Patient-Derived Explants of Colorectal Cancer: Histopathological and Molecular Analysis of Long-Term Cultures.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Although short-term cultures of tumour sections and xenotransplants have been used to determine drug efficacy, the results frequently fail to confer clinically useful information. Biomarker discovery has changed the paradigm for advanced CRC, though the presence of a biomarker does not necessarily translate into therapeutic success. To improve clinical outcomes, translational models predictive of drug response are needed. We describe a simple method for the fast establishment of CRC patient-derived explant (CRC-PDE) cultures from different carcinogenesis pathways, employing agitation-based platforms. A total of 26 CRC-PDE were established and a subset was evaluated for viability (n = 23), morphology and genetic key alterations (n = 21). CRC-PDE retained partial tumor glandular architecture and microenvironment features were partially lost over 4 weeks of culture. Key proteins (p53 and Mismatch repair) and oncogenic driver mutations of the original tumours were sustained throughout the culture. Drug challenge (n = 5) revealed differential drug response from distinct CRC-PDE cases. These findings suggest an adequate representation of the original tumour and highlight the importance of detailed model characterisation. The preservation of key aspects of the CRC microenvironment and genetics supports CRC-PDE potential applicability in pre- and co-clinical settings, as long as temporal dynamics are considered.

Venous thromboembolism and prostate cancer: what about genetic markers?

Aim & methods: To review the existing literature concerning the relationship between venous thromboembolism (VTE) and prostate cancer (PC) and explore the putative biological and clinical implications of VTE genetic markers on PC patients by screening the PubMed database. Results: Considering the roles of VTE genome-wide association studies-identified genetic determinants in disease development in the general population, these variants might also underlie the susceptibility for PC-related VTE. Therefore, they could help to identify those with a positive benefit-to-harm ratio for thromboprophylaxis approaches during cancer therapy management, thereby improving patient's prognosis. Conclusion: Future studies are mandatory to explore the relationship between VTE and PC and dissect the predictive value of VTE genome-wide association studies-identified genetic determinants in PC patients, given their clinical implications.

Patient-derived ovarian cancer explants: preserved viability and histopathological features in long-term agitation-based cultures.

Ovarian carcinoma (OvC) remains a major therapeutic challenge due to its propensity to develop resistance after an initial response to chemotherapy. Interactions of tumour cells with the surrounding microenvironment play a role in tumour survival, invasion capacity and drug resistance. Cancer models that retain tissue architecture and tumour microenvironment components are therefore essential to understand drug response and resistance mechanisms. Herein, our goal was to develop a long-term OvC patient-derived explant (OvC-PDE) culture strategy in which architecture and cell type heterogeneity of the original tumour would be retained. Samples from 25 patients with distinct OvC types and one with a benign tumour, were cultured for 30 days in agitation-based culture systems with 100% success rate. OvC-PDE cultures retained the original tumour architecture and main cellular components: epithelial cells, fibroblasts and immune cells. Epithelial cells kept their original levels of proliferation and apoptosis. Moreover, the major extracellular components, such as collagen-I and -IV, were retained in explants. OvC-PDE cultures were exposed to standard-of-care chemotherapeutics agents for 2 weeks, attesting the ability of the platform for drug assays employing cyclic drug exposure regimens. We established an OvC-PDE dynamic culture in which tumour architecture and cell type heterogeneity were preserved for the different OvC types, replicating features of the original tumour and compatible with long-term drug exposure for drug efficacy and resistance studies.

[From simulation to research to improve practices]. / De la simulation à la recherche pour améliorer les pratiques.

How can we respond differently to the demographic challenge of the growing population of seniors with cognitive-behavioural problems? The gerontology pole of the University Hospital Centre of Nîmes has installed a simulation room through a call for projects that has made it possible to develop nursing skills to improve care.

Cysteine allows ovarian cancer cells to adapt to hypoxia and to escape from carboplatin cytotoxicity.

