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James P. Quigley - One of the best experts on this subject based on the ideXlab platform.
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LTBP3 promotes early metastatic events during cancer cell dissemination
Oncogene, 2018Co-Authors: Elena I. Deryugina, Ewa Zajac, Lior Zilberberg, Tomoki Muramatsu, Grishma Joshi, Branka Dabovic, Daniel Rifkin, James P. QuigleyAbstract:Latent transforming growth factor β (TGFβ)-binding proteins (LTBPs) are important for the secretion, activation, and function of mature TGFβ, especially so in cancer cell physiology. However, specific roles of the LTBPs remain understudied in the context of the primary tumor microenvironment. Herein, we investigated the role of LTBP3 in the distinct processes involved in cancer metastasis. By using three human tumor cell lines of different tissue origin (epidermoid HEp-3 and prostate PC-3 carcinomas and HT-1080 fibrosarcoma) and several metastasis models conducted in both mammalian and avian settings, we show that LTBP3 is involved in the early dissemination of primary cancer cells, namely in the Intravasation step of the metastatic cascade. Knockdown of LTBP3 in all tested cell lines led to significant inhibition of tumor cell Intravasation, but did not affect primary tumor growth. LTBP3 was dispensable in the late steps of carcinoma cell metastasis that follow tumor cell Intravasation, including vascular arrest, extravasation, and tissue colonization. However, LTBP3 depletion diminished the angiogenesis-inducing potential of HEp-3 cells in vivo, which was restorable by exogenous delivery of LTBP3 protein. A similar compensatory approach rescued the dampened Intravasation of LTBP3-deficient HEp-3 cells, suggesting that LTBP3 regulates the induction of the Intravasation-supporting angiogenic vasculature within developing primary tumors. Using our recently developed microtumor model, we confirmed that LTBP3 loss resulted in the development of intratumoral vessels with an abnormal microarchitecture incompatible with efficient Intravasation of HEp-3 carcinoma cells. Collectively, these findings demonstrate that LTBP3 represents a novel oncotarget that has distinctive functions in the regulation of angiogenesis-dependent tumor cell Intravasation, a critical process during early cancer dissemination. Our experimental data are also consistent with the survival prognostic value of LTBP3 expression in early-stage head and neck squamous cell carcinomas, further indicating a specific role for LTBP3 in cancer progression toward metastatic disease.
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Tumor angiogenesis: MMP-mediated induction of Intravasation- and metastasis-sustaining neovasculature.
Matrix Biology, 2015Co-Authors: Elena I. Deryugina, James P. QuigleyAbstract:Metastasis is a distinct stage of cancer progression that requires the development of angiogenic blood vessels serving as conduits for tumor cell dissemination. An accumulated body of evidence indicates that metastasis-supporting neovasculature should possess certain structural characteristics allowing for the process of tumor cell Intravasation, an active entry of cancer cells into the vessel interior. It appears that the development of tumor vessels with lumens of a distinctive size and support of these vessels by a discontinuous pericyte coverage constitute critical microarchitectural requirements to: (a) provide accessible points for vessel wall penetration by primary tumor cells; (b) provide enough lumen space for a tumor cell or cell aggregate upon Intravasation; and (c) allow for sufficient rate of blood flow to carry away intravasated cells from the primary tumor to the next, proximal or distal site. This review will primarily focus on the functional roles of matrix metalloproteinases (MMPs), which catalytically trigger the development of an Intravasation-sustaining neovasculature at the early stages of tumor growth and are also required for the maintenance of a metastasis-supporting state of blood vessels at later stages of cancer progression.
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abstract a06 the role of the epidermal growth factor receptor egfr in primary tumor cell escape and development of an Intravasation sustaining vasculature
Cancer Research, 2015Co-Authors: Petra Minder, Elena I. Deryugina, James P. QuigleyAbstract:Metastasis of malignant cells is regarded as the leading cause of cancer-related deaths. Metastasis is a complex process whereby a subset or individual cancer cells disseminate from the primary tumor to distant/secondary sites. Intravasation, which is the entry of escaping tumor cells into the vasculature, is a critical and rate-limiting early event in the metastatic cascade. The complex process of Intravasation is difficult to model and quantify in vivo, and therefore it remains the least studied step of the metastatic cascade. To study Intravasation, we employ the chick embryo model involving grafting of human cancer cells on the chorioallantoic membrane (CAM), the highly vascularized tissue capable of supporting primary tumor growth and development of angiogenic vasculature. The epidermal growth factor receptor (EGFR) is known to play an important role in cancer progression and EGFR-induced signaling has been associated with tumor invasion and metastasis. In this study, we investigated the specific roles of EGFR in the early steps of tumor metastasis such as escape of cancer cells from the primary tumor and development of angiogenic vasculature, the two critical events required for tumor cell Intravasation. For this purpose we employed the EGFR-overexpressing highly disseminating variant of human fibrosarcoma HT-1080 cell line, HT-hi/diss, in several independent in vivo CAM models. In the spontaneous metastasis model, siRNA silencing of EGFR significantly reduced the levels of HT-hi/diss Intravasation, suggesting a role of EGFR overexpression in tumor cell escape and/or development of Intravasation-sustaining vasculature. We have demonstrated that EGFR silencing prevented HT-hi/diss cell escape from the primary microtumors in the intramesodermal model, linking EGFR-regulated cell motility to tumor cell Intravasation. We further investigated the role of EGFR in development of tumor-associated vasculature. By using our newly developed topical microtumor model, we have demonstrated that both control and EGFR-silenced cells attract comparable numbers of angiogenic vessels converging to developing primary microtumors. However, epifluorescence microscopy revealed dramatic difference in the microarchitecture of blood vessels within the two types of microtumors. Whereas control microtumors exhibited well-developed networks of intratumoral blood vessels with lumen diameter of 15-30 µm, EGFR-silenced microtumors contained only thin blood vessels with lumen diameter less than 15 µm. This lack of appropriately dilated intratumoral vasculature in EGFR-silenced tumors directly correlated with a significant decrease in vascular permeability concomitant with the suppression of tumor cell Intravasation. These data highlight a specific aspect of EGFR functionality that is different from its well-established role in transcriptional regulation of genes required for tumor growth and suggest a novel mechanism whereby tumor cell EGFR regulates microenvironmental cues for induction, development, and functionality of an Intravasation-sustaining vasculature. Citation Format: Petra Minder, Elena I. Deryugina, James P. Quigley. The role of the epidermal growth factor receptor (EGFR) in primary tumor cell escape and development of an Intravasation-sustaining vasculature. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A06. doi:10.1158/1538-7445.CHTME14-A06
