Gene Expression In Stromal Cells Research Articles

Abstract P2-21-08: Digital spatial profiling of RNA and protein in the invasive lobular breast cancer microenvironment

Abstract Invasive lobular breast cancer (ILC) is an understudied subtype of breast cancer with late recurrence, metastasis to serosal surfaces, such as the peritoneum, and dismal long-term outcome. We previously reported better patient outcome in ER+ breast cancers with high glucocorticoid receptor (GR) expression (Pan et al 2011). Preliminary data suggests more favorable patient outlook in the ILC subtype when tumors express higher levels of GR. The dynamic interaction between a tumor and its microenvironment leads to phenotypic changes in stromal cells and the extracellular matrix to promote growth or invasion of malignant cells. ILC is histologically distinct from invasive ductal carcinoma and is characterized by discohesive tumor cells that grow as “Indian files” due to lack of the cell adhesion molecule E-cadherin. Because of these differences we expect the tumor microenvironment (TME) to be quite unique in ILC compared to other breast cancer subtypes. We hypothesized that differing levels of expression of nuclear receptors by tumor cells would impact cells residing within the stroma, presumably through paracrine signaling. Immunohistochemistry for 25 ILC biopsies revealed a wide variation in GR expression, ranging from completely negative to strongly positive. Our goal was to gain insight into how GR presence or absence in ILC cells might impact gene and protein expression in malignant cells and their TME. We were inspired to examine how crosstalk between the GR-positive or GR-negative cancer cells and their respective tumor microenvironment (TME) differentially impact stromal cell gene expression as well as the immune cell milieu. Bulk expression profiling is inadequate to examine molecular profiles of the tumor and stroma separately. Therefore, we used nanoString GeoMx® high-plex digital spatial profiling of RNA and protein expression in GR-positive and GR-negative primary ILC. We first performed spatial gene expression using the Whole Transcriptome Atlas (WTA) for six primary ILC, two of which were strongly positive for GR, two GR-negative and 2 tumors harboring both GR-positive and GR-negative regions. To examine the tumor cells and the stroma independently we segmented regions into panCK-positive and panCK-negative segments and analyzed those separately. Intriguing differences were observed between GR-positive and negative ILC, for the tumor cells as well as for the TME. Implementation of the Spatial Deconvolution script embedded within the GeoMx® software revealed striking differences in the abundance of immune cells, endothelial cells and fibroblasts in the stroma of GR-positive vs GR-negative ILC. Macrophages and other myeloid subsets were present in significantly higher numbers in the TME of GR-positive compared to GR-negative ILC. The contribution of B- and T-cell subsets as well as endothelial cells and fibroblasts were also notably different. To more precisely define the spatial distribution and abundance of various cell subtypes and their association with tumor cells, we are including protein DSP as an added layer to complement the WTA. We are also performing RNA and protein DSP for an additional ten GR-positive and ten GR-negative ILC. Thus, we will test our initial observations and expand our investigation to generate comprehensive molecular profiles of ILC tumors and stromal cells and acquire a deeper understanding of how GR expression/activation influences the crosstalk between ILC cells and their TME. We anticipate that differences in gene and protein expression will provide clues as to how GR activation affects proliferative and adhesive properties of ILC through modification of the TME. Citation Format: Lynda B. Bennett, Candace Frerich, Sunati Sahoo, Cheryl Lewis, Indu Raman, Min Xu, Guanchun Chen, Suzanne D. Conzen. Digital spatial profiling of RNA and protein in the invasive lobular breast cancer microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-08.

An optimised tissue disaggregation and data processing pipeline for characterising fibroblast phenotypes using single-cell RNA sequencing

Single-cell RNA sequencing (scRNA-Seq) provides a valuable platform for characterising multicellular ecosystems. Fibroblasts are a heterogeneous cell type involved in many physiological and pathological processes, but remain poorly-characterised. Analysis of fibroblasts is challenging: these cells are difficult to isolate from tissues, and are therefore commonly under-represented in scRNA-seq datasets. Here, we describe an optimised approach for fibroblast isolation from human lung tissues. We demonstrate the potential for this procedure in characterising stromal cell phenotypes using scRNA-Seq, analyse the effect of tissue disaggregation on gene expression, and optimise data processing to improve clustering quality. We also assess the impact of in vitro culture conditions on stromal cell gene expression and proliferation, showing that altering these conditions can skew phenotypes.