Ovarian cancer is the second most common gynaecologic malignancy and the main cause of death from gynaecologic cancer, due to late diagnosis and chemoresistance. Studies have reported the role of cysteine in cancer, by contributing for hydrogen sulphide (H2S) generation and as a precursor of glutathione (GSH). However, the role of cysteine in the adaptation to hypoxia and therapy response remains unclear. We used several ovarian cancer cell lines, ES2, OVCAR3, OVCAR8, A2780 and A2780cisR, to clarify cysteine relevance in ovarian cancer cells survival upon hypoxia and carboplatin. Results show that ES2 and OVCAR8 cells presented a stronger dependence on cysteine availability upon hypoxia and carboplatin exposure than OVCAR3 cells. Interestingly, the A2780 cisR, but not A2780 parental cells, benefits from cysteine upon carboplatin exposure, showing that cysteine is crucial for chemoresistance. Moreover, GSH degradation and subsequent cysteine recycling pathway is associated with ovarian cancer as seen in peripheral blood serum from patients. Higher levels of total free cysteine (Cys) and homocysteine (HCys) were found in ovarian cancer patients in comparison with benign tumours and lower levels of GSH were found in ovarian neoplasms patients in comparison with healthy individuals. Importantly, the total and S-Homocysteinylated levels distinguished blood donors from patients with neoplasms as well as patients with benign from patients with malignant tumours. The levels of S-cysteinylated proteins distinguish blood donors from patients with neoplasms and the free levels of Cys in serum distinguish blood from patients with benign tumours from patients with malignant tumours. Herein we disclosed that cysteine contributes for a worse disease prognosis, allowing faster adaptation to hypoxia and protecting cells from carboplatin. The measurement of serum cysteine levels can be an effective tool for early diagnosis, for outcome prediction and follow up of disease progression.

Modulation of membrane properties of lung cancer cells by azurin enhances the sensitivity to EGFR-targeted therapy and decreased ß1 integrin-mediated adhesion.

In lung cancer, the Epidermal Growth Factor Receptor (EGFR) is one of the main targets for clinical management of this disease. The effectiveness of therapies toward this receptor has already been linked to the expression of integrin receptor subunit ß1 in NSCLC A549 cells. In this work we demonstrate that azurin, an anticancer therapeutic protein originated from bacterial cells, controls the levels of integrin ß1 and its appropriate membrane localization, impairing the intracellular signaling cascades downstream these receptors and the invasiveness of cells. We show evidences that azurin when combined with gefitinib and erlotinib, tyrosine kinase inhibitors which targets specifically the EGFR, enhances the sensitivity of these lung cancer cells to these molecules. The broad effect of azurin at the cell surface level was examined by Atomic Force Microscopy. The Young 's module (E) shows that the stiffness of A549 lung cancer cells decreased with exposure to azurin and also gefitinib, suggesting that the alterations in the membrane properties may be the basis of the broad anticancer activity of this protein. Overall, these results show that azurin may be relevant as an adjuvant to improve the effects of other anticancer agents already in clinical use, to which patients often develop resistance hampering its full therapeutic response.

Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models.

Currently there is an effort toward the development of in vitro cancer models more predictive of clinical efficacy. The onset of advanced analytical tools and imaging technologies has increased the utilization of spheroids in the implementation of high throughput approaches in drug discovery. Agitation-based culture systems are commonly proposed as an alternative method for the production of tumor spheroids, despite the scarce experimental evidence found in the literature. In this study, we demonstrate the robustness and reliability of stirred-tank cultures for the scalable generation of 3D cancer models. We developed standardized protocols to a panel of tumor cell lines from different pathologies and attained efficient tumor cell aggregation by tuning hydrodynamic parameters. Large numbers of spheroids were obtained (typically 1000-1500 spheroids/mL) presenting features of native tumors, namely morphology, proliferation and hypoxia gradients, in a cell line-dependent mode. Heterotypic 3D cancer models, based on co-cultures of tumor cells and fibroblasts, were also established in the absence or presence of additional physical support from an alginate matrix, with maintenance of high cell viability. Altogether, we demonstrate that 3D tumor cell model production in stirred-tank culture systems is a robust and versatile approach, providing reproducible tools for drug screening and target verification in pre-clinical oncology research.

The influence of gait cadence on the ground reaction forces and plantar pressures during load carriage of young adults.

Biomechanical gait parameters--ground reaction forces (GRFs) and plantar pressures--during load carriage of young adults were compared at a low gait cadence and a high gait cadence. Differences between load carriage and normal walking during both gait cadences were also assessed. A force plate and an in-shoe plantar pressure system were used to assess 60 adults while they were walking either normally (unloaded condition) or wearing a backpack (loaded condition) at low (70 steps per minute) and high gait cadences (120 steps per minute). GRF and plantar pressure peaks were scaled to body weight (or body weight plus backpack weight). With medium to high effect sizes we found greater anterior-posterior and vertical GRFs and greater plantar pressure peaks in the rearfoot, forefoot and hallux when the participants walked carrying a backpack at high gait cadences compared to walking at low gait cadences. Differences between loaded and unloaded conditions in both gait cadences were also observed.

