CD4/CD8 co-expression shows independent prognostic impact in resected non-small cell lung cancer patients treated with adjuvant radiotherapy
Sigurd M. HaldRoy M. BremnesKhalid Al‐ShibliSamer Al‐SaadSigve AndersenHelge StenvoldLill‐Tove BusundTom Dønnem
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Stromal-epithelial cell interaction is very important for the development of human benign prostatic hyperplasia (BPH). Growth factors and their receptors in the prostate are thought to mediate the cell communication and play some roles in the development of BPH. Among many growth factors, fibroblast growth factor (FGF) family members have received the most intensive study, and mRNAs for acidic FGF (aFGF), basic FGF (bFGF) and keratinocyte growth factor (KGF) have been identified in rat or human prostate. However, synthesis sites and roles of them in stromal-epithelial cell interaction remain to be undefined. In the present study, to define the mechanisms for the regulation of prostatic cell growth by stromal cells in human BPH, we established the method for isolation and culture of epithelial cells as well as stromal cells from human BPH tissue. Using these primary cultured prostatic cells, we evaluated the effects of stromal cell conditioned medium (SCM) and stromal cell extract (SCE) on the growth of stromal cells and epithelial cells by [3H]-thymidine incorporation assay. We also examined the expression of mRNAs for aFGF, bFGF, KGF, FGF receptor 1 (FGFR1) and FGFR2 in epithelial and stromal cells using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis. As the results, both SCM and SCE stimulated the growth of stromal cells, and the growth promoting effects of them to stromal cells were completely suppressed by anti-bFGF neutralizing antibody, but not by anti-aFGF neutralizing antibody at all. SCM also stimulated the growth of epithelial cells. The growth promoting effect of it to epithelial cells was not completely suppressed by anti-bFGF neutralizing antibody, and not by anti-aFGF neutralizing antibody at all. Furthermore, RT-PCR analysis demonstrated the expression of mRNAs for bFGF, KGF and FGFR1 in stromal cells and that of FGFR2 in epithelial cells. These findings suggest that bFGF produced by stromal cells acts on stromal cells through FGFR1 by an autocrine mechanism, KGF produced by stromal cells acts on epithelial cells through FGFR2 by a paracrine manner in human BPH. These mechanisms for the regulation of cell growth by stromal cells were thought to contribute to the development of human BPH.
Keratinocyte growth factor
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Objective To search a simple making mothd of tissue microarray and evaluate its validation. Methods A new, simple and easy method was designed to improve the technology of TMA during the experiment. Tissue microarrays with 1.6 mm in diameter tissue core were constructed from 124 cases of breast cancer. The tissue microarrays were stained for oestrogen receptor(ER),progesterone receptor(PR)and c-erBb2 using immunohistochemistry.At the same time the stains on the tissue microarrays were compared with that from the full tissue sections. Results A highly significant association was observed between the staining scores(0 ～ 7)obtained from the full tissue sections and from the tissue microarrays. Concordance for the receptor status(positive / negative)of the whole section and the tissue core were 94.35% for ER,93.55% for PR.and 91.94% for c-erBb2.The discordance between full section and tissue microarrays was studied using Two - Related - Samples tests and the result had no statistical significance(P0.05) . Conclusion Tissue microarays with 1.6 mm in diameter tissue core made by simple method can represent full tissue section on immunohistochemistry study.We believe our method of tissue microarray is a reliable and valid
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Oestrogen receptor
Breast tissue
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To explore the biological effect of prostate peripheral zones (PZs) stromal cells on the proliferation of prostate cells by overexpression of LMO2 gene.Genes expressional distinction of different prostate stromal cells was screened by gene expression arrays. To validate the microarray data, real time-polymerase chain reaction (RT-PCR), Western blotting analysis were used to check the over expression of LMO2 in PZs cells.To compare the effect of stromal cells which overexpressed LMO2 gene on in vitro proliferation ability of BPH-1 and PC3 cell lines, cell proliferation was measured by CCK-8 and EdU assay. Cytokines chip was used to screen expression of cytokines in WPMY-1-LMO2 conditioned medium. The changes of BPH-1 and PC3 proliferation associated proteins were assessed by Western blotting.A total of 512 genes were identified as markedly differentially expressed in stromal cells originated from different zones. Among these genes, LMO2 gene was overexpression in peripheral zones stromal cells, and confirmed by RT-PCR and Western blotting. Expression level of LMO2 gene was significantly up-regulated in peripheral zones stromal cells compared with transitional zones stromal cells, increased by 3.36 folds on average (P<0.01). The proliferation of both PC3 and BPH-1 were found increased and STAT3 phosphorylation and CCND1 expression were increased after cultured in conditioned medium from stromal cells which stably expressed LMO2. Cytokines chip found increased FGF-9 and IL-11 expression in the medium supernatant reserved from LMO2-overexpressed stromal cell line.Distinct gene expression exists among prostate stromal cells originated from different zones. LMO2 overexpressed stromal cells can induce prostate epithelial cell growth via paracrine of FGF-9, IL-11 or other cytokines.
