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Background: Comprehensive tumor analysis by Next Generation Sequencing (NGS) is imperative for targeted and immunotherapy related biomarkers’ analysis in metastatic breast cancer enabling personalization of cancer treatment in these patients. In parallel with targeted therapies, immunotherapies are also evolving, revolutionizing cancer therapy. Thus, biomarkers such as. Microsatellite Instability (MSI), and Tumor Mutational Burden (TMB) are commonly employed in NGS analysis due to their correlation to Immune Checkpoint inhibitors treatment.Methods: In the present study, tumor molecular profile analysis was performed in 73 metastatic breast cancer patients using a broad NGS assay, which analyzes more than 500 unique cancer related genes and is able to detect relevant single nucleotide variations (SNVs), indels, copy number variations (CNVs) and gene fusions, in addition to TMB and MS) simultaneously. Results: An actionable alteration was detected in 79.45% of the patients. 35.62% of them had at least one alteration with an on label treatment associated and 10.96% received information related to an off-label treatment (Tiers 1A.1 and 2C.1 respectively). Additionally, 31.51% of the tumors harbored an alteration that could be used for clinical trial inclusion (Table 1). The most prevalent altered gene in these patients was the PIK3CA gene, with 27.40% mutation rate. Moreover, in 11 cases (15.07%) the mutation detected was in a gene involved in the Homologous recombination (HR) pathway, with evidence of response to PARP inhibitors. BRCA1/2 genes were as expected the most prominent HR mutated genes (5 out of 11 HR mutations), while the remaining HR mutations involved other HR genes (ATM, BLM, CHEK2, FANCM, RAD50). 24.66% of the tumors had a TMB value >10muts/MB and thus were eligible for ICI treatment. The addition of TMB to targeted biomarkers’ analysis increased the number of patients with an on-label treatment recommendation by 8.22%. Conclusions: Tumor molecular profile analysis using NGS, in real world samples, is a first-tier method for metastatic breast cancer and provides important information for decision making in the treatment of such patients. Importantly, simultaneous analysis for targeted therapy and immunotherapy biomarkers could lead to better tumor characterization and offered actionable information in 9 out of 10 patients tested. However, appropriate interpretation of results from such analysis is essential for implementation in clinical practice and accurate refinement of the treatment strategy.. Table 1.Biomarker's summary in the 73 patients included in the studyPercentage of patients with Tier1 variantsPercentage of patients with 2C.1 variantsPercentage of patients with 2C.2/2D variantsPercentage of patients with Tier 3 variantsPercentage of patients with TMB>10muts/MBMEDIAN TMBPercentage of MSI-High patients35.62% (26/73)10.96% (8/73)12.33% (9/73)0% (0/73)24.66% (18/73)5.050% (0/73) Citation Format: Eirini Papadopoulou, Nikolaos Tsoulos, Vasiliki Metaxa-Mariatou, Aikaterini Tsantikidi, Georgios Kapetsis, Chrysiida Florou-Chatzigiannidou, Sonia Maravelaki, Evgenia Bourkoula, Dimitrios Fotiou, Georgios Tsaousis, Nikolaos Touroutoglou, Dimitrios Trafalis, Ioannis Boukovinas, Ioannis Varthalitis, Zacharenia Saridaki, Charalampos Zoublios, Eleni Galani, George Papatsibas, Christos Papadimitriou, Tania Zlatintsi, Polixenia Iorga, Bülent Orhan, Sualp Tansan, Tahsin Özatlı, George Nasioulas. Comprehensive tumor analysis by NGS in metastatic breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-01.