Heparin Differentially Impacts Gene Expression of Stromal Cells from Various Tissues

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.

Transcript abundance of stromal and thecal cell related genes during bovine ovarian development.

Movement and expansion of mesonephric-derived stroma appears to be very important in the development of the ovary. Here, we examined the expression of 24 genes associated with stroma in fetal ovaries during gestation (n = 17; days 58–274) from Bos taurus cattle. RNA was isolated from ovaries for quantitative RT-PCR. Expression of the majority of genes in TGFβ signalling, stromal transcription factors (NR2F2, AR), and some stromal matrix genes (COL1A1, COL3A1 and FBN1, but not FBN3) showed a positive linear increase with gestational age. Expression of genes associated with follicles (INSL3, CYP17A1, CYP11A1 and HSD3B1), was low until mid-gestation and then increased with gestational age. LHCGR showed an unusual bimodal pattern; high levels in the first and last trimesters. RARRES1 and IGFBP3 also increased with gestational age. To relate changes in gene expression in stromal cells with that in non stromal cells during development of the ovary we combined the data on the stromal genes with another 20 genes from non stromal cells published previously and then performed hierarchical clustering analysis. Three major clusters were identified. Cluster 1 genes (GATA4, FBN3, LHCGR, CYP19A1, ESR2, OCT4, DSG2, TGFB1, CCND2, LGR5, NR5A1) were characterised by high expression only in the first trimester. Cluster 2 genes (FSHR, INSL3, HSD3B1, CYP11A1, CYP17A1, AMH, IGFBP3, INHBA) were highly expressed in the third trimester and largely associated with follicle function. Cluster 3 (COL1A1, COL3A1, FBN1, TGFB2 TGFB3, TGFBR2, TGFBR3, LTBP2, LTBP3, LTBP4, TGFB1I1, ALDH1A1, AR, ESR1, NR2F2) had much low expression in the first trimester rising in the second trimester and remaining at that level during the third trimester. Cluster 3 contained members of two pathways, androgen and TGFβ signalling, including a common member of both pathways namely the androgen receptor cofactor TGFβ1 induced transcript 1 protein (TGFB1I1; hic5). GATA4, FBN3 and LHCGR, were highly correlated with each other and were expressed highly in the first trimester during stromal expansion before follicle formation, suggesting that this could be a critical phase in the development of the ovarian stroma.

Efficient Delivery of Antiapoptotic Signals from Marrow Stromal Cells to Acute Lymphoblastic Leukemia Cells Is Associated with Differential Expression of Genes Related to the Membrane and to Regulation of Apoptosis and Is Dependent in Part on Gap Junction Function