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EGFR Regulates the Development and Microarchitecture of Intratumoral Angiogenic Vasculature Capable of Sustaining Cancer Cell Intravasation
Neoplasia (New York N.Y.), 2015Co-Authors: Petra Minder, Ewa Zajac, James P. Quigley, Elena I. DeryuginaAbstract:Many malignant characteristics of cancer cells are regulated through pathways induced by the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Herein, we show that besides directly affecting the biology of cancer cells per se, EGFR also regulates the primary tumor microenvironment. Specifically, our findings demonstrate that both the expression and signaling activity of EGFR are required for the induction of a distinct intratumoral vasculature capable of sustaining tumor cell Intravasation, a critical rate-limiting step in the metastatic cascade. An Intravasation-sustaining mode of intratumoral angiogenic vessels depends on high levels of tumor cell EGFR and the interplay between EGFR-regulated production of interleukin 8 by tumor cells, interleukin-8–induced influx of tumor-infiltrating neutrophils delivering their unique matrix metalloproteinase-9, and neutrophil matrix metalloproteinase-9–dependent release of the vascular permeability and endothelial growth factor, VEGF. Our data indicate that through VEGF-mediated disruption of endothelial layer integrity and increase of intratumoral vasculature permeability, EGFR activity significantly facilitates active Intravasation of cancer cells. Therefore, this study unraveled an important but overlooked function of EGFR in cancer, namely, its ability to create an Intravasation-sustaining microenvironment within the developing primary tumor by orchestrating several interrelated processes required for the initial steps of cancer metastasis through vascular routes. Our findings also suggest that EGFR-targeted therapies might be more effective when implemented in cancer patients with early-staged primary tumors containing a VEGF-dependent angiogenic vasculature. Accordingly, early EGFR inhibition combined with various anti-VEGF approaches could synergistically suppress tumor cell Intravasation through inhibiting the highly permeable angiogenic vasculature induced by EGFR-overexpressing aggressive cancer cells.
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Abstract PR14: Quantitative and multiparameter analysis of tumor cell Intravasation
Emerging Technologies and Advances in Modeling, 2014Co-Authors: Elena I. Deryugina, William B. Kiosses, James P. QuigleyAbstract:Multiple steps in cancer metastasis, including tumorigenesis, tumor angiogenesis, invasion and colonization, can be successfully examined with well-established models. However, due to deficiency in experimental model systems that would accurately recapitulate tumor cell entry into the host vasculature, Intravasation remains the least studied step in the metastatic cascade. Our laboratory has developed several independent avian and murine model systems to investigate the complex process of Intravasation in a quantifiable and mechanistic manner. Chick embryo models of Intravasation are based on the ability of a highly vascularized tissue, the chorioallantoic membrane (CAM), to sustain primary tumor development and spontaneous metastasis. Furthermore, the CAM serves as a repository of newly intravasated cells, which are quantified by human-specific Alu-qPCR. By employing different CAM models, the early events in spontaneous metastasis that ultimately lead to Intravasation can be dissected and their individual input to overall Intravasation can be quantified. These early processes include cell escape from the primary tumor, establishment of intratumoral and extratumoral angiogenic networks, invasion of adjacent stroma by escaped tumor cells, and migration of escaped tumor cells along tumor-associated blood vessels. In conjunction with confirmatory models of cancer metastasis in mice, we have identified several molecular systems that functionally contribute to the Intravasation process. To investigate the specific mechanisms whereby tumor cells enter the angiogenic blood vessels, we have developed a new high-resolution confocal microscopy model based on acquisition and multiparameter analysis of primary microtumors imaged as entire units. This model was adapted to primary tumors developing within the mesoderm of the CAM as well as primary tumors developing in the dermis of mouse ear. In both avian and murine models, the volume of microtumors, the number of tumor cells per tumor, and the total volume of tumor-associated vasculature can be determined from 3D-reconstructed Z-stacks. Most importantly, the volume of intravascular tumor cells can be quantified by colocalizing tumor-vascular signals with a stringent threshold of a minimum volume of an individual tumor cell so that the cells that are localized within the vessels could be discriminated from the cells adherent to the abluminal vessel surface. In addition, high-resolution imaging of whole microtumors allows to visualize and quantify the tumor cells that appear as entering into the vasculature or in close association with the abluminal surface of the vessels. This approach allowed us for the fist time to visualize the Intravasation events in numbers rendering statistical significance. By employing unique cell and protein markers in this novel system, the vascular cells interacting with the intravasating tumor cells can be identified and the candidate molecules involved in the close tumor-vascular interactions can be probed by treating the developing microtumors with specific reagents and small molecule inhibitors. By using these novel high-resolution models for Intravasation, we have documented a majority of Intravasation events within the primary tumor or in the nearest proximity to the primary tumor border. These data challenge the widely accepted concept that tumor cell escape, stromal invasion, and entry of escaped tumor cells into blood vessels adjacent to the primary tumor, are prerequisites of tumor cell Intravasation. In contrast, our findings indicate that the bulk of cell Intravasation events can take place within the primary tumor and prior to significant stromal invasion and migration of escaped tumor cells towards and along tumor-associated blood vessels. Our data suggest that tumor cell Intravasation and metastatic spread can occur much earlier than commonly acknowledged by oncologists based on histological examination of resected tumors. This abstract is also presented as Poster A24. Citation Format: Elena I. Deryugina, William B. Kiosses, James P. Quigley. Quantitative and multiparameter analysis of tumor cell Intravasation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR14.
