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Supplementary Figure 2 - Patient characteristics in 6 cohorts analysed

Supplementary Figure 3 - Clinical characteristics of EphA2high and EphA2low patients in cohort 1 to 6

Purpose: EphA2 receptor is involved in multiple cross-talks with other cellular networks, including EGFR, FAK, and VEGF pathways, with which it collaborates to stimulate cell migration, invasion, and metastasis. Colorectal cancer (CRC) EphA2 overexpression has also been correlated to stem-like properties of cells and tumor malignancy. We investigated the molecular cross-talk and miRNAs modulation of the EphA2 and EGFR pathways. We also explored the role of EphA2/EGFR pathway mediators as prognostic factors or predictors of cetuximab benefit in patients with CRC.Experimental Design: Gene expression analysis was performed in EphA2high cells isolated from CRC of the AOM/DSS murine model by FACS-assisted procedures. Six independent cohorts of patients were stratified by EphA2 expression to determine the potential prognostic role of a EphA2/EGFR signature and its effect on cetuximab treatment response.Results: We identified a gene expression pattern (EphA2, Efna1, Egfr, Ptpn12, and Atf2) reflecting the activation of EphA2 and EGFR pathways and a coherent dysregulation of mir-26b and mir-200a. Such a pattern showed prognostic significance in patients with stage I–III CRC, in both univariate and multivariate analysis. In patients with stage IV and WT KRAS, EphA2/Efna1/Egfr gene expression status was significantly associated with poor response to cetuximab treatment. Furthermore, EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutation status.Conclusions: These results suggest that EphA2/Efna1/Egfr genes, linked to a possible control by miR-200a and miR-26b, could be proposed as novel CRC prognostic biomarkers. Moreover, EphA2 could be linked to a mechanism of resistance to cetuximab alternative to KRAS mutations. Clin Cancer Res; 23(1); 159–70. ©2016 AACR.

Supplementary Table 3 - Univariate and multivariate analyses of factors affecting PFS in patients who received Cetuximab monotherapy belonging to EphA2high group (cohort 5, N=40).

Kaplan-Meier survival curves of EGFRhigh (dashed line) versus EGFRlow (solid line) for cohort 1, 2 and 3. P-values were calculated using log-rank tests. Tick marks represent censored data. Expression value thresholds for determining high and low groups were found through maxstat R package. For the analysis conducted only for patients belonging to EphA2high group (below figures) Hazard Ratio and 95% Confidence Intervals are reported below each cohort analysed.

Supplementary Table 2A. KRAS mutational status vs. EphA2 expression (N = 70; P = 0.133{section sign}); 2B. Response rate vs. EphA2 expression (N = 68; P = 0.33{section sign}); 2C. Disease control rate vs. EphA2 expression (N = 68; P = 0.012{section sign}); 2D. Response rate vs. EphA2 expression (In KRAS WT patients; N = 39; P = 0.574{section sign}); 2E. Disease control rate vs. EphA2 expression (In KRAS WT patients; N = 39; P = 0.008{section sign})

Supplementary Table 1 - Univariate and multivariate analyses of factors affecting DFS of patients with EphA2high from pooled cohorts 1, 2, and 3 (N = 455).

Kaplan-Meier survival curves of (A) Ptpn12 (high versus low for EphA2high group) (B) Atf2 (high versus low for EphA2high group) and (C) Pik3CG (high versus low for EphA2high group) for cohorts in which results were significant. P-values were calculated using log-rank tests. Expression value thresholds for determining high and low groups were determined through maxstat R package. Tick marks represent censored data.

Supplementary Figure 1 - TaqMan assays ID (Applied Biosystems)

Aim: Cutaneous squamous cell carcinoma (cSCC) is a common disease in patients exposed to UV-light and human papillomavirus. Electrochemotherapy, a well-established treatment modality with minimum side effects in human and veterinary medicine, circumvents chemoresistance to bleomycin by the use of electric fields. However, patients are sensitive to the trauma produced by the insertion of the needles that lengthen recovery times, particularly cats with nasal planum cSCC. To address this matter, we developed thin-needles electrodes. Methods: Thin-needles electrodes developed using computer simulations and plant tissue models were compared to standard electrodes. A prospective non-randomized study recruiting 52 feline patients with nasal planum cSCC was performed. Local response, anorexia, and overall survival were evaluated. Results: Computer simulations and plant model experiments showed satisfactory results with both electrodes. The patients treated with the thin-needle electrode obtained similar local response rates compared to the standard group, OR 97.3% vs. 80%, respectively (P < 0.067). Most patients in the thin-needle group resumed eating in less than 48 h, as the anorexia was significantly lower (P < 0.0001). Using the standard electrode, most patients took 3 to 5 days to resume normal feeding. The electric current circulating in the standard electrode was 44% higher, contributing to a longer duration of anorexia due to tissue damage. The overall survival in both groups was similar. Conclusion: Electrochemotherapy using thin-needle electrodes provides equivalent local response rates and overall survival compared with standard electrodes but significantly reduced return to appetite after the treatment. These results may be useful in the development of new electrodes for human patients.

Purpose: Although early detection and high-quality treatment options, metastatic colorectal cancer (mCRC) is the main cause of CRC-related mortality. Dissecting the mechanisms underlying CRC progression may identify new accurate markers for invasive tumors, which can also be used as druggable targets, providing further improvement in the patients clinical outcomes. YKL-40 is a glycosyl hydrolase functionally linked to increased proliferation, angiogenesis, migration, and invasion of tumor cells. Because in colorectal cancer (CRC), YKL-40 serological level may serve as a risk predictor and prognostic biomarker, we investigated the underlying mechanisms by which it may contribute to tumor progression and the clinical significance of its tissue expression in metastatic CRC. Experimental Design: HCT116 and Caco2 cells genetically engineered to achieve YKL-40 silencing/overexpression were used for cell motility, invasion and proliferation assays, and to measure EMT markers. The YKL-40 expression level was assessed in the early and late tumor phases obtained in the AOM/DSS mouse model, as well as in tumors and sera from CRC patients. Six independent cohorts of CRC patients were stratified by YKL-40 tissue expression to define its prognostic role and its effect on cetuximab and oxaliplatin treatment response. Results: YKL-40 high-expressing HCT116 and Caco2 cells showed increased motility, invasion and proliferation. YKL-40 up-regulation was associated with EMT signaling activation. In murine and human samples, elevated YKL-40 levels correlated with high-grade tumors. In retrospective analyses, YKL-40 elevated expression correlated with shorter survival in patients with advanced CRC. Strikingly, YKL-40 high tissue levels showed a predictive value for better response to cetuximab, even in patients with stage IV CRC and mutant KRAS, and worse sensitivity to oxaliplatin. Conclusions: Our study recognizes a novel role of YKL-40 tissue expression in promoting CRC metastatic potential, through EMT signaling activation, and provides significant clinical implications that may impact the risk prediction of patients with mCRC. YKL-40 high tissue levels also strengthen a predictive value for better cetuximab responsiveness, even in patients with KRAS mutations. Since resistance to cetuximab remains one of the greatest challenges in treating CRC, our findings may be crucial for developing novel YKL-40-targeted therapy approaches. Citation Format: Mariangela De Robertis, Maria Raffaella Greco, Rosa Angela Cardone, Tommaso Mazza, Flaviana Marzano, Nikolay Mehterov, Maria Kazakova, Nikolay Belev, Apollonia Tullo, Graziano Pesole, Victoria Sarafian, Emanuela Signori. YKL-40 overexpression enhances metastatic potential and is a novel prognostic candidate and predictive biomarker for patients with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2465.

