Transcription profiling of pancreatic tumor cell invasion and angiogenesis in the PDAC-CAM model
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ABSTRACT: Human pancreatic adenocarcinoma cells were grafted on the chick chorioallantoic membrane (CAM). Human and chicken GeneChips were used simultaneously to study gene regulation during PDAC cell invasion. Experiment Overall Design: Pooled RNA from T1 (n=3) and T6 (n=3) CAMs were extracted using the RNeasy mini kit (Qiagen). T6 was compared to T1 on human GeneChips and in parallel, on chicken GeneChips.
Project description:Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. We investigated vascular development in vivo by determining global gene regulation throughout the formation of the chick chorio-allantoic membrane (CAM). Our study provides a comprehensive molecular map of vascular maturation during developmental angiogenesis and might thus be a valuable resource to streamline further research of candidates susceptible to mediate pathological angiogenesis. Experiment Overall Design: The developmental stage of the embryos was determined after isolation of the CAM according to Hamburger & Hamilton (HH) (1992). CAMs were isolated from embryos at developmental day E5 (HH26), E7 (HH30), E10 (HH>35) and E14 (HH40), (n=3 embryos/day). mRNA was isolated and hybridized to Affymetrix chicken GeneChips using the Affymetrix standard protocol. We compared one embryo at a lower HH stage to three embryos at a more advanced HH stage and repeated the comparison with two more embryos. Overall, we compared expression between E7 and E5, E10 and E5, E14 and E5, E10 and E7 and E14 and E10.
Project description:Cancer-associated fibroblasts are a major component of the cancer stroma. Here we focus on gastric cancer associated myofibroblasts (CAMs). CAMs secrete factors that increase the migration, invasion and proliferation of cancer cells when compared to adjacent tissue myofibroblasts (ATMs), or normal tissue myofibroblasts (NTMs). In this study we identified and quantified the proteins secreted by normoxic (21% O2) and hypoxic (1% O2) myofibroblast cells.
Project description:Glioblastoma multiforme (GBM) brain tumours have one of the shortest mean survival times (<1 year). In vivo models of GBM in mice and rats have been developed to study aspects of glioma that cannot be observed in cell culture such as angiogenesis, invasion and metastasis. Gliomas can be induced by implantation of rodent glioma cell lines into the brain or flank of nude mice. The disadvantages of rodent models however include variable growth rate and poor penetrance, which leads to difficulties in collecting clearly graded samples (pre-vascular/vascular). Bikfalvi et al have previously established a human GBM model that addresses these issues based on the chicken egg chorio-allantoic membrane (CAM), a highly vascularised extra-embryonic tissue. We have used DNA microarrays and a CAM model of GBM to study gene expression during the recruitment and development of the tumour vasculature. Over a 5 day period samples were taken every 12 hours from the tumour implantation site consisting of tumour cells and stroma cells, and also from a site distant from the implantation site consisting of just CAM cells. This study will shed light on the dynamic transcriptional signature of pathological angiogenesis. On day 10 of embryonic development 3-5 million U87 cells were deposited onto the surface of the CAM after gentle laceration. The cells were contained within a plastic ring and each tumour was size matched based on its volume. Tumour/stroma and distant CAM samples were cut out at 12 hour intervals post implantation for 5 days, equalling 10 time points. Each time point consisted of three replicates. U87 cells in culture (pre-implantation) were also included in the study in triplicate.
Project description:Human pancreatic adenocarcinoma cells were grafted on the chick chorioallantoic membrane (CAM). Human and chicken GeneChips were used simultaneously to study gene regulation during PDAC cell invasion.
Project description:mRNA was sampled during exponential growth phase (T1), beginning of stationary/production phase (T2), middle of production phase (T3-T4) and end of production phase (T5-T6) strains: Y. lipolytica Af4 - DHA producer (Gemperlein et al., 2019) and Y. lipolytica Po1h - wild type
Project description:CAM reagent was synthesized and used to uniquely convert cysteine residues, under mild conditions, into trypsin-recognsable sites. We apply this cysteine-capping methodology in the identification of missense variants in HCT-15 cell lines.
Project description:Comparative profiling of fresh-cut lettuce samples treated with an alternative washing solution (PAA) with respect to standard treated samples STD (SH-treated).We generated a comprehensive repertory of transcripts useful to study the global change induced by two sanitizer in different time points of storage (T1, T3, and T6).
Project description:In chickens, embryonic development begins upon egg formation and lasts for 21 days of incubation until hatching. The CAM is an extraembryonic membrane that serves a critical role in acid-base balance, gaseous exchange, calcium solubilization, and antimicrobial protection. Comparative proteomic analyses of CAM at two developmental stages (ED12 and ED19), in comparison to the proteome of embryonic blood serum, revealed protein groups that are relatively or highly specific to the CAM. The specific CAM functions include gaseous exchange, Ca2+ transport, vasculature development, and protection against pathogen invasion. Overall, our results highlight the structure-function relationship of the CAM protein constituents that potentially could expand its biomedical applications.
Project description:Functional targeted therapy has unfortunately failed to improve the outcome of glioblastoma patients. Success stories evidenced by the use of antibody-drug conjugates in other tumor types are encouraging, but targets specific to glioblastoma and accessible through the blood stream remain scarce. In the current work we have identified and characterized novel and accessible proteins using an innovative proteomic approach on 6 human glioblastomas. We have also analysed U87 tumors grown of chicken chorioallantoic membrane (CAM) and normal brains from mice as controls. Both were processed in the same fashion as the human tumors.
Project description:Age as the primary rise factor could be play an important role in incidence and development of osteoarthritis. Several studies have confirmed some tissue specific microRNA were associated with development of osteoarthritis. But if age related microRNA or miRNA cluster would be involved in pivotal post-transcriptional gene regulation in osteoarthritis is unclear. In view of this, we have an idea that several age-related miRNAs would be screened from the rat knee cartilage at different development ages by miRNAs Microarray analysis. We used microarrays to detail the global programme of gene expression underlying the rat knee cartilage and identified distinct classes of age-related miRNAs during this process. The rat knee articular cartilage were selected at successive stages of the rat developmental for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of cartilage at each developmental stage in order to increase the temporal resolution of expression profiles. To that end, we hand-selected cartilage according to the rat developmental stages, i.e. seven time-points: newborn (T0), childhood (T1), youth(T2), adult (T3), middle-aged (T4) early-stage elderly(T5) and latter-stage elderly(T6). The objective of the study is to identify miRNA profile of knee articular cartilage at different developmental ages in rats. Total RNA were extracted from the knee articular cartilage of Sprague-Dawley rats at postnatal day 0(T0), week1(T1), week 4(T2), mon3(T3), mon 6(T4), mon 12(T5), and mon 18(T6). The microRNA profile in the specimens was detected with the Affymetrix GeneChip® miRNA 3.0 Array.