Elena I. Deryugina - One of the best experts on this subject based on the ideXlab platform.
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LTBP3 promotes early metastatic events during cancer cell dissemination
Oncogene, 2018Co-Authors: Elena I. Deryugina, Ewa Zajac, Lior Zilberberg, Tomoki Muramatsu, Grishma Joshi, Branka Dabovic, Daniel Rifkin, James P. QuigleyAbstract:Latent transforming growth factor β (TGFβ)-binding proteins (LTBPs) are important for the secretion, activation, and function of mature TGFβ, especially so in cancer cell physiology. However, specific roles of the LTBPs remain understudied in the context of the primary tumor microenvironment. Herein, we investigated the role of LTBP3 in the distinct processes involved in cancer metastasis. By using three human tumor cell lines of different tissue origin (epidermoid HEp-3 and prostate PC-3 carcinomas and HT-1080 fibrosarcoma) and several metastasis models conducted in both mammalian and avian settings, we show that LTBP3 is involved in the early dissemination of primary cancer cells, namely in the Intravasation step of the metastatic cascade. Knockdown of LTBP3 in all tested cell lines led to significant inhibition of tumor cell Intravasation, but did not affect primary tumor growth. LTBP3 was dispensable in the late steps of carcinoma cell metastasis that follow tumor cell Intravasation, including vascular arrest, extravasation, and tissue colonization. However, LTBP3 depletion diminished the angiogenesis-inducing potential of HEp-3 cells in vivo, which was restorable by exogenous delivery of LTBP3 protein. A similar compensatory approach rescued the dampened Intravasation of LTBP3-deficient HEp-3 cells, suggesting that LTBP3 regulates the induction of the Intravasation-supporting angiogenic vasculature within developing primary tumors. Using our recently developed microtumor model, we confirmed that LTBP3 loss resulted in the development of intratumoral vessels with an abnormal microarchitecture incompatible with efficient Intravasation of HEp-3 carcinoma cells. Collectively, these findings demonstrate that LTBP3 represents a novel oncotarget that has distinctive functions in the regulation of angiogenesis-dependent tumor cell Intravasation, a critical process during early cancer dissemination. Our experimental data are also consistent with the survival prognostic value of LTBP3 expression in early-stage head and neck squamous cell carcinomas, further indicating a specific role for LTBP3 in cancer progression toward metastatic disease.
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Intratumoral Cancer Cell Intravasation Can Occur Independent of Invasion into the Adjacent Stroma
Cell reports, 2017Co-Authors: Elena I. Deryugina, William B. KiossesAbstract:Intravasation, active entry of cancer cells into the circulation, is often considered to be a relatively late event in tumor development occurring after stromal invasion. Here, we provide evidence that Intravasation can be initiated early during tumor development and proceed in parallel to or independent of tumor invasion into surrounding stroma. By applying direct and unbiased Intravasation-scoring methods to two histologically distinct human cancer types in live-animal models, we demonstrate that Intravasation takes place almost exclusively within the tumor core, involves intratumoral vasculature, and does not involve vasculotropic cancer cells invading tumor-adjacent stroma and migrating along tumor-converging blood vessels. Highlighting an additional role for EGFR in cancer, we find that EGFR is required for the development of an Intravasation-sustaining intratumoral vasculature. Intratumoral localization of Intravasation supports the notion that overt metastases in cancer patients could be initiated much earlier during cancer progression than appreciated within conventional clinical tumor staging systems.
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Tumor angiogenesis: MMP-mediated induction of Intravasation- and metastasis-sustaining neovasculature.
Matrix Biology, 2015Co-Authors: Elena I. Deryugina, James P. QuigleyAbstract:Metastasis is a distinct stage of cancer progression that requires the development of angiogenic blood vessels serving as conduits for tumor cell dissemination. An accumulated body of evidence indicates that metastasis-supporting neovasculature should possess certain structural characteristics allowing for the process of tumor cell Intravasation, an active entry of cancer cells into the vessel interior. It appears that the development of tumor vessels with lumens of a distinctive size and support of these vessels by a discontinuous pericyte coverage constitute critical microarchitectural requirements to: (a) provide accessible points for vessel wall penetration by primary tumor cells; (b) provide enough lumen space for a tumor cell or cell aggregate upon Intravasation; and (c) allow for sufficient rate of blood flow to carry away intravasated cells from the primary tumor to the next, proximal or distal site. This review will primarily focus on the functional roles of matrix metalloproteinases (MMPs), which catalytically trigger the development of an Intravasation-sustaining neovasculature at the early stages of tumor growth and are also required for the maintenance of a metastasis-supporting state of blood vessels at later stages of cancer progression.