Oral malignant melanoma is the most common, but aggressive oral cancer in dogs with poor prognosis. Electrochemotherapy (ECT) has therapeutic potential in such tumors as effective local treatment. Therefore, the aim of this prospective clinical study was to evaluate treatment effectiveness of ECT in as first line treatment for canine oral malignant melanoma, and search for factors influencing treatment outcome. Sixty-seven canines with primary oral malignant melanoma, non-candidates for first-line therapy, were enrolled. All dogs received ECT and follow-up exams for the span of two years. Based on RECIST criteria, the objective response rate was 100%, 89.5%, 57.7%, and 36.4%, in stage I, II, III and IV, respectively. Only patients in stage I, II and III with partial or complete response improved their quality of life. The median time to progression was 11, 7, 4 and 4 months, and median survival time after the treatment was 16.5, 9.0, 7.5 and 4.5 months, for patients in stage I, II, III and IV, respectively. Significantly better was local response in stage I and II disease (p = 0.0013), without the bone involvement (p = 0.043) Electrochemotherapy is effective local treatment of oral canine malignant melanoma when no alternative treatment is available. Better response is expected in stage I and II patients with tumors without bone involvement. Fil: Tellado, Matías Nicolás. No especifíca; Fil: Maglietti, Felipe Horacio. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Michinski, Sebastián Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentina Fil: Marshall, Guillermo Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentina Fil: Signori, Emanuela. Centre National de la Recherche Scientifique; Francia

IL-6 detection is highly desirable since can monitor many diseases in humans and assess the response to treatments. Herein, two novel label-free voltammetric immunosensors for rapid and accurate interleukin-6 (IL-6) detection in human serum are presented. The immunosensors are fabricated by immobilising two different IL-6 antibodies, identified as mAb-IL-6 clone-5 and clone-7, on in-house produced screen-printed electrodes modified with inexpensive recycling biochar (Bio-SPEs). To ensure high structural fidelity and performance, an in-depth electrochemical characterization of the layer-by-layer assembly of the immunosensor was conducted by cyclic voltammetry (CV) and sensing was performed using square wave voltammetry (SWV). The two immunosensors showed good analytical performances in human serum, exhibiting a wide linear range (LR) between 26-125 and 30-138 pg/mL, a good limit of detection (LOD) of 4.8 and 5.4 pg/mL and selectivity for IL-6 over other common cytokines, including IL-1β and TNF-α. Performance comparison of IL-6 immunosensors with those of a commercial spectrophotometric ELISA kit (LOD of 20 pg/mL, RSD% of 15%) denotes a better sensitivity and reproducibility of the proposed label-free devices, associated with a reduced detection time (30 min instead of more than 3 h for ELISA test). Furthermore, the proposed immunosensors were successfully applied in blood samples (with only a dilution of 1:100 v/v in PBS and without additional treatments) with good sensitivity (LOD of 14.3 pg/mL) and reproducibility (RSD% 11%), thus paving the way for their application as viable diagnostic and therapeutic point-of-care tools alternative to the IL-6 detection techniques routinely used (ELISA and Western Blot).

Reversible electropermeabilization (RE) is an ultrastructural phenomenon that transiently increases the permeability of the cell membrane upon application of electrical pulses. The technique was described in 1972 by Neumann and Rosenheck and is currently used in a variety of applications, from medicine to food processing. In oncology, RE is applied for the intracellular transport of chemotherapeutic drugs as well as the delivery of genetic material in gene therapies and vaccinations. This review summarizes the physical changes of the membrane, the particularities of bleomycin, and the immunological aspects involved in electrochemotherapy and gene electrotransfer, two important EP-based cancer therapies in human and veterinary oncology.

Gene therapy has become an important approach for treating cancer, and electroporation represents a technology for introducing therapeutic genes into a cell. An example of cancer gene therapy relying on gene electrotransfer is the use of immunomodulatory cytokines, such as interleukin 2 (IL-2) and 12 (IL-12), which directly stimulate immune cells at the tumour site. The aim of our study was to determine the effects of gene electrotransfer with two plasmids encoding IL-2 and IL-12 in vitro and in vivo. Two different pulse protocols, known as EP1 (600 V/cm, 5 ms, 1 Hz, 8 pulses) and EP2 (1300 V/cm, 100 %s, 1 Hz, 8 pulses), were assessed in vitro for application in subsequent in vivo experiments. In the in vivo experiment, gene electrotransfer of pIL-2 and pIL-12 using the EP1 protocol was performed in B16.F10 murine melanoma. Combined treatment of tumours using pIL2 and pIL12 induced significant tumour growth delay and 71% complete tumour regression. Furthermore, in tumours coexpressing IL-2 and IL-12, increased accumulation of dendritic cells and M1 macrophages was obtained along with the activation of proinflammatory signals, resulting in CD4 + and CD8 + T-lymphocyte recruitment and immune memory development in the mice. In conclusion, we demonstrated high antitumour efficacy of combined IL-2 and IL-12 gene electrotransfer protocols in low-immunogenicity murine B16.F10 melanoma. Bibliografija na koncu prispevka. Abstract.

Screen-printed electrodes are the most recent generation of low-cost, mass-produced, sensitive and portable devices for the measurement of analytes of interest. The responses of these platforms, in terms of current intensity and reproducibility, are strongly influenced by factors such as printing procedures, type of ink, substrates, etc. In this paper, an improved inverse-designed screen-printed electrode (IDSPE) is proposed. The electrochemical performance is compared with that obtained using classical screen printed electrodes (SEPs), showing enhanced sensitivity and signal-to-noise ratio (background current minimization 32 ± 3nA and 0.64 ± 0.01nA, for SPE and IDSPE, respectively). A full comparison between inverse and classical screen-printed electrodes is carried out using various electroactive species (potassium ferricyanide, ascorbic acid, hexaammineruthenium(III) chloride and NADH) and two different electrochemical techniques (cyclic and square-wave voltammetry). In tests conducted with potassium ferricyanide the sensitivity of the IDSPE shows a nearly four-fold improvement, and a limit of detection three times better than the values obtained employing the classical SPE. The reproducibility (RSD%) in tests conducted with ascorbic acid is 6% and 12% for IDSPE and SPE, respectively. Moreover, surface modification of both screen-printed electrodes (SPE and IDSPE) with biochar obtained from recycled brewers waste (Bio-SPEs and Bio-IDSPEs), further improves their electrochemical performance, in terms, for example, of the heterogeneous electron transfer constant (0.0024 and 0.0018 for Bio-SPE and Bio-IDSPE, respectively).