In-shoe plantar pressures and ground reaction forces during overweight adults' overground walking.

PURPOSE: Because walking is highly recommended for prevention and treatment of obesity and some of its biomechanical aspects are not clearly understood for overweight people, we compared the absolute and normalized ground reaction forces (GRF), plantar pressures, and temporal parameters of normal-weight and overweight participants during overground walking. METHOD: A force plate and an in-shoe pressure system were used to record GRF, plantar pressures (foot divided in 10 regions), and temporal parameters of 17 overweight adults and 17 gender-matched normal-weight adults while walking. RESULTS: With high effect sizes, the overweight participants showed higher absolute medial-lateral and vertical GRF and pressure peaks in the central rearfoot, lateral midfoot, and lateral and central forefoot. However, analyzing normalized (scaled to body weight) data, the overweight participants showed lower vertical and anterior-posterior GRF and lower pressure peaks in the medial rearfoot and hallux, but the lateral forefoot peaks continued to be greater compared with normal-weight participants. Time of occurrence of medial-lateral GRF and pressure peaks in the midfoot occurred later in overweight individuals. CONCLUSIONS: The overweight participants adapted their gait pattern to minimize the consequences of the higher vertical and propulsive GRF in their musculoskeletal system. However, they were not able to improve their balance as indicated by medial-lateral GRF. The overweight participants showed higher absolute pressure peaks in 4 out of 10 foot regions. Furthermore, the normalized data suggest that the lateral forefoot in overweight adults was loaded more than the proportion of their extra weight, while the hallux and medial rearfoot were seemingly protected.

High-throughput molecular profiling of a P-cadherin overexpressing breast cancer model reveals new targets for the anti-cancer bacterial protein azurin.

Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherin-overexpressing models, a bad prognosis marker in breast cancer increasing invasion and other malignant features, azurin decreases the invasion of cancer cells. We performed a microarray analysis to compare the expression profile of azurin treated cells with different P-cadherin expression levels. Azurin up-regulated apoptosis mediated by p53 protein, endocytosis and vesicle-mediated transport. In the contrary, in invasive MCF-7/AZ.Pcad cells, azurin decreased the expression of genes associated with cell surface receptors and signal transduction, as well as biological adhesion. Further, azurin decreased adhesion of cells to proteins from the extracellular matrix (ECM) and altered protein expression of integrins α6, ß4 and ß1 and interfered with the ability of these cells to form mammospheres. Altogether, our results further enlighten the anti-cancer effects mediated by azurin in P-cadherin overexpression breast cancer models.

Influence of pressure-relief insoles developed for loaded gait (backpackers and obese people) on plantar pressure distribution and ground reaction forces.

The aims of this study were to test the effects of two pressure relief insoles developed for backpackers and obese people on the ground reaction forces (GRF) and plantar pressure peaks during gait; and to compare the GRF and plantar pressures among normal-weight, backpackers, and obese participants. Based on GRF, plantar pressures, and finite element analysis two insoles were manufactured: flat cork-based insole with (i) corkgel in the rearfoot and forefoot (SLS1) and with (ii) poron foam in the great toe and lateral forefoot (SLS2). Gait data were recorded from 21 normal-weight/backpackers and 10 obese participants. The SLS1 did not influence the GRF, but it relieved the pressure peaks for both backpackers and obese participants. In SLS2 the load acceptance GRF peak was lower; however, it did not reduce the plantar pressure peaks. The GRF and plantar pressure gait pattern were different among the normal-weight, backpackers and obese participants.

The bacterial protein azurin impairs invasion and FAK/Src signaling in P-cadherin-overexpressing breast cancer cell models.

P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50-100 µM) also caused a specific decrease on P-cadherin protein levels from 30-50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.

Ground reaction forces and plantar pressure distribution during occasional loaded gait.

This study compared the ground reaction forces (GRF) and plantar pressures between unloaded and occasional loaded gait. The GRF and plantar pressures of 60 participants were recorded during unloaded gait and occasional loaded gait (wearing a backpack that raised their body mass index to 30); this load criterion was adopted because is considered potentially harmful in permanent loaded gait (obese people). The results indicate an overall increase (absolute values) of GRF and plantar pressures during occasional loaded gait (p < 0.05); also, higher normalized (by total weight) values in the medial midfoot and toes, and lower values in the lateral rearfoot region were observed. During loaded gait the magnitude of the vertical GRF (impact and thrust maximum) decreased and the shear forces increased more than did the proportion of the load (normalized values). These data suggest a different pattern of GRF and plantar pressure distribution during occasional loaded compared to unloaded gait.

