Protocol for constructing tissue arrays by cutting edge matrix assembly.

We present a protocol for construction of high-density tissue microarrays, cutting edge matrix assembly, which is based on repetitive sectioning and bonding of tissues. Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are bonded edge-to-edge, resulting in minimal wasted space between samples. Construction of the tissue array blocks from paraffin-embedded tissue involves initial bonding of primary tissue plates into multiple primary tissue stacks. This is achieved by taking a shaving of desired thickness from the face of each specimen block, trimming the shavings into a set of rectangular primary tissue plates, and bonding multiple plates into primary stacks of tissue. Each resulting primary tissue stack is then transversely cut to produce a set of secondary tissue plates that contains elements of each tissue represented in the primary stacks. Secondary plates from multiple primary sample stacks are then restacked and bonded into a secondary stack. The assembled secondary stack represents a laminate of laminates, which becomes the final array block. The final array block is then reembedded in paraffin and can be sectioned transversely using a microtome to yield micrometer thin sections that are transferred to glass slides for array display and analysis. This technology has facilitated the construction of arrays containing more than 10,000 tissue features on a standard glass slide.

Genetic ablation of caveolin-1 drives estrogen-hypersensitivity and the development of DCIS-like mammary lesions.

Caveolin-1 (Cav-1) loss-of-function mutations are exclusively associated with estrogen receptor-positive (ER(+)) human breast cancers. To dissect the role of Cav-1 loss-of-function in the pathogenesis of human breast cancers, we used Cav-1(-/-) null mice as a model system. First, we demonstrated that Cav-1(-/-) mammary epithelia overexpress two well-established ER co-activator genes, CAPER and Foxa1, in addition to ER-alpha. Thus, the functional loss of Cav-1 may be sufficient to confer estrogen-hypersensitivity in the mammary gland. To test this hypothesis directly, we subjected Cav-1(-/-) mice to ovariectomy and estrogen supplementation. As predicted, Cav-1(-/-) mammary glands were hyper-responsive to estrogen and developed dysplastic mammary lesions with adjacent stromal angiogenesis that resemble human ductal carcinoma in situ. Based on an extensive biomarker analysis, these Cav-1(-/-) mammary lesions contain cells that are hyperproliferative and stain positively with nucleolar (B23/nucleophosmin) and stem/progenitor cell markers (SPRR1A and beta-catenin). Genome-wide transcriptional profiling identified many estrogen-related genes that were over-expressed in Cav-1(-/-) mammary glands, including CAPER--an ER co-activator gene and putative stem/progenitor cell marker. Analysis of human breast cancer samples revealed that CAPER is overexpressed and undergoes a cytoplasmic-to-nuclear shift during the transition from pre-malignancy to ductal carcinoma in situ. Thus, Cav-1(-/-) null mice are a new preclinical model for studying the molecular paradigm of estrogen hypersensitivity and the development of estrogen-dependent ductal carcinoma in situ lesions.

Cyclin D1b is aberrantly regulated in response to therapeutic challenge and promotes resistance to estrogen antagonists.

Cyclin D1 is a key mediator of cell cycle progression that is aberrantly regulated in multiple cancers, especially in breast cancers. A number of studies have indicated that a polymorphism in a splice donor site in the cyclin D1 gene is associated with alternative splicing and the production of the alternative cyclin D1b transcript. Furthermore, this polymorphism is selectively associated with disease outcomes. However, relatively little is known regarding the protein product of the alternatively spliced message, cyclin D1b. Using antibodies specific for cyclin D1b, it was found that this protein is readily detectable in a number of cancer cell lines and primary breast cancers. Whereas cyclin D1b interacts with cyclin-dependent kinase 4 (CDK4), it is relatively inefficient at mediating RB phosphorylation and cell cycle progression in model systems due to the lack of exon 5 of cyclin D1-encoded sequences. However, cyclin D1b protein levels are not significantly attenuated by DNA damage or antiestrogen treatment, indicating that the protein may have significant effect on the response to such therapeutic modalities. Whereas enforced expression of cyclin D1b was not sufficient to abrogate DNA damage checkpoint responses, it did efficiently overcome cell cycle arrest mediated by antiestrogen therapeutics. This action of cyclin D1b was not associated with effects on estrogen receptor activity, but was rather dependent on functional association with CDK4. Combined, these studies indicate that the cyclin D1b protein is aberrantly regulated and could contribute to therapeutic failure in the context of ER-positive breast cancer.