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abstract a06 the role of the epidermal growth factor receptor egfr in primary tumor cell escape and development of an Intravasation sustaining vasculature
Cancer Research, 2015Co-Authors: Petra Minder, Elena I. Deryugina, James P. QuigleyAbstract:Metastasis of malignant cells is regarded as the leading cause of cancer-related deaths. Metastasis is a complex process whereby a subset or individual cancer cells disseminate from the primary tumor to distant/secondary sites. Intravasation, which is the entry of escaping tumor cells into the vasculature, is a critical and rate-limiting early event in the metastatic cascade. The complex process of Intravasation is difficult to model and quantify in vivo, and therefore it remains the least studied step of the metastatic cascade. To study Intravasation, we employ the chick embryo model involving grafting of human cancer cells on the chorioallantoic membrane (CAM), the highly vascularized tissue capable of supporting primary tumor growth and development of angiogenic vasculature. The epidermal growth factor receptor (EGFR) is known to play an important role in cancer progression and EGFR-induced signaling has been associated with tumor invasion and metastasis. In this study, we investigated the specific roles of EGFR in the early steps of tumor metastasis such as escape of cancer cells from the primary tumor and development of angiogenic vasculature, the two critical events required for tumor cell Intravasation. For this purpose we employed the EGFR-overexpressing highly disseminating variant of human fibrosarcoma HT-1080 cell line, HT-hi/diss, in several independent in vivo CAM models. In the spontaneous metastasis model, siRNA silencing of EGFR significantly reduced the levels of HT-hi/diss Intravasation, suggesting a role of EGFR overexpression in tumor cell escape and/or development of Intravasation-sustaining vasculature. We have demonstrated that EGFR silencing prevented HT-hi/diss cell escape from the primary microtumors in the intramesodermal model, linking EGFR-regulated cell motility to tumor cell Intravasation. We further investigated the role of EGFR in development of tumor-associated vasculature. By using our newly developed topical microtumor model, we have demonstrated that both control and EGFR-silenced cells attract comparable numbers of angiogenic vessels converging to developing primary microtumors. However, epifluorescence microscopy revealed dramatic difference in the microarchitecture of blood vessels within the two types of microtumors. Whereas control microtumors exhibited well-developed networks of intratumoral blood vessels with lumen diameter of 15-30 µm, EGFR-silenced microtumors contained only thin blood vessels with lumen diameter less than 15 µm. This lack of appropriately dilated intratumoral vasculature in EGFR-silenced tumors directly correlated with a significant decrease in vascular permeability concomitant with the suppression of tumor cell Intravasation. These data highlight a specific aspect of EGFR functionality that is different from its well-established role in transcriptional regulation of genes required for tumor growth and suggest a novel mechanism whereby tumor cell EGFR regulates microenvironmental cues for induction, development, and functionality of an Intravasation-sustaining vasculature. Citation Format: Petra Minder, Elena I. Deryugina, James P. Quigley. The role of the epidermal growth factor receptor (EGFR) in primary tumor cell escape and development of an Intravasation-sustaining vasculature. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A06. doi:10.1158/1538-7445.CHTME14-A06
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EGFR Regulates the Development and Microarchitecture of Intratumoral Angiogenic Vasculature Capable of Sustaining Cancer Cell Intravasation
Neoplasia (New York N.Y.), 2015Co-Authors: Petra Minder, Ewa Zajac, James P. Quigley, Elena I. DeryuginaAbstract:Many malignant characteristics of cancer cells are regulated through pathways induced by the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Herein, we show that besides directly affecting the biology of cancer cells per se, EGFR also regulates the primary tumor microenvironment. Specifically, our findings demonstrate that both the expression and signaling activity of EGFR are required for the induction of a distinct intratumoral vasculature capable of sustaining tumor cell Intravasation, a critical rate-limiting step in the metastatic cascade. An Intravasation-sustaining mode of intratumoral angiogenic vessels depends on high levels of tumor cell EGFR and the interplay between EGFR-regulated production of interleukin 8 by tumor cells, interleukin-8–induced influx of tumor-infiltrating neutrophils delivering their unique matrix metalloproteinase-9, and neutrophil matrix metalloproteinase-9–dependent release of the vascular permeability and endothelial growth factor, VEGF. Our data indicate that through VEGF-mediated disruption of endothelial layer integrity and increase of intratumoral vasculature permeability, EGFR activity significantly facilitates active Intravasation of cancer cells. Therefore, this study unraveled an important but overlooked function of EGFR in cancer, namely, its ability to create an Intravasation-sustaining microenvironment within the developing primary tumor by orchestrating several interrelated processes required for the initial steps of cancer metastasis through vascular routes. Our findings also suggest that EGFR-targeted therapies might be more effective when implemented in cancer patients with early-staged primary tumors containing a VEGF-dependent angiogenic vasculature. Accordingly, early EGFR inhibition combined with various anti-VEGF approaches could synergistically suppress tumor cell Intravasation through inhibiting the highly permeable angiogenic vasculature induced by EGFR-overexpressing aggressive cancer cells.
John S Condeelis - One of the best experts on this subject based on the ideXlab platform.