Background Tumor molecular profile analysis by Next Generation Sequencing technology is currently widely applied in clinical practice and has enabled the detection of predictive biomarkers of response to targeted treatment. In parallel with targeted therapies, immunotherapies are also evolving, revolutionizing cancer therapy, with Programmed Death-ligand 1 (PD-L1), Microsatellite instability (MSI), and Tumor Mutational Burden (TMB) analysis being the biomarkers employed most commonly. Methods In the present study, tumor molecular profile analysis was performed using a 161 gene NGS panel, containing the majority of clinically significant genes for cancer treatment selection. A variety of tumor types have been analyzed, including aggressive and hard to treat cancers such as pancreatic cancer. Besides, the clinical utility of immunotherapy biomarkers (TMB, MSI, PD-L1), was also studied. Results Molecular profile analysis was conducted in 610 cancer patients, while in 393 of them a at least one biomarker for immunotherapy response was requested. An actionable alteration was detected in 77.87% of the patients. 54.75% of them received information related to on-label or off-label treatment (Tiers 1A.1, 1A.2, 2B, and 2C.1) and 21.31% received a variant that could be used for clinical trial inclusion. The addition to immunotherapy biomarker to targeted biomarkers’ analysis in 191 cases increased the number of patients with an on-label treatment recommendation by 22.92%, while an option for on-label or off-label treatment was provided in 71.35% of the cases. Conclusions Tumor molecular profile analysis using NGS is a first-tier method for a variety of tumor types and provides important information for decision making in the treatment of cancer patients. Importantly, simultaneous analysis for targeted therapy and immunotherapy biomarkers could lead to better tumor characterization and offer actionable information in the majority of patients. Furthermore, our data suggest that one in two patients may be eligible for on-label ICI treatment based on biomarker analysis. However, appropriate interpretation of results from such analysis is essential for implementation in clinical practice and accurate refinement of treatment strategy.

Additional file 1: Table S1. Title: Genomic Regions and fusions analyzed with the 24 and 50 gene panels. Additional file 2: Table S2. Title: Gene alterations detected by the 161 gene panel. Description: The frequency of the different mutation types (SNVs, indels, CNV, fusions) for each gene is reported. Additional file 3: Table S3. Title: Alterations identified by the 161 gene panel in the 610 patients analyzed. Description: Genomic alterations and level of evidence of the variants detected in the 610 patients analyzed. The tumor type, age of diagnosis and gender are also reported. Additional file 4: Table S4. Title: Biomarker's summary in the 610 patients included in the study. Description: Patients' categorization based on TIER classification of their most clinically significant variant and immunotherapy biomarkers’ results are reported. Additional file 5: Figure S1. Title and description: Pancreatic cancer patients' categorization based on TIER classification of their most clinically significant variant. A. Pancreatic cancer patients' categorization based on TIER classification of their most clinically significant variant. Patients were categorized in the following categories: No Biomarker: Patients with no biomarker available, 1B: Patients harboring biomarkers with strong evidence of correlation to treatment, 2C.1 KRAS: Patients with a single finding in the KRAS gene, 2C.1: Patients with biomarkers related to off-label treatment. B. Percentage of patients with On-label and off-label mutations identified and the type of alterations detected. Genes of the homologous recombination complex are labeled in blue. Additional file 6: Figure S2. Title and description: Lung cancer patients' categorization based on TIER classification. A. Lung cancer patients' categorization based on TIER classification of their most clinically significant variant. The following categories were used:, 1A.1: Patients with biomarkers related to on-label treatment, 1B: Patients harboring biomarkers with strong evidence of correlation to treatment, 2C.1: Patients with biomarkers related to off-label treatment, 1A.2R; 2C.1: Patients harboring a KRAS mutation related to resistance to treatment plus an off-label, 1A.2R: Patients harboring a KRAS mutation related to EGFR TKIs resistance, 2.C.2: Patients B. % of patients with On-label and off-label mutations identified and the type of alterations detected. Genes of the homologous recombination complex are labeled in blue. Additional file 7: Figure S3. Title and description: Breast cancer patients' categorization based on the TIER classification. A. Patients' categorization based on TIER classification of their most clinically significant variant. The following categories were used: No Biomarker: Patients with no biomarker available, 1A.1: Patients with biomarkers related to on-label treatment, 2C.1: Patients with biomarkers related to off-label treatment, 2C.2: Patients with biomarkers related to clinical trials, 2D: Patients with biomarkers with preclinical evidence. B. Percentage of patients with On-label and off-label mutations identified and the type of alterations detected. Genes of the homologous recombination complex are labeled in blue. Additional file 8: Figure S4. Title and description: Colorectal cancer patients' categorization based on TIER classification. A. Patients' categorization based on TIER classification of their most clinically significant variant. Patients were categorized in the following categories: No Mutation, 1A.1: Patients with no mutation in KRAS/NRAS genes with or without other variations, 1A.2: Patients with biomarkers included in professional guidelines, 1A.1R: Patients harboring either KRAS or NRAS mutations related to resistance to treatment. B. % of patients with on-label and off-label mutations identified and the type of alterations detected. Additional file 9: Table S5. Title and description: Alterations that would have been detected if two hotspot panels of 24 and 50 genes respectively, had been used in the 610 patients analyzed. Additional file 10: Table S6. Title and description: Simulation results of the alterations that would have been identified if the gene set of the 161 gene NGS panel was used in the PCAWG samples. Additional file 11: Table S7. Title and description: Simulation results of the alterations that would have been identified if the gene set of the 514 gene NGS panel was used in the PCAWG samples. Additional file 12: Table S8. Title and Description: TMB, PD-L1 and MSI results in the 395 patients with at least one immunotherapy biomarker requested. Additional file 13: Table S9. Title and Description: Patients with suspicious hereditary pathogenic findings detected in tumor tissue.