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Abstract Purpose There has been a resurgence of interest in the tumor stroma in recent years. Whether the relative abundance of various stromal cells can be used as a classification system for muscle-invasive bladder cancer (MIBC) remain elusive.Methods We applied single-cell RNA-sequence (scRNA-seq) data from two MIBCs to identify stromal cell (CD45 negative cells) clusters and the marker gene set of each cell cluster. The single sample gene set enrichment analysis method is used to estimate the relative abundance of the cell clusters in each MIBC sample from The Cancer Genome Atlas. Subsequently, k-means clustering was performed to cluster the MIBCs. Prognosis, oncogenic pathway enrichment score, epithelial-mesenchymal transition (EMT) score, gene mutation frequency, and tumor infiltrating lymphocytes (TILs) were compared among the stromal component-based types of MIBC.ResultsIn the scRNA-seq analysis, a total of nine cell clusters mainly composed of four types of cells were identified. Cell clusters 0, 1, 2, 3, 4, and 7 were considered as bladder epithelial cells, the cells in clusters 5 and 8 were mainly recognized as stem cells and fibroblasts, and the cell cluster 6 was recognized were endothelial cells. The 408 MIBC samples were classified into three types. Type 1 and 3, the “stromal-sufficient” type I and II with higher stromal cells, and Type 2, the “stromal-desert” type with lower stromal cells. The stromal-desert type had significantly better overall survival. As the tumor progresses, the stromal component of the stromal-desert type increases and change into the stromal-sufficient type. Increased stromal cells may come from EMT. The enrichment scores of multiple oncogenic pathways in stromal-sufficient types were significantly higher than those in stromal-desert type. More TILs were found in stromal-sufficient type, but their function may be inhibited by stromal cells. In addition, the three stromal types of MIBC may have specific gene mutation characteristics.Conclusions We proposed a novel stromal component-based classification system to divided into MIBC three phenotype. The three types of MIBC differ in various biological characteristics. The progress of MIBC may be summarized as the process of gradually increasing stromal components and constructing a microenvironment suitable for cancer cells.
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The immune status is important in cancer patients. Tissue microarrays from 249 patients with soft tissue sarcomas were constructed. Immunohistochemistry was used to evaluate the CD3+, CD4+, CD8+, CD20+ and CD45+ lymphocytes in tumors. High density of CD20+ lymphocytes is an independent positive prognostic indicator for these patients.
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Objective To evaluate the validation of tissue microarrays.Methods Tissue microarrays with 2 mm in diameter tissue core were constructed from seventy cases of breast cancer.The tissue microarrays were stained for oestrogen receptor(ER) and progesterone receptor(PR) using immunohistochemistry.At the same time the stains on the tissue microarrays were compared with that from the full tissue sections. Results A highly significant association was observed between the staining scores(0～7) obtained from the full tissue sections and from the tissue microarrays.Concordance for the receptor status(positive/negative) of the whole section and the tissue core were 96.67% for ER,93.93% for PR.The discordance between full section and tissue microarrays was studied using Two-Related-Samples tests and the result had no statistical significance(P0.05). Conclusion Tissue microarays with 2 mm in diameter tissue core can represent full tissue section on immunohistochemistry study,if the tissue microarrays are constructed as standard method.We believe tissue microarray is a reliable and highthroughout tool for research.
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Bone marrow stromal cells have well documented effects on the production of B lymphocytes, but whether or not stromal cell signals are involved in the pre-B to B cell transition is unclear. The potential of two stromal cell lines, S10 and S17, in this process was examined. Initial experiments, using a short term liquid culture, indicated that S10 and S17 stroma efficiently supported the generation of clonable B cells (B lymphocyte CFU) from their immediate precursors in fresh bone marrow. The contribution of macrophages and other accessory cells in those experiments was minimized through use of a colony assay system that permits the direct effects of stromal cell signals on single B cell progenitors to be evaluated. The results indicated that soluble mediators from the S10 and S17 lines could support colony formation from fresh or cultured surface Ig- bone marrow cells. Colonies supported by S17 stroma appeared on day 15 and contained cells that expressed the B220 Ag; surface IgM expression was never observed. S10 supported colonies appeared on day 7 and routinely included surface IgM+ cells. Individual colonies were capable of undergoing additional growth when picked and replated directly onto the different stroma. Those colonies replated onto S10 stroma generated surface IgM expressing cells in up to 60% of experiments, but colonies transferred onto the S17 cell line included B cells only 10% of the time. These data demonstrate that stromal cells alone can provide the signals necessary for generating a surface IgM+ B cell from precursors but that not all stromal cell lines are equally efficient at doing so.
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The effects of in vitro irradiation on proliferation and hematopoietic supportive functions of stromal cells were studied. To assess the effects of radiation on stromal cells proliferation, marrow cells were exposed to a single dose of gamma radiation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that stromal cell proliferation was significantly suppressed after radiation in a dose-dependent manner. Stromal layers obtained from irradiated marrow cells failed to establish adherent layers after 6, 8, or 10 Gy of radiation. To assess the functions of stromal cells that survived radiation, stromal layers derived from irradiated marrow cells were cocultured with freshly isolated autologous hematopoietic cells and assayed for their capacity to support prolonged granulocyte-macrophage progenitors (CFU-GM) production. Stromal layers derived from 2-Gy-irradiated marrow cells resulted in similar CFU-GM production as control cells, while stromal layers derived from 4- to 10-Gy-irradiated marrow cells significantly decreased CFU-GM production. To study the influence of radiation on hematopoietic supportive capacity in established stromal layers, stromal layers generated from non-irradiated marrow cells were irradiated and cocultured with freshly isolated autologous hematopoietic cells. Established stromal layers irradiated up to 10 Gy sustained prolonged CFU-GM production, suggesting that hematopoietic stromal supportive functions remained intact at this dose of radiation. In conclusion, our results indicated that proliferation of stromal cells and bone-marrow stromal layer formation from stromal cells are sensitive to radiation in vitro, while established bone-marrow stromal layer originating from stromal cells is relatively resistant to radiation. Data generated may have implications in future bone-marrow transplantation research.
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