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The role of the tumor microenvironment in tumor cell Intravasation and dissemination
European journal of cell biology, 2020Co-Authors: Lucia Borriello, Maja H Oktay, David Entenberg, George S. Karagiannis, Camille L. Duran, Anouchka Coste, John S CondeelisAbstract:Metastasis, a process that requires tumor cell dissemination followed by tumor growth, is the primary cause of death in cancer patients. An essential step of tumor cell dissemination is Intravasation, a process by which tumor cells cross the blood vessel endothelium and disseminate to distant sites. Studying this process is of utmost importance given that Intravasation in the primary tumor, as well as the secondary and tertiary metastases, is the key step in the systemic spread of tumor cells, and that this process continues even after removal of the primary tumor. High-resolution intravital imaging of the tumor microenvironment of breast carcinoma has revealed that tumor cell Intravasation exclusively occurs at doorways, termed "Tumor MicroEnvironment of Metastasis" (TMEM), composed of three different cell types: a Tie2high/VEGFhigh perivascular macrophage, a Mena overexpressing tumor cell, and an endothelial cell, all in direct contact. In this review article, we discuss the interactions between these cell types, the subsequent signaling events which lead to tumor cell Intravasation, and the role of invadopodia in supporting tumor cell invasion and dissemination. We end our review by discussing how the knowledge acquired from the use of intravital imaging is now leading to new clinical trials targeting tumor cell dissemination and preventing metastatic progression.
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A Unidirectional Transition from Migratory to Perivascular Macrophage Is Required for Tumor Cell Intravasation
Cell reports, 2018Co-Authors: Esther N Arwert, Yarong Wang, Allison S Harney, David Entenberg, Jeffrey W Pollard, Erik Sahai, John S CondeelisAbstract:Tumor-associated macrophages (TAMs) are critical for tumor metastasis. Two TAM subsets support cancer cell Intravasation: migratory macrophages guide cancer cells toward blood vessels, where sessile perivascular macrophages assist their entry into the blood. However, little is known about the inter-relationship between these functionally distinct TAMs or their possible inter-conversion. We show that motile, streaming TAMs are newly arrived monocytes, recruited via CCR2 signaling, that then differentiate into the sessile perivascular macrophages. This unidirectional process is regulated by CXCL12 and CXCR4. Cancer cells induce TGF-β-dependent upregulation of CXCR4 in monocytes, while CXCL12 expressed by perivascular fibroblasts attracts these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiate into perivascular macrophages, promoting vascular leakiness and Intravasation.
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abstract lb 006 macrophage dependent activation of notch1 signaling regulates breast tumor cell Intravasation
Cancer Research, 2016Co-Authors: Jeanine Pignatelli, Jose Javier Bravocordero, Minna Rohjohnson, Saumil J Gandhi, Yarong Wang, Robert H Singer, Louis Hodgson, Maja H Oktay, John S CondeelisAbstract:The process of Intravasation, a key component to the metastatic cascade, remains poorly understood. How the multi-cell type tumor microenvironment facilitates tumor cell Intravasation is largely unknown. Intravital imaging of rodent mammary tumors has shown that direct contact between a Mena expressing tumor cell, a perivascular macrophage and an endothelial cell forms a microanatomical structure named TMEM (Tumor Microenvironment of Metastasis) which is the site where Intravasation occurs in mammary tumors. Clinical studies have shown that the number of TMEM is correlated with increased risk of developing distant metastasis in breast cancer patients. While TMEM is an excellent prognostic marker for predicting metastasis, the mechanisms of TMEM assembly and function are not well understood. Recently, we showed that heterotypic cell contact between tumor cells and macrophages induces the formation of invadopodia in tumor cells, invasive structures necessary for matrix degradation and required for tumor cell Intravasation. In further work we found that Notch1, a known receptor involved in heterotypic cell contact signaling, is involved. In the absence of Notch1 signaling, macrophage-induced invadopodium formation and tumor cell Intravasation are abolished. This heterotypic tumor cell - macrophage interaction regulates the expression profile of Mena in the tumor cell. Upon touching, the transcription of Mena shows a rapid kinetic response, with detectable changes in single transcriptome activity within 1 hour. Inhibition of Notch1 in vivo results in decreased Intravasation of mammary tumor cells. Our findings indicate that Notch1 signaling regulates heterotypic cell contact mediated invadopodium formation, transendothelial migration and Intravasation and reveals a novel Notch1/Mena pathway as a molecular target to prevent TMEM function and therefore metastasis. Citation Format: Jeanine Pignatelli, Jose Javier Bravo-Cordero, Minna Roh-Johnson, Saumil Gandhi, Yarong Wang, Robert Singer, Louis Hodgson, Maja Oktay, John Condeelis. Macrophage-dependent activation of Notch1 signaling regulates breast tumor cell Intravasation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-006.