[Evaluation of the performance of three spacers]. / Avaliação do Desempenho de Três Câmaras de Expansão.

INTRODUCTION: Several aspects are known to influence the drug distribution within the low respiratory tract, with particular emphasis on those related to the inhalation device. The aim of this work was to assess the performance of three spacers in the drug release, and also the quantity of active agent deposited inside these devices. MATERIALS AND METHODS: In order to evaluate the behaviour of particles in suspension delivered through the Ventilan®HFA inhaler coupled to three different spacers (Volumatic®, AeroChamber MAX® and NebuChamber®) the Multistage Liquid Impinger (MSLI) was used, according to the Portuguese Pharmacopoeia. The mass of salbutamol sulphate deposited on the different impinger compartments and inside the spacer was determined by spectrophotometry, with the purpose of determining the percentage of cumulative mass for each spacer, and then the fine particle fraction. The results were compared statistically using a one-way analysis of variance (one-way ANOVA) with a Bonferroni post-hoc test. RESULTS: About 40 to 50% of salbutamol sulphate was found deposited in the body of the three spacers. This deposition was slightly lower for NebuChamber® (average ± standard deviation of 43.8 % ± 11.6 %), in relation to Volumatic® (p=0.351) or AeroChamber MAX® (p=0.115). The fine particle fraction reached values of 28.2 ± 4.1%, 29.6 ± 2.4% and 30.9 ± 6.7% for Volumatic®, AeroChamber MAX® and NebuChamber®, respectively. CONCLUSION: The spacers showed to have similar efficiencies in the delivery of salbutamol sulphate in the last stages, and there was no relation between the results and the spacers characteristics such as volume, shape and material. Therefore, Volumatic® appears to be perfect for hospital use, since its big volume does not constitute a disadvantage, and its lower cost, when compared to the remaining two spacers, represents an advantage of utmost importance for public hospitals.

Atribuições causais e nível educativo familiar na compreensão do desempenho escolar em alunos portugueses / Causal attributions and educational level of the family in understanding school performance in Portuguese students / Atribuciones causales y nivel educativo familiar en la comprensión del desempeño escolar en alumnos portugueses

Este artigo analisa o contributo específico do nível educativo familiar e das atribuições causais para o bom e fraco rendimento escolar dos alunos na explicação do seu desempenho nas disciplinas de Língua Portuguesa e de Matemática numa amostra de 2.082 alunos do ensino público de 11 escolas de Portugal, incluindo as Regiões Autónomas dos Açores e da Madeira. As atribuições causais foram avaliadas por meio do Questionário das Atribuições Causais para os Resultados Escolares (QARE). O nível educativo familiar foi estimado levando em conta o nível escolar do progenitor com maior habilitação escolar. As classificações obtidas pelos alunos nas disciplinas de língua portuguesa e matemática foram também examinadas. Os resultados apontam para correlações estatisticamente significativas, destacando as atribuições na capacidade para a explicação do rendimento escolar, situação que contrasta com a atribuição dos níveis de rendimento a variáveis externas ao aluno. A análise de regressão permite associar 34,5% da variância no rendimento conjunto em Língua Portuguesa e em Matemática no ensino básico, assim como 21,3% no ensino secundário, às dimensões atribucionais e ao nível educativo familiar. Implicações educacionais são derivadas a partir dos achados.

This paper analyzes the contribution of both family educational level and causal attributions in explaining students´ academic performance in Mathematics and Portuguese Language in a sample of 2.082 public school students from mainland Portugal and the Azores and Madeira regions. Causal attributions were assessed by the Causal Attributions Questionnaire for School Results (QARE). The family educational level was estimated taking into account the educational level of the parent with higher academic degree. The academic grades obtained by students in mathematics and portuguese language were also considered. The results show statistically significant correlations between variables. Internal causal attributions such as capacity and study methods were predominant to explain school performance in the sample and stood out in contrast to external attributions explanations. Regression analyses revealed that 34.5% of the variance in the combined achievement on Portuguese Language and Mathematics in primary education and 21.3% in secondary education could be explained by both the attributional dimensions and family education level. Some educational implications are present taking these data.

