Ultrastructural localization of extracellular matrix proteins of the lymph node cortex: evidence supporting the reticular network as a pathway for lymphocyte migration

<p>Abstract</p> <p>Background</p> <p>The lymph node (LN) is a crossroads of blood and lymphatic vessels allowing circulating lymphocytes to efficiently recognize foreign molecules displayed on antigen presenting cells. Increasing evidence indicates that after crossing high endothelial venules, lymphocytes migrate within the node along the reticular network (RN), a scaffold of fibers enwrapped by fibroblastic reticular cells (FRC). Light microscopy has shown that the RN contains specific extracellular matrix (ECM) proteins, which are putative molecular "footholds" for migration, and are known ligands for lymphocyte integrin adhesion receptors.</p> <p>Results</p> <p>To investigate whether ECM proteins of the RN are present on the outer surface of the FRC and are thus accessible to migrating lymphocytes, ultrastructural immunohistochemical staining of cynomolgus monkey LN was performed using antibodies to human ECM proteins that were successfully employed at the light microscopic level. The fibrillar collagens I and III were observed primarily within the reticular network fibers themselves. In contrast, the matrix proteins laminin, fibronectin, collagen IV, and tenascin were observed within the reticular fibers and also on the outer membrane surface of the FRC.</p> <p>Conclusions</p> <p>These findings suggest a molecular basis for how the RN functions as a pathway for lymphocyte migration within the lymph node.</p

Expression profiling of human renal carcinomas with functional taxonomic analysis

BACKGROUND: Molecular characterization has contributed to the understanding of the inception, progression, treatment and prognosis of cancer. Nucleic acid array-based technologies extend molecular characterization of tumors to thousands of gene products. To effectively discriminate between tumor sub-types, reliable laboratory techniques and analytic methods are required. RESULTS: We derived mRNA expression profiles from 21 human tissue samples (eight normal kidneys and 13 kidney tumors) and two pooled samples using the Affymetrix GeneChip platform. A panel of ten clustering algorithms combined with four data pre-processing methods identified a consensus cluster dendrogram in 18 of 40 analyses and of these 16 used a logarithmic transformation. Within the consensus dendrogram the expression profiles of the samples grouped according to tissue type; clear cell and chromophobe carcinomas displayed distinctly different gene expression patterns. By using a rigorous statistical selection based method we identified 355 genes that showed significant (p < 0.001) gene expression changes in clear cell renal carcinomas compared to normal kidney. These genes were classified with a tool to conceptualize expression patterns called "Functional Taxonomy". Each tumor type had a distinct "signature," with a high number of genes in the categories of Metabolism, Signal Transduction, and Cellular and Matrix Organization and Adhesion. CONCLUSIONS: Affymetrix GeneChip profiling differentiated clear cell and chromophobe carcinomas from one another and from normal kidney cortex. Clustering methods that used logarithmic transformation of data sets produced dendrograms consistent with the sample biology. Functional taxonomy provided a practical approach to the interpretation of gene expression data

Acquisition of mitochondrial dysregulation and resistance to mitochondrial-mediated apoptosis after genotoxic insult in normal human fibroblasts: A possible model for early stage carcinogenesis