Gene Electro-Transfer (GET) is a powerful method of DNA delivery with great potential for medical applications. Although GET has been extensively studied in vitro and in vivo, the optimal parameters remain controversial. 2D cell cultures have been widely used to investigate GET protocols, but have intrinsic limitations, whereas 3D cultures may represent a more reliable model thanks to the capacity of reproducing the tumor architecture. Here we applied two GET protocols, using a plate or linear electrode, on 3D-cultured HCC1954 and MDA-MB231 breast cancer cell lines grown on a novel collagen-free 3D scaffold and compared results with conventional 2D cultures. To evaluate the electrotransfer efficiency, we used the plasmid pEGFP-C3 encoding the enhanced green fluorescent protein (EGFP) reporter gene. The novel 3D scaffold promoted extracellular matrix deposition, which particularly influences cell behavior in both in vitro cell cultures and in vivo tumor tissue. While the transfection efficiency was similar in the 2D-cultures, we observed significant differences in the 3D-model. The transfection efficiency in the 3D vs 2D model was 44% versus 15% (p &lt, 0.01) and 24% versus 17% (p &lt, 0.01) in HCC1954 and MDA-MB231 cell cultures, respectively. These findings suggest that the novel 3D scaffold allows reproducing, at least partially, the peculiar morphology of the original tumor tissues, thus allowing us to detect meaningful differences between the two cell lines. Following GET with plate electrodes, cell viability was higher in 3D-cultured HCC1954 (66%) and MDA-MB231 (96%) cell lines compared to their 2D counterpart (53% and 63%, respectively, p &lt, 0.001). Based on these results, we propose the novel 3D scaffold as a reliable support for the preparation of cell cultures in GET studies. It may increase the reliability of in vitro assays and allow the optimization of GET parameters of in vivo protocols.

Tumor electroporation (EP) refers to the permeabilization of the cell membrane by means of short electric pulses thus allowing the potentiation of chemotherapeutic drugs. Standard plate adhesion 2D cell cultures can simulate the in vivo environment only partially due to lack of cell&ndash, cell interaction and extracellular matrix (ECM). In this study, we assessed a novel 3D scaffold for cell cultures based on hyaluronic acid and ionic-complementary self-assembling peptides (SAPs), by studying the growth patterns of two different breast carcinoma cell lines (HCC1569 and MDA-MB231). This 3D scaffold modulates cell shape and induces extracellular matrix deposit around cells. In the MDA-MB 231 cell line, it allows three-dimensional growth of structures known as spheroids, while in HCC1569 it achieves a cell organization similar to that observed in vivo. Interestingly, we were able to visualize the electroporation effect on the cells seeded in the new scaffold by means of standard propidium iodide assay and fluorescence microscopy. Thanks to the presence of cell&ndash, cell and cell&ndash, ECM interactions, the new 3D scaffold may represent a more reliable support for EP studies than 2D cancer cell cultures and may be used to test new EP-delivered drugs and novel EP protocols.

International audience; Background and Objective: The paper focuses on the numerical strategies to optimize a plasmid DNA delivery protocol, which combines hyaluronidase and electroporation. Methods: A well-defined continuum mechanics model of muscle porosity and advanced numerical optimization strategies have been used, to propose a substantial improvement of a pre-existing experimental protocol of DNA transfer in mice. Our work suggests that a computational model might help in the definition of innovative therapeutic procedures, thanks to the fine tuning of all the involved experimental steps. This approach is particularly interesting in optimizing complex and costly protocols, to make in vivo DNA therapeutic protocols more effective. Results: Our preliminary work suggests that computational model might help in the definition of innovative therapeutic protocol, thanks to the fine tuning of all the involved operations. Conclusions: This approach is particularly interesting in optimizing complex and costly protocols for which the number of degrees of freedom prevents a experimental test of the possible configuration.

Electrochemotherapy is a treatment modality which has been increasingly used in veterinary and human medicine for treating cutaneous and subcutaneous tumors. In this prospective work we evaluated the outcome of using electrochemotherapy as a first-line treatment for canine oral melanoma in different stages, with the aim of determining predictive factors of response to the treatment. Mucosal melanoma is the most common cause of oral cancer in dogs. Canine oral malignant melanoma is very similar to human oral melanoma in many aspects, being a very good translational model for studying response to this treatment. Sixty-seven canine patients were treated. Intravenous bleomycin was the preferred drug, and the standard operating procedures for electrochemotherapy were followed. The patients were followed-up for two years. According to WHO criteria, the objective response per stage was: stage I 100%, stage II 89.5%, stage III 57.7% and, stage IV 36.4%. The overall median survival was 7.5 months (2-30 months, mean 9.1 months). Median overall survival of patients in stage I was 16.5 months, in stage II was 9 months, in stage III 7.5 months, and in stage IV 4.5 months. The average number of electrochemotherapy sessions was 1.5 for every stage. The incidence of new metastases among treated patients was 28.4%. Patients in advanced stages, with bone involvement, and caudal location of the tumor had poorer response rates and shorter overall survival times. The treatment greatly improved the quality of life of the patients. Electrochemotherapy is an important technique in the oncological armamentarium against melanoma, and these results can be used to predict human response to this therapy in each stage.