Background: Tumor molecular profile is of great importance for the detection of biomarkers of response to targeted treatment due to the increased availability, with concomitant reduction of cost, of Next Generation Sequencing technology (NGS). In parallel to targeted therapies’, immunotherapies are also evolving, revolutionizing cancer therapy, with Programmed Death-ligand 1 (PD-L1), Microsatellite Instability (MSI), and Tumor Mutational Burden (TMB) analysis being the biomarkers employed most commonly.Methods: In the present study, a 161 gene NGS panel, containing the majority of clinically significant genes for cancer treatment selection, was used for tumor molecular profile analysis. A variety of tumor types have been analyzed, including aggressive and hard to treat cancers such as pancreatic cancer. Besides, the clinical utility of immunotherapy biomarkers (TMB, MSI, PD-L1), was also studied.Results: Molecular profile analysis was conducted in 610 cancer patients, while in 393 of them a at least one biomarker for immunotherapy response was requested. At least one actionable alteration was detected in 77.70% of the patients. 54.59% of them received information related to on-label or off-label treatment (Tiers 1A.1, 1A.2, 2B, and 2C.1) and 21.31% received a variant that could be used for clinical trial inclusion. The addition to immunotherapy biomarker to targeted biomarkers’ analysis in 191 cases increased the number of patients with an on-label treatment recommendation by 22.40%, while an option for on-label or off-label treatment was provided in 71.35% of the cases. Conclusions: Tumor molecular profile analysis by NGS is a first-tier methodology for a variety of tumor types, which provides critical information for treatment decision making in cancer patients. Importantly, simultaneous analysis for targeted therapy and immunotherapy biomarkers could lead to a better tumor characterization and provide actionable information in the majority of patients. Moreover, our results indicate that one in two patients is eligible for ICI treatment based on the biomarkers’ analysis. However, when these analyses are performed, the challenge is their implementation in clinical practice. Multidisciplinary patient management is critical to the refinement of the strategy incorporating such information.

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

Gastrointestinal malignancies are among the leading causes of cancer-related deaths worldwide. Like all human malignancies they are characterized by accumulation of mutations which lead to inactivation of tumor suppressor genes or activation of oncogenes. Advances in Molecular Biology techniques have allowed for more accurate analysis of tumors' genetic profiling using new breakthrough technologies such as next generation sequencing (NGS), leading to the development of targeted therapeutical approaches based upon biomarker-selection. During the last 10 years tremendous advances in the development of targeted therapies for patients with advanced cancer have been made, thus various targeted agents, associated with predictive biomarkers, have been developed or are in development for the treatment of patients with gastrointestinal cancer patients. This review summarizes the advances in the field of molecular biomarkers in tumors of the gastrointestinal tract, with focus on the available NGS platforms that enable comprehensive tumor molecular profile analysis.

