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PDF file - 85K, Supplementary Figure S2.Representation of the results of the class comparison analysis of the gene expression data. In total, 3,078 genes were differentially expressed in the PP samples compared with all other SNBs. In the analysis, 333 genes were differentially expressed between the PN and the PP tumor-positive SNB samples, 58 genes were differentially expressed between all tumor-positive SNBs and the N samples, and 18 genes were differentially expressed between the PN and N samples.

XLSX file - 202K, Supplementary Table S3. Results generated by GSEA comparing the PN and the PP samples.

XLSX file - 132K, Supplementary Table S5.CD30 expression in the PP.

XLSX file - 15K, Supplementary Table S1.Clinicopathological data for the SNB samples analyzed in the training and validation studies.

PDF file - 107K, Supplementary Figure S3.Comparative gene expression analysis by qRT-PCR of the genes that were differently expressed between the PP and the PN groups. A. Genes upregulated in the PP samples (DEFA1, IL17B, and CST6) (p

PDF file - 68K, Supplementary Figure S1.Expression levels of melanoma genes in the SNB samples, as indicated by the array data. The genes and SNB groups are indicated, and the intensity values (minimum to maximum) and the median are shown. The values for the PN or PP samples in comparison with the N samples are indicated (*, p

Sentinel lymph nodes set the stance of the immune system to a localized tumor and are often the first site to be colonized by neoplastic cells that metastasize. To investigate how the presence of neoplastic cells in sentinel lymph nodes may trigger pathways associated with metastatic progression, we analyzed the transcriptional profiles of archival sentinel node biopsy specimens obtained from melanoma patients. Biopsies from positive nodes were selected for comparable tumor infiltration, presence or absence of further regional node metastases, and relapse at 5-year follow-up. Unsupervised analysis of gene expression profiles revealed immune response to be a major gene ontogeny represented. Among genes upregulated in patients with progressing disease, the TNF receptor family member CD30/TNFRSF8 was confirmed in biopsy specimens from an independent group of patients. Immunohistochemical analysis revealed higher numbers of CD30+ lymphocytes in nodes from progressing patients compared with nonprogressing patients. Phenotypic profiling demonstrated that CD30+ lymphocytes comprised a broad population of suppressive or exhausted immune cells, such as CD4+Foxp3+ or PD1+ subpopulations and CD4−CD8− T cells. CD30+ T lymphocytes were increased in peripheral blood lymphocytes of melanoma patients at advanced disease stages. Our findings reinforce the concept that sentinel nodes act as pivotal sites for determining progression patterns, revealing that the presence of CD30+ lymphocytes at those sites associate positively with melanoma progression. Cancer Res; 74(1); 130–40. ©2014 AACR.

XLSX file - 2088K, Supplementary Table S2.Differentially expressed gene lists.

PDF file - 93K

XLSX file - 198K, Supplementary Table S4. Genes found to be differentially expressed between the PP and the PN samples, as confirmed by qPCR.

Magnetic hyperthermia has the potential to play an important role in cancer therapy and its efficacy relies on the nanomaterials selected. Superparamagnetic iron oxide nanoparticles (SPIONs) are excellent candidates due to the ability of producing enough heat to kill tumor cells by thermal ablation. However, their heating properties depend strongly on crystalline structure and size, which may not be controlled and tuned during the synthetic process; therefore, a postprocessing is needed. We show how thermal annealing can be simultaneously coupled with ligand exchange to stabilize the SPIONs in polar solvents and to modify their crystal structure, which improves hyperthermia behavior. Using high-resolution transmission electron microscopy, X-ray diffraction, Mössbauer spectroscopy, vibrating sample magnetometry, and lock-in thermography, we systematically investigate the impact of size and ligand exchange procedure on crystallinity, their magnetism, and heating ability. We describe a valid and simple approach to optimize SPIONs for hyperthermia by carefully controlling the size, colloidal stability, and crystallinity.