AbstractAcquisition of death-resistance is critical in the evolution of neoplasia. Our aim was to model the early stages of carcinogenesis by examining intracellular alterations in cells that have acquired apoptosis-resistance after exposure to a complex genotoxin. We previously generated sub-populations of BJ-hTERT human diploid fibroblasts, which have acquired death-resistance following exposure to hexavalent chromium [Cr(VI)], a broad-spectrum genotoxicant. Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis. Here, we report on the death-sensitivity of subclonal populations derived from clonogenic survivors of BJ-hTERT cells treated with 5μM Cr(VI) (DR1, DR2), or selected by dilution-based cloning without treatment (CC1). Following Cr(VI) treatment, CC1 cells downregulated expression of the anti-apoptotic protein Bcl-2 and exhibited extensive expression of cleaved caspase 3. In contrast, the DR cells exhibited no cleaved caspase 3 expression and maintained expression of Bcl-2 following recovery from 24h Cr(VI) exposure. The DR cells also exhibited attenuated mitochondrial-membrane depolarization and mitochondrial retention of cytochrome c and SMAC/DIABLO following Cr(VI) exposure. The DR cells exhibited less basal mtDNA damage, as compared to CC1 cells, which correlates with intrinsic (non-induced) death-resistance. Notably, there was no difference in p53 protein expression before or after treatment among all cell lines. Taken together, our data suggest the presence of more resilient mitochondria in death-resistant cells, and that death-resistance can be acquired in normal human cells early after genotoxin exposure. We postulate that resistance to mitochondrial-mediated cell death and mitochondrial dysregulation may be an initial phenotypic alteration observed in early stage carcinogenesis

The influence of tumor size and environment on gene expression in commonly used human tumor lines

BACKGROUND: The expression profiles of solid tumor models in rodents have been only minimally studied despite their extensive use to develop anticancer agents. We have applied RNA expression profiling using Affymetrix U95A GeneChips to address fundamental biological questions about human tumor lines. METHODS: To determine whether gene expression changed significantly as a tumor increased in size, we analyzed samples from two human colon carcinoma lines (Colo205 and HCT-116) at three different sizes (200 mg, 500 mg and 1000 mg). To investigate whether gene expression was influenced by the strain of mouse, tumor samples isolated from C.B-17 SCID and Nu/Nu mice were also compared. Finally, the gene expression differences between tissue culture and in vivo samples were investigated by comparing profiles from lines grown in both environments. RESULTS: Multidimensional scaling and analysis of variance demonstrated that the tumor lines were dramatically different from each other and that gene expression remained constant as the tumors increased in size. Statistical analysis revealed that 63 genes were differentially expressed due to the strain of mouse the tumor was grown in but the function of the encoded proteins did not link to any distinct biological pathways. Hierarchical clustering of tissue culture and xenograft samples demonstrated that for each individual tumor line, the in vivo and in vitro profiles were more similar to each other than any other profile. We identified 36 genes with a pattern of high expression in xenograft samples that encoded proteins involved in extracellular matrix, cell surface receptors and transcription factors. An additional 17 genes were identified with a pattern of high expression in tissue culture samples and encoded proteins involved in cell division, cell cycle and RNA production. CONCLUSIONS: The environment a tumor line is grown in can have a significant effect on gene expression but tumor size has little or no effect for subcutaneously grown solid tumors. Furthermore, an individual tumor line has an RNA expression pattern that clearly defines it from other lines even when grown in different environments. This could be used as a quality control tool for preclinical oncology studies

Upregulation of Poly (ADP-Ribose) Polymerase-1 (PARP1) in Triple-Negative Breast Cancer and Other Primary Human Tumor Types

Poly (ADP-ribose) polymerase-1 (PARP1) is a key facilitator of DNA repair and is implicated in pathways of tumorigenesis. PARP inhibitors have gained recent attention as rationally designed therapeutics for the treatment of several malignancies, particularly those associated with dysfunctional DNA repair pathways, including triple-negative breast cancer (TNBC). We investigated the PARP1 gene expression profile in surgical samples from more than 8,000 primary malignant and normal human tissues. PARP1 expression was found to be significantly increased in several malignant tissues, including those isolated from patients with breast, uterine, lung, ovarian, and skin cancers, and non-Hodgkin’s lymphoma. Within breast infiltrating ductal carcinoma (IDC) samples tested, mean PARP1 expression was significantly higher relative to normal breast tissue, with over 30% of IDC samples demonstrating upregulation of PARP1, compared with 2.9% of normal tissues. Because of known DNA repair defects, including BRCA1 dysfunction, associated with TNBC, exploration of PARP1 expression in breast cancers related to expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) led to the observation that negative expression of any of the 3 receptors was associated with upregulation of PARP1 expression, compared with receptor-positive tissues. To validate these observations, an independent set of breast adenocarcinomas was evaluated and demonstrated >2-fold upregulation of PARP1 in approximately 70% of primary breast adenocarcinomas, including TNBC, compared with syngeneic nonmalignant breast tissues. Immunohistochemistry (IHC) showed that upregulation of the PARP1 gene was consistent with increased protein expression in TNBC. These analyses suggest a potential biological role for PARP1 in several distinct malignancies, including TNBC. Further investigation of PARP1 as a biomarker for the therapeutic activity of PARP inhibitor-based therapy is warranted

