Project description:We used a microarray to compare bulk gene expression differences between ASD+/+ and ASD+/- tumor stroma. We observed enrichment for neutrophil transcripts in ASD+/- stroma compared to ASD+/+
Project description:We used scRNA-seq to compare intratumoral heterogeniety between ASD+/+ and ASD+/- tumors. We found that RMS populations were similar between genotypes and that neutrophils were strongly enriched in ASD+/- tumor stroma
Project description:Gene expression profile at single cell level of purified "Tumor" and "Stroma" cells in ASD+/+ and ASD+/- murine RMS tumors [scRNA-seq]
Project description:Background: The molecular determinants of carcinogenesis, tumor progression and patient prognosis can be deduced from simultaneous comparison of thousands of genes by microarray analysis. However, the presence of stroma cells in surgically excised carcinoma tissues might obscure the tumor cell-specific gene expression profiles of these samples. To circumvent this complication, laser microdissection can be performed to separate tumor epithelium from the surrounding stroma and healthy tissue. In this report, we compared RNAs isolated from macrodissected, of which only surrounding healthy tissue had been removed, and microdissected rectal carcinoma samples by microarray analysis in order to determine the most reliable approach to detect the expression of tumor cell-derived genes by microarray analysis. Results: As microdissection yielded low tissue and RNA quantities, extra rounds of mRNA amplification were necessary to obtain sufficient RNA for microarray experiments. These second rounds of amplification influenced the gene expression profiles. Moreover, the presence of stroma cells in macrodissected samples had a minor contribution to the tumor cell gene expression profiles, which can be explained by the observation that more RNA is extracted from tumor epithelial cells than from stroma. Conclusion: These data demonstrate that the more convenient procedure of macrodissection can be adequately used and yields reliable data regarding the identification of tumor cell-specific gene expression profiles. Keywords: macrodissection, microdissection, RNA amplification, stroma, rectal carcinoma
Project description:A study on a newly developed tumor model to elucidated molecular changes in the stroma versus the tumor compartment by hyperbaric oxygen (HBO) treatment
Project description:Objective: Adult Still’s disease (ASD) is a systemic disorder of unknown etiology characterized by high spiking fever, rash and arthritis. The purpose of this study was to determine the pathogenic roles of specific genes in ASD. Methods: Differentially expressed genes (DEGs) were examined by DNA microarray and validated by quantitative PCR using monocytes isolated from patients with active-ASD, inactive-ASD and healthy controls. The correlation between validated DEGs and ASD activity was analyzed. After inflammasome activation with LPS and Nigericin, the production of IL-1β, IL-18, inflammasome and autophagy related proteins in DEGs-overexpressing THP-1 cells was carried out by ELISA or western blotting. DEGs-overexpressing THP-1 cells were treated with an inhibitor of autophagy followed by assessment of IL-1β and IL-18 production by ELISA and western blotting method.Conclusions: The overexpression of PLAC8 in monocytes might play a regulatory role in the production of IL-1β and IL-18 by the enhancement of autophagy, resulting in the suppression of ASD. Results:A total of 68 genes were highly expressed in monocytes isolated from active-ASD patients, relative to their expression in inactive-ASD patients and healthy controls. After validation of expression of 13 genes (CLU, FCGR1B, PLAC8, TLR1, S100A12, CD55, PIM1, BCL2A1, SOD2, PLSCR1, CYP1B1, STEAP4, IL1RN), the expression of PLAC8 was significantly higher in active-ASD patients than the other groups. In ASD, PLAC8 expression level correlated with serum levels of CRP, ferritin and IL-18. Stimulation of monocytes with lipopolysaccharide resulted in PLAC8 upregulation. LPS or Nigericin stimulation of PLAC8-overexpressing THP-1, but not THP-1 cells< was associated with significant decrease in IL-1β and IL-18 production. PLAC8 overexpressing in THP-1 cells was associated with enhanced autophagy and suppression of IL-1β and IL-18 production. Conclusions: PLAC8 upregulation in monocytes seemed to play a regulatory role in the production of IL-1β and IL-18 through enhanced autophagy, resulting in suppression of ASD. The results highlight the role of PLAC8 in the pathogenesis of ASD and suggest its potential suitability as a therapeutic target in ASD.
Project description:Intrahepatic cholangiocarcinoma (ICC) is the second most common type of primary cancer in the liver. ICC is an aggressive cancer with poor prognosis and limited therapeutic strategies. The identification of new drug targets and prognostic biomarkers is an important clinical challenge for ICC. The presence of an abundant stroma is a histological hallmark of ICC. Given the well established role of the stromal compartment in the progression of cancer diseases, we hypothesized that relevant biomarkers could be identified by analyzing the stroma of ICC. By combining laser capture microdissection and gene expression profiling we demonstrated that ICC stromal cells exhibit dramatic genomic changes. We identified a signature of 1,073 non-redundant genes that significantly discriminate the tumor stroma from non tumor fibrous tissue. Functional analysis of differentially expressed genes demonstrated that up-regulated genes in the stroma of ICC were related to cell cycle, extracellular matrix and Transforming Growth Factor beta (TGFM-NM-2) pathways. Tissue microarray analysis using an independent cohort of 40 ICC patients validated at a protein level the increase expression of Collagen 4, Laminin, Osteopontin/SPP1, KIAA0101 and TGFM-NM-22 genes in the stroma of ICC. Statistical analysis of clinical and pathological features demonstrated that the expression of Osteopontin, TGFM-NM-22 and Laminin in the stroma of ICC was significantly correlated with patient overall survival. More importantly, multivariate analysis demonstrated that the stromal expression of Osteopontin was an independent prognostic marker for overall and disease-free survival. Conclusion: The study identifies clinically relevant genomic alterations in the stroma of ICC, including candidates biomarkers for prognosis, supporting the idea that tumor stroma is an important factor for ICC onset and progression. 20 RNA samples were analyzed, from 10 patients with ICC. For each patient, RNA were isolated from laser capture microdissected (LCM) stroma from tumor and non tumor areas.
Project description:Stratification of breast cancers into subtypes are generally based on immune assays on tumor cells and/or mRNA expression of tumor cell enriched tissues. Here, we have laser microdissected tumor epithelium and tumor stroma from 24 breast cancer biopsies (12 luminal-like and 12 basal-like). We hypothesized that the stromal proteome would separate patients with breast into groups independently of the traditional epithelial based subtypes.