Tumor Cell Intravasation Research Articles

Epithelial-to-mesenchymal transition, circulating tumor cells and cancer metastasis: Mechanisms and clinical applications.

Epithelial-to-mesenchymal transition (EMT) endows epithelial cells with enhanced motility and invasiveness, allowing them to participate in many physiological and pathological processes. Epithelial-to-mesenchymal transition contributes to the generation of circulating tumor cells (CTCs) in epithelial cancers because it increases tumor cell invasiveness, promotes tumor cell intravasation and ensures tumor cell survival in the peripheral system. Although the contribution of epithelial-to-mesenchymal transition to tumor cell invasiveness has been confirmed, the role epithelial-to-mesenchymal transition plays in metastasis remains debated. As a favorable material for a “liquid biopsy”, circulating tumor cells have been shown to have promising values in the clinical management of tumors. Furthermore, an increasing number of studies have begun to explore the value of CTC-related biomarkers, and some studies have found that the expression of EMT and stemness markers in circulating tumor cells, in addition to CTC detection, can provide more information on tumor diagnosis, treatment, prognosis and research.

Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel or eribulin in patients with metastatic breast cancer (MBC).

TPS2611 Background: TMEM (Tumor Microenvironment of Metastasis) are microanatomic structures formed by a Mena-expressing tumor cell, Tie2-expressing macrophage, and endothelial cell in direct content, which serve as the primary portal for tumor cell intravasation into the circulation and subsequent metastasis. Paclitaxel (P) induces the formation of TMEM in the primary tumors of patients treated with neoadjuvant chemotherapy (NAC), and in the primary tumor and distant metastases in the PyMT/PDX models. Tumor cell intravasation is mediated by release of VEGF at TMEM sites from TMEM-associated Tie2HI/VEGFHI macrophages upon binding of the Tie2 receptor to angiopoietin. The Tie2 inhibitor rebastinib (R) inhibits intravasation at TMEM sites, reduces circulating tumor cell (CTC) burden, prevents distant metastases, and improves survival in breast cancer animal models when added to either P or eribulin (E). We hypothesize that the addition of R to antitubulin therapy in patients with HER2-negative MBC will prevent hematogenous dissemination and distant metastasis by inhibiting TMEM function, reduce CTC burden; and improve clinical outcomes. Methods: Primary objective of this phase Ib study (NCT02824575) is to evaluate safety and tolerability of R in two dose levels (DL) (50mg or 100mg PO BID) combined with IV P 80mg/m2 (day 1, 8 and 15) or E 1.4mg/m2 (day1 and 8) for four 21-day cycles. Key eligibility includes histologically confirmed HER2 negative MBC, ≤ 2 non-taxane chemotherapy regimens for R plus P arm or ≥ 2 chemotherapy regimens (including a taxane) for E plus R arm, ≥2 endocrine therapies ( including CDK4/6 inhibitor) for ER positive patients, ECOG PS 0 or 1; and normal organ and marrow function. Exclusion criteria include significant ocular or cardiac disease. Pharmacodynamic biomarkers to be measured during cycle 1-3 include CTCs, angiopoietin 1/2 levels and Tie-2 expressing monocytes. Tissue biopsy after two treatment cycles in 6 patients will be performed to evaluate TMEM score and function. With two DL of rebastinib, and 3-6 patients at each DL, it is anticipated that 6-12 patients will be required. This trial has enrolled two patients assigned to P arm combined with R 50mg BID. Clinical trial information: NCT02824575.

Gold nanoparticles attenuate metastasis by tumor vasculature normalization and epithelial-mesenchymal transition inhibition.

Angiogenesis is a process by which vessels are formed through preexisting ones, and this plays a key role in the progression of solid tumors. However, tumor vessels are influenced by excessive pro-angiogenic factors, resulting in deformed structures that facilitate the intravasation of tumor cells into the circulation and subsequent metastasis. Moreover, abnormal tumor vessels have low blood perfusion and thereby decreased oxygen infusion into tumors. This results in a hostile microenvironment that promotes epithelial–mesenchymal transition (EMT), a process in which epithelial cells lose their polarity and gain increased motility, which is associated with metastasis and invasion. Here, we demonstrate that gold nanoparticles (AuNPs) facilitate tumor vasculature normalization, increase blood perfusion and alleviate hypoxia in melanoma tumors. Additionally, AuNPs were observed to reverse EMT in tumors, accompanied by the alleviation of lung metastasis. These AuNPs inhibited the migration of B16F10 cells and reversed EMT in B16F10 cells, indicating that AuNPs could directly regulate EMT independent of improvements in hypoxia. Taken together, our data demonstrated that AuNPs could induce tumor vasculature normalization and reverse EMT, resulting in decreased melanoma tumor metastasis.

