U87 cells implanted on a chick CAM (Chorioallantoic Membrane)
Ontology highlight
ABSTRACT: 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: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.
Project description:Platinum-based neoadjuvant chemotherapy (NAC) prior to radical cystectomy is the preferred treatment for muscle-invasive bladder cancer (MIBC) despite modest survival benefit and significant associated toxicities. Here, we profiled the global proteome of MIBC tumours pre- and post-NAC treatment using archival formalin-fixed paraffin-embedded tissue. We identified four pre-NAC proteomic clusters with distinct biology and response to therapy, and overlaid these with transcriptomic subtypes and immunohistochemistry. We observed proteomic plasticity post-NAC that was associated with increased extracellular matrix and reduced keratinization compared to pre-NAC. Post-NAC clusters appeared to be differentially enriched for druggable proteins. For example, MTOR, and PARP were over-expressed at the protein level in tumours identified as neuronal-like. In addition, we determined that high intra-tumour proteome heterogeneity in pre-NAC tissue was associated with worse prognosis. Our work highlights new aspects of MIBC tumour biology associated with clinical outcomes, and suggests new therapeutic targets based on proteomic clusters.
Project description:In this study the transcription profile of bovine trophoblastic cells infected with Brucella samples was evaluated. Chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), and to compare with the transcription profile of trophoblastic cells infected with mutant strains lacking virB2 or btpB.M-NM-^TvirB2 or M-NM-^TbtpB by microarray analysis at 4 hours post infection. Genes with significant variation in levels of transcripts (fold change > 2 and P < 0.05) were functionally classified, and transcripts related to defense and inflammation were assessed by quantitative real time RT-PCR. Chorioallantoic membrane explant culture (CAM) were obtained from 7 intact pregnant bovine uteruses at the final third of gestation. Three-condition experiment, wild type B. abortus 2308, DvirB2 or DbtpB in 4 control replicates and Microarray analysis.
Project description:A cell line (MFD-1) was derived from a 55-year old male with oesophageal adenocarcinoma. Using different sources of genetic material from normal and tumour tissue surgically resected, peripheral blood and the derived cell line a high concordance of genotypes calls across the whole genome confirms MFD-1 was derived from parent tumour. The SNP6 array contained 906,000 probes for the genotyping of SNPs and 946,000 probes for the genotyping of non-polymorphic copy number. Affymetrix CEL files were analysed using the tool PICNIC2 (predicting absolute allele copy number variation with microarray cancer data).
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: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:Gene expression microarray profiling on glioblastoma intra-tumour regions, where the study hypothesis states that the infiltrative tumour margin harbours a distinct transcriptomic profile from all non-infiltrative tumour regions. Data is from three patients (patient 9, 14 and 15) where regions 1-4 per patient were obtained from non-infiltrative intra-tumour regions, and region 5 was obtained from the infiltrative margin. All patients underwent craniotomy with intra-operative image guidance and visualization of 5ALA induced fluorescence to obtain infiltrative margin biopsies.
Project description:Cancer stem cells (CSCs) have been reported in various cancers including skin squamous cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here, we found that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells (SCs), was the most upregulated transcription factor in CSCs of squamous skin tumours. Sox2 is absent in normal epidermis and begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which Sox2 is frequently genetically amplified, the expression of Sox2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis dramatically decreases skin tumour formation following chemical induced carcinogenesis. Using Sox2-GFP knockin mice, we showed that Sox2 expressing cells in invasive SCC are greatly enriched in tumour propagating cells (TPCs) that further increase upon serial transplantations. Lineage ablation of Sox2 expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of Sox2 expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to their regression and decreases their ability to be propagated upon transplantation into immunodeficient mice, supporting the essential role of Sox2 in regulating CSC functions. Transcriptional profiling of Sox2-GFP expressing CSC and upon Sox2 deletion uncovered a gene network regulated by Sox2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct Sox2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion, and paraneoplastic syndrome. Altogether, our study demonstrates that Sox2, by marking and regulating the functions of skin tumour initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours. We used microarrays to profile tumour epithelial cells upon Sox2 deletion to uncover a gene network regulated by Sox2 in primary tumour cells in vivo. Microarray analysis was performed on FACS isolated Epcam+ a6+ TECs from 3 different biological experiments following TAM administration to K14CREER:SOX2fl/fl and control mice. Total RNA was analysed using Mouse whole genome 430 2.0 array from Affymetrix.
Project description:Cancer stem cells (CSCs) have been reported in various cancers including skin squamous cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here, we found that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells (SCs), was the most upregulated transcription factor in CSCs of squamous skin tumours. Sox2 is absent in normal epidermis and begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which Sox2 is frequently genetically amplified, the expression of Sox2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis dramatically decreases skin tumour formation following chemical induced carcinogenesis. Using Sox2-GFP knockin mice, we showed that Sox2 expressing cells in invasive SCC are greatly enriched in tumour propagating cells (TPCs) that further increase upon serial transplantations. Lineage ablation of Sox2 expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of Sox2 expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to their regression and decreases their ability to be propagated upon transplantation into immunodeficient mice, supporting the essential role of Sox2 in regulating CSC functions. Transcriptional profiling of Sox2-GFP expressing CSC and upon Sox2 deletion uncovered a gene network regulated by Sox2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct Sox2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion, and paraneoplastic syndrome. Altogether, our study demonstrates that Sox2, by marking and regulating the functions of skin tumour initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours. We used microarrays to profile Sox2-GFP expressing CSC to uncover a gene network regulated by Sox2 in primary tumour cells in vivo. Two different biological replicates from SOX2-GFP+ and SOX2-GFP- TECs (control) from 2 different SCC from 2 different mice were analysed. Total RNA was analysed using Mouse whole genome 430 2.0 array from Affymetrix.
Project description:Membrane adenylyl cyclases (ACs) catalyze the conversion of ATP to cAMP, a second messenger involved in different signaling pathways. AC8 is one of the 9 membrane-bound isoforms, present in the brain and implicated in cognitive functions. AC8 is regulated by Ca2+/CaM and different G proteins but structural evidence on its regulation is scarce. We solved the structure of full-length AC8 in complex with Gas and CaM. ACs contain large stretches of disordered/highly flexible domains that cannot be resolved with cryo-EM. To overcome this limitation, we have studied AC8's interaction with CaM, Gas and Gbg using crosslinking mass spectrometry (XL-MS).