Intravital Imaging Illuminates Transforming Growth Factor β Signaling Switches during Metastasis

Giampieri, Silvia; Pinner, Sophie

doi:10.1158/0008-5472.can-10-0466

Cited by 53 publications

(47 citation statements)

References 25 publications

(31 reference statements)

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“…Although intravital imaging and use of organotypic bioreactors has improved the ability to visualize metastasis at various stages, the phenotypic plasticity exhibited during EMT and MErT is nonetheless difficult to capture [24][25][26]. Evidence of EMT and MErT in clinical specimens is rare and has been used as an argument that cancer-associated EMT does not occur during the course of disease.…”

Section: Discussionmentioning

confidence: 99%

Partial Mesenchymal to Epithelial Reverting Transition in Breast and Prostate Cancer Metastases

Chao

Acquafondata

et al. 2011

Cancer Microenvironment

140

136

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Epithelial to mesenchymal transition (EMT) is an oft-studied mechanism for the initiation of metastasis. We have recently shown that once cancer cells disseminate to a secondary organ, a mesenchymal to epithelial reverting transition (MErT) may occur, which we postulate is to enable metastatic colonization. Despite a wealth of in vitro and in vivo studies, evidence supportive of MErT in human specimens is rare and difficult to document because clinically detectable metastases are typically past the micrometastatic stage at which this transition is most likely evident. We obtained paired primary and metastatic tumors from breast and prostate cancer patients and evaluated expression of various epithelial and mesenchymal markers by immunohistochemistry. The metastases exhibited increased expression of membranous E-cadherin compared to primary tumors, consistent with EMT at the primary site and MErT at the metastatic site. However, the re-emergence of the epithelial phenotype was only partial or incomplete. Expression of epithelial markers connexins 26 and/or 43 was also increased on the majority of metastases, particularly those to the brain. Despite the upregulation of epithelial markers in metastases, expression of mesenchymal markers vimentin and FSP1 was mostly unchanged. We also examined prostate carcinoma metastases of varied sizes and found that while E-cadherin expression was increased compared to the primary lesion, the expression inversely correlated with size of the metastasis. This not only suggests that a second EMT may occur in the ectopic site for tumor growth or to seed further metastases, but also provides a basis for the failure to discern epithelial phenotypes in clinically examined macrometastases. In summary, we report increased expression of epithelial markers and persistence of mesenchymal markers consistent with a partial MErT that readily allows for a second EMT at the metastatic site. Our results suggest that cancer cells continue to display phenotypic plasticity beyond the EMT that initiates metastasis.

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Section: Discussionmentioning

confidence: 99%

Partial Mesenchymal to Epithelial Reverting Transition in Breast and Prostate Cancer Metastases

Chao

Acquafondata

et al. 2011

Cancer Microenvironment

140

136

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“…IHC analysis has shown increased levels of activated nuclear pSmad2 at the invasive front of human breast tumors (Kang et al 2005), and intravital microscopy using a fluorescent reporter to track Smad activation in live mouse tumors reveals the dynamic nature of this activity within breast cancer cells in vivo (Giampieri et al 2010). The latter study shows that TGF-b signaling promotes single cell motility and metastasis in a transient manner.…”

Section: Ligand Expression and Tgf-b Responsiveness In Human Tumorsmentioning

confidence: 99%

“…The latter study shows that TGF-b signaling promotes single cell motility and metastasis in a transient manner. Importantly, TGF-b signaling is down-regulated at the destination site of metastasis, favoring outgrowth of secondary tumors (Giampieri et al 2010). Clearly, active TGF-b signaling depends not only on the tumor genetics but also, in a dynamic fashion, on a cell state that fluctuates between active and inactive signaling.…”

Section: Ligand Expression and Tgf-b Responsiveness In Human Tumorsmentioning

confidence: 99%

Targeting TGF-β Signaling for Therapeutic Gain

Akhurst

2017

Cold Spring Harb Perspect Biol

162

133

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Transforming growth factor bs (TGF-bs) are closely related ligands that have pleiotropic activity on most cell types of the body. They act through common heterotetrameric TGF-b type II and type I transmembrane dual specificity kinase receptor complexes, and the outcome of signaling is context-dependent. In normal tissue, they serve a role in maintaining homeostasis. In many diseased states, particularly fibrosis and cancer, TGF-b ligands are overexpressed and the outcome of signaling is diverted toward disease progression. There has therefore been a concerted effort to develop drugs that block TGF-b signaling for therapeutic benefit. This review will cover the basics of TGF-b signaling and its biological activities relevant to oncology, present a summary of pharmacological TGF-b blockade strategies, and give an update on preclinical and clinical trials for TGF-b blockade in a variety of solid tumor types.

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“…First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor [4][5][6] . Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells [7][8][9] .…”

mentioning

confidence: 96%

<em>In vivo</em> Dual Substrate Bioluminescent Imaging

Molter¹,

Flask²,

Schiemann³

2011

JoVE

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Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies [1][2][3] . Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4 . Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor [4][5][6] . Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells [7][8][9] . Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis. Video LinkThe video component of this article can be found at https://www.jove.com/video/3245/ Protocol Stable Expression of CMV-Driven Renilla Luciferase1. Transfection and selection of a stable clonal population is the preferred method for expression of this renilla luciferase reporter. This approach yi...

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Intravital Imaging Illuminates Transforming Growth Factor β Signaling Switches during Metastasis

Cited by 53 publications

References 25 publications

Partial Mesenchymal to Epithelial Reverting Transition in Breast and Prostate Cancer Metastases

Partial Mesenchymal to Epithelial Reverting Transition in Breast and Prostate Cancer Metastases

Targeting TGF-β Signaling for Therapeutic Gain

<em>In vivo</em> Dual Substrate Bioluminescent Imaging

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