Magnetosomes are near-perfect intracellular magnetite nanocrystals found in magnetotactic bacteria. Their synthetic imitation, known as superparamagnetic iron oxide nanoparticles (SPIONs), have found applications in a variety of (nano)medicinal fields such as magnetic resonance imaging contrast agents, multimodal imaging and drug carriers. In order to perform these functions in medicine, shape and size control of the SPIONs is vital. We sampled SPIONs at ten-minutes intervals during the high-temperature thermal decomposition reaction. Their shape (sphericity and anisotropy) and geometric description (volume and surface area) were retrieved using three-dimensional imaging techniques, which allowed to reconstruct each particle in three dimensions, followed by stereological quantification methods. The results, supported by small angle X-ray scattering characterization, reveal that SPIONs initially have a spherical shape, then grow increasingly asymmetric and irregular. A high heterogeneity in volume at the initial stages makes place for lower particle volume dispersity at later stages. The SPIONs settled into a preferred orientation on the support used for transmission electron microscopy imaging, which hides the extent of their anisotropic nature in the axial dimension, there by biasing the interpretation of standard 2D micrographs. This information could be feedback into the design of the chemical processes and the characterization strategies to improve the current applications of SPIONs in nanomedicine.

Magnetic nanoparticles and their ability to convert electromagnetic energy into heat are of explicit interest for various applications. However, precise quantification of their heating efficiency is not always upfront, and several parameters render comparative studies challenging. This paper describes the theory behind lock-in thermography, a new technique for quantifying the heating properties of magnetic nanoparticles. This technique allows the investigation of some of the potential sources of variability: key factors such as magnetic field inhomogeneity and its effects on the heating power are explored in detail. The presented results, obtained from various nanoparticle batches of different origins, highlight the importance of pursuing a standardized and systematic approach when quantifying the heating efficiency of magnetic nanoparticles.

Lock-in thermography (LIT) is a sensitive imaging technique generally used in engineering and materials science ( e.g. detecting defects in composite materials). However, it has recently been expanded for investigating the heating power of nanomaterials, such as superparamagnetic iron oxide nanoparticles (SPIONs). Here we implement LIT as a rapid and reproducible method that can evaluate the heating potential of various sizes of SPIONs under an alternating magnetic field (AMF), as well as the limits of detection for each particle size. SPIONs were synthesized via thermal decomposition and stabilized in water via a ligand transfer process. Thermographic measurements of SPIONs were made by stimulating particles of varying sizes and increasing concentrations under an AMF. Furthermore, a commercially available SPION sample was included as an external reference. While the size dependent heating efficiency of SPIONs has been previously described, our objective was to probe the sensitivity limits of LIT. For certain size regimes it was possible to detect signals at concentrations as low as 0.1 mg Fe/mL. Measuring at different concentrations enabled a linear regression analysis and extrapolation of the limit of detection for different size nanoparticles.

Journal of Magnetism and Magnetic Materials, 427, ISSN:0304-8853

Abstract: Understanding the interaction between cells and their environment is fundamental for mechanobiology. To mimic the behavior of cells in physiological and pathological conditions, synthetic substrates must have topographical and/or mechanical properties that evolve in time. Dynamic substrates mainly rely on stimuli-responsive materials where an external stimulus induces controlled variations in topography or mechanics. Herein, we describe the development of a dynamic cell culture substrate where mechanical properties are reversibly tuned in situ using magnetically-responsive superparamagnetic iron oxide nanoparticles (SPIONs).