Background While recent genome-wide association studies have suggested novel low-grade glioma (LGG) stratification models based on a molecular classification, we explored the potential clinical utility of patient-derived cells. Specifically, we assayed glioma-associated stem cells (GASC) that are patient-derived and representative of the glioma microenvironment. Methods By next-generation sequencing, we analyzed the transcriptional profile of GASC derived from patients who underwent anaplastic transformation either within 48 months (GASC-BAD) or ≥7 years (GASC-GOOD) after surgery. Gene set enrichment and pathway enrichment analyses were applied. The prognostic role of a nuclear factor-kappaB (NF-κB) signature derived from GASC-BAD was tested in 530 newly diagnosed diffuse LGG patients comprised within The Cancer Genome Atlas (TCGA) database. The prognostic value of the GASC upstream regulator p65 NF-κB was assessed, by univariate and multivariate Cox analyses, in a single center case study, including 146 grade II LGGs. Results The key elements differentiating the transcriptome of GASC isolated from LGG with different prognoses were mostly related to hallmarks of cancer (eg, inflammatory/immune process, NF-κB activation). Consistently, the NF-κB signature extrapolated from the GASC study was prognostic in the dataset of TCGA. Finally, the nuclear expression of the NF-kB-p65 protein, assessed using an inexpensive immunohistochemical method, was an independent predictor of both overall survival and malignant progression-free survival in 146 grade II LGGs. Conclusion This study demonstrates for the first time the independent prognostic role of NF-kB activation in LGG and outlines the role of patient-based stem cell models as a tool for precision medicine approaches.

Plasma etching has evolved in an important technology for rapid and cost-effective generation of random or quasi-ordered nanostructures in large areas and in a repeatable manner, if properly controlled. It simultaneously affects the chemical composition of etched surfaces. Thus, plasma etching finds numerous applications in the areas of biomaterials and biomicrosystems, since surface chemistry and topography are proven to influence strongly cell–substrate interactions. Herein, we briefly review published studies addressing cell–surface interactions, especially those focusing on optimal surface properties favoring cell adhesion and proliferation. We show that plasma-based micro- and nano-texturing of polymeric surfaces provides a unique, simple and yet versatile tool for tuning the physicochemical properties of polymeric surfaces to those favoring cell cultures. Plasma etching and nanotexturing is proven indispensable also for the patterning on the same substrate of different chemical and/or topographical areas to induce preferential cell adhesion in predefined areas. In this respect, the implementation of surfaces with extreme wettabilities (superhydrophobic/superhydrophilic patterns) is highly valued and when integrated inside microchannels can add new potential to the current archery of microanalytical devices. The paper concludes with the authors’ view to the future outlook of the niche area of plasma nanotextured polymer surfaces.

Non‑small cell lung cancer (NSCLC) is the most common type of lung cancer and a tumor with a broad spectrum of targeted therapies already available or in clinical trials. Thus, molecular characterization of the tumor using next generation sequencing (NGS) technology, has become a key tool for facilitating treatment decisions and the clinical management of NSCLC patients. The performance of a custom 23 gene multiplex amplification hot spot panel, based on Ion AmpliSeq™ technology, was evaluated for the analysis of tumor DNA extracted from formalin-fixed and paraffin-embedded (FFPE) tissues. Furthermore, the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was used for fusion RNA transcript analysis. The mutation spectrum of the tumors was determined in a cohort of 502 patients with NSCLC using the aforementioned targeted gene panels. The panel used for tumor DNA analysis in this study exhibited high rates (100%) of sensitivity, specificity and reproducibility at a mutation allelic frequency of 3%. At least one DNA mutation was detected in 374 patients (74.5%) and an RNA fusion was identified in 16 patients, (3.2%). In total, alterations in a cancer-driver gene were identified (including point mutations, gene rearrangements and MET amplifications) in 77.6% of the tumors tested. Among the NSCLC patients, 23% presented a mutation in a gene associated with approved or emerging targeted therapy. More specifically, 13.5% (68/502) presented a mutation in a gene with approved targeted therapy (EGFR, ALK, ROS1) and 9.4% (47/502) had an alteration in a gene related to emerging targeted therapies (ERBB2, BRAF, MET and RET). Furthermore, 51.6% of the patients had a mutation in a gene that could be related to an off label therapy or indicative for access to a clinical trial. Thus, the targeted NGS panel used in this study is a reliable approach for tumor molecular profiling and can be applied in personalized treatment decision making for NSCLC patients.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and a tumor with a broad spectrum of targeted therapies already available or in clinical trials. Thus, molecular characterization of the tumor using next generation sequencing (NGS) technology, has become a key tool for facilitating treatment decisions and the clinical management of NSCLC patients. The performance of a custom 23 gene multiplex amplification hot spot panel, based on Ion AmpliSeq™ technology, was evaluated for the analysis of tumor DNA extracted from formalin-fixed and paraffinembedded (FFPE) tissues. Furthermore, the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was used for fusion RNA transcript analysis. The mutation spectrum of the tumors was determined in a cohort of 502 patients with NSCLC using the aforementioned targeted gene panels. The panel used for tumor DNA analysis in this study exhibited high rates (100%) of sensitivity, specificity and reproducibility at a mutation allelic frequency of 3%. At least one DNA mutation was detected in 374 patients (74.5%) and an RNA fusion was identified in 16 patients, (3.2%). In total, alterations in a cancer-driver gene were identified (including point mutations, gene rearrangements and MET amplifications) in 77.6% of the tumors tested. Among the NSCLC patients, 23% presented a mutation in a gene associated with approved or emerging targeted therapy. More specifically, 13.5% (68/502) presented a mutation in a gene with approved targeted therapy (EGFR, ALK, ROS1) and 9.4% (47/502) had an alteration in a gene related to emerging targeted therapies (ERBB2, BRAF, MET and RET). Furthermore, 51.6% of the patients had a mutation in a gene that could be related to an off label therapy or indicative for access to a clinical trial. Thus, the targeted NGS panel used in this study is a reliable approach for tumor molecular profiling and can be applied in personalized treatment decision making for NSCLC patients.

