Caracterización de células madre tumorales en cáncer de pulmón no microcítico

[EN] Background: Despite the advances in the molecular characterization of lung cancer, chemoresistance, tumor progression and metastasis make of lung cancer the first cause of death cancer-related worldwide. Cancer stem cells (CSCs) are small subpopulations of stem-like cells with self-renewal, differentiation and tumorigenic properties that constitute a promising target, but remain largely unknown. The aim of this study was to isolate and analyze gene expression of CSCs from lung cancer cell-lines and tumor-tissue from resectable NSCLC patients. Methods: This study was performed on cells from NSCLC tumor samples and cell lines (H1650, H1993, A549 and PC9) grown in monolayer and as spheroids. The expression of: CSC-markers (CD133, EPCAM1, ALDH1A1, CD166, ABCG2, CD44, MUC1, BMI1); pluripotency (KLF4, OCT4, NANOG, SOX2, MYC, CCND1); cell cycle (CDKN1A, CDKN2A, MDM2, WEE1); invasiveness (CDH1, VIM, SNAI1, MMP2, MMP9, CEACAM5); Notch pathway (NOTCH1, NOTCH2, NOTCH3, DLL1, DLL4, HEY1, HES1); Wnt pathway (WNT1, WNT2, WNT3, WNT5A, CTNBB1, DKK1, FZD7) and Hedgehog pathway (SMO, PTCH1, SHH, GLI1) were analyzed by quantitative real-time PCR (qPCR). Housekeeping genes ACTB, CDKN1B and GUSB were used as endogenous controls for relative expression calculation. Results: Lung tumorspheres had increased expression of EPCAM1, CD44, ALDH1A1 and CDKN1A (p= 0.028, p= 0.021, p= 0.043 and p= 0.021, respectively) when compared to their paired-adherent cells. In addition, epithelial to mesenquimal transition (EMT) inducer SNAI1 was overexpressed (p= 0.011) in tumorspheres. Regarding Notch pathway, DLL4, NOTCH1 and NOTCH2 showed higher expression in spheroids (p= 0.028, p= 0.038 and p= 0.036, respectively). In Wnt pathway, we found higher expression levels of WNT3, CTNBB1 and GSK3B (p= 0.021, p= 0.008 and p= 0.021, respectively) in lungspheres, whereas the activator of the non-canonical Wnt pathway, WNT5A, tended to be less expressed in spheroids compared to adherent-cultured cells. No significant differences were found in other analyzed genes. Conclusions: Lung spheroids from cancer cell lines and primary tumors showed increased levels of CSC-markers. Genes related to Notch and Wnt were found to be more expressed in tumorspheres, suggesting these pathways as interesting lung-CSC targets.[ES] Introducción: A pesar de los avances en la caracterización molecular del cáncer de pulmón, la resistencia a la quimioterapia, la progresión tumoral y la metástasis hacen del mismo la primera causa de muerte debida a cáncer a nivel mundial. Las células madre tumorales (CSC) son pequeñas subpoblaciones de células con capacidad de autorenovación, diferenciación y tumorigenicidad que constituyen una diana terapéutica prometedora, pero cuya caracterización es aún un campo poco explorado. El objetivo de este trabajo es aislar y analizar la expresión génica de CSCs procedentes de líneas celulares de cáncer de pulmón y de tejido tumoral de pacientes con cáncer de pulmón no microcítico (CPNM) en estadios resecables. Material y Métodos: El estudio se realizó en líneas celulares (H1650, H1993, A549 and PC9) y muestras tumorales de pacientes resecados con CPNM crecidas en monocapa y en placas de baja adherencia con medio sin suero (tumoresferas). La expresión de marcadores de CSC (CD133, EPCAM1, ALDH1A1, CD166, ABCG2, CD44, MUC1, BMI1), genes de pluripotencia (KLF4, OCT4, NANOG, SOX2, MYC, CCND1), genes reguladores del ciclo celular (CDKN1A, CDKN2A, MDM2, WEE1), genes asociados a metástasis (CDH1, VIM, SNAI1, MMP2, MMP9, CEACAM5); y genes de las vías de señalización Notch (NOTCH1, NOTCH2, NOTCH3, DLL1, DLL4, HEY1, HES1); Wnt (WNT1, WNT2, WNT3, WNT5A, CTNBB1, DKK1, FZD7) y Hedgehog (SMO, PTCH1, SHH, GLI1) fue analizada mediante PCR cuantitativa a tiempo real (qPCR), normalizándose frente a la expresión de tres genes controles seleccionados: ACTB, CDKN1B y GUSB , utilizados para el cálculo de la expresión relativa. Resultados: Las tumoresferas de pulmón presentan una expresión incrementada de EPCAM1, CD44, ALDH1A1 y CDKN1A (p= 0.028, p= 0.021, p= 0.043 and p= 0.021, respectivamente) comparadas con sus correspondientes células crecidas en adherencia. Además, el inductor de la transición epitelio-mesenquimal (EMT), SNAI1, está sobreexpresado (p= 0.011) en tumoresferas. Los genes de la vía Notch: DLL4, NOTCH1 y NOTCH2 también muestran mayor expresión en esferoides (p= 0.028, p= 0.038 and p= 0.036, respectivamente) que en células crecidas en monocapas. En cuanto a los genes de la ruta de Wnt, se observan mayores niveles de expresión de WNT3, CTNBB1 and GSK3B (p= 0.021, p= 0.008 and p= 0.021, respectivamente) en esferoides, mientras que el activador de la vía no canónica de Wnt, WNT5A, tiende a estar menos expresado en las células cultivadas en suspensión frente a las células cultivadas en adherencia. No se encontraron diferencias significativas en el resto de genes analizados. Conclusiones: Las tumoresferas de pulmón obtenidas a partir de líneas celulares y tumores primarios de pacientes muestran mayores niveles de marcadores de CSC. Además, genes pertenecientes a las rutas de señalización de Notch y Wnt están mayoritariamente expresados en tumoresferas, sugiriendo estas vías de señalización como dianas potenciales contra las CSC en cáncer de pulmón.Herreros Pomares, A. (2016). Characterization of cancer stem cells from non-small cell lung cancer. http://hdl.handle.net/10251/146816TFG