Tumor heterogeneity and the presence of stem-like cells have been identified as key features for resistance to anticancer treatments including targeted therapy. The Eph receptors comprise a large family of receptor tyrosine kinases that marks stem-like cells in different tissues. In colorectal cancer (CRC), EphA2 receptor overexpression has been linked to stem-like properties of cells and malignancy. In particular, EphA2 is involved in multiple cross-talks with other cellular networks including EGFR, FAK and VEGF pathways, with which it collaborates to stimulate cell migration, invasion and metastasis. We investigated the molecular crosstalk and miRNAs modulation of the EphA2 and EGFR pathways in CRC. We also explored the role of EphA2/EGFR pathway mediators as prognostic factors or predictors of cetuximab benefit in CRC patients. We used a strategy to uncover in murine homogeneous tumor EphA2high cells a novel potential molecular signature involving EphA2 and EGFR pathways. Gene expression analysis was performed in EphA2high cells isolated from colorectal adenocarcinoma obtained from the AOM/DSS-induced CRC murine model. Six independent cohorts of patients were analyzed to determine the potential prognostic role of a EphA2/EGFR signature and its effect on cetuximab treatment response. We identified a gene expression pattern, including a coherent miRNAs dysregulation, reflecting the activation of EphA2 and EGFR pathways. Such pattern showed prognostic significance in stage I-III CRC patients, in both univariate and multivariate analysis. In patients with stage IV and WT KRAS, EphA2/Efna1/EGFR gene expression status was significantly associated with poor response to cetuximab. Furthermore, EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutation status. Collectively, our data indicate that EphA2/Efna1/EGFR genes, linked to a possible control by miRNAs, could be proposed as novel prognostic biomarkers in CRC. Moreover, EphA2 could be linked to a mechanism of resistance to cetuximab alternative to KRAS mutations. Since cetuximab resistance, associated to an intrinsic genetic heterogeneity, remains the most critical issue in treating CRC, this study sheds light on new potential biomarkers and therapeutically actionable kinase targets in the EphA2/EGFR-linked pathway. Citation Format: Mariangela De Robertis, Luisa Loiacono, Caterina Fusilli, Maria Luana Poeta, Tommaso Mazza, Massimo Sanchez, Luigi Marchionni, Emanuela Signori, Giuseppe Lamorte, Angelo Luigi Vescovi, Jesus Garcia-Foncillas, Vito Michele Fazio. The EphA2/EGFR pathway dysregulation associates with poor prognosis and cetuximab treatment response in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4296.

Cancer vaccines based on plasmid DNA represent a good therapeutic perspective, despite their low potency. Animal-derived hyaluronidases (Hyals) are employed in oncological clinical practice. Hyal has been also demonstrated to be a good enhancer of intramuscular Gene Electro-Transfer (GET) efficiency in anti-cancer preclinical protocols, with increased transfected cells and higher expression of the encoded genes. Nevertheless, the use of animal-derived Hyals results limited respect to their potentialities, since such preparations could be affected by low purity, variable potency and uncertain safety. To improve the delivery of intramuscular GET-based protocols in mouse, we investigated a new recombinant Hyal, the rHyal-sk, to assess in vivo safety and activity of this treatment at cellular and biochemical levels. We evaluated the cellular events and the inflammation chemical mediators involved at different time points after rHyal-sk administration plus GET. Our results demonstrated the in vivo safety and efficacy of rHyal-sk when injected once intramuscularly in association with GET, with no toxicity, good plasmid in-take ability, useful inflammatory response activation, and low immunogenicity. Following these findings, we would recommend the use of the new rHyal-sk for the delivery of DNA-based vaccines and immunotherapy, as well as into clinical practice, for tumor disease treatments.

EphB2 and EphA2 control stemness and differentiation in the intestinal mucosa, but the way they cooperate with the complex mechanisms underlying tumor heterogeneity and how they affect the therapeutic outcome in colorectal cancer (CRC) patients, remain unclear. MicroRNA (miRNA) expression profiling along with pathway analysis provide comprehensive information on the dysregulation of multiple crucial pathways in CRC. Through a network-based approach founded on the characterization of progressive miRNAomes centered on EphA2/EphB2 signaling during tumor development in the AOM/DSS murine model, we found a miRNA-dependent orchestration of EphB2-specific stem-like properties in earlier phases of colorectal tumorigenesis and the EphA2-specific control of tumor progression in the latest CRC phases. Furthermore, two transcriptional signatures that are specifically dependent on the EphA2/EphB2 signaling pathways were identified, namely EphA2, miR-423-5p, CREB1, ADAMTS14, and EphB2, miR-31-5p, mir-31-3p, CRK, CXCL12, ARPC5, SRC. EphA2- and EphB2-related signatures were validated for their expression and clinical value in 1663 CRC patients. In multivariate analysis, both signatures were predictive of survival and tumor progression. The early dysregulation of miRs-31, as observed in the murine samples, was also confirmed on 49 human tissue samples including preneoplastic lesions and tumors. In light of these findings, miRs-31 emerged as novel potential drivers of CRC initiation. Our study evidenced a miRNA-dependent orchestration of EphB2 stem-related networks at the onset and EphA2-related cancer-progression networks in advanced stages of CRC evolution, suggesting new predictive biomarkers and potential therapeutic targets. Electronic supplementary material The online version of this article (10.1186/s12943-018-0912-z) contains supplementary material, which is available to authorized users.

Cancer stem cells (CSCs) in colorectal tumorigenesis are suggested to be responsible for initiation, development and propagation of colorectal cancer (CRC) and have been extensively characterized by the expression of phenotypic determinants, such as surface or intracellular proteins. The generation of CSCs is likely due to a dysregulation of the signaling pathways that principally control self-renewal and pluripotency in normal intestinal stem cells (ISCs) through different (epi)genetic changes that define cell fate, identity, and phenotype of CSCs. These aspects are currently under intense investigation. In the framework of the oncogenic signaling pathways controlled by microRNAs (miRNAs) during CRC development, a plethora of data suggests that miRNAs can play a key role in several regulatory pathways involving CSCs biology, epithelial-mesenchymal transition (EMT), angiogenesis, metastatization, and pharmacoresistance. This review examines the most relevant evidences about the role of miRNAs in the etiology of CRC, through the regulation of colon CSCs and the principal differences between colorectal CSCs and benign stem cells. In this perspective, the utility of the principal CSCs-related miRNAs changes is explored, emphasizing their use as potential biomarkers to aid in diagnosis, prognosis and predicting response to therapy in CRC patients, but also as promising targets for more effective and personalized anti-CRC treatments.

