FOXO transcriptional activity is associated with response to chemoradiation in EAC.

In this study we aimed to investigate signaling pathways that drive therapy resistance in esophageal adenocarcinoma (EAC). Paraffin-embedded material was analyzed in two patient cohorts: (i) 236 EAC patients with a primary tumor biopsy and corresponding post neoadjuvant chemoradiotherapy (nCRT) resection; (ii) 66 EAC patients with resection and corresponding recurrence. Activity of six key cancer-related signaling pathways was inferred using the Bayesian inference method. When assessing pre- and post-nCRT samples, lower FOXO transcriptional activity was observed in poor nCRT responders compared to good nCRT responders (p = 0.0017). This poor responder profile was preserved in recurrences compared to matched resections (p = 0.0007). PI3K pathway activity, inversely linked with FOXO activity, was higher in CRT poor responder cell lines compared to CRT good responders. Poor CRT responder cell lines could be sensitized to CRT using PI3K inhibitors. To conclude, by using a novel method to measure signaling pathway activity on clinically available material, we identified an association of low FOXO transcriptional activity with poor response to nCRT. Targeting this pathway sensitized cells for nCRT, underlining its feasibility to select appropriate targeted therapies.

The effects of human skin fibroblast monolayers on human sperm motility and mouse zygote development.

A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified.

The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation.

In a human in-vitro fertilization (IVF) programme, the effect of co-culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.