Methylation of NOTCH genes in normal and at-risk colorectal epithelium

PhD ThesisIntroduction Colorectal cancer (CRC) arises from genetic defects in stem cells. NOTCH signalling plays a key role in stem cell replication control. NOTCH-related genes are overexpressed in CRC. The mechanism for this is not known but could include epigenetic activation of NOTCH oncogenes via promoter hypomethylation. Methylation can be modulated by environmental stimuli including dietary factors such as butyrate, produced by bacterial fermentation of non-digestible carbohydrates in the colon. Butyrate exerts potent anti-oncogenic effects in the colorectal mucosa. Methods Participants were recruited at endoscopy and included those at normal risk of CRC (n=75), or higher risk of CRC because of previous adenomatous polyps (n=28) or ulcerative colitis (n=12). Participants provided 9 rectal biopsies. Normal risk participants were randomised to resistant starch (Hi-maize 260) or polydextrose supplementation in a 2x2 factorial placebo controlled trial for 50 days. Methylation of several CpG sites in the promoters of JAG1 (NOTCH pathway ligand) and RBP-J (NOTCH intracellular activator) was quantified using pyrosequencing. Results For JAG1 there was trend towards lower methylation at all CpG sites in those at higher CRC risk. Methylation at RBP-J CpG 11 was lower in polyp patients than in controls (18.0(1.5) vs. 23.6% (0.8), p=0.011). At JAG1 CpG 4, methylation increased following polydextrose supplementation compared to placebo (3.1(0.4) vs. 1.7%(0.4), p=0.009). A similar, but non-significant, trend was observed at other CpG sites for JAG1. Conclusions DNA methylation of NOTCH signalling genes is altered in macroscopically normal colorectal mucosa of patients at higher CRC risk. The observed changes in JAG1 methylation after polydextrose supplementation are consistent with a protective effect against carcinogenesis.Northumbria Healthcare NHS Foundation Trust: The BBSRC (grant reference BH090948)