Display Omitted Lithographic processes for strippable photoresists directly on polymeric substrates.Plasma nanotexturing to etch, roughen, and chemically modify polymeric substrates and microfluidics.Fabrication of microchannel surfaces with patterns ranging from superhydrophilic to superhydrophobic.Enhanced cell attachment on nanotextured hydrophilic surfaces.On-off cell patterning by highly selective cell adhesion on the hydrophilic versus the superhydrophobic areas. We propose a planar technology for fabrication and surface modification of disposable, polymeric, microfluidic devices, and show applications in cell patterning. By planar technology we mean lithography directly on a polymeric plate followed by plasma etching of the plate. In this context, we developed lithographic processes directly on the polymeric substrate, employing easily strippable photoresists, for which stripping is performed without attacking the polymeric substrate. We then applied oxygen plasma etching to transfer the pattern and chemically modify the polymeric substrates, followed by optional fluorocarbon plasma deposition through stencil masks, producing microfluidic channels with desired geometrical and wetting characteristics. We tested various organic photoresists such as AZ 15nXT, AZ 9260, maP-1275, a silicon containing resist Ormocomp? on an organic strippable underlayer (LOR?), as well as metal masks in order achieve high resolution (1-4µm), plasma etch resistance, and stripability for photoresists with thickness of ~5-20µm (for details see Supporting information). We selected the Ormocomp? stack as the best candidate to define microfluidics with plasma etching onto PMMA, PEEK and COP substrates containing hydrophilic and superhydrophobic areas. We demonstrate significantly increased cell attachment on the plasma treated PMMA areas compared to untreated ones, and highly selective cell attachment (on-off) onto hydrophilic versus the superhydrophobic areas using a particular cell line. Such control of cell attachment and growth on plasma nanotextured surfaces can be applied to creation of microdevices aiming to cell patterning, cell isolation, as well as cell arrays.

The role of Wilms' tumor suppressor 1 (WT1) in leukemogenesis has been investigated mostly in acute (AML) and chronic (CML) myeloid leukemias. So far, its oncogenic role has been controversially discussed because both overexpression and inactivating mutations are found. A recent study on primary samples from patients with acute T-cell leukemia (T-ALL) revealed that most of them do not express WT1 proteins although they express WT1 mRNA. In our study, we investigated WT-1 expression in ten T-ALL cell lines established from leukemia/lymphoma patients. We show that consistent with the finding in primary T-ALL cells, most of the leukemic T-cell lines tested do not overexpress WT1 proteins. We found that leukemic T-cells overexpressing WT1 protein produce higher levels of CD95L and show elevated CD95L-mediated activation-induced cell death (AICD) compared to cells lacking or expressing low levels of WT1. Ectopic expression of WT1 in the WT1-nonexpressing leukemic T-cell line increases CD95L expression and elevates activation-induced apoptosis, whereas silencing WT1 expression in the WT1-overexpressing leukemic T-cell line by siRNA confers reduced CD95L expression and reduction in AICD. Chromatin immunoprecipitation and luciferase-promoter reporter analysis demonstrate that WT1 binds to and enhances CD95L promoter activity through the Egr-binding sites. Our study provides a new role of WT1 in regulation of CD95L-mediated cell death.