Nonmalignant marrow stroma provides a favorable microenvironment for leukemia cell survival. The mechanisms of support are not well understood. Some studies have identified roles for some stromal derived molecules including some growth factors (EGF, FGF, PDGF), adhesion molecules (VCAM, selectins, osteopontins, integrins, fibronectin), cytokines (IL3, IL6, IL7) and others (KITLG, ANGPT1, CXCL12, LGALS3). It is clearly a multifactorial mechanism. We have developed a simple but powerful experimental system of human marrow derived stromal cells co-cultured with primary human high risk ALL cells that allows us to study the molecular mechanisms.Efficient delivery of the antiapoptotic signals requires leukemia cell contact with living, metabolically active stromal cells. Figure 1 shows several conditions in which stromal cell support for ALL is abrogated: (a) when ALL contact with stroma is prevented by a transwell membrane that allows free flow of extracellular medium; (b) after formalin fixation of stromal cells and their matrix; (c) when live stromal cell protein synthesis is prevented by pretreatment of stromal cells with G418 at nonlethal concentrations; (d) when live stromal cell RNA transcription is impaired by pretreatment with triptolide.Efficient delivery of antiapoptotic signals to leukemia cells is associated with differential of genes related to the membrane and extracellular region and to regulation of cell death. We performed we performed RNASeq studies of gene expression in stromal cells and ALL cells. We made comparisons (a) between supporting cells that could (n=17) and could not (n=7) provide antiapoptotic support to ALL, and (b) between ALL cells that were (n=3) and were not (n=3) supported by stromal cells. We identified 409 genes differentially expressed in stromal cells that prevent ALL apoptosis including genes known to play roles in cell surface interactions, cell matrix interactions, growth factor binding and regulation of cell death (Table 1). We identified 458 genes upregulated in ALL cells following contact with stromal cells including genes related to interactions with the extracellular environment, purine metabolism and regulation of apoptosis (Table 2). We identified 494 genes upregulated in stromal cells following contact with ALL related to extracellular interactions, regulation of oxidation, regulation of daunorubicin, regulation of NADP and ethanol metabolism, interferon signaling and regulation of cell proliferation (Table 3).Based on these discoveries we hypothesized that important mechanisms of stromal cell support of ALL might involve direct intercellular exchange of small molecules related to energy metabolism, nucleic metabolism, and messengers regulating cell proliferation and cell apoptosis. Possible mechanisms could include gap junctions, nanotubules or exosomes. The predominant gap junction in human cells is connexin 43 (GJA1). We found that GJA1 is constitutively expressed at high levels by stromal cells, and that GJA1 expression is increased ten-fold in ALL cells upon contact with stromal cells.Rapid bidirectional exchange of intracellular molecules occurs between stromal cells and ALL. We loaded stromal cells with fluorescent calcien AM which once inside a cell cannot diffuse through the membrane but can pass through gap junctions. We cocultured ALL cells with labelled stromal for 16 hr. We observed calcien in ALL cells in physical contact with stroma, but not in ALL cells separated from labelled stroma by a transwell. When leukemia cells were labeled with calcien we saw transfer of calcien to stroma in contact with ALL but not when separated by a transwell.Interference with gap junction function increases ALL cell death. GAP27 is an 11-amino acid peptide that specifically interferes with GJA1/connexin 43 gap junctions. Figure 2 shows substantial reduction of ALL survival in cocultures containing 1 mM GAP27 peptide. We observed similar results with use of 75 micromolar carbenoxolone, an FDA approved drug that impairs gap junction function. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Noncanonical Wnt signaling in stromal cells regulates B-lymphogenesis through interleukin-7 expression

The regulation of early B cell development and the interaction of hematopoietic precursors with stromal cells in the bone marrow (BM) are controlled by various secreted signaling molecules. Several recent studies showed Wnt signaling involved in B-lymphogenesis through stromal cells. However, the molecules modulated by Wnt signaling in stromal cells regulating B-lymphogenesis have not been identified yet. Interleukin (IL)-7 and CXC chemokine ligand (CXCL) 12 are known to be express in stromal cells, and both molecules are essential for B-lymphogenesis. In the present study, we examined the role of Wnt signaling in regulating IL-7 and CXCL12 expression and in affecting B-lymphogenesis. In mouse stromal ST2 cells, expression of IL-7 and CXCL12 mRNA was augmented by noncanonical Wnt5a. When mouse BM-derived cells were cultured on Wnt5a-overexpressing ST2 cells, an increased number of B220+/IgM- B-lymphoid precursor cells was observed. These results show that Wnt5a regulates IL-7 gene expression in stromal cells and suggest the possibility that noncanonical Wnt regulates B-lymphogenesis via IL-7 expression in stromal cells.

Association of stromal cell gene expression with response to neoadjuvant chemotherapy in locally advanced breast cancer.

11129 Background: There is increasing evidence that breast cancer behavior is a reflection of an interactive signaling between the malignant epithelial compartment and the surrounding microenvironment. A high stromal gene expression, characterizing a reactive stroma, was associated with resistance to neoadjuvant chemotherapy, in indirect analysis. Our aim was to detect a stromal cell signature associated with response to neoadjuvant chemotherapy in locally advanced breast cancer, using a direct approach, consisting of stromal cell selection, through laser microdissection. Methods: Twenty one patients treated at Hospital de Câncer de Barretos were included. Patients were diagnosed with invasive ductal carcinoma, except one (lobular invasive carcinoma); median age was 44.5 years; all presented stage III disease; mean tumor dimension was 6.89 cm. Eleven tumors were luminal HER2 negative; 5 HER2 positive [3 ER(+); 2 ER(-)] and 5 triple negative. All patients were submitted to neoadjuvant chemotherapy (4AC fol...