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abstract b05 invadopodium formation is enriched in perivascular niches and leads to cancer cell Intravasation
Cancer Research, 2016Co-Authors: Aviv Bergman, John S Condeelis, Bojana GligorijevicAbstract:Invadopodia are actin-rich protrusions used for cancer cell movement across obstacles. In the past, they were mostly studied in cell cultures, where they form in contact with extracellular matrix. Our past work has suggested that elimination of key molecular components of invadopodia in cancer cells can lead to elimination of metastasis. In the current study, we use high resolution multiphoton microscopy to understand how the tumor microenvironment controls formation of invadopodia, followed by cancer cell locomotion, Intravasation and metastasis. We demonstrate that the motile cancer cells in breast carcinoma in vivo can exhibit either invadopodium-related, slow locomotion or invadopodium-independent rapid locomotion along collagen fibers. These two phenotypes occur in spatially separate tumor regions, the location of which can be predicted from measurements of microenvironmental features. For example, cancer cells form invadopodia predominantly in perivascular niches, regions surrounding major blood vessels and characterized by cross-linked collagen fibers and dense macrophages. Invadopodia in vivo extend towards blood vessels and their density decreases with blood vessel distance. We observe that only cells with invadopodia are capable of Intravasation, while fast locomotion results in cell dispersal throughout tumor. Further, either invadopodium-specific TkS5 knock-down or short-term inhibition of metalloproteases eliminate invadopodia and stop dissemination to lung. Interestingly, external modulation of collagen cross-linking also inhibits invadopodia, switching the cancer cell phenotype to fast locomotion. Our results suggest that the perivascular niche is the site of invadopodium formation and cancer cell Intravasation. Moreover, invadopodia and hence metastasis, can be inhibited by external modulation of the microenvironment. In summary, the presence of invadopodia is a great candidate for a novel early-metastasis marker, while their elimination via microenvironment modulation has therapeutic potential. Citation Format: Aviv Bergman, John Condeelis, Bojana Gligorijevic. Invadopodium formation is enriched in perivascular niches and leads to cancer cell Intravasation. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B05.
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Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA
Cancer discovery, 2015Co-Authors: Allison S Harney, Yarong Wang, Maja H Oktay, Esther N Arwert, David Entenberg, Peng Guo, Binzhi Qian, Jeffrey W Pollard, Joan G Jones, John S CondeelisAbstract:Dissemination of tumor cells is an essential step in metastasis. Direct contact between a macrophage, mammalian-enabled (MENA)–overexpressing tumor cell, and endothelial cell [Tumor MicroEnvironment of Metastasis (TMEM)] correlates with metastasis in breast cancer patients. Here we show, using intravital high-resolution two-photon microscopy, that transient vascular permeability and tumor cell Intravasation occur simultaneously and exclusively at TMEM. The hyperpermeable nature of tumor vasculature is described as spatially and temporally heterogeneous. Using real-time imaging, we observed that vascular permeability is transient, restricted to the TMEM, and required for tumor cell dissemination. VEGFA signaling from TIE2hi TMEM macrophages causes local loss of vascular junctions, transient vascular permeability, and tumor cell Intravasation, demonstrating a role for the TMEM within the primary mammary tumor. These data provide insight into the mechanism of tumor cell Intravasation and vascular permeability in breast cancer, explaining the value of TMEM density as a predictor of distant metastatic recurrence in patients. Significance: Tumor vasculature is abnormal with increased permeability. Here, we show that VEGFA signaling from TIE2hi TMEM macrophages results in local, transient vascular permeability and tumor cell Intravasation. These data provide evidence for the mechanism underlying the association of TMEM with distant metastatic recurrence, offering a rationale for therapies targeting TMEM. Cancer Discov; 5(9); 932–43. ©2015 AACR . See related commentary by Kadioglu and De Palma, [p. 906][1] . This article is highlighted in the In This Issue feature, [p. 893][2] [1]: /lookup/volpage/5/906?iss=9 [2]: /lookup/volpage/5/893?iss=9
Jeffrey E. Segall - One of the best experts on this subject based on the ideXlab platform.
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Tumor cell Intravasation
American journal of physiology. Cell physiology, 2016Co-Authors: Serena P. H. Chiang, Ramon M. Cabrera, Jeffrey E. SegallAbstract:The process of entering the bloodstream, Intravasation, is a necessary step in the development of distant metastases. The focus of this review is on the pathways and molecules that have been identified as being important based on current in vitro and in vivo assays for Intravasation. Properties of the vasculature which are important for Intravasation include microvessel density and also diameter of the vasculature, with increased Intravasation correlating with increased vessel diameter in some tumors. TGFB signaling can enhance Intravasation at least in part through induction of EMT, and we discuss other TGFB target genes that are important for Intravasation. In addition to TGFB signaling, a number of studies have demonstrated that activation of EGF receptor family members stimulates Intravasation, with downstream signaling through PI3K, N-WASP, RhoA, and WASP to induce invadopodia. With respect to proteases, there is strong evidence for contributions by uPA/uPAR, while the roles of MMPs in Intravasation may be more tumor specific. Other cells including macrophages, fibroblasts, neutrophils, and platelets can also play a role in enhancing tumor cell Intravasation. The technology is now available to interrogate the expression patterns of circulating tumor cells, which will provide an important reality check for the model systems being used. With a better understanding of the mechanisms underlying Intravasation, the goal is to provide new opportunities for improving prognosis as well as potentially developing new treatments.