Optimal gene electrotransfer (GET) requires a compromise between maximum transgene expression and minimal tissue damage. GET in skeletal muscle can be improved by pretreatment with hyaluronidase which contributes to maximize transgene uptake and expression. Nevertheless, tissue damage remains severe close to the electrodes, with a concomitant loss of GET efficiency. Here we analyze the role of pH in tissue damage in GET protocols through in vivo modeling using a transparent chamber implanted into the dorsal skinfold of a mouse (DSC) and intravital microscopy, and in silico modeling using the Poisson–Nernst–Planck equations for ion transport. DSC intravital microscopy reveals the existence of pH fronts emerging from both electrodes and that these fronts are immediate and substantial thus giving rise to tissue necrosis. Theoretical modeling confirms experimental measurements and shows that in GET protocols whether with or without hyaluronidase pretreatment, pH fronts are the principal cause of muscle damage near the electrodes. It also predicts that an optimal efficiency in GET protocols, that is a compromise between obtaining maximum electroporated area and minimal tissue damage, is achieved when the electric field applied is near 183 V/cm in a GET protocol and 158 V/cm in a hyaluronidase + GET protocol. Fil: Olaiz, Nahuel Manuel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Signori, Emanuela. Istituto di Farmacologia Traslazionale; Italia Fil: Maglietti, Felipe Horacio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soba, Alejandro. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Suárez, Cecilia Ana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Turjanski, Pablo Guillermo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Michinski, Sebastián Diego. Instituto Tecnológico de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marshall, Guillermo Ricardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Computación; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Gene electrotransfer (GET) protocols, based on the introduction into the cells of genes encoding immunomodulatory molecules, constitute a safe and powerful strategy for inducing an immune response against cancer. But GET efficiency can be significantly affected by damage due to the products of electrolysis, in particular, pH fronts. To elucidate the role of pH fronts and damage in GET efficiency we present an analysis of the pH fronts-tissue natural buffer interaction through a theoretical model using the Nernst-Planck equations for ion transport assuming a tissue with a bicarbonate buffering system and its validation with experimental measurements. pH front-buffer interaction measurements unveil a remarkable behavior tuned by pulse length and frequency: during the ON pulse critical pH front trajectories (pH=8.5 or 5.5) jump forward, during the OFF pulse, they recede due to tissue natural buffer attenuation. Theory shows that they are intimately related to ion transport mode: during the ON pulse, ion transport is mainly governed by migration and trajectories jump forward in time; during the OFF pulse, migration ceases, ion transport is governed solely by diffusion and trajectories recede due to buffer attenuation. Experiments and theory show that regardless of the presence of buffer attenuation, pH fronts remain during several minutes in a non-physiological state after the treatment. These results suggest that regions enclosed by pH fronts trajectories (thus subjected to non-physiological pH values during a sufficiently long time) may be subjected to plasmid damage during a GET treatment. Ways to minimize this effect, thus optimizing GET efficiency are suggested. Fil: Marino, M. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Olaiz, Nahuel Manuel. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentina Fil: Signori, Emanuela. Consiglio Nazionale delle Ricerche; Italia Fil: Maglietti, Felipe Horacio. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentina Fil: Suárez, Cecilia Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentina Fil: Michinski, Sebastián Diego. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentina Fil: Marshall, Guillermo Ricardo. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentina

Cancer immunotherapy is currently one of the leading approaches in cancer treatment. Gene electrotransfer of plasmids encoding interleukin 12 (IL-12) into the cells leads to the production of IL-12, which drives immune cell polarization to an antitumoral response. One of the cell types that shows great promise in targeting tumor cells under the influence of IL-12 cytokine milieu is that of macrophages. Therefore, the aim of this study was to evaluate gene electrotransfer of antibiotic resistance-free plasmid DNA-encoding murine IL-12 (mIL-12) in mice bearing aggressive B16F10 murine melanoma. IL-12 electrotransfer resulted in the complete long-term eradication of the tumors. Serum mIL-12 and murine interferon γ (mIFNγ) were increased after IL-12 gene electrotransfer. Further on, hematoxylin and eosin (HE) staining showed increased infiltration of immune cells that lasted from day 4 until day 14. Immunohistochemistry (IHC) staining of F4/80, MHCII, and CD11c showed higher positive staining in the IL-12 gene electrotransfer group than in the control groups. Immune cell infiltration into the tumors and the high density of MHCII- and CD11c-positive cells suggest an antitumor polarization of macrophages and the presence of antigen-presenting cells that contributes to the important antitumor effectiveness of IL-12.

Since conventional therapeutic approaches in cancer are highly invasive they hardly prolong patient survival for more than few months. Having the ability to stimulate both cellular and humoural immune responses, immunisation with naked plasmid DNA encoding tumour-associated antigens or tumour-specific antigens has recently reported a plethora of advantages, and the improvement of vaccine efficacy has emerged as a goal in the development of DNA vaccination as anti-tumour therapy. Nevertheless, because of their poor immunogenicity when administered as unformulated intramuscular injections, plasmid DNA vaccines need to be improved. Recent data suggest that the DNA vaccine efficacy may significantly be increased by electroporation. This review highlights the recent literature that supports electroporation as an effective strategy to improve DNA-based vaccination protocols, investigating the most relevant studies, recently developed for the applications of DNA vaccine electrotransfer against tumours in pre-clinical and clinical studies.