A Novel Platform For The Identification Of Stromal-Derived Factors That Enhance Acute Lymphoblastic Leukemia Cell Survival In The Context Of Maintenance Chemotherapy

IntroductionChildhood B-lineage acute lymphoblastic leukemia (ALL) is the most common form of malignancy in children. ALL is highly responsive to intensive, multi-agent chemotherapy, and most children enter remission; 25% of children relapse. Furthermore, cure requires an additional two years of low intensity maintenance chemotherapy with 6-mercaptopurine (6-MP) and methotrexate. It is known that serum levels of maintenance chemotherapy drugs are not highly cytotoxic for ALL cells, and it is thought that bone marrow stromal cells (BM-SC) provide trophic signals to leukemia cells that result in increased resistance to standard therapy. If anti-apoptotic signals from stromal cells to leukemia cells were known, new molecular targets for leukemia therapy could be developed. We are developing a conceptually simple screening system that will identify stromal cell-derived molecules that support ALL cell survival in the setting of 6MP and methotrexate. Our hypothesis is based on the observation that in serum-free conditions, BMSC prevent apoptosis of primary ALL cells. We reason that if we interfere with the production of key BMSC-derived trophic factors, an increase in ALL cell apoptosis in vitro will be seen. MethodsThe system has 3 components: (1) human marrow stromal cells; (2) primary ALL cells (not established cell lines); and (3) siRNAs to knockdown candidate stromal genes. We employ a human mesenchymal stromal cell (MSC) line immortalized with a human TERT gene that has been shown to be representative of primary human stroma. 20,000 BMSC are placed in wells of a 96-well plate and treated with siRNA. 48 hours later, 30,000 primary human ALL cells are added in serum-free media. After 5 days, viable ALL cells are measured by flow cytometry. Results(1) The addition of pharmacological concentrations of 6-MP to BMSC/ALL co-cultures does not significantly increase primary ALL apoptosis. Co-cultures were established as above and 6-MP was added at 1 micromolar. ALL survival was measured at 5 days. No increase in ALL survival was seen in the 6-MP containing cultures. On unmanipulated stroma cultured without 6-MP, 1948.8 (± 397.0) viable ALL cells were recovered; on manipulated stroma in the presence of 6-MP, 1698.5 (±299.3) (p value = 0.12). (2) Interference with stromal cell protein synthesis significantly increases ALL cell apoptosis. We hypothesize that interference with new protein synthesis might reduce stromal support of ALL cells. To test this we treated BMSC for 6 hours with 25 micrograms of G418 which irreversibly blocks polypeptide synthesis. Wells were then washed with serum-free medium. Stromal cells remained viable for up to 1 week. However, ALL cells apoptosis was much higher on such treated stromal cells (32.7±18.5 viable ALL cells on G418-treated stroma versus 1948.8 ± 397.0 on unmanipulated stroma, p value< 0.001)(Figure 1). (3) Interference with global stromal cell RNA transcription significantly increased ALL cell apoptosis. Triptolide is a molecule that irreversibly inhibits RNA transcription. BMSC were treated for 6 hr with 1 microgram triptolide and washed with serum-free medium. Stromal cells remain viable for up to 1 week. ALL cell apoptosis was significantly reduced by triptolide treatment of stroma (not shown in Figure 1). The number of viable ALL cells recovered from triptolide-treated stroma was 252.3±113.2, compared to 996.6±239.2 on unmanipulated stroma (p< 0.001). (4) Knockdown of CXCL12 gene expression in stromal cells signficantly increase ALL cell apoptosis. For proof of principle, we have started with CXCL12, a gene shown in CLL to contribute to leukemia cell survival. BMSC were treated with 0.3 picomolar anti-CXCL12 siRNA for 24 hr. As measured by quantitative RT-PCR, CXCL12 gene transcription was reduced by 80%. Moreover, there was a 53.5% reduction in ALL cell viability in ALL cells co-cultured with CXCL12 siRNA-treated stroma compared to ALL cells co-cultured with untreated stroma (p value< 0.001) (Figure 1). [Display omitted] ConclusionsThis BMSC cell/primary ALL cell system can be used to identify stromal cell genes that prevent apoptosis of primary ALL cells. The simple system can be scaled up to allow high throughput screening of candidate genes that have antagonistic or additive effects to maintenance chemotherapy drugs. Disclosures:No relevant conflicts of interest to declare.