As a highly interdisciplinary field, working with nanoparticles in a biomedical context requires a robust understanding of soft matter physics, colloidal behaviors, nano-characterization methods, biology, and bio-nano interactions. When reporting results, it can be easy to overlook simple, seemingly trivial experimental details. In this context, we set out to understand how in vitro technique, specifically the way we administer particles in 2D culture, can influence experimental outcomes. Gold nanoparticles coated with poly(vinylpyrrolidone) were added to J774A.1 mouse monocyte/macrophage cultures as either a concentrated bolus, a bolus then mixed via aspiration, or pre-mixed in cell culture media. Particle-cell interaction was monitored via inductively coupled plasma-optical emission spectroscopy and we found that particles administered in a concentrated dose interacted more with cells compared to the pre-mixed administration method. Spectroscopy studies reveal that the initial formation of the protein corona upon introduction to cell culture media may be responsible for the differences in particle-cell interaction. Modeling of particle deposition using the in vitro sedimentation, diffusion and dosimetry model helped to clarify what particle phenomena may be occurring at the cellular interface. We found that particle administration method in vitro has an effect on particle-cell interactions (i.e. cellular adsorption and uptake). Initial introduction of particles in to complex biological media has a lasting effect on the formation of the protein corona, which in turn mediates particle-cell interaction. It is of note that a minor detail, the way in which we administer particles in cell culture, can have a significant effect on what we observe regarding particle interactions in vitro.

Users of dynamic light scattering (DLS) are challenged when a sample of nanoparticles (NPs) contains dust. This is a frequently inevitable scenario and a major problem that critically affects the reproducibility and accuracy of DLS measurements. Current methods approach this problem via photon correlation spectroscopy, but remedy exists only for a few special cases. We introduce here a general criterion and a clearly defined measure to discriminate between NPs and dust particles. The experimental results show that, in contrast to photon correlation spectroscopy, hypothesis testing and the statistical moment analysis of the photon count distribution provides an accurate and precise way to characterize NPs and Brownian dynamics in the presence of dust. To demonstrate, analyses of silica, iron oxide, and gold NPs of low polydispersity are presented.

Taylor dispersion analysis (TDA) is an analytical method that has so far mainly been utilized to determine the diffusion coefficient of small molecules, and proteins. Due to increasing interest in nanoscience, some research has been done on the applicability of TDA towards characterizing nanoparticles. This work aims to expand this knowledge and give insight into the range for which TDA can be used for nanoparticle characterization, focusing on various materials and sizes. The TDA setup shown in this work was successful in characterizing all engineered metallic, non-metallic nanoparticles, and proteins tested in this work. Results were compared to dynamic light scattering and electron microscopy, and were in good agreement with both methods. Taking into consideration the wide range of nanoparticle sizes that can be characterized, the minimal sample preparation, and sample volume, required and the simplicity of the method, TDA can be considered as a valuable technique for nanoparticle characterization.

Thermo- and magneto-responsive shape-memory bionanocomposites based on a bio-based polyurethane and magnetite nanoparticles were prepared. Due to the structure of the reactants, the behavior of the polyurethane matrix differs from common polyurethanes, since the soft segment was formed by a diisocyanate and a chain extender, whereas the macrodiol served as hard segment. The influence of the magnetite nanoparticles on the thermal and mechanical properties and the shape-memory behavior was studied. It was observed that magnetite nanoparticles interacted with macrodiol-rich domains and decreased the overall crystallinity of the material, although their presence did not affect the mechanical properties to a great extent. At the same time, the magnetite nanoparticles increased the shape fixity and contributed to shape recovery. The bionanocomposites exhibited magnetic behavior and could be easily heated in an alternating magnetic field, allowing fast and almost complete shape recovery. Preliminary cytotoxicity, hemocompatibility, and cell adhesion analysis suggest that the new materials are benign and potentially useful for biomedical applications.

We perform a comparative study of dynamic force measurements using an Atomic Force Microscope (AFM) on the same soft polymer blend samples in both air and liquid environments. Our quantitative analysis starts with calibration of the same cantilever in both environments. Intermodulation AFM (ImAFM) is used to measure dynamic force quadratures on the same sample. We validate the accuracy of the reconstructed dynamic force quadratures by numerical simulation of a realistic model of the cantilever in liquid. In spite of the very low quality factor of this resonance, we find excellent agreement between experiment and simulation. A recently developed moving surface model explains the measured force quadrature curves on the soft polymer, in both air and liquid.