Induction and metastasis of cancer stem cells in pancreatic cancer

Pancreatic cancer is one of the most lethal of all types of cancer with an overall 5-year survival rate of less than 8%. Cancer cells in pancreatic tumor are heterogeneous, and it is poorly understood which population is most responsible for the cancer initiation, progression and metastasis. Recent studies provide evidence for the existence of a highly tumorigenic and metastatic cells within a heterogeneous tumor known as the cancer stem cells (CSCs). Studies also provided ample evidence for the existence of distinct types of CSC populations in a heterogeneous tumor with type specific genotypic, phenotypic and functional characteristics. But, it is not clear how CSCs are induced, and how these cells are involved in the metastasis process. Several external factors such as cigarette smoking and alcohol consumption have been shown to induce CSCs in lung and breast cancers. Cigarette smoking is also associated with 30% of pancreatic cancer cases. The association of smoking with CSC induction is also well appreciated in lung cancer and other cancers. Based on these studies, we aimed to investigate whether and how cigarette smoke induce CSCs in pancreatic cancer in the first part of my dissertation. My second goal primarily explores the metastatic function of CSCs in pancreatic cancer. Previous studies have shown the existence of distinct CSC populations in a heterogeneous tumor. For instance, breast tumors consist of two distinct CSC populations such as ALDH+ and CD44+CD24-. The study also demonstrated that ALDH+ cells rely on oxidative phosphorylation for their energy needs, whereas CD44+CD24- population rely on glycolysis. Recent studies also proposed the propensity of a specific cancer cell with specific metabolic profile metastasizes to specific organ. For instance, liver has a high glycolytic environment, and a metastatic cell required to be highly glycolytic in nature to overcome the glycolytic barrier in the liver. Based on these studies, we have hypothesized that distinct types of CSC populations with type specific stemness and metabolic profiles metastasize to specific organ. Overall, my dissertation is broadly divided into two major chapters. We experimentally demonstrated that cigarette smoke induces pancreatic CSC populations by activating CHRNA7 mediated FOSL1 signaling. We also demonstrated for the first time that pancreatic tumors consist of distinct sub-types of CSC populations with sub-type specific stem cell and metabolic features. I also experimentally proved that distinct sub-types of CSC populations with type-specific metabolic profiles are involved in organ-specific metastasis. Taken together, our findings demonstrate a clear mechanism of CSC induction. In addition, our investigation suggests the existence of distinct sub-types of CSC populations with specific metabolic, stemness and metastatic profiles. In conclusion, our work provides a solid foundation for the understanding of CSCs in pancreatic cancer thereby opening new avenues for further research in developing CSC-targeted therapy for pancreatic cancer and its metastasis

Carcinogen

During the last decades, cancer diseases have increased all over the world. The low quality of food and strong pollution of environment are the main prerequisites for carcinogenesis. The main problem for scientists is to find strategy for prevention of cancer diseases. Therefore, the information about the models for studying carcinogenesis and mutagens which appear during cooking, environmental pollutants, and tests for specific detection of carcinogens is particularly important. The book "Carcinogen" is intended for biologists, researchers, students in medical sciences and professionals interested in associated areas

Accumulation of Somatic Mutations in Normal Human Colonic Epithelium

I investigated stem cell dynamics in normal human colon by detecting somatic variants affecting X-linked and autosomal genes using immunohistochemistry. Applying neutral clonal marks to a large cohort of patients to interpret age-related trends in clone frequencies has established the baseline stem cell dynamics of the tissue. Analysis of a number of new clonal marks showing biased behaviours suggests that different gene-specific mutations can subvert constraints resulting from the tissue architecture in different ways. In respect of intra-gland competition of stem cell derived clones, a disadvantage is observed for the histone modifier HDAC6, while at the other end of the spectrum, loss of the cohesin member STAG2 strongly advantages affected stem cells. Subsequent clonal expansion beyond the boundary of a single crypt is recognised by clones occupying multi-crypt patches. Quantification of such events allows the rate of lateral expansion for different mutations to be measured. Moderate effects were found for PTEN, p53 and STAG2, while mutations in the histone demethylase KDM6A generate very large areas of mutant epithelium in aged humans. Further, targeted sequencing revealed dramatic expansion of KRAS-mutant clones in histologically normal colon. Patches may arise from crypt fission and fusion events. Using a clonal mark based on mild periodic acid-Schiff staining, the neutral crypt fission and fusion rates were quantified. Against this baseline, it was found that KDM6A-mutant clones expand mainly by fission, while fusion remains at homeostatic levels. The emerging picture is that of the aged human colon consisting of a mosaic of different mutations. The work presented in this thesis offers detailed insights into the rates at which different gene-specific mutations arising in colonic stem cells can become fixed within individual crypts and undergo subsequent lateral expansion through crypt fission and fusion events. This defines the timeframe taken for cancer drivers to achieve high mutant allele burden within the tissue, which can serve as a basis for cancer prevention strategies.Wellcome Trus