Erythropoietin Gene Expression in the Stromal Cell Increased by Silica to Induce Erythrocyte Differentiation

Silica containing materials are often applied in bone tissue engineering, which may contact with bone marrow cells. However, the biological effects have not always been observed in studies of bone marrow cells exposed to silica. In this experiment, the relevant biological effects were evaluated. Bone marrow cells and stromal cells treated with silica particles (0.5-10 μm) were applied to investigate the possible interaction mechanism. HEL-92 cells were culture with the condition medium of stromal cells treated with or without silica particles. The erythrogenesis of bone marrow cells treated with silica particles was increased significantly. The expression level of glycophorin A the erythroid marker in HEL-92 cells treated by condition medium was higher than control group. The silica particles could also up-regulate the erythropoietin gene expression of stromal cells. The results indicate that bone marrow cells can be stimulated by silica particles to differentiate into erythrocytes. Our results suggest that silica particles can stimulate bone marrow cells to differentiate erythrocytes possibly via enhancing gene expression of erythropoietin.

Abstract 4277: Comparative gene expression analysis of proliferating stromal cells from pancreatic ductal adenocarcinoma, pancreatitis and normal pancreas

Abstract Introduction: Stromal cells associated with cancer cells are considered an important component of tumor microenvironment of pancreatic cancer. The intratumoral desmoplasia characteristic of pancreatic cancer is a result of growing carcinoma paracrine action on surrounding normal tissue cells. Selective targeting of tumor stroma cells can markedly increase the effectiveness of currently used chemotherapeutic agents against pancreatic cancer. Therefore, a search for new targets in stromal cells is an important part of developing new combined therapies of this disease. Our work was aimed at comparative studying of gene expression in stromal cells of normal pancreas, pancreatitis and pancreatic ductal adenocarcinoma tissues. Methods: Cultured stromal cells were obtained from samples of normal pancreatic (n=3), pancreatitis (n=3) and ductal adenocarcinoma tissues (n=4). The cell cultures obtained were preliminary characterized by immunofluorescence, RT-PCR and western blotting. A full genome gene expression analysis was performed using a modified SAGE technique. Expression of selected dysregulated genes was analyzed by quantitative PCR. Results: Morphological and immunofluorescent analyses of the obtained primary stromal cultures indicated that they could be assigned to pancreatic stellate cells (PSC). The SAGE analysis allowed to quantitatively estimate expression of 9860 genes in normal PSC, 8744 PSC genes in pancreatitis, and 9311 genes in tumor PSC. A comparison of normal and tumor PSC revealed statistically significant (P&amp;lt;0.001) differences in expression of 270 genes. Of them, 146 genes were upregulated and 124 genes downregulated in tumor PSC as compared to normal PSC. An expression analysis of selected dysregulated genes by RT-PCR detected changes in the activity of some genes involved in tumor progression of pancreatic cancer. In particular, the genes of two chemokines (CXCL12 and CX3CL1) and the JAG1 Notch ligand were upregulated in tumor PSC. A similar comparison of tumor PSC and PSC from pancreatitis tissue detected 68 differentially expressed genes (P&amp;lt;0.001), 40 of which were upregulated and 28 downregulated in tumor PSC as compared to pancreatitis PSC. Expression of 23 genes was upregulated in tumor PSC as compared with PSC both from pancreas and pancreatitis. On the whole, the expression profile of tumor PSC was markedly more similar to that of PSC from pancreatitis tissues than from normal PSC. Conclusions: The results obtained in our work revealed statistically significant changes in the expression of some PSC genes in stroma of ductal pancreatic adenocarcinoma as compared to normal PSC. The similarity of tumor and pancreatitis PSC expression profiles may suggest mutual mechanisms of forming the fibrous tissue of pancreatitis and tumor stroma of pancreatic carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4277. doi:1538-7445.AM2012-4277

Monitoring mmp-9 gene expression in stromal cells using a novel transgenic mouse model.