Background Glioblastoma stem cells (GSC) have been extensively recognized as a plausible cause of glioblastoma resistance to therapy and recurrence resulting in high glioblastoma mortality. Abnormalities in the DNA repair pathways might be responsible for the inability of the currently used chemotherapeutics to eliminate the (GSC) subpopulation. Methods In this work, we compared the expression of sixty DNA repair related genes between primary glioblastoma cell cultures and the glioblastoma enriched stem cell primary cultures. MTT test was used to analyze the effect of selected drugs and immunofluorescence to evaluate the load of DNA damage. Results We found several differentially expressed genes and we identified topoisomerase IIβ (Top2β) as the gene with highest up-regulation in GSC. Also among the tested cell lines the expression of Top2β was the highest in NCH421k cells, a well-characterized glioblastoma cell line with all the stemness characteristics. On the other hand, Top2β expression markedly decreased upon the induction of differentiation by all trans-retinoic acid. Depletion of Top2β increased the sensitivity of NCH421k cells to replication stress inducing drugs, such as cisplatin, methyl-methanesulfonate, hydrogen peroxide, and temozolomide. Consistently, we found an increased load of DNA damage and increased Chk1 activation upon Top2β depletion in NCH421k cells. Conclusion We suggest that Top2β may represent a new target for gene therapy in glioblastoma. In addition, the other genes that we found to be up-regulated in GSC versus glioblastoma primary cells should be further investigated as glioblastoma theranostics. Electronic supplementary material The online version of this article (doi:10.1186/s12935-016-0339-9) contains supplementary material, which is available to authorized users.

Additional file 1: Table S1. Full list of genes on the PCR array.

Background: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-the-art markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. Methods and Findings: We isolated glioma-associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASC-based score was the only independent predictor of overall survival and malignant progression free-survival. Conclusions: The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma-initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma. Stem Cells 2014;32:1239–1253

In this work, silicon substrates with poly(vinyl alcohol) (PVA) patterns created by a simple, low-cost and high-fidelity photolithographic procedure were evaluated with respect to cell adhesion and alignment, viability, metabolic activity, proliferation and cell cycle progression using the human glioblastoma cell-line U87MG and human skin fibroblasts. In addition, rat adrenal pheochromocytoma cells (PC-12) were employed to evaluate a modified photolithographic protocol appropriate for adhesion of cells requiring extracellular matrix components to adhere on the surface and to demonstrate that the proposed patterned substrates could provide unhindered cell differentiation. Regarding U87MG cells and skin fibroblasts, it was found that as the stripes width increased from 10 to 50 μm, the percentage of cells attached to Si versus the total area (Si + PVA) increased from 78% and 72% to 98.5% and 94.5% (p 0.05), for U87MG cells and skin fibroblasts, respectively, with optimum cell alignment (≥95% of adherent cells with fidelity between 0.90 and 1.0; p 0.05) for stripes width ranging between 20 and 22.5 μm. Concerning the viability, metabolic activity and proliferation of adherent cells, no statistically significant differences were observed compared to cells cultured onto non-patterned surfaces. Regarding PC-12 cells, a modification of the patterning procedure was followed involving coating of the substrate with type IV collagen prior to the photolithographic procedure, since they could not adhere on plain Si substrates. It was found that PC-12 cells adhere selectively (95%) to collagen-coated Si stripes when the pattern width was equal to or wider than 10 μm. Following treatment with nerve growth factor, approximately 80% (p 0.05) of the adherent cells differentiated to neuron-like cells extending neurites exclusively within the pattern. Given that the proposed patterning procedure allows highly selective cell adhesion without affecting cell proliferation, metabolic activity, and differentiation it could serve as a useful tool in various fields including tissue engineering, cell-based sensors and analytical microsystems.

