Next-generation Sequencing

A Powerful Tool for the Discovery of Molecular Markers in Breast Ductal Carcinoma In Situ

Hitchintan Kaur; Shihong Mao; Seema Shah; David H Gorski; Stephen A Krawetz; Bonnie F Sloane; Raymond R Mattingly

Disclosures

Expert Rev Mol Diagn. 2013;13(2):151-165. 

In This Article

Experimental Models of DCIS

Comprehensive molecular analyses of DCIS have been limited in part by the small size of DCIS lesions and thus tissue available for study, as well as the few experimental model systems that recapitulate human disease.[24] Xenograft models in which human DCIS cells are directly injected into mammary fat pads or the flank of athymic nude mice are available.[25] These xenografts are amenable to hormonal manipulation and have been widely used to study the effects of estrogen and antiestrogen therapies on epithelial cell proliferation.[26,27] These models can be used for screening of chemopreventive or chemotherapeutic agents, and for the analysis of gene expression and signaling pathways. Despite their advantages, these xenograft models, when used to study progression to invasive cancer, do not fully recapitulate the complexity associated with the DCIS lesions. Even the humanized fat-pad transplantation models do not represent the true intraductal microenvironment. Nevertheless, subcutaneous implantation of MCF10.DCIS cells produces lesions that recapitulate human high-grade comedo DCIS by both histological and molecular markers.[28] Efforts are ongoing to develop better models of disease progression that represent the diversity of DCIS lesions within the context of innate surroundings. In this regard, a human in mouse intraductal (MIND) transplantation model has been developed using human DCIS cell lines such as MCF10.DCIS and SUM225, as well as primary human DCIS cells (FSK-H7).[29] The MIND xenografts maintain histology and biomarker expression comparable to the original patient samples.[30] These models reflect some of the diversity of DCIS lesions because the MCF10.DCIS line represents basal-like DCIS, whereas SUM225 and FSK-H7 derive from Her-2 overexpressing DCIS. The MIND system utilizes transplantation into the mammary ducts of immunocompromised NSG (NOD-SCID IL-2Rγ null) mice for primary human cells. A major limitation of all xenograft models for DCIS to date is that the role of the interaction with immune cells in invasive progression cannot be assessed. The future application of humanized NSG mice to studies of DCIS may potentially address this drawback.[31]

Transgenic mice are an alternative approach to study DCIS development and progression. Several models of transgenic mice are available that are predisposed to develop mammary carcinoma with various latencies due to induced expression of oncogenes, including Her-2, polyoma middle T antigen driven by mouse mammary tumor virus or SV40 large T antigen driven by the whey-acid protein promoter. As part of the progression of disease, these mice develop DCIS lesions with distinct morphological and cytological features that somewhat resemble those observed in DCIS patients. These transgenic models have improved the understanding of the genetic events and molecular mechanisms underlying DCIS progression[32–35] and offer opportunities to evaluate hypotheses for the role of DCIS in the progression of breast cancer (Figure 1).

Researchers have also focused efforts toward the development of cellular model systems that would recapitulate the architecture and complexity of DCIS in the human breast. Earlier studies for preclinical therapeutic identification and development were commonly based on conventional cell culture systems on plastic dishes. These monolayer cultures do not provide the environment as it exists in vivo, lacking natural cues for hallmarks of differentiated phenotypes such as polarity, and thus exhibiting altered cell signaling and gene expression.[36] 3D matrices, such as reconstituted basement membrane (rBM), are more physiologically relevant substrates, and cancer cells grown in rBM exhibit responses and resistance to drugs that are closer to those observed in vivo.[37–42] Another advantage of 3D rBM cultures is that they provide a source for high-quality RNA[43] without concomitant stromal cell RNA contamination. This is hard to achieve in the isolation of RNA from microscopic clinical DCIS specimens or from formalin-fixed paraffin-embedded tissues, although the use of laser capture and new sequencing technologies for fragmented samples are becoming available.

Heterotypic 3D coculture models of DCIS, such as mammary architecture and microenvironment engineering (Figure 2), enable live-cell, real-time imaging of cell–cell and cell–matrix interactions. These models allow for the analysis of interactions between the breast epithelial cells and various stromal cells such as macrophages, fibroblasts and lymphatic and blood vessel microvascular endothelial cells. The individual cell populations can be manipulated and the contribution of each component to the progression to IDC can be characterized.[44] These models have:

Figure 2.

Mammary architecture and microenvironment models for functional, live-cell imaging of ductal carcinoma in situ–stromal interactions. (A) MCF10.DCIS human ductal carcinoma in situ cells in an upper layer of reconstituted basement membrane (rBM) + 2% rBM overlay form dysplastic structures. Cancer-associated fibroblasts are in a lower layer of collagen I. (B) Human macrophages are added to the rBM layer. (C) Human microvascular endothelial cells are added to the rBM layer.

  • Revealed roles for cathepsin B in pericellular proteolysis and invasiveness of premalignant epithelial and carcinoma cells;[45]

  • Identified a proteolytic pathway, involving caveolar localization of cathepsin B and urokinase plasminogen activator receptor, in pericellular proteolysis and invasiveness of triple-negative inflammatory breast cancer cells;[46]

  • Demonstrated roles for cancer-associated fibroblasts (CAFs) and CAF secretion of HGF in increased proteolysis and invasiveness of DCIS cells;[47]

  • Shown a role for acidic pH in pericellular proteolysis and invasiveness,[48] which is linked to the secretion of cathepsin B and induction of chronic autophagy.[49]

Using similar heterotypic 3D coculture models, Dang et al. have shown differential interactions of basal and luminal subtypes with CAFs.[50] They report that both basal and luminal type breast cancer cells when cultured alone formed noninvasive DCIS spheroids. In contrast, when cocultured with CAFs, the basal type showed invasive outgrowths and the luminal type spheroids formed noninvasive duct-like structures.[50]

Other recent developments include computer modeling of the biomechanical forces influencing growth and necrosis in comedo-type DCIS with the goal of improving the prognostic value of mammographic results,[51] and a microfluidic 3D compartmentalized system that enables control of both spatial and temporal aspects within the microenvironment of cell cultures. This compartmentalized model system allows sampling of secreted molecules and facilitates inhibitor screening and studies of cell signaling pathways involved in the progression of DCIS.[52] Further optimization and validation of organotypic culture models will need to consider challenges such as adaptability to high-throughput screening.

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