Matrix metalloproteinase (MMP)-9 (gelatinase B) is involved in extracellular matrix degradation in the context of the motility and in in vivo migration of normal and malignant cells. Accordingly, its expression is highly regulated at the transcriptional level. In several types of human cancers, MMP-9 expression is abnormally elevated and has been associated with poor prognosis. Such high levels of MMP-9 expression are found in tumor cells and in stromal components. Therefore, it is important to understand the spatiotemporal expression pattern of MMP-9 in tissues for the development of effective therapeutic strategies that are aimed at suppressing mmp-9 gene activation. In the present work, we describe a transgenic mouse model harboring a luciferase gene under the control of the murine mmp-9 promoter. We found that the expression pattern of the transgene was similar to that of the endogenous mmp-9 gene either constitutively or following inflammatory stimuli. A constitutive transgene expression was observed in the bone marrow, consistent with the observed high levels of endogenous mmp-9 gene expression normally found in the bone. LPS injection in mice also induced a consistent and significant increase in bioluminescent signals in the liver, which is a major target of LPS-induced septic shock. Finally, we further used the model to provide evidence that mmp-9 is activated in stromal cells of the lung and spleen in melanoma tumor-bearing mice. This bioluminescent imaging model may facilitate in vivo monitoring of MMP-9 activation in stromal cells in tumor progression and inflammatory diseases.

Abstract 2406: Molecular characterization of ciclooxigenase-2 in canine proliferative inflammatory atrophy of the prostate

Abstract Due to the importance and incidence of prostatic carcinoma in man, some animals are being used as a model in the investigation of the pathogenesis and development of this lesion. Similarly to men, the dog presents a spontaneous development of prostate cancer, which is locally invasive and can develop bone metastasis. Proliferative inflammatory atrophy (PIA) is considered a pre-neoplastic lesion in men. Overexpression of COX-2 has been associated with PIA, PIN and prostatic carcinoma playing a role in angiogenesis, apoptosis inhibition, increasing of invasive capacity, metastasis and inflammation in different prostatic affections. The aim of this study was to assess the protein and gene expression of COX-2 in laser capture microdissected stroma and epithelial cells of PIA foci in canine prostate by immunohistochemistry (IHC) and qRT-PCR. For IHC, 38 PIA lesions arranged in a tissue microarray were evaluated. Frozen fragments from 17 PIA and 3 normal prostatic tissues were used for COX2 gene expression. Our results showed increased COX2 gene expression in stromal cells compared to epithelial cells. High level of COX-2 protein expression was observed in 87% of PIA lesions, despite epithelial or stromal compartment. The In the present study, the stroma presented an important role, since the COX-2 gene expression in canine PIA lesions was increased in this cell compartment. To the best of our knowledge, this is the first report of COX2 gene expression in canine PIA. Since high levels of COX-2 have been associated to the risk of developing of prostate cancer in man, dog can be used as a natural model for this disease. The stroma can act as paracrine way in the epithelial cells, stimulating epithelial proliferation, reducing the apoptosis of this compartment and promoting angiogenesis in the microenvironment, factor that contribute to prostatic carcinogenesis and that the protein expression is increased in this lesion. Sponcer: Fapesp (Fundação de Apoio a Pesqusia do Estado de São Paulo) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2406. doi:10.1158/1538-7445.AM2011-2406

Metformin modulates IL-8, IL-1β, ICAM and IGFBP-1 expression in human endometrial stromal cells

To evaluate the effects of metformin on endometrial stromal cell gene expression and on the decidualization process, endometrial biopsies were collected from five healthy, regularly cycling women. Stromal cell culture was performed and decidualized with oestrogen/progesterone in the presence or absence of metformin and thereafter stimulated with insulin. The effect of metformin on decidualization was analysed by prolactin determination in the cell culture supernatant. Gene expression of insulin-like growth factor binding protein 1 (IGFBP-1), interleukin (IL) 8 and 1β and intercellular adhesion molecule (ICAM) was analysed by real-time PCR. Decidualization was significantly diminished in cells incubated with metformin ( P < 0.05) accompanied by a significant reduction of prolactin secretion in the supernatant (day 10: 2.2 fold, P < 0.05; day 15: 3.1 fold, P < 0.05). IGFBP-1 gene expression was reduced after long-term metformin exposure (7.7 fold, P < 0.05). The negative effect of insulin on IL-8 (4.8 fold) and IL-1β (9.3 fold) gene expression was similarly found in cells incubated with metformin. As far as is known, this is the first demonstration of a change in endometrial gene and protein expression after in-vitro stimulation with metformin, including a diminished decidualization process and changes in genes relevant to implantation. This study evaluated the effects of metformin (an antidiabetic drug) on cells of the inner lining of the uterus (stromal cells) and their process of transformation, called decidualization, as well as their gene expression during implantation. Stromal cell culture was performed and cells were stimulated with oestrogen/progesterone in the presence or absence of metformin. Effects of metformin on decidualization were analysed by prolactin, a decidualization marker, in stromal cell culture supernatant. Furthermore, gene expression of insulin-like growth factor binding protein 1 (IGFBP1), interleukin (IL) 8 and 1β and intercellular adhesion molecule (ICAM) before and after insulin stimulation was analysed by quantitative real-time polymerase chain reaction. Over time, decidualization was significantly diminished in cells incubated with metformin leading to a 3.1 fold reduction after 15 days ( P < 0.05). IGFBP-1 gene expression was reduced after long-term metformin exposure (7.7 fold, P < 0.05). Looking at implantation markers, 48-h metformin incubation increases IL-8 gene expression (31.1 fold, P = 0.059) and IL-1β gene expression (8.2 fold, P < 0.05), while it does not change ICAM gene expression. The negative effect of insulin on IL-8 (4.8 fold) and IL-1β (9.3 fold) gene expression was similarly found in cells incubated with metformin. We were able to show for the first time a change in endometrial gene and protein expression after in-vitro stimulation with metformin, including a diminished decidualization process and changes in genes relevant to implantation. This may potentially affect the rate of implantation as well as pregnancy outcome in women under metformin treatment.