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abstract 4019 examining mechanisms by which macrophages enhance Intravasation in breast cancer
Cancer Research, 2015Co-Authors: Serena Chiang, Zhenni Zhou, Jeffrey E. SegallAbstract:Breast cancer is the most common invasive malignancy and a leading cause of cancer-associated deaths in women worldwide. Metastasis is the major cause of morbidity and mortality in women afflicted with breast cancer. In the metastatic process, Intravasation is an important step and largely dependent on interactions between tumor cells and stromal cells in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs), an integral component of the TME, have been found to associate and co-migrate with tumor cells during Intravasation. Invasive TAMs that co-migrate with tumor cells in response to epidermal growth factor display a unique transcriptome in comparison to the general TAM population found in the TME. Furthermore, intercellular signaling between tumor cells and TAMs generates a positive feedback loop that promotes tumor cell invasion and Intravasation. Chemokines play a major role in cell migration and are an important component of the TME. In particular, chemokine (C-X-C motif) receptor 2 (CXCR2) has been shown to be important in tumor growth and angiogenesis. MIF and IL8, two ligands of CXCR2, correlate with poor outcome in breast cancer. Our objective is to elucidate the role of macrophage CXCR2 in tumor cell Intravasation. We have used flow cytometry to characterize the basal expression of CXCR2 in a murine macrophage cell line, BAC-1.2F5 (BAC). We have also analyzed mRNA expression of CXCR2 in BAC and primary TAMs isolated from a murine model of breast cancer, the polyoma middle T oncoprotein (PyMT), by qRT-PCR. To test the functional significance of CXCR2 in Intravasation, we have examined the effects of an inhibitor of CXCR2 on the ability of tumor cells to migrate through the endothelium using an in vitro transendothelial migration assay (iTEM). In this assay, MDA-MB-231 tumor cells, a human breast cancer cell line, are co-cultured with BAC cells and subsequently placed in an environment that is in contact with the basal surface of endothelial cells. We have observed that BAC cells produce CXCR2 mRNA and express surface CXCR2. We have also found elevated expression of CXCR2 in invasive TAMs as compared to the general TAM population. When MDA-MB-231 cells are co-cultured with BAC cells in the iTEM assay in the presence of SB265610, an inhibitor of CXCR2, we have found a significant reduction (p Citation Format: Serena Chiang, Zhenni Zhou, Jeffrey E. Segall. Examining mechanisms by which macrophages enhance Intravasation in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4019. doi:10.1158/1538-7445.AM2015-4019
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Intravital Imaging and Photoswitching in Tumor Invasion and Intravasation Microenvironments.
Microscopy today, 2010Co-Authors: Bojana Gligorijevic, Jeffrey E. Segall, Dmitriy Kedrin, David Entenberg, Jacco Van Rheenen, John S CondeelisAbstract:Our group studies the spread of cells in primary breast tumors to distant sites, a process that is called metastasis. In order to metastasize, cells migrate and invade the surrounding areas (invasion) and enter the blood (Intravasation). Previously, we developed techniques to visualize these cell movements in mammary tumors of living mice at single-cell resolution [1-3]. These techniques involved intravital imaging of carcinomas in which tumor cells express genetically encoded fluorophores such as CFP or GFP [4] using multiphoton microscopy. In contrast to widefield or confocal microscopy, high-contrast images of features several cell diameters deep inside the tissue can be acquired with multiphoton microscopy without tumor dissection and removal from the animal. Using this technology, we were able to simultaneously monitor tumor cells and several components of the tumor microenvironment, such as collagen fibers, blood vessels, and several subpopulations of macrophages. The results of our studies demonstrated that the primary tumor is not just a collection of cells where every cell is equally able to metastasize, but that only a small population of tumor cells is capable of moving rapidly. These fast, invasive cells were seen to invade surrounding tissue, crawl along collagen fibers [5], interact with perivascular macrophages forming a paracrine loop [2], as well as enter into blood vessels (Intravasation) under the guidance of macrophages [1]. Based on these data, we were able to identify two separate microenvironments in the mammary carcinoma: the invasion and Intravasation microenvironments.
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erbb1 and erbb2 have distinct functions in tumor cell invasion and Intravasation
Clinical Cancer Research, 2009Co-Authors: Dmitriy Kedrin, Jeffrey B. Wyckoff, Pamela Boimel, Salvatore J. Coniglio, Nancy E. Hynes, Carlos L. Arteaga, Jeffrey E. SegallAbstract:Purpose: The epidermal growth factor receptor (ERBB1) and related family member HER-2/ neu (ERBB2) are often overexpressed in aggressive breast cancers and their overexpression is correlated with poor prognosis. Clinical studies using ERBB inhibitors have focused on tumor growth effects, but ERBBs can contribute to malignancy independent of their effects on tumor growth. Our studies were designed to evaluate the effect of ERBB inhibition on tumor cell motility and Intravasation in vivo using clinically relevant small-molecule inhibitors. Experimental Design: Using in vivo mouse models of breast cancer, we test the effects of ERBB1 and ERBB2 inhibitors AC480 and lapatinib, ERBB1 inhibitor gefitinib, and ERBB2 inhibitor AG825 on in vivo tumor cell invasive properties in mammary fat pad tumors. Results: ERBB1 and ERBB2 inhibition rapidly (within 3 h) inhibits both tumor cell motility and Intravasation. Using gefitinib, ERBB1 inhibition rapidly inhibits tumor cell motility and invasion but not Intravasation, whereas ERBB2 inhibition by AG825 rapidly blocks Intravasation. Conclusions: ERBB1 and ERBB2 inhibition can rapidly block tumor cell invasive properties. In addition, we differentiate for the first time the contributions of ERBB1 and ERBB2 to the key metastatic properties of in vivo tumor cell invasion and Intravasation. These experiments temporally and molecularly separate two key stages in tumor cell entry into blood vessels: invasion and Intravasation. These results indicate that ERBB inhibition should be considered for blocking other tumor cell malignant properties besides growth.