The ability to detect and accurately characterize particles is required by many fields of nanotechnology, including materials science, nanotoxicology, and nanomedicine. Among the most relevant physicochemical properties of nanoparticles, size and the related surface-to-volume ratio are fundamental ones. Taylor dispersion combines three independent phenomena to determine particle size: optical extinction, translational diffusion, and sheer-enhanced dispersion of nanoparticles subjected to a steady laminar flow. The interplay of these defines the apparent size. Considering that particles in fact are never truly uniform nor monodisperse, we rigorously address particle polydispersity and calculate the apparent particle size measured by Taylor dispersion analysis. We conducted case studies addressing aqueous suspensions of model particles and large-scale-produced “industrial” particles of both academic and commercial interest of various core materials and sizes, ranging from 15 to 100 nm. A comparison with particle sizes determined by transmission electron microscopy confirms that our approach is model-independent, non-parametric, and of general validity that provides an accurate account of size polydispersity—independently on the shape of the size distribution and without any assumption required a priori.

Background: Debate remains around the accuracy and prognostic implications of sentinellymph node biopsy (SLNB) for melanoma arising in the head and neck (HN) areas becauseseveral analyses have shown discordances between clinically predicted lymphaticdrainage pathways and those identified by lymphoscintigraphy. This study assesses theaccuracy and prognostic value of SLNB in this critical anatomic region.Methods: Retrospective review of a prospectively collected melanoma database identified331 patients with HN melanomas from January 2000 to December 2012. Primary end pointsincluded SLNB result, time to recurrence, site of recurrence, and survival. Multivariatemodels were constructed for analyses.Results: A sentinel lymph node (SLN) was identified in all 331 patients. There were 59 pa-tients with a positive SLN (17.8%) with a recurrence rate of 88.1% compared with 22.4% inSLN-negative patients (P < 0.0001). The 5-y overall survival was 91.2% for SLN-negativepatients and 48.7% for SLN-positive patients (P < 0.0001). Patients with scalp melanomahad thicker lesions and an elevated risk of SLN positivity, recurrence, and death comparedwith those with other sites. Among the 272 SLN-negative patients, four patients developedregional nodal disease in the same basin and had undergone a previous SLNB procedure fora false-omission rate of 1.45%. Risks for false-negative SLN occurrences included thick andscalp melanomas. Multivariate analysis on prognostic factors affecting relapse-free sur-vival showed positive SLNB status to be the most prognostic clinicopathologic predictor ofrecurrence (hazard ratio, 20.56; P < 0.0001).Conclusions: SLNB for patients with HN melanomas is an accurate procedure and hasprognostic value.a 2013 Elsevier Inc. All rights reserved.* Corresponding author. Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Giacomo Venezian 1, 20133 Milan,Italy. Tel.: þ39 02 23902874; fax: þ39 02 23902404.E-mail address: andrea.maurichi@istitutotumori.mi.it (A. Maurichi).

Sentinel lymph nodes set the stance of the immune system to a localized tumor and are often the first site to be colonized by neoplastic cells that metastasize. To investigate how the presence of neoplastic cells in sentinel lymph nodes may trigger pathways associated with metastatic progression, we analyzed the transcriptional profiles of archival sentinel node biopsy specimens obtained from melanoma patients. Biopsies from positive nodes were selected for comparable tumor infiltration, presence or absence of further regional node metastases, and relapse at 5-year follow-up. Unsupervised analysis of gene expression profiles revealed immune response to be a major gene ontogeny represented. Among genes upregulated in patients with progressing disease, the TNF receptor family member CD30/TNFRSF8 was confirmed in biopsy specimens from an independent group of patients. Immunohistochemical analysis revealed higher numbers of CD30+ lymphocytes in nodes from progressing patients compared with nonprogressing patients. Phenotypic profiling demonstrated that CD30+ lymphocytes comprised a broad population of suppressive or exhausted immune cells, such as CD4+Foxp3+ or PD1+ subpopulations and CD4−CD8− T cells. CD30+ T lymphocytes were increased in peripheral blood lymphocytes of melanoma patients at advanced disease stages. Our findings reinforce the concept that sentinel nodes act as pivotal sites for determining progression patterns, revealing that the presence of CD30+ lymphocytes at those sites associate positively with melanoma progression. Cancer Res; 74(1); 130–40. ©2014 AACR.