Biophysical and chemical effects of fibrin on mesenchymal stromal cell gene expression

Mesenchymal stromal cells (MSCs) are multipotent cells that have high expansion yields and fibrin is a native extracellular matrix (ECM) material widely used for cell delivery and surgery. MSCs and fibrin have tremendous potential for tissue engineering applications, but the effect of fibrin on MSCs is not well characterized. The purpose of this study was to analyze the role of fibrin in modulating MSC phenotype by gene expression analysis. The results demonstrate that fibrin up-regulated MSC gene expression of vasculogenic (FLK1, ACTA2, VECAD, SM22 and CNN1), myogenic (MYF5 and MYH13), neurogenic (TH and GFAP) and chondrogenic (COL2A1) markers after 5 days incubation. These gene expression results were supported by induction of expression on the protein level for early lineage-specific markers such as ACTA2, FLK1 and MYF5. The ability of fibrin to modulate MSC gene expression was not affected by matrix pore size (80-110 microm diameter) or Young's modulus (5-25k Pa) and the differential expression of some phenotypic markers could be partially mimicked by other ECM proteins, such as fibronectin and collagen I. In some cases the inductive effect of fibrin on gene expression could be further augmented by the treatment with growth factors such as nerve growth factor. However, the effect of fibrin appeared to be limited, as MSCs did not differentiate into fully mature cells based on immunofluorescence staining after 12 days. This body of work provides a rational approach for studying the interactions of MSC with fibrin, which has important therapeutic implications for the delivery of stem cells.

Analysis of gene expression in prostate cancer epithelial and interstitial stromal cells using laser capture microdissection

BackgroundThe prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM).MethodsLCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry.ResultsThe two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues.ConclusionsThe prostate represents a complex mix of cell types and there is a need to analyze distinct cell populations to better understand their potential interactions. In the present study, LCM and microarray analysis were used to identify novel gene expression patterns in prostate cell populations, including identification of WT1 expression in epithelial cells. The relevance of WT1 expression in prostate cancer was confirmed by analysis of tumor tissue and cell lines, suggesting a potential role for WT1 in prostate tumorigenesis.

1001 Stromal cell gene expression changes by secreted factors from cancer cells

1001 Stromal cell gene expression changes by secreted factors from cancer cells

CONVERSION OF PROSTATE STROMAL CELL GENE EXPRESSION BY EMBRYONAL CARCINOMA FACTORS TO THAT OF TUMOR-ASSOCIATED STROMAL CELLS

You have accessJournal of Urology1 Apr 2009CONVERSION OF PROSTATE STROMAL CELL GENE EXPRESSION BY EMBRYONAL CARCINOMA FACTORS TO THAT OF TUMOR-ASSOCIATED STROMAL CELLS Eneida F Vencio, Laura E Pascal, Kai Wang, and Alvin Y Liu Eneida F VencioEneida F Vencio More articles by this author , Laura E PascalLaura E Pascal More articles by this author , Kai WangKai Wang More articles by this author , and Alvin Y LiuAlvin Y Liu More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(09)60130-0AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "CONVERSION OF PROSTATE STROMAL CELL GENE EXPRESSION BY EMBRYONAL CARCINOMA FACTORS TO THAT OF TUMOR-ASSOCIATED STROMAL CELLS." The Journal of Urology, 181(4S), p. 42 © 2009 by American Urological AssociationFiguresReferencesRelatedDetails Volume 181Issue 4SApril 2009Page: 42 Advertisement Copyright & Permissions© 2009 by American Urological AssociationMetricsAuthor Information Eneida F Vencio More articles by this author Laura E Pascal More articles by this author Kai Wang More articles by this author Alvin Y Liu More articles by this author Expand All Advertisement PDF downloadLoading ...