Under a Creative Commons license.-- Review.-- et al., Targeted therapies and the consequent adoption of >personalized> oncology have achieved notablesuccesses in some cancers; however, significant problems remain with this approach. Many targetedtherapies are highly toxic, costs are extremely high, and most patients experience relapse after a fewdisease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistantimmortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are notreliant upon the same mechanisms as those which have been targeted). To address these limitations, aninternational task force of 180 scientists was assembled to explore the concept of a low-toxicity >broad-spectrum> therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspectsof relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a widerange of high-priority targets (74 in total) that could be modified to improve patient outcomes. For thesetargets, corresponding low-toxicity therapeutic approaches were then suggested, many of which werephytochemicals. Proposed actions on each target and all of the approaches were further reviewed forknown effects on other hallmark areas and the tumor microenvironment. Potential contrary or procar-cinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixedevidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of therelationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. Thisnovel approach has potential to be relatively inexpensive, it should help us address stages and types ofcancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for futureresearch is offered., Amr Amin was funded by Terry Fox Foundation Grant # TF-13-20 and UAEU Program for Advanced Research (UPAR) #31S118; Jack Arbiser was funded by NIHAR47901; Alexandra Arreola was funded by NIH NRSA Grant F31CA154080; Alla Arzumanyan was funded by NIH (NIAID) R01: Combination therapies for chronic HBV, liver disease, and cancer (AI076535); Work in the lab of Asfar S. Azmi is supported by NIH R21CA188818 as well as from Sky Foundation Inc. Michigan; Fabian Benencia was supported by NIH Grant R15 CA137499-01; Alan Bilsland was supported by the University of Glasgow, Beatson Oncology Centre Fund, CRUK (www.cancerresearchuk.org) Grant C301/A14762; Amancio Carnero was supported by grants from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-6844 and CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). His work on this project has also been made possible thanks to the Grant PIE13/0004 co-funded by the ISCIII and FEDER funds; Stephanie C. Casey was supported by NIH Grant F32CA177139; Mrinmay Chakrabarti was supported by the United Soybean Board; Rupesh Chaturvedi was supported by an NIH NCCAM Grant (K01AT007324); Georgia Zhuo Chen was supported by an NIH NCI Grant (R33 CA161873-02); Helen Chen acknowledges financial support from the Michael Cuccione Childhood Cancer Foundation Graduate Studentship; Sophie Chen acknowledges financial support from the Ovarian and Prostate Cancer Research Trust, UK; Yi Charlie Chen acknowledges financial support from the West Virginia Higher Education Policy Commission/Division of Science Research, his research was also supported by NIH grants (P20RR016477 and P20GM103434) from the National Institutes of Health awarded to the West Virginia IDeA Network of Biomedical Research Excellence; Maria Rosa Ciriolo was partially supported by the Italian Association for Cancer Research (AIRC) Grants #IG10636 and #15403; Helen M. Coley acknowledges financial support from the GRACE Charity, UK and the Breast Cancer Campaign, UK; Marisa Connell was supported by a Michael Cuccione Childhood Cancer Foundation Postdoctoral Fellowship; Sarah Crawford was supported by a research grant from Connecticut State University; Charlotta Dabrosin acknowledges financial support from the Swedish Research Council and the Swedish Research Society; Giovanna Damia gratefully acknowledges the generous contributions of The Italian Association for Cancer Research (IG14536 to G.D.), Santanu Dasgupta gratefully acknowledges the support of the University of Texas Health Science Centre at Tyler, Elsa U. Pardee Foundation; William K. Decker was supported in part by CPRIT, the Cancer Prevention and Research Institute of Texas; Anna Mae E. Diehl was supported by NIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the NIH National Institute on Alcohol Abuse and Alcoholism (NIAAA), Gilead and Shire Pharmaceuticals; Q. Ping Dou was partially supported by NIH/NCI (1R01CA20009, 5R01CA127258-05 and R21CA184788), and NIH P30 CA22453 (to Karmanos Cancer Institute); Janice E. Drew was supported by the Scottish Government's Rural and Environment Science and Analytical Services Division; Eyad Elkord thanks the National Research Foundation, United Arab Emirates University and the Terry Fox Foundation for supporting research projects in his lab; Bassel El-Rayes was supported by Novartis Pharmaceutical, Aveo Pharmaceutical, Roche, Bristol Myers Squibb, Bayer Pharmaceutical, Pfizer, and Kyowa Kirin; Mark A. Feitelson was supported by NIH/NIAID Grant AI076535, Dean W. Felsher was supported by NIH grants (R01CA170378, U54CA149145, and U54CA143907); Lynnette R Ferguson was financially supported by the Auckland Cancer Society and the Cancer Society of New Zealand; Gary L. Firestone was supported by NIH Public Service Grant CA164095 awarded from the National Cancer Institute; Christian Frezza “would like to acknowledge funding from a Medical Research Council CCU-Program Grant on cancer metabolism, and a unique applicant AICR project grant”; Mark M. Fuster was supported by NIH Grant R01-HL107652; Alexandros G. Georgakilas was supported by an EU Marie Curie Reintegration Grant MC-CIG-303514, Greek National funds through the Operational Program ‘Educational and Lifelong Learning of the National Strategic Reference Framework (NSRF)-Research Funding Program THALES (Grant number MIS 379346) and COST Action CM1201 ‘Biomimetic Radical Chemistry’; Michelle F. Green was supported by a Duke University Molecular Cancer Biology T32 Training Grant; Brendan Grue was supported by a National Sciences Engineering and Research Council Undergraduate Student Research Award in Canada; Dorota Halicka was supported by by NIH NCI grant NCI RO1 28704; Petr Heneberg was supported by the Charles University in Prague projects UNCE 204015 and PRVOUK P31/2012, by the Czech Science Foundation projects 15-03834Y and P301/12/1686, by the Czech Health Research Council AZV project 15-32432A, and by the Internal Grant Agency of the Ministry of Health of the Czech Republic project NT13663-3/2012; Matthew D. Hirschey wishes to acknowledge Duke University Institutional Support, the Duke Pepper Older Americans Independence Center (OAIC) Program in Aging Research supported by the National Institute of Aging (P30AG028716-01) and NIH/NCI training grants to Duke University (T32-CA059365-19 and 5T32-CA059365), Lorne J. Hofseth was supported by NIH grants (1R01CA151304, 1R03CA1711326, and 1P01AT003961); Kanya Honoki was supported in part by the grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 24590493); Hsue-Yin Hsu was supported in part by grants from the Ministry of Health and Welfare (CCMP101-RD-031 and CCMP102-RD-112) and Tzu-Chi University (61040055-10) of Taiwan; Lasse D. Jensen was supported by Svenska Sallskapet for Medicinsk Forskning, Gosta Fraenkels Stiftelse, Ak.e Wibergs Stiftelse, Ollie och Elof Ericssons Stiftelse, Linkopings Universitet and the Karolinska Institute, Sweden; Wen G. Jiang wishes to acknowledge the support by Cancer Research Wales, the Albert Hung Foundation, the