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ERBB1 and ERBB2 Have Distinct Functions in Tumor Cell Invasion and Intravasation
Clinical cancer research : an official journal of the American Association for Cancer Research, 2009Co-Authors: Dmitriy Kedrin, Jeffrey B. Wyckoff, Pamela Boimel, Salvatore J. Coniglio, Nancy E. Hynes, Carlos L. Arteaga, Jeffrey E. SegallAbstract:The epidermal growth factor receptor (ERBB1) and related family member HER-2/neu (ERBB2) are often overexpressed in aggressive breast cancers and their overexpression is correlated with poor prognosis. Clinical studies using ERBB inhibitors have focused on tumor growth effects, but ERBBs can contribute to malignancy independent of their effects on tumor growth. Our studies were designed to evaluate the effect of ERBB inhibition on tumor cell motility and Intravasation in vivo using clinically relevant small-molecule inhibitors. Using in vivo mouse models of breast cancer, we test the effects of ERBB1 and ERBB2 inhibitors AC480 and lapatinib, ERBB1 inhibitor gefitinib, and ERBB2 inhibitor AG825 on in vivo tumor cell invasive properties in mammary fat pad tumors. ERBB1 and ERBB2 inhibition rapidly (within 3 h) inhibits both tumor cell motility and Intravasation. Using gefitinib, ERBB1 inhibition rapidly inhibits tumor cell motility and invasion but not Intravasation, whereas ERBB2 inhibition by AG825 rapidly blocks Intravasation. ERBB1 and ERBB2 inhibition can rapidly block tumor cell invasive properties. In addition, we differentiate for the first time the contributions of ERBB1 and ERBB2 to the key metastatic properties of in vivo tumor cell invasion and Intravasation. These experiments temporally and molecularly separate two key stages in tumor cell entry into blood vessels: invasion and Intravasation. These results indicate that ERBB inhibition should be considered for blocking other tumor cell malignant properties besides growth.
Heike Allgayer - One of the best experts on this subject based on the ideXlab platform.
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Src induces urokinase receptor gene expression and invasion/Intravasation via activator protein-1/p-c-Jun in colorectal cancer.
Molecular cancer research : MCR, 2007Co-Authors: Jörg Hendrik Leupold, Irfan A. Asangani, Gabriele D. Maurer, Ernst Lengyel, Stefan Post, Heike AllgayerAbstract:The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region -152/-135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator protein-1 (AP-1) motif at -190/-171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR-mediated invasion, in particular Intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1-related transcriptional mediators are required for Src-induced u-PAR-gene expression, (b) to show in vivo relevance of AP-1-mediated Src-induced u-PAR gene expression for invasion/Intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region -190/-171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho-c-Jun, in addition to JunD and Fra-1, bound to region -190/-171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser(73) and also Ser(63), which was paralleled by increased phospho-c-jun-NH(2)-kinase. Significant decreases of invasion/in vivo Intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR-small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region -190/-171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/Intravasation in vivo is also mediated via AP-1 region -190/-171, especially bound with c-Jun phosphorylated at Ser(73/63), and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential.
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src induces urokinase receptor gene expression and invasion Intravasation via activator protein 1 p c jun in colorectal cancer
Molecular Cancer Research, 2007Co-Authors: Jörg Hendrik Leupold, Irfan A. Asangani, Gabriele D. Maurer, Ernst Lengyel, Stefan Post, Heike AllgayerAbstract:The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region -152/-135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator protein-1 (AP-1) motif at -190/-171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR-mediated invasion, in particular Intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1-related transcriptional mediators are required for Src-induced u-PAR-gene expression, (b) to show in vivo relevance of AP-1-mediated Src-induced u-PAR gene expression for invasion/Intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region -190/-171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho-c-Jun, in addition to JunD and Fra-1, bound to region -190/-171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser(73) and also Ser(63), which was paralleled by increased phospho-c-jun-NH(2)-kinase. Significant decreases of invasion/in vivo Intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR-small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region -190/-171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/Intravasation in vivo is also mediated via AP-1 region -190/-171, especially bound with c-Jun phosphorylated at Ser(73/63), and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential.
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Tumor suppressor Pdcd4 inhibits invasion/Intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors
Oncogene, 2007Co-Authors: Jörg Hendrik Leupold, Irfan A. Asangani, Stefan Post, Hsin-sheng Yang, Nancy H. Colburn, Heike AllgayerAbstract:Tumor suppressor Pdcd4 has recently been shown to inhibit invasion by activating activator protein-1 (AP-1); however, little is known of the functionally significant Pdcd4-target genes. The urokinase receptor (u-PAR) promotes invasion/metastasis, and is associated with poor cancer-patient survival. The present study was conducted (1) to investigate a role for Pdcd4 in Intravasation, invasion and u-PAR regulation, and (2) to describe mechanisms by which this is achieved. Fourteen cell lines showed reciprocal expression of u-PAR/Pdcd4. Resected tumor/normal tissues of 29 colorectal cancer patients demonstrated a significant inverse correlation between Pdcd4/u-PAR. siRNA-Pdcd4-transfected GEO cells significantly increased endogenous u-PAR mRNA/protein. A u-PAR-promoter-chloramphenicol acetyl transferase (CAT)-reporter was reduced in activity with increasing Pdcd4 expression in RKO. Deletion of a putative Sp-1-binding site (-402/-350) inhibited u-PAR promoter regulation by Pdcd4, this being paralleled by a reduction of Sp1 binding to this region in pdcd4-transfected cells. Pdcd4-transfected cells showed an increase in Sp3 binding to u-PAR promoter region -152/-135, the deletion of which reduces the ability of Pdcd4 to suppress u-PAR promoter activity. Surprisingly, the u-PAR-AP-1 site was not targeted by Pdcd4. Finally, RKO cells overexpressing Pdcd4 showed an inhibition of invasion/Intravasation (chicken embryo metastasis assay). These data suggest Pdcd4 as a new negative regulator of Intravasation, and qas the invasion-related gene u-PAR. It is the first study to implicate Pdcd4 regulation of gene expression via Sp1/Sp3.