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Frost, Andra R.; Sparks, Dierick; Grizzle, William E.

doi:10.1097/00022744-200009000-00011

Cited by 31 publications

(4 citation statements)

References 29 publications

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“…The immunostaining was semi-quantified using a scoring system that combines the number of cells or area stained and the intensity of the staining. This scoring system has been used by us and others previously [14–17]. Stroma surrounding histologically normal breast epithelium and within breast cancers was immunoscored.…”

Section: Resultsmentioning

confidence: 99%

“…Our methods of performing immunohistochemistry have been reported in the literature [14–17]. For estrogen receptor (ER) and Ki-67 staining, sections (5 μm thick) were subjected to low temperature antigen retrieval with enzymatic pretreatment, which consists of pre-digestion in 0.1% trypsin (Type II-S from porcine pancreas, Sigma Chemicals, St. Louis, MO) in phosphate buffered saline for 15 min in a 37°C oven followed by incubation in 10 mM citrate buffer, pH 6, for 0 h at 80°C, as previously described [14]. Sections for FBLN1 staining did not require antigen retrieval.…”

Section: Methodsmentioning

confidence: 99%

“…To semi-quantify FBLN1 immunostaining, a scoring system based on both staining intensity and percentage of cells or area stained was utilized, as previously described [14, 15, 17]. In this system, the intensity of staining is graded from 0 (no staining) to 4 (greatest staining possible).…”

Section: Methodsmentioning

confidence: 99%

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Identification of Molecular Distinctions Between Normal Breast-Associated Fibroblasts and Breast Cancer-Associated Fibroblasts

Sadlonova

Bowe

Novák

et al. 2009

Cancer Microenvironment

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Stromal fibroblasts influence the behavior of breast epithelial cells. Fibroblasts derived from normal breast (NAF) inhibit epithelial growth, whereas fibroblasts from breast carcinomas (CAF) have less growth inhibitory capacity and can promote epithelial growth. We sought to identify molecules that are differentially expressed in NAF versus CAF and potentially responsible for their different growth regulatory abilities. To determine the contribution of soluble molecules to fibroblast–epithelial interactions, NAF were grown in 3D, transwell or direct contact co-cultures with MCF10AT epithelial cells. NAF suppressed proliferation of MCF10AT in both direct contact and transwell co-cultures, but this suppression was significantly greater in direct co-cultures, indicating involvement of both soluble and contact factors. Gene expression profiling of early passage fibroblast cultures identified 420 genes that were differentially expressed in NAF versus CAF. Of the eight genes selected for validation by real-time PCR, FIBULIN 1, was overexpressed in NAF, and DICKKOPF 1, NEUREGULIN 1, PLASMINOGEN ACTIVATOR INHIBITOR 2, and TISSUE PLASMINOGEN ACTIVATOR were overexpressed in CAF. A higher expression of FIBULIN 1 in normal- than cancer-associated fibroblastic stroma was confirmed by immunohistochemistry of breast tissues. Among breast cancers, stromal expression of Fibulin 1 protein was higher in estrogen receptor α-positive cancers and low stromal expression of Fibulin 1 correlated with a higher proliferation of cancer epithelial cells. In conclusion, expression profiling of NAF and CAF cultures identified many genes with potential relevance to fibroblast–epithelial interactions in breast cancer. Furthermore, these early passage fibroblast cultures can be representative of gene expression in stromal fibroblasts in vivo.Electronic supplementary materialThe online version of this article (doi:10.1007/s12307-008-0017-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Identification of Molecular Distinctions Between Normal Breast-Associated Fibroblasts and Breast Cancer-Associated Fibroblasts

Sadlonova

Bowe

Novák

et al. 2009

Cancer Microenvironment

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“…Immunohistochemistry. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 4-6 m. For some immunohistochemistry, sections were pretreated in water in a microwave (19). A guinea pig polyclonal antibody to porcine pancreatic insulin (Dako) was used at a 1:500 dilution, and rabbit anti-human somatostatin, glucagon, and gastrin were all used at 1:1,000 dilution (Dako).…”