Fong Family Foundation, and Welsh Government A4B scheme; Lee W. Jones was supported in part by grants from the NIH NCI; W Nicol Keith was supported by the University of Glasgow, Beatson Oncology Centre Fund, CRUK (www.cancerresearchuk.org) Grant C301/A14762; Sid P. Kerkar was supported by the NIH Intramural Research Program; Rob J. Kulathinal was supported by the National Science Foundation, and the American Cancer Society; Byoung S. Kwon was supported in part by National Cancer Center (NCC-1310430-2) and National Research Foundation (NRF-2005-0093837); Anne Le was supported by Sol Goldman Pancreatic Cancer Research Fund Grant 80028595, a Lustgarten Fund Grant 90049125 and Grant NIHR21CA169757 (to Anne Le); Michael A. Lea was funded by the The Alma Toorock Memorial for Cancer Research; Ho-Young Lee., This work was supported by grants from the National Research Foundation of Korea (NRF), the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea (Nos. 2011-0017639 and 2011-0030001) and by a NIH Grant R01 CA100816; Liang-Tzung Lin was supported in part by a grant from the Ministry of Education of Taiwan (TMUTOP103005-4); Jason W. Locasale acknowledges support from NIH awards (CA168997 and AI110613) and the International Life Sciences Institute; Bal L. Lokeshwar was supported in part by United States’ Public Health Services Grants: NIH R01CA156776 and VA-BLR&D Merit Review Grant No. 5I01-BX001517-02; Valter D. Longo acknowledges support from NIH awards (P01AG034906 and R01AG020642) and from the V Foundation; Costas A. Lyssiotis was funded in part by the Pancreatic Cancer Action Network as a Pathway to Leadership Fellow and through a Dale F. Frey Breakthrough award from the Damon Runyon Cancer Research Foundation; Karen L. MacKenzie wishes to acknowledge the support from the Children's Cancer Institute Australia (affiliated with the University of New South Wales, Australia and the Sydney Children's Hospital Network); Maria Marino was supported by grant from University Roma Tre to M.M. (CLA 2013) and by the Italian Association for Cancer Research (AIRC-Grant #IG15221), Ander Matheu is funded by Carlos III Health Institute (AM: CP10/00539), Basque Foundation for Science (IKERBASQUE) and Marie Curie CIG Grant (AM: 2012/712404); Christopher Maxwell was supported by funding from the Canadian Institutes of Health Research, in partnership with the Avon Foundation for Women (OBC-134038) and the Canadian Institutes of Health Research New Investigator Salary Award (MSH-136647); Eoin McDonnell received Duke University Institutional Support; Kapil Mehta was supported by Bayer Healthcare System G4T (Grants4Targets); Gregory A. Michelotti received support from NIH NIDDK, NIH NIAAA, and Shire Pharmaceuticals; Vinayak Muralidhar was supported by the Harvard-MIT Health Sciences and Technology Research Assistantship Award; Elena Niccolai was supported by the Italian Ministry of University and the University of Italy; Virginia R. Parslow gratefully acknowledges the financial support of the Auckland Cancer Society Research Centre (ACSRC); Graham Pawelec was supported by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) Grant number 16SV5536K, and by the European Commission (FP7 259679 “IDEAL”); Peter L. Pedersen was supported by NIH Grant CA-10951; Brad Poore was supported by Sol Goldman Pancreatic Cancer Research Fund Grant 80028595, the Lustgarten Fund Grant 90049125, and Grant NIHR21CA169757 (to Anne Le); Satya Prakash was supported by a Canadian Institutes of Health Research Grant (MOP 64308); Lizzia Raffaghello was supported by an NIH Grant (P01AG034906-01A1) and Cinque per Mille dell’IRPEF–Finanziamento della Ricerca Sanitaria; Jeffrey C. Rathmell was supported by an NIH Grant (R01HL108006); Swapan K. Ray was supported by the United Soybean Board; Domenico Ribatti received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under Grant agreement n°278570; Luigi Ricciardiello was supported by the AIRC Investigator Grants 10216 and 13837, and the European Community's Seventh Framework Program FP7/2007–2013 under Grant agreement 311876; Francis Rodier acknowledges the support of the Canadian Institute for Health Research (FR: MOP114962, MOP125857), Fonds de Recherche Québec Santé (FR: 22624), and the Terry Fox Research Institute (FR: 1030), Gian Luigi Russo contributed to this effort while participating in the Fulbright Research Scholar Program 2013–14; Isidro Sanchez-Garcia is partially supported by FEDER and by MICINN (SAF2012-32810), by NIH Grant (R01 CA109335-04A1), by Junta de Castilla y León (BIO/SA06/13) and by the ARIMMORA project (FP7-ENV-2011, European Union Seventh Framework Program). Isidro Sanchez-Garcia's lab is also a member of the EuroSyStem and the DECIDE Network funded by the European Union under the FP7 program; Andrew J. Sanders wishes to acknowledge the support by Cancer Research Wales, the Albert Hung Foundation, the Fong Family Foundation, and Welsh Government A4B scheme; Neeraj K. Saxena was supported by grant funding from NIH NIDDK (K01DK077137, R03DK089130); Dipali Sharma was partially funded by NIH NCI grants (R01CA131294, R21 CA155686), the Avon Foundation and a Breast Cancer Research Foundation Grant (90047965); Markus David Siegelin received funding from National Institute of Health, NINDS Grant K08NS083732, and the 2013 AACR-National Brain Tumor Society Career Development Award for Translational Brain Tumor Research, Grant Number 13-20-23-SIEG; Neetu Singh was supported by funds from the Department of Science and Technology (SR/FT/LS-063/2008), New Delhi, India; Carl Smythe was supported by Yorkshire Cancer Research and The Wellcome Trust, UK; Carmela Spagnuolo was supported by funding from Project C.I.S.I.A., act n. 191/2009 from the Italian Ministry of Economy and Finance Project CAMPUS-QUARC, within program FESR Campania Region 2007/2013, objectives 2.1, 2.2; Diana M. Stafforini was supported by grants from the National Cancer Institute (5P01CA073992), IDEA Award W81XWH-12-1-0515 from the Department of Defense, and by the Huntsman Cancer Foundation; John Stagg was supported by the Canadian Institutes of Health Research; Pochi R. Subbarayan was supported by the University of Miami Clinical and Translational Science Institute (CTSI) Pilot Research Grant (CTSI-2013-P03) and SEEDS You Choose Awards; Phuoc T. Tran was funded by the DoD (W81XWH-11-1-0272 and W81XWH-13-1-0182), a Kimmel Translational Science Award (SKF-13-021), an ACS Scholar award (122688-RSG-12-196-01-TBG) and the NIH (R01CA166348); Kathryn E. Wellen receives funding from the National Cancer Institute, Pancreatic Cancer Action Network, Pew Charitable Trusts, American Diabetes Association, and Elsa U. Pardee Foundation; Huanjie Yang was partially supported by the Scientific Research Foundation for the Returned Oversea Scholars, State Education Ministry and Scientific and Technological Innovation Project, Harbin (2012RFLXS011), Paul Yaswen was supported by funding from the United States National Institutes of Health (ES019458) and the California Breast Cancer Research Program (17UB-8708); Clement Yedjou was supported by a grant from the National Institutes of Health (Grant # G1200MD007581), through the RCMI-Center for Environmental Health; Xin Yin was supported by NIH/National Heart, Lung, and Blood Institute Training Grant T32HL098062.; Jiyue Zhu was supported by NIH Grant R01GM071725; Massimo Zollo was supported by the European FP7-TuMIC HEALTH-F2-2008-201662, the Italian Association for Cancer research (AIRC) Grant IG # 11963 and the Regione Campania L.R:N.5, the European National Funds PON01-02388/1 2007-2013.