Introduction Autologous fat grafting is commonly used to correct soft-tissue contour deformities. However, results are impaired by a variable and unpredictable resorption rate. Autologous adipose-derived stromal cells in combination with lipoinjection (cell-assisted lipotransfer) seem to favor a long-term persistence of fat grafts, thus fostering the development of devices to be used in the operating room at the point of care, to isolate the stromal vascular fraction (SVF) and produce SVF-enhanced fat grafts with safe and standardized protocols. Focusing on patients undergoing breast reconstruction by lipostructure, we analyzed a standard technique, a modification of the Coleman’s procedure, and three different commercially available devices (Lipokit, Cytori, Fastem), in terms of 1) ability to enrich fat grafts in stem cells and 2) clinical outcome at 6 and 12 months. Methods To evaluate the ability to enrich stem cells, we compared, for each patient (n = 20), the standard lipoaspirate with the respective stem cell-enriched one, analyzing yield, immunophenotype and colony-forming capacity of the SVF cells as well as immunophenotype, clonogenicity and multipotency of the obtained adipose stem cells (ASCs). Regarding the clinical outcome, we compared, by ultrasonography imaging, changes at 6 and 12 months in the subcutaneous thickness of patients treated with stem-cell enriched (n = 14) and standard lipoaspirates (n = 16). Results Both methods relying on the enzymatic isolation of primitive cells led to significant increase in the frequency, in the fat grafts, of SVF cells as well as of clonogenic and multipotent ASCs, while the enrichment was less prominent for the device based on the mechanical isolation of the SVF. From a clinical point of view, patients treated with SVF-enhanced fat grafts demonstrated, at six months, a significant superior gain of thickness of both the central and superior-medial quadrants with respect to patients treated with standard lipotransfer. In the median-median quadrant the effect was still persistent at 12 months, confirming an advantage of lipotransfer technique in enriching improving long-term fat grafts. Conclusions This comparative study, based on reproducible biological and clinical parameters and endpoints, showed an advantage of lipotransfer technique in enriching fat grafts in stem cells and in favoring, clinically, long-term fat grafts. Electronic supplementary material The online version of this article (doi:10.1186/scrt536) contains supplementary material, which is available to authorized users.

© 2015, Spyropoulos-Antonakakis et al.; licensee Springer. Photodynamic therapy (PDT) involves the action of photons on photosensitive molecules, where atomic oxygen or OH− molecular species are locally released on pathogenic human cells, which are mainly carcinogenic, thus causing cell necrosis. The efficacy of PDT depends on the local nanothermodynamic conditions near the cell/nanodrug system that control both the level of intracellular translocation of nanoparticles in the pathogenic cell and their agglomeration on the cell membrane. Dendrimers are considered one of the most effective and promising drug carriers because of their relatively low toxicity and negligible activation of complementary reactions. Polyamidoamine (PAMAM) dendrite delivery of PDT agents has been investigated in the last few years for tumour selectivity, retention, pharmacokinetics and water solubility. Nevertheless, their use as drug carriers of photosensitizing molecules in PDT for cardiovascular disease, targeting the selective necrosis of macrophage cells responsible for atheromatous plaque growth, has never been investigated. Furthermore, the level of aggregation, translocation and nanodrug delivery efficacy of PAMAM dendrimers or PAMAM/zinc phthalocyanine (ZnPc) conjugates on human atheromatous tissue and endothelial cells is still unknown. In this work, the aggregation of PAMAM zero generation dendrimers (G0) acting as drug delivery carriers, as well as conjugated G0 PAMAM dendrimers with a ZnPc photosensitizer, to symptomatic and asymptomatic human carotid tissues was investigated by using atomic force microscopy (AFM). For the evaluation of the texture characteristics of the AFM images, statistical surface morphological and fractal analytical methodologies and Minkowski functionals were used. All statistical quantities showed that the deposition of nanodrug carriers on healthy tissue has an inverse impact when comparing to the deposition on atheromatous tissue with different aggregation features between G0 and G0/ZnPc nanoparticles and with considerably larger G0/ZnPc aggregations on the atheromatous plaque. The results highlight the importance of using PAMAM dendrimer carriers as a novel and promising PDT platform for atherosclerosis therapies.