Chapter Membrane Dynamics of Spermatozoa during Capacitation: New Insight in Germ Cells Signalling

The study of germline stem cells and of germline cells has deep implications for the understanding of fertility, development and cancer. Nowadays, we are experiencing the very fascinating challenge of application of –OMICS technologies to this issue, which is opening new and unexpected horizons in virtually all the branches of biology. Here, we carried out a review of signalling systems involved in maturation of male germ cells and in the process that leads them to become fully fertile. In particular, we discuss the control mechanisms involved in capacitation and acrosome reaction that act at membrane level. Indeed, spermatozoa membranes play key roles in determining the achievement of fertility: they are the interface with the surrounding environment, they locate the signal transduction systems and they are active in recognizing and binding the oocyte. In addition, we discuss the effect of several compounds that could exert a negative effect on reproductive activity, by interfering with the endocrine axis, the so-called endocrine disruptors

Tumorspheres as an in vitro model for cancer stem-like cell characterization in non-small cell lung cancer. Prognostic implications

[ES] El cáncer de pulmón es el tipo de cáncer más frecuentemente diagnosticado y la principal causa de muerte debida a cáncer en el mundo, con sólo un 15% de pacientes con una supervivencia mayor a 5 años tras el diagnóstico. La resección quirúrgica es el tratamiento estándar para los pacientes en estadios tempranos con un buen ECOG, pero el 75% de los pacientes son diagnosticados en estadios avanzados, cuando la intervención quirúrgica no es posible y entre un 35% y un 50% de los pacientes operados recaen tras una cirugía aparentemente exitosa. En los últimos años, se han logrado importantes avances en el desarrollo de la inmunoterapia y de tratamientos contra mutaciones conductoras, pero muchos pacientes todavía desarrollan resistencia, progresan y mueren. Esta resistencia terapéutica ha sido asociada a las células madre tumorales (CMTs), una población tumoral con propiedades de célula madre capaz de sobrevivir a las terapias convencionales y regenerar el tumor incluso cuando son indetectables. En esta tesis doctoral, se establecieron cultivos primarios de pacientes de cáncer de pulmón no microcítico (CPNM) resecados, usando ensayos de formación de tumoresferas para el enriquecimiento en CMTs y condiciones de adherencia para los controles. Las tumoresferas derivadas de pacientes mostraron capacidad de autorenovación y crecimiento exponencial ilimitado, alta resistencia a agentes quimioterápicos, capacidad de invasión y diferenciación in vitro y un elevado potencial tumorigénico in vivo. Usando PCR cuantitativa, se analizaron los perfiles de expresión de los cultivos y se determinó que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 e ITGA6 eran los genes más diferencialmente expresados entre tumoresferas y células adherentes. Los análisis de inmunoblot e inmunofluorescencia confirmaron que las proteínas codificadas por estos genes se encuentran aumentadas en tumoresferas de los pacientes con adenocarcinoma y mostraron patrones de expresión y localización diferencial entre éstas y los controles en adherencia. El valor pronóstico de los genes significativamente sobreexpresados en tumoresferas fue evaluado in silico en una cohorte de 661 pacientes con CPNM procedente del TCGA. De todos ellos, CDKN1A, SNAI1 y ITGA6 mostraron estar relacionados con el pronóstico de los pacientes de acuerdo a un análisis de regresión de Cox y fueron seleccionados para construir una firma de expresión génica, denominada firma de CMTs. Los análisis de supervivencia por Kaplan-Meier mostraron que los pacientes con valores elevados de la firma tienen una supervivencia global (SG) menor para la cohorte completa de CPNM [37,7 vs. 60,40 meses, p = 0,001] y para la subcohorte de adenocarcinoma (ADC) [36,6 vs. 53,5 meses, p = 0,003], pero no para la de los epidermoides. Además, el análisis multivariante mostró que la firma de CMTs es un marcador pronóstico independiente para la SG de los pacientes en la cohorte completa [hazard ratio (HR): 1,498; intervalo de confianza (IC) 95%, 1,167-1,922; p = 0,001] y la subcohorte de ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Esta firma fue también analizada en un grupo independiente de 245 pacientes procedentes del Consorci Hospital General Universitari de València, confirmando su valor pronóstico en los pacientes con ADC [42,90 vs. no alcanzado (NA) meses, p = 0,020]. En resumen, nuestros hallazgos aportan información pronóstica relevante para los pacientes con ADC de pulmón y establecen las bases para el desarrollo de nuevos tratamientos.