INTRODUCTIONMelanoma with recurrent loco-regional metastases to limbs often makes difficult a second surgical approach because of the adhesions affecting the vascular access. Our aim was to evaluate whether the placement of a polytetrafluoroethylene (PTFE) membrane around vessels might facilitate a surgical re-approach.PRESENTATION OF CASEWe reported a case of a 64-year-old male with a melanoma on the left foot who developed in transit metastases after LND. While performing the inguinopelvic LND we coated the iliac vessels with PTFE patch to facilitate the vascular access in case of re-intervention for a ILP. In the second surgical approach we made a cutaneous incision in the left iliac region and we proceeded through the subcutaneous tissue until detection of iliac vessels, more clearly visible because of the PTFE patch fixed around vascular walls to minimize adhesions. We removed the PTFE coating and easily performed arteriotomy and venotomy for the completion of the ILP.DISCUSSIONThis case report seems to demonstrate the efficacy of a PTFE membrane applied in a patient around iliac vessels during inguinopelvic dissection, to reduce adhesion density. In fact this membrane provided a barrier to adhesions of the iliac vessels, decreasing the risk of vascular injury thereby facilitating a subsequent vascular access. Re-coating of the iliac vessels with PTFE could be preparatory to a better identification of the vascular structures in cases of a surgical re-approach.CONCLUSIONThe use of PTFE effectively simplifies the second approach to vessels in event of a melanoma metastasizing to limbs.

We propose a new methodology based on lock-in thermography to study and quantify the heating power of magnetic nanoparticles. Superparamagnetic iron oxide nanoparticles exposed to a modulated alternating magnetic field were used as model materials to demonstrate the potency of the system. Both quantitative and qualitative information on their respective heating power was extracted at high thermal resolutions under increasingly complex conditions, including nanoparticles in the liquid, solid and aggregated states. Compared to conventional techniques, this approach offers a fast, sensitive and non-intrusive alternative to investigate multiple and dilute specimens simultaneously, which is essential for optimizing and accelerating screening procedures and comparative studies.

The aim of this retrospective study was to analyze the incidence of further nonsentinel node metastases at completion lymphadenectomy of the groin after a positive sentinel node biopsy to evaluate whether radical dissection remains the treatment of choice for these patients. Patients treated at the National Cancer Institute of Milan between January 1999 and December 2006 were reviewed retrospectively. The analysis included patients with a diagnosis of positive sentinel node biopsy of the groin (clinically negative) who underwent completion groin, iliac, and obturatory dissections. The primary melanoma was located on the lower extremities and trunk in 82.5 and 17.5%, respectively. The median follow-up was more than 30 months. The number of positive sentinel nodes was considered, as well as the size and location of the metastases (micro vs. macro). After radical dissection, the number and the location (groin, iliac, or groin+iliac nodes) of further nonsentinel node metastases were analyzed. The frequency of further nonsentinel node metastases at completion of groin dissection was correlated to Breslow's thickness and to the characteristics of the positive sentinel node biopsy. A total of 1581 patients with primary melanoma (>1 mm, or Clark's IV-V) underwent lymphatic mapping and sentinel node biopsy: 752 patients had sentinel node biopsy at the groin basin; among these, 150 (20%) patients presented positive sentinel node biopsy and underwent completion radical dissection (groin, obturatory, and external iliac+obturatory radical node dissections). We found further positive nonsentinel node metastases in 36 of 150 (24%) patients, 69% (25 of 36) of which were located in the iliac-obturator area and 31% in the groin area only: 16 patients (44.4%) had one additional metastatic node and seven patients (19.4%) had two, whereas 13 (36.1%) had three or more. In 22 cases (61.1%), the sentinel node showed a macrometastasis (>2 mm deposit in the node) and in 14 cases (38.9%) a micrometastasis (