Este artículo analiza la contribución específica del nivel educativo familiar y de las atribuciones causales para el buen y el flaco rendimiento escolar de los alumnos en la explicación de su desempeño en las disciplinas de Lengua Portuguesa y Matemáticas en una muestra de 2.082 alumnos de enseñanza pública de 11 escuelas de Portugal, incluyendo las Regiones Autónomas de Açores y Madeira. Las atribuciones causales fueron evaluadas por medio del "Questionário das Atribuições Causais para os Resultados Escolares" (QARE). El nivel educativo familiar fue estimado teniendo en cuenta el nivel escolar del progenitor con mayor habilitación escolar. Las clasificaciones obtenidas por los alumnos en las disciplinas de lengua portuguesa y matemáticas fueron también examinadas. Los resultados señalaron correlaciones estadísticamente significativas, destacando las atribuciones en la capacidad para la explicación del rendimiento escolar, situación que contrasta con la atribución de los niveles de rendimiento a variables externas al alumno. El análisis de regresión permite asociar 34.5% de la variancia en el rendimiento conjunto de la Lengua Portuguesa y las Matemáticas en la enseñanza básica, así como 21.3% en la enseñanza secundaria, a las dimensiones de atribución y al nivel educativo familiar. Implicaciones educacionales son derivadas a partir de los hallazgos.

Atribuições causais e nível educativo familiar na compreensão do desempenho escolar em alunos portugueses / Causal attributions and educational level of the family in understanding school performance in Portuguese students / Atribuciones causales y nivel educativo familiar en la comprensión del desempeño escolar en alumnos portugueses

Este artigo analisa o contributo específico do nível educativo familiar e das atribuições causais para o bom e fraco rendimento escolar dos alunos na explicação do seu desempenho nas disciplinas de Língua Portuguesa e de Matemática numa amostra de 2.082 alunos do ensino público de 11 escolas de Portugal, incluindo as Regiões Autónomas dos Açores e da Madeira. As atribuições causais foram avaliadas por meio do Questionário das Atribuições Causais para os Resultados Escolares (QARE). O nível educativo familiar foi estimado levando em conta o nível escolar do progenitor com maior habilitação escolar. As classificações obtidas pelos alunos nas disciplinas de língua portuguesa e matemática foram também examinadas. Os resultados apontam para correlações estatisticamente significativas, destacando as atribuições na capacidade para a explicação do rendimento escolar, situação que contrasta com a atribuição dos níveis de rendimento a variáveis externas ao aluno. A análise de regressão permite associar 34,5% da variância no rendimento conjunto em Língua Portuguesa e em Matemática no ensino básico, assim como 21,3% no ensino secundário, às dimensões atribucionais e ao nível educativo familiar. Implicações educacionais são derivadas a partir dos achados.(AU)

This paper analyzes the contribution of both family educational level and causal attributions in explaining students´ academic performance in Mathematics and Portuguese Language in a sample of 2.082 public school students from mainland Portugal and the Azores and Madeira regions. Causal attributions were assessed by the Causal Attributions Questionnaire for School Results (QARE). The family educational level was estimated taking into account the educational level of the parent with higher academic degree. The academic grades obtained by students in mathematics and portuguese language were also considered. The results show statistically significant correlations between variables. Internal causal attributions such as capacity and study methods were predominant to explain school performance in the sample and stood out in contrast to external attributions explanations. Regression analyses revealed that 34.5% of the variance in the combined achievement on Portuguese Language and Mathematics in primary education and 21.3% in secondary education could be explained by both the attributional dimensions and family education level. Some educational implications are present taking these data.(AU)

Este artículo analiza la contribución específica del nivel educativo familiar y de las atribuciones causales para el buen y el flaco rendimiento escolar de los alumnos en la explicación de su desempeño en las disciplinas de Lengua Portuguesa y Matemáticas en una muestra de 2.082 alumnos de enseñanza pública de 11 escuelas de Portugal, incluyendo las Regiones Autónomas de Açores y Madeira. Las atribuciones causales fueron evaluadas por medio del "Questionário das Atribuições Causais para os Resultados Escolares" (QARE). El nivel educativo familiar fue estimado teniendo en cuenta el nivel escolar del progenitor con mayor habilitación escolar. Las clasificaciones obtenidas por los alumnos en las disciplinas de lengua portuguesa y matemáticas fueron también examinadas. Los resultados señalaron correlaciones estadísticamente significativas, destacando las atribuciones en la capacidad para la explicación del rendimiento escolar, situación que contrasta con la atribución de los niveles de rendimiento a variables externas al alumno. El análisis de regresión permite asociar 34.5% de la variancia en el rendimiento conjunto de la Lengua Portuguesa y las Matemáticas en la enseñanza básica, así como 21.3% en la enseñanza secundaria, a las dimensiones de atribución y al nivel educativo familiar. Implicaciones educacionales son derivadas a partir de los hallazgos.(AU)