[CA] El càncer de pulmó és el tipus de càncer més diagnosticat i la principal causa de mort deguda a càncer en el món, amb només un 15% de pacients amb una supervivència major a 5 anys després del diagnòstic. La resecció quirúrgica és el tractament estàndard per als pacients en estadis primaris amb un bon ECOG, però el 75% dels pacients són diagnosticats en estadis avançats, quan la intervenció quirúrgica no és possible i entre un 35% i un 50% dels pacients operats recauen després d'una cirurgia aparentment satisfactòria. En els últims anys, s'han aconseguit importants avanços en el desenvolupament de la immunoteràpia i de tractaments contra mutacions conductores, però molts pacients encara desenvolupen resistència, progressen i moren. Aquesta resistència a les teràpies ha estat relacionada amb les cèl·lules mare tumorals (CMTs), una població tumoral amb propietats de cèl·lula mare capaç de sobreviure a les teràpies convencionals i regenerar el tumor fins i tot quan són indetectables. En aquesta tesi doctoral, es van establir cultius primaris de pacients de càncer de pulmó no microcític (CPNM) ressecats, usant assajos de formació de tumoresferes per a l'enriquiment en CMTs i condicions d'adherència per als controls. Les tumoresferes derivades de pacients van mostrar capacitat d'autorenovació, creixement exponencial il·limitat, alta resistència a agents quimioteràpics, capacitat d'invasió i diferenciació in vitro i un elevat potencial tumorigènic in vivo. Usant PCR quantitativa, es van analitzar els perfils d'expressió dels cultius i es va determinar que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 i ITGA6 eren els gens més diferencialment expressats entre tumoresferes i cèl·lules adherents. Les anàlisis de immunoblot i immunofluorescència van confirmar que les proteïnes codificades per aquests gens es troben augmentades en tumoresferes dels pacients amb adenocarcinoma i van mostrar patrons d'expressió i localització diferencial entre aquestes i els controls en adherència. El valor pronòstic dels gens significativament sobreexpressats en tumoresferes va ser avaluat in silico en una cohort de 661 pacients amb CPNM procedent del TCGA. De tots ells, CDKN1A, SNAI1 i ITGA6 van mostrar estar relacionats amb el pronòstic dels pacients d'acord a una anàlisi de regressió de Cox i van ser seleccionats per a construir una signatura d'expressió gènica, denominada signatura de CMTs. Les anàlisis de supervivència per Kaplan-Meier van mostrar que els pacients amb valors elevats de la signatura tenen una supervivència global (SG) menor per a la cohort completa de CPNM [37,7 vs. 60,40 mesos, p = 0,001] i per a la subcohort d'adenocarcinoma (ADC) [36,6 vs. 53,5 mesos, p = 0,003], però no per a la dels escamosos. A més, l'anàlisi multivariant va mostrar que la signatura de CMTs és un marcador pronòstic independent per a la SG dels pacients en la cohort completa [hazard ratio, (HR): 1,498; interval de confiança (IC) 95%, 1,167-1,922; p = 0,001] i la subcohort d'ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Aquesta signatura va ser també analitzada en un grup independent de 245 pacients procedents del Consorci Hospital General Universitari de València, confirmant el seu valor pronòstic en els pacients amb ADC [42,90 vs. no arribat (NA) mesos, p = 0,020]. En resum, els nostres resultats aporten informació pronòstica rellevant per als pacients amb ADC de pulmó i estableixen les bases per al desenvolupament de nous tractaments.[EN] Lung cancer is the most commonly diagnosed type of cancer and the leading cause of cancer-related death worldwide, with approximately 15% of patients surviving 5 years after diagnosis. Curative surgery is the standard of care for early-stage patients with a good performance status, but 75% are diagnosed at advances stages, when surgery is not possible, and 35-50% of the resected patients relapse after an apparently successful surgical treatment. Significant advances in the development of therapies against driver mutations and immune-based treatments for these patients have been achieved in recent years, but many patients still develop treatment resistance, progress, and die. The high resistance against these therapies has been associated to cancer stem-like cells (CSCs), a population with stem properties which is able to survive after conventional treatments and regenerate tumor even when are undetectable. In this thesis, primary cultures from early-stage non-small cell lung cancer (NSCLC) patients were established, using sphere-forming assays for CSCs enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using RTqPCR, gene expression profiles were analyzed, and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected as the best contributors to distinguish tumorspheres from adherent cells. Immunoblot and immunofluorescence analyses confirmed that proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The prognostic role of genes significantly overexpressed in tumorspheres was evaluated in silico in a cohort of 661 NSCLC patients from TCGA. Based on a Cox regression analysis, CDKN1A, SNAI1 and ITGA6 were found to be associated with prognosis and used to calculate a gene expression score, named CSCs score. Kaplan-Meier survival analysis showed that patients with high CSCs score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months, p = 0.001] and in the adenocarcinoma (ADC) subcohort [36.6 vs. 53.5 months, p = 0.003], but not in the squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score is an independent biomarker of prognosis for OS in both, the entire cohort [hazard ratio (HR): 1.498; 95% confidence interval (CI), 1.167-1.922; p = 0.001], and the ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p = 0.001]. This score was also analyzed in an independent group of 245 patients from Consorci Hospital General Universitari de València, confirming its prognostic value in the ADC subtype [42.90 vs. not reached (NR) months, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung ADC patients and the basis for developing novel therapies.Herreros Pomares, A. (2020). Tumorspheres as an in vitro model for cancer stem-like cell characterization in non-small cell lung cancer. Prognostic implications [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137036TESI