Vaccination is historically one of the most important methods for preventing infectious diseases in humans and animals. Due to recent advances in understanding the biology of the immune system, a more rational design of vaccines and vaccination strategies such as those based on gene transfer have been proposed. In particular, naked DNA vaccination is emerging as a promising approach for introducing foreign antigens into the host, inducing protective immunity against infectious diseases and malignant tumours. Plasmid DNA vaccines offer several advantages in comparison to traditional vaccines such as safety, tolerability and feasibility in manufacture. Nevertheless, because of their poor immunogenicity, plasmid DNA vaccines need further implementation. Recent data suggest electroporation as useful strategy to improve DNA-based vaccination protocols, being able to stimulate both the humoural and cellular immune responses. In pre-clinical trials, electroporation is successfully used in prime-boost combination protocols and its efficacy and tolerability has been demonstrated in Phase I clinical trials. Since these initial results appear promising, in the next future we will assist to further developments of naked DNA vaccination associated to the electroporation technology. This approach not only provides the basis for human studies but also a practical application to veterinary medicine.

Eradication of cancer cells is imperative for a successful treatment of tumours. In addition to the existent chemotherapy or radiation therapy, other novel immunotherapeutic strategies to fight tumours are currently under investigation. One of these is cancer vaccination, an approach aimed at inducing effective immune responses in the host against defined tumour antigens. Among several classes of cancer vaccines, the subunit vaccines based on the single and multi epitope-approach are worthy of note as they offer an exquisite specificity in targeting only tumour cells. In this review we will focus on the significant advances made in the development and use of epitope-based cancer vaccines, reporting a selection of important and recent patents on tumour antigen discovery and epitope design for immunotherapy of cancer.

Vaccines represent one of the most successful strategies in medical science. From the mechanistic perspective, vaccination works by manipulating the immune response through selecting, activating and expanding the memory of B and T cells. To determine the magnitude and quality of immune response, suitable vaccine adjuvants are required; therefore, much effort is going into finding new, effective and non-toxic adjuvant formulations focussed on the activation of key immune targets for inducing a long-term, potent and safe immune response. Significant research is being done in this area, to develop new classes of vaccines for use not only against infectious diseases, but also in the treatment of autoimmune disorders, allergies, chronic inflammatory diseases and cancer. Here the authors review and classify some of the main vaccine adjuvants on the basis of their immunomodulating properties on the immune system.

In a series of invasive ductal breast carcinoma, we investigated the status of chromosomal and intrachromosomal instability by fluorescence in situ hybridisation and determined the level of mRNA expression for two genes involved in the mitotic spindle checkpoint pathway, BUBIB and MAD2L1. All breast cancers demonstrated higher chromosomal instability rates in tumor samples (average: 56.86%, range: 36.24-76.78%) than in controls (average: 11.54%, range: 9.91-14.84%) (P < 0.0001). As well as intrachromosomal instability rates were elevated in tumor (average: 18.45% range: 8.34-35.8%) as compared with controls (average: 4.18% range: 3.47-4.81%) (P < 0.0001). An increase in BUBIB and MAD2L1 transcripts was demonstrated in the majority of the tumor tested. BUBIB mRNA levels but not MAD2L1 levels correlated with intrachromosomal instability (r = 0.722, P = 0.018).

Electroporation is a platform technology for drug and gene delivery. When applied to cell in vitro or tissues in vivo, it leads to an increase in membrane permeability for molecules which otherwise cannot enter the cell (e.g., siRNA, plasmid DNA, and some chemotherapeutic drugs). The therapeutic effectiveness of delivered chemotherapeutics or nucleic acids depends greatly on their successful and efficient delivery to the target tissue. Therefore, the understanding of different principles of drug and gene delivery is necessary and needs to be taken into account according to the specificity of their delivery to tumors and/or normal tissues. Based on the current knowledge, electrochemotherapy (a combination of drug and electric pulses) is used for tumor treatment and has shown great potential. Its local effectiveness is up to 80 % of local tumor control, however, without noticeable effect on metastases. In an attempt to increase systemic antitumor effectiveness of electrochemotherapy, electrotransfer of genes with immunomodulatory effect (immunogene electrotransfer) could be used as adjuvant treatment. Since electrochemotherapy can induce immunogenic cell death, adjuvant immunogene electrotransfer to peritumoral tissue could lead to locoregional effect as well as the abscopal effect on distant untreated metastases. Therefore, we propose a combination of electrochemotherapy with peritumoral IL-12 electrotransfer, as a proof of principle, using electrochemotherapy boosted with immunogene electrotransfer as in situ vaccination for successful tumor treatment. Fil: Sersa, Gregor. Institute of Oncology Ljubljana; Eslovenia Fil: Teissie, Justin. Centre National de la Recherche Scientifique; Francia Fil: Cemazar, Maja. Institute of Oncology Ljubljana; Eslovenia. University of Primorska; Eslovenia Fil: Signori, Emanuela. Istituto di Farmacologia Traslazionale ; Italia Fil: Kamensek, Urska. Institute of Oncology Ljubljana; Eslovenia Fil: Marshall, Guillermo Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física del Plasma. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física del Plasma; Argentina Fil: Miklavcic, Damijan. University of Ljubljana; Eslovenia

Cancer immune evasion is a major stumbling block in designing effective anticancer therapeutic strategies. Although considerable progress has been made in understanding how cancers evade destructive immunity, measures to counteract tumor escape have not kept pace. There are a number of factors that contribute to tumor persistence despite having a normal host immune system. Immune editing is one of the key aspects why tumors evade surveillance causing the tumors to lie dormant in patients for years through “equilibrium” and “senescence” before re-emerging. In addition, tumors exploit several immunological processes such as targeting the regulatory T cell function or their secretions, antigen presentation, modifying the production of immune suppressive mediators, tolerance and immune deviation. Besides these, tumor heterogeneity and metastasis also play a critical role in tumor growth. A number of potential targets like promoting Th1, NK cell, γδ T cell responses, inhibiting Treg functionality, induction of IL-12, use of drugs including phytochemicals have been designed to counter tumor progression with much success. Some natural agents and phytochemicals merit further study. For example, use of certain key polysaccharide components from mushrooms and plants have shown to possess therapeutic impact on tumor-imposed genetic instability, anti-growth signaling, replicative immortality, dysregulated metabolism etc. In this review, we will discuss the advances made toward understanding the basis of cancer immune evasion and summarize the efficacy of various therapeutic measures and targets that have been developed or are being investigated to enhance tumor rejection.

Several protein and DNA-based methods have been previously described for the identification of apolipoprotein E isoforms or genotypes. However, all of them generate frequently false-positive results. The purpose of this study was to set up a new, simple, and effective method for the analysis of the apoE polymorphism. A total of 1,253 subjects previously examined for the apolipoprotein E polymorphism by restriction fragment length polymorphism were reanalyzed by our new method based on Taq DNA polymerase's inability to correctly initiate the replication in the presence of a mismatch at the 3' end of the primer. We conceived a combination of 4 specific primers in 3 different pairs sharing the same stringent polymerase chain reaction conditions to directly detect the presence/absence of polymerase chain reaction products, and thus reveal the 6 apolipoprotein E genotypes. We confirm our previous results in 1,171 subjects, whereas in 82 subjects out of 1,253 (about 6%), the results have been reinterpreted. The final analysis revealed a total of 12 homozygotic subjects for the e2 allele (1.0%), 874 homozygotes for the e3 allele (69.8 %), and 8 homozygotes for the e4 allele (0.6 %). The frequence of heterozygotes was 8.7% for the e2/e3 genotype (n=109), 1.4% for the e2/e4 genotype (n=17), and 0.6% for the e3/e4 genotype (n=8). Relative allele frequencies were e2=0.060, e3=0.834, and e4=0.106. We describe a new, simple, unequivocal, and nonexpensive method for the identification of the 6 apoE genotypes.