a statistical approach for sensitive and specific end-point detection of immunoreactions.

In the last decade, microcantilever biosensors have shown enormous potential for highly sensitive label-free detection of nucleic acid and proteins. Despite the enormous advances, the promise of applications of this technology in the biomedical field has been frustrated because of its low reproducibility. Here we tackle the reproducibility issue in microcantilever biosensors and provide the guidelines to minimize the deviations in the biosensor response between different assays. We use as a model system the label-free end-point detection of horseradish peroxidase. We choose the end-point detection mode because of its suitability for implementation in the clinical field that requires simplicity and point-of-care capability. Our study comprises the analysis of 1012 cantilevers with different antibody surface densities, two blocking strategies based on polyethylene-glycol (PEG) and bovine serum albumin (BSA) and stringent controls. The study reveals that the performance of the assay critically depends on both antibody surface density and blocking strategies. We find that the optimal conditions involve antibody surface densities near but below saturation and blocking with PEG. We find that the surface stress induced by the antibody-antigen binding is significantly correlated with the surface stress generated during the antibody attachment and blocking steps. The statistical correlation is harnessed to identify immobilization failure or success, and thus enhancing the specificity and sensitivity of the assay. This procedure enables achieving rates of true positives and true negatives of 90% and 91% respectively. The detection limit is of 10 ng mL(-1) (250 pM) that is similar to the detection limit obtained in our enzyme-linked immunosorbent assay (ELISA) and at least two orders of magnitude smaller than that achieved with well-established label-free biosensors such as a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) sensor.

Lurbinectedin (PM01183), a new DNA minor groove binder, inhibits growth of orthotopic primary graft of cisplatin-resistant epithelial ovarian cancer.

PURPOSE: Epithelial ovarian cancer (EOC) is the fifth leading cause of death in women diagnosed with gynecologic malignancies. The low survival rate is because of its advanced-stage diagnosis and either intrinsic or acquired resistance to standard platinum-based chemotherapy. So, the development of effective innovative therapeutic strategies to overcome cisplatin resistance remains a high priority. EXPERIMENTAL DESIGN: To investigate new treatments in in vivo models reproducing EOCs tumor growth, we generated a preclinical model of ovarian cancer after orthotopic implantation of a primary serous tumor in nude mice. Further, matched model of acquired cisplatin-resistant tumor version was successfully derived in mice. Effectiveness of lurbinectedin (PM01183) treatment, a novel marine-derived DNA minor groove covalent binder, was assessed in both preclinical models as a single and a combined-cisplatin agent. RESULTS: Orthotopically perpetuated tumor grafts mimic the histopathological characteristics of primary patients' tumors and they also recapitulate in mice characteristic features of tumor response to cisplatin treatments. We showed that single lurbinectedin or cisplatin-combined therapies were effective in treating cisplatin-sensitive and cisplatin-resistant preclinical ovarian tumor models. Furthermore, the strongest in vivo synergistic effect was observed for combined treatments, especially in cisplatin-resistant tumors. Lurbinectedin tumor growth inhibition was associated with reduced proliferation, increased rate of aberrant mitosis, and subsequent induced apoptosis. CONCLUSIONS: Taken together, preclinical orthotopic ovarian tumor grafts are useful tools for drug development, providing hard evidence that lurbinectedin might be a useful therapy in the treatment of EOC by overcoming cisplatin resistance.

Epigenetic disruption of cadherin-11 in human cancer metastasis.

Little is known about the molecular events occurring in the metastases of human tumours. Epigenetic alterations are dynamic lesions that change over the natural course of the disease, and so they might play a role in the biology of cancer cells that have departed from the primary tumour. Herein, we have adopted an epigenomic approach to identify some of these changes. Using a DNA methylation microarray platform to compare paired primary tumour and lymph node metastatic cell lines from the same patient, we observed cadherin-11 promoter CpG island hypermethylation as a likely target of the process. We found that CDH11 DNA methylation-associated transcriptional silencing occurred in the corresponding lymph node metastases of melanoma and head and neck cancer cells but not in the primary tumours. Using in vitro and in vivo cellular and mouse models for depleted or enhanced CDH11 activity, we also demonstrated that CDH11 acts as an inhibitor of tumour growth, motility and dissemination. Most importantly, the study of CDH11 5'-CpG island hypermethylation in primary tumours and lymph node metastases of cancer patients showed this epigenetic alteration to be significantly confined to the disseminated cells. Overall, these results indicate the existence of metastasis-specific epigenetic events that might contribute to the progression of the disease.

Specific peptides as alternative to antibody ligands for biomagnetic separation of Clostridium tyrobutyricum spores.