Development of micro fluidic based devices for studying tumour biology and evaluating treatment response in head and neck cancer biopsies

Head and Neck Squamous Cell Carcinoma (HNSCC) presents particular challenges to both the researcher and the clinician. Encompassing a group of tumours with distinct epidemiological and oncological behaviour, each sub site differs greatly in its management and prognosis. However, due to insufficient tissue quantities and culture techniques, research studies commonly group together different sub sites, limiting our understanding of the biological behaviour and treatment response of tumours from distinct sites.Micro fluidics relates to the science of systems that process or manipulate small amounts of fluids within micro channels. When applied in biology, the technology enables the production of simple, robust and highly versatile systems for studying cells and tissues. The aim of the work in this thesis was to maintain small biopsies of tumour in a physiological state, more comparable to the in vivo environment than traditional tissue culture techniques, for up to 9 days. This recreation of the ‘tumour microenvironment’ in vitro provided a platform for the testing of chemotherapy agents and analysing individual tumour behaviour. Initial optimisation studies were performed to demonstrate tissue viability within this novel culture method. Based on LDH excretion as a marker of cell death and WST-1 metabolism as a marker of viability, tumour and nodal biopsies from a variety of sub sites remained viable within the device until the addition of cell lysis buffer at 68 h. Histo-architectural examination of tissue incubated within the device for 96 h demonstrated that original tissue structure is largely maintained. In addition, comparison of viability between fresh and frozen tissues showed little difference, thus the clinical applicability of the technique was significantly enhanced, as biopsies could be collected and stored prior to use at a later date.Using clinically relevant combinations of chemotherapy drugs, nodal biopsies (n=50 micro chips from n=2 tumours with all experiments duplicated) were interrogated with cisplatin, 5-Fluorouracil and docetaxel within the micro device for up to 9 days. The addition of each chemotherapeutic agent resulted in increased cell death compared to control, with a synergistic effect seen when agents were given in combination; results in agreement with clinical trial data.This study demonstrates a robust and reproducible system for the maintenance and ‘interrogation’ of individual tumour biopsies. The innovative model provides a new platform for testing individual patient responses to chemotherapy, paving the way for ‘personalised’ treatment regimens

Membrane Dynamics of Spermatozoa during Capacitation: New Insight in Germ Cells Signalling

The study of germline stem cells and of germline cells has deep implications for the understanding of fertility, development and cancer. Nowadays, we are experiencing the very fascinating challenge of application of –OMICS technologies to this issue, which is opening new and unexpected horizons in virtually all the branches of biology. Here, we carried out a review of signalling systems involved in maturation of male germ cells and in the process that leads them to become fully fertile. In particular, we discuss the control mechanisms involved in capacitation and acrosome reaction that act at membrane level. Indeed, spermatozoa membranes play key roles in determining the achievement of fertility: they are the interface with the surrounding environment, they locate the signal transduction systems and they are active in recognizing and binding the oocyte. In addition, we discuss the effect of several compounds that could exert a negative effect on reproductive activity, by interfering with the endocrine axis, the so-called endocrine disruptors