Progress in the field of nanoparticles has enabled the rapid development of multiple products and technologies; however, some nanoparticles can pose both a threat to the environment and human health. To enable their safe implementation, a comprehensive knowledge of nanoparticles and their biological interactions is needed. In vitro and in vivo toxicity tests have been considered the gold standard to evaluate nanoparticle safety, but it is becoming necessary to understand the impact of nanosystems on cell mechanics. Here, the interaction between particles and cells, from the point of view of cell mechanics (i.e., bionanomechanics), is highlighted and put in perspective. Specifically, the ability of intracellular and extracellular nanoparticles to impair cell adhesion, cytoskeletal organization, stiffness, and migration are discussed. Furthermore, the development of cutting-edge, nanotechnology-driven tools based on the use of particles allowing the determination of cell mechanics is emphasized. These include traction force microscopy, colloidal probe atomic force microscopy, optical tweezers, magnetic manipulation, and particle tracking microrheology.

Aims and background Because of its high thickness, nodular melanoma often bears a poor prognosis. Thus, an earlier diagnosis of this type of lesion while it is still thin would be an important step in secondary prevention. The principal aim of the present study was to better define the initial clinical features of nodular melanoma to allow an early diagnosis. A secondary aim was to establish the prognosis of this type of lesion. Methods We retrospectively studied and illustrated the clinical features of 11 small (≤6 mm maximum diameter) cutaneous nodular melanomas seen and treated during a 10-year period. Prognostic characteristics of the various lesions were also described. Results The results of the study help to describe a small nodular melanoma as a dark and/or pink/red raised lesion, which may be evenly or unevenly colored, with well-defined borders, that often appears de novo. A correct clinical diagnosis was made in 7 of the cases. During a median follow-up of 6 years, none of the patients had local or distant relapses. Conclusions Detection of small nodular melanoma is feasible by accurate visual inspection, provided that physicians are aware of this type of lesion and maintain the index of suspicion at a high level to bring about curative surgery. Free full text available at www.tumorionline.it

The purpose of the study was to evaluate the excision margin necessary for desmoplastic melanoma (DM).DM consists of 2 histologic subtypes, pure DM (PDM) and mixed DM (MDM), differing in extent of fibrotic component. We investigated clinical and therapeutic determinants of prognosis in these DM entities.We reviewed 118 PDM and 124 MDM treated at our Institute over 25 years. Local relapse, distant metastasis, and survival were studied.Most (91.7%) distant metastases in PDM developed after 1 or more local recurrences; whereas distant metastasis usually (79.6%) occurred as first event in MDM. Overall mortality trends in relation to lesion-thickness-plus-excision-width differed for PDM (P = 0.014) but not MDM (P = 0.185). For PDM, 5-year crude cumulative incidence (CCI) of mortality was higher (40.0%) for thin tumors (≤ 2 mm thick) excised with 1 cm margin than those excised with 2 cm (14.8%); CCI of mortality for PDM2 mm thick excised with 2 cm margins (13.4%) was similar to that for thin PDM lesions excised with 2 cm (14.8%). CCI of local recurrence was also greater in PDM excised with 1 cm margins. In MDM, mortality increased with stage but was independent of excision width (CCI: 29.4% for ≤ 2 mm/2 cm, 31.3% for ≤ 2 mm/1 cm, and 48.3% for2 mm/2 cm); a similar trend was found for MDM distant metastases.In PDM, limited excision width is associated with significantly greater local recurrence and mortality; treatment should be excision with 2 cm margins even for thin lesions. MDM behaves similarly to other melanomas; treatment should follow guidelines on melanoma management.