Characteristic gene expression in stromal cells of gastric cancers among atomic‐bomb survivors

To elucidate the mechanism of radiation-induced cancers, molecular analysis of cancers in atomic-bomb survivors is important. In our study, we developed a custom oligonucleotide array of 208 genes. We analyzed gene expression profiles of gastric cancers (GCs) from atomic-bomb survivors and identified 9 genes with significantly lower expression in GCs from exposed patients than in GCs from nonexposed patients. Among these 9 genes, expression of versican and osteonectin was investigated in greater detail using immunohistochemistry in 116 GCs from 64 exposed and 52 nonexposed patients who developed GC after the bombing. In the Stage I/II GCs, the clinicopathologic, phenotypic and proliferative characteristics of GCs from exposed and nonexposed patients did not differ significantly; however, versican and osteonectin were expressed at much lower levels in the area of tumor-associated stroma of exposed patients than in nonexposed patients (p = 0.026 and p = 0.024, respectively). These results suggest that the characteristics of tumor-associated stromal cells differ between GCs from exposed and nonexposed patients.

ID: 281 An in vitro model for tumor cell - stromal cell crosstalk in ovarian cancer. Induction of uPA gene expression in stromal cells involves multiple signaling molecules, and is augmented by a 3D extra-cellular matrix.

ID: 281 An in vitro model for tumor cell - stromal cell crosstalk in ovarian cancer. Induction of uPA gene expression in stromal cells involves multiple signaling molecules, and is augmented by a 3D extra-cellular matrix.

Gene Expression Profiling of Human Endometrial-Trophoblast Interaction in a Coculture Model

Investigating the interaction of human endometrium and trophoblast during implantation is difficult in vitro and impossible in vivo. This study was designed to analyze the effect of trophoblast on endometrial stromal cells during implantation by comprehensive gene profiling. An in vitro coculture system of endometrial stromal cells with first-trimester trophoblast explants was established. Trophoblast and endometrial stromal cells were separated after 24 h. Gene expression of endometrial stromal cells after coculture was compared with the gene expression of endometrial stromal cells cultured alone by microarray analysis. We confirmed the expression of distinct genes using real-time PCR. Genes up-regulated included those for inflammatory response, immune response, and chemotaxis (pentraxin-related gene 3, chemokine ligands, IL-8, IL-1 receptors, IL-18 receptor, IL-15, IL-15 receptor, TNF-alpha-induced protein 6, and IL-6 signal transducer), regulators of cell growth (IGF-binding proteins 1 and 2) and signal transduction. Also up-regulated were genes for growth and development, glucose metabolism, and lipid metabolism: DKK-1, WISP, IGF-II, hydroxysteroid 11beta-dehydrogenase 1, hydroxyprostaglandin dehydrogenase 15, prostaglandin E synthase, prostaglandin F receptor, aldehyde dehydrogenase 1 family, member A3 and phosphatidic acid phosphatase type 2B. Other genes included genes for cell-cell signaling (pre-B-cell colony-enhancing factor 1), proteolysis, calcium ion binding, regulation of transcription, and others. Down-regulated genes included genes for proteolysis (MMP-11 and mitochondrial intermediate peptidase), genes for cell death (caspase 6, death-associated protein kinase 1, and histone deacetylase 5), transcription factors (sex determining region Y-box 4, dachshund homolog 1, ets variant gene 1, and zinc finger protein 84 and 435), and genes for humoral immune response (CD24 antigen). Trophoblast has a significant impact on endometrial stromal cell gene expression. Some of the genes regulated by trophoblast in endometrial stromal cells are already known to be regulated by progesterone and show the endocrine function of trophoblast during pregnancy. Others are genes so far unknown to play a role in endometrial-trophoblast interaction and open a wide field of investigation.