Hierarchical clustering analysis showing the structure within the data of the 13 tissue culture samples (suffix – TC) and 8 xenograft samples (suffix – X)

<p><b>Copyright information:</b></p><p>Taken from "The influence of tumor size and environment on gene expression in commonly used human tumor lines"</p><p>BMC Cancer 2004;4():35-35.</p><p>Published online 15 Jul 2004</p><p>PMCID:PMC493269.</p><p>Copyright © 2004 Gieseg et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Samples are displayed vertically, genes are displayed horizontally. A dendrogram of relatedness of the samples is at the top in green. For any two samples, the vertical distance from the sample roots to the first node joining them is a measure of their similarity; the shorter the distance the more similar. The color in each cell of the table represents the median adjusted expression value of each gene. The color scale used to represent the expression ratios is shown on the right, with yellow indicating increased expression relative to the median and blue decreased

Circulating tumor cell investigation in breast cancer patient-derived xenograft models by automated immunofluorescence staining, image acquisition, and single cell retrieval and analysis

Abstract Background Breast cancer patient-derived xenograft (BC-PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs) for studying their role in tumor biology and metastasis. We have previously shown the utility of BC-PDX models in the study of CTCs by immunohistochemistry (IHC) on serial paraffin sections and manual microscopic identification of cytokeratin-positive cells, a method that is both low-throughput and labor-intensive. We therefore aimed to identify and characterize CTCs from small volume mouse blood samples and examined its practical workflow in a study of BC-PDX mice treated with chemotherapy using an automated imaging platform, the AccuCyte®–CyteFinder® system. Methods CTC analysis was conducted using blood from non-tumor bearing SCID/Beige mice spiked with human breast cancer cells, BC-PDX-bearing mice, and BC-PDX mice treated with vehicle or chemotherapeutic agent(s). After red blood cell lysis, nucleated cells were mixed with transfer solution, processed onto microscope slides, and stained by immunofluorescence. The CyteFinder automated scanning microscope was used to identify CTCs, defined as nucleated cells that were human cytokeratin-positive, and mouse CD45-negative. Disaggregated primary BC-PDX tumors and lung metastatic nodules were processed using the same immunostaining protocol. Collective expression of breast cancer cell surface markers (EpCAM, EGFR, and HER2) using a cocktail of target-specific antibodies was assessed. CTCs and disaggregated tumor cells were individually retrieved from slides using the CytePicker® module for sequence analysis of a BC-PDX tumor-specific PIK3CA mutation. Results The recovery rate of human cancer cells spiked into murine blood was 83 ± 12%. CTC detection was not significantly different from the IHC method. One-third of CTCs did not stain positive for cell surface markers. A PIK3CA T1035A mutation present in a BC-PDX tumor was confirmed in isolated single CTCs and cells from dissociated metastatic nodules after whole genome amplification and sequencing. CTC evaluation could be simply implemented into a preclinical PDX therapeutic study setting with substantial improvements in workflow over the IHC method. Conclusions Analysis of small volume blood samples from BC-PDX-bearing mice using the AccuCyte–CyteFinder system allows investigation of the role of CTCs in tumor biology and metastasis independent of surface marker expression