Section: Time Study͞autopsy͞blood Chemistry Heterozygous Men1mentioning

confidence: 99%

A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors

Crabtree

Scacheri

Ward

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

414

352

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Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome, characterized primarily by multiple tumors in the parathyroid glands, endocrine pancreas, and anterior pituitary. Other tumors, including gastrinoma, carcinoid, adrenal cortical tumors, angiofibroma, collagenoma, and lipoma, also occur in some patients. Individuals with MEN1 almost always have loss-of-function mutations in the MEN1 gene on chromosome 11, and endocrine tumors arising in these patients usually show somatic loss of the remaining wild-type allele. To examine the role of MEN1 in tumor formation, a mouse model was generated through homologous recombination of the mouse homolog Men1. Homozygous mice die in utero at embryonic days 11.5-12.5, whereas heterozygous mice develop features remarkably similar to those of the human disorder. As early as 9 months, pancreatic islets show a range of lesions from hyperplasia to insulin-producing islet cell tumors, and parathyroid adenomas are also frequently observed. Larger, more numerous tumors involving pancreatic islets, parathyroids, thyroid, adrenal cortex, and pituitary are seen by 16 months. All of the tumors tested to date show loss of the wild-type Men1 allele, further supporting its role as a tumor suppressor gene.M ultiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome characterized by multiple tumors of the parathyroid, endocrine pancreas, and the anterior pituitary. Additional tumors have been associated with MEN1, including foregut carcinoid, adrenal cortical tumors, angiofibroma, collagenoma, and lipoma (1, 2). Linkage studies in affected families mapped the MEN1 locus to chromosome 11q13 (3), and the responsible gene, MEN1, was identified by positional cloning in 1997 (4). Germline mutations in MEN1 have been identified in almost all MEN1 kindreds (5-7), and somatic mutations in MEN1 have been reported in sporadic parathyroid adenomas, pituitary tumors, insulinomas, gastrinomas, and lung carcinoids (8 -12). Over 70% of germline mutations are nonsense and frameshifts, predicting truncation or absence of the resulting protein.Missense mutations and in-frame deletions account for the remaining 30% of the almost 250 unique mutations identified to date. Hence, MEN1 appears to be a classic tumor suppressor gene with tumors in affected patients showing somatic loss of the wild-type allele.The MEN1 gene consists of 10 exons (the first of which is untranslated), spanning 7.2 kb of genomic sequence and encoding a protein of 610 amino acids. The protein product, menin, does not reveal homologies to any other known proteins or possess notable motifs from which the putative function of the protein could be deduced. Menin RNA and protein apparently are expressed in all tissues (13), leaving unexplained the basis for endocrine predominance of neoplasia. Menin is found in the nucleus (14) and binds to the AP1 transcription factor JunD (15). This finding suggests a role in transcriptional regulation, although a detailed model for menin tumor suppression ...

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Laser Microdissection

Frost

Eltoum

Siegal

et al. 2015

CP Molecular Biology

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Laser microdissection (LM) offers a relatively rapid and precise method of isolating and removing specified cells from complex tissues for subsequent analysis of their RNA, DNA, protein or metabolite content, thereby allowing assessment of the role of different cell types in the normal physiological or disease processes being studied. In this unit, protocols for the preparation of mammalian frozen tissues, fixed tissues, and cytologic specimens for LM, including tissue freezing, tissue processing and paraffin embedding, histologic sectioning, cell processing, hematoxylin and eosin staining, immunohistochemistry, and image-guided cell targeting are presented. Also provided are recipes for generating lysis buffers for the recovery of nucleic acids and proteins. The Commentary section addresses the types of specimens that can be utilized for LM and approaches to staining of specimens for cell visualization. Emphasis is placed on the preparation of tissue or cytologic specimens as this is critical to effective LM.

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Untitled

Cited by 31 publications

References 29 publications

Identification of Molecular Distinctions Between Normal Breast-Associated Fibroblasts and Breast Cancer-Associated Fibroblasts

Identification of Molecular Distinctions Between Normal Breast-Associated Fibroblasts and Breast Cancer-Associated Fibroblasts

A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors

Laser Microdissection

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