Nowadays, the reference method for the detection of Clostridium tyrobutyricum in milk is the most-probable-number method, a very time-consuming and non-specific method. In this work, the suitability of the use of superparamagnetic beads coated with specific antibodies and peptides for bioseparation and concentration of spores of C. tyrobutyricum has been assessed. Peptide or antibody functionalized nanoparticles were able to specifically bind C. tyrobutyricum spores and concentrate them up to detectable levels. Moreover, several factors, such as particle size (200 nm and 1 µm), particle derivatization (aminated and carboxylated beads), coating method, and type of ligand have been studied in order to establish the most appropriate conditions for spore separation. Results show that concentration of spore is favored by a smaller bead size due to the wider surface of interaction in relation to particle volume. Antibody orientation, related to the binding method, is also critical in spore recovery. However, specific peptides seem to be a better ligand than antibodies, not only due to the higher recovery ratio of spores obtained but also due to the prolonged stability over time, allowing an optimal recovery of spores up to 3 weeks after bead coating. These results demonstrate that specific peptides bound to magnetic nanoparticles can be used instead of traditional antibodies to specifically bind C. tyrobutyricum spores being a potential basis for a rapid method to detect this bacterial target.

Taking advantage of unspecific interactions to produce highly active magnetic nanoparticle-antibody conjugates.

Several strategies for linking antibodies (Abs) through their Fc region in an oriented manner have been proposed at the present time. By using these strategies, the Fab region of the Ab is available for antigen molecular recognition, leading to a more efficient interaction. Most of these strategies are complex processes optimized mainly for the functionalization of surfaces or microbeads. These methodologies imply though the Ab modification through several steps of purification or the use of expensive immobilized proteins. Besides, the functionalization of magnetic nanoparticles (MNPs) turned out to be much more complex than expected due to the lack of stability of most MNPs at high ionic strength and non-neutral pH values. Therefore, there is still missing an efficient, easy and universal methodology for the immobilization of nonmodified Abs onto MNPs without involving their Fab regions during the immobilization process. Herein, we propose the functionalization of MNPs via a two-steps strategy that takes advantage of the ionic reversible interactions between the Ab and the MNP. These interactions make possible the orientation of the Ab on the MNP surface before being attached in an irreversible way via covalent bonds. Three Abs (Immunoglobulin G class) with very different isoelectric points (against peroxidase, carcinoembryonic antigen, and human chorionic gonadotropin hormone) were used to prove the general applicability of the strategy here proposed and its utility for the development of more bioactive NPs.

Small molecule enoxacin is a cancer-specific growth inhibitor that acts by enhancing TAR RNA-binding protein 2-mediated microRNA processing.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at the posttranscriptional level and are critical for many cellular pathways. The disruption of miRNAs and their processing machineries also contributes to the development of human tumors. A common scenario for miRNA expression in carcinogenesis is emerging that shows that impaired miRNA production and/or down-regulation of these transcripts occurs in many neoplasms. Several of these lost miRNAs have tumor-suppressor features, so strategies to restore their expression globally in malignancies would be a welcome addition to the current therapeutic arsenal against cancer. Herein, we show that the small molecule enoxacin, a fluoroquinolone used as an antibacterial compound, enhances the production of miRNAs with tumor suppressor functions by binding to the miRNA biosynthesis protein TAR RNA-binding protein 2 (TRBP). The use of enoxacin in human cell cultures and xenografted, orthotopic, and metastatic mouse models reveals a TRBP-dependent and cancer-specific growth-inhibitory effect of the drug. These results highlight the key role of disrupted miRNA expression patterns in tumorigenesis, and suggest a unique strategy for restoring the distorted microRNAome of cancer cells to a more physiological setting.

Slc7a9 knockout mouse is a good cystinuria model for antilithiasic pharmacological studies.

Cystinuria is a hereditary disorder caused by a defect in the apical membrane transport system for cystine and dibasic amino acids in renal proximal tubules and intestine, resulting in recurrent urolithiasis. Mutations in SLC3A1 and SLC7A9 genes, that codify for rBAT/b(0,+)AT transporter subunits, cause type A and B cystinuria, respectively. In humans, cystinuria treatment is based on the prevention of calculi formation and its dissolution or breakage. Persistent calculi are treated with thiols [i.e., d-penicillamine (DP) and mercaptopropionylglycine (MPG)] for cystine solubilization. We have developed a new protocol with DP to validate our Slc7a9 knockout mouse model for the study of the therapeutic effect of drugs in the treatment of cystine lithiasis. We performed a 5-wk treatment of individually caged lithiasic mutant mice with a previously tested DP dose. To appraise the evolution of lithiasis throughout the treatment a noninvasive indirect method of calculi quantification was developed: calculi mass was quantified by densitometry of X-ray images from cystinuric mice before and after treatment. Urine was collected in metabolic cage experiments to quantify amino acids in DP-treated and nontreated, nonlithiasic mutant mice. We found significant differences between DP-treated and nontreated knockout mice in calculi size and in urinary cystine excretion. Histopathological analysis showed that globally nontreated mutant mice had more severe and diffuse urinary system damage than DP-treated mice. Our results validate the use of this mouse model for testing the efficacy of potential new drugs against cystinuria.