© Springer International Publishing Switzerland 2015. The efficiency of penetration of nanodrugs through cell membranes imposes further complexity due to nanothermodynamic and entropic potentials at interfaces. Action of nanodrugs is effective after cell membrane penetration. Contrary to diffusion of water diluted common molecular drugs, nanosize imposes an increasing transport complexity at boundaries and interfaces (e.g., cell membrane). Indeed, tiny dimensional systems brought the concept of “nanothermodynamic potential,” which is proportional to the number of nanoentities in a macroscopic system, from either the presence of surface and edge effects at the boundaries of nanoentities or the restriction of the translational and rotational degrees of freedom of molecules within them. The core element of nanothermodynamic theory is based on the assumption that the contribution of a nanosize ensemble to the free energy of a macroscopic system has its origin at the excess interaction energy between the nanostructured entities. As the size of a system is increasing, the contribution of the nanothermodynamic potential to the free energy of the system becomes negligible. Furthermore, concentration gradients at boundaries, morphological distribution of nanoentities, and restriction of the translational motion from trapping sites are the source of strong entropic potentials at the interfaces. It is evident therefore that nanothermodynamic and entropic potentials either prevent or allow enhanced concentration very close to interfaces and thus strongly modulate nanoparticle penetration within the intracellular region. In this work, it is shown that nano-sized polynuclear iron (III)-hydroxide in sucrose nanoparticles have a nonuniform concentration around the cell membrane of macrophages in vivo, compared to uniform concentration at hydrophobic prototype surfaces. The difference is attributed to the presence of entropic and nanothermodynamic potentials at interfaces.

e23193 Background: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and a tumor with broad spectrum of targeted therapies already available or in clinical trials. Thus, molecular characterization of the tumor using the Next Generation Sequencing (NGS) technology, has become a key tool for NSCLC patients’ treatment decision and clinical management. Methods: The performance of a custom 23 gene multiplex amplification hot spot panel, based on Ion AmpliSeq Technology, was evaluated for the analysis of tumor DNA extracted from FFPE (Formalin Fixed Parraffin Embedded) tissue. Furthermore the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was used for fusion RNA transcript analysis. Tumors’ mutation spectrum was determined in a cohort of 502 patients with NSCLC using the aforementioned targeted gene panels. Results: The panel used for tumor DNA analysis in this study exhibit high rates (100%) of sensitivity, specificity and reproducibility at a mutation frequency of 3%. At least one DNA mutation was detected in 374 patients (74.5%) and an RNA fusion was identified in 16 patients, (3.2%). In total, alterations in a cancer driver gene were identified (including point mutations, gene rearrangements and MET amplifications) in 77.6% of the tumors tested. Among the NSCLC patients, 13.5% (68/502) presented a mutation in a gene with approved targeted therapy (EGFR, ALK ROS1) and 9.4% had an alteration in a gene related to emerging targeted therapies according the NCCN guidelines. These alterations include ERBB2, BRAF and MET mutations, MET amplification and RET rearrangements. The remaining 51.6% of the patients had a mutation in a gene that could be related to an off label therapy or give them access to a clinical trial. Conclusions: Thus the NGS panel validated is a reliable approach of clinical applicability for tumor molecular profile detection in NSCLC patients.

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.

The efficiency of penetration of nanodrugs through cell membranes imposes further complexity due to nanothermodynamic and entropic potentials at interfaces. Action of nanodrugs is effective after cell membrane penetration. Contrary to diffusion of water diluted common molecular drugs, nanosize imposes an increasing transport complexity at boundaries and interfaces (e.g., cell membrane). Indeed, tiny dimensional systems brought the concept of “nanothermodynamic potential,” which is proportional to the number of nanoentities in a macroscopic system, from either the presence of surface and edge effects at the boundaries of nanoentities or the restriction of the translational and rotational degrees of freedom of molecules within them. The core element of nanothermodynamic theory is based on the assumption that the contribution of a nanosize ensemble to the free energy of a macroscopic system has its origin at the excess interaction energy between the nanostructured entities. As the size of a system is increasing, the contribution of the nanothermodynamic potential to the free energy of the system becomes negligible. Furthermore, concentration gradients at boundaries, morphological distribution of nanoentities, and restriction of the translational motion from trapping sites are the source of strong entropic potentials at the interfaces. It is evident therefore that nanothermodynamic and entropic potentials either prevent or allow enhanced concentration very close to interfaces and thus strongly modulate nanoparticle penetration within the intracellular region. In this work, it is shown that nano-sized polynuclear iron (III)-hydroxide in sucrose nanoparticles have a nonuniform concentration around the cell membrane of macrophages in vivo, compared to uniform concentration at hydrophobic prototype surfaces. The difference is attributed to the presence of entropic and nanothermodynamic potentials at interfaces.