Elevated collagen-I augments tumor progressive signals, intravasation and metastasis of prolactin-induced estrogen receptor alpha positive mammary tumor cells

BackgroundThe development and progression of estrogen receptor alpha positive (ERα+) breast cancer has been linked epidemiologically to prolactin. However, activation of the canonical mediator of prolactin, STAT5, is associated with more differentiated cancers and better prognoses. We have reported that density/stiffness of the extracellular matrix potently modulates the repertoire of prolactin signals in human ERα + breast cancer cells in vitro: stiff matrices shift the balance from the Janus kinase (JAK)2/STAT5 cascade toward pro-tumor progressive extracellular regulated kinase (ERK)1/2 signals, driving invasion. However, the consequences for behavior of ERα + cancers in vivo are not known.MethodsIn order to investigate the importance of matrix density/stiffness in progression of ERα + cancers, we examined tumor development and progression following orthotopic transplantation of two clonal green fluorescent protein (GFP) + ERα + tumor cell lines derived from prolactin-induced tumors to 8-week-old wild-type FVB/N (WT) or collagen-dense (col1a1tm1Jae/+) female mice. The latter express a mutant non-cleavable allele of collagen 1a1 “knocked-in” to the col1a1 gene locus, permitting COL1A1 accumulation. We evaluated the effect of the collagen environment on tumor progression by examining circulating tumor cells and lung metastases, activated signaling pathways by immunohistochemistry analysis and immunoblotting, and collagen structure by second harmonic generation microscopy.ResultsERα + primary tumors did not differ in growth rate, histologic type, ERα, or prolactin receptor (PRLR) expression between col1a1tm1Jae/+ and WT recipients. However, the col1a1tm1Jae/+ environment significantly increased circulating tumor cells and the number and size of lung metastases at end stage. Tumors in col1a1tm1Jae/+ recipients displayed reduced STAT5 activation, and higher phosphorylation of ERK1/2 and AKT. Moreover, intratumoral collagen fibers in col1a1tm1Jae/+ recipients were aligned with tumor projections into the adjacent fat pad, perpendicular to the bulk of the tumor, in contrast to the collagen fibers wrapped around the more uniformly expansive tumors in WT recipients.ConclusionsA collagen-dense extracellular matrix can potently interact with hormonal signals to drive metastasis of ERα + breast cancers.

MenaINV dysregulates cortactin phosphorylation to promote invadopodium maturation.

Invadopodia, actin-based protrusions of invasive carcinoma cells that focally activate extracellular matrix-degrading proteases, are essential for the migration and intravasation of tumor cells during dissemination from the primary tumor. We have previously shown that cortactin phosphorylation at tyrosine residues, in particular tyrosine 421, promotes actin polymerization at newly-forming invadopodia, promoting their maturation to matrix-degrading structures. However, the mechanism by which cells regulate the cortactin tyrosine phosphorylation-dephosphorylation cycle at invadopodia is unknown. Mena, an actin barbed-end capping protein antagonist, is expressed as various splice-isoforms. The MenaINV isoform is upregulated in migratory and invasive sub-populations of breast carcinoma cells, and is involved in tumor cell intravasation. Here we show that forced MenaINV expression increases invadopodium maturation to a far greater extent than equivalent expression of other Mena isoforms. MenaINV is recruited to invadopodium precursors just after their initial assembly at the plasma membrane, and promotes the phosphorylation of cortactin tyrosine 421 at invadopodia. In addition, we show that cortactin phosphorylation at tyrosine 421 is suppressed by the phosphatase PTP1B, and that PTP1B localization to the invadopodium is reduced by MenaINV expression. We conclude that MenaINV promotes invadopodium maturation by inhibiting normal dephosphorylation of cortactin at tyrosine 421 by the phosphatase PTP1B.

Oriented collagen fibers direct tumor cell intravasation.

In this work, we constructed a Collagen I-Matrigel composite extracellular matrix (ECM). The composite ECM was used to determine the influence of the local collagen fiber orientation on the collective intravasation ability of tumor cells. We found that the local fiber alignment enhanced cell-ECM interactions. Specifically, metastatic MDA-MB-231 breast cancer cells followed the local fiber alignment direction during the intravasation into rigid Matrigel (∼10 mg/mL protein concentration).

Endosialin-Expressing Pericytes Promote Metastatic Dissemination.

Metastasis is a multistep process that is critically dependent on the interaction of metastasizing tumor cells with cells in the local microenvironment. Within this tumor stroma, vessel-associated pericytes and myofibroblasts share a number of traits, including the upregulated expression of the transmembrane receptor endosialin (CD248). Comparative experiments in wild-type and endosialin-deficient mice revealed that stromal endosialin does not affect primary tumor growth but strongly promotes spontaneous metastasis. Mechanistically, endosialin-expressing pericytes in the primary tumor facilitate distant site metastasis by promoting tumor cell intravasation in a cell contact-dependent manner, resulting in elevated numbers of circulating tumor cells. Corresponding to these preclinical experiments, in independent cohorts of primary human breast cancers, upregulated endosialin expression significantly correlates with increased metastasis and poorer patient survival. Together, the data demonstrate a critical role for endosialin-expressing primary tumor pericytes in mediating metastatic dissemination and identify endosialin as a promising therapeutic target in breast cancer. Cancer Res; 76(18); 5313-25. ©2016 AACR.

Discontinuation of anti-VEGF cancer therapy promotes metastasis through a liver revascularization mechanism.

The impact of discontinuation of anti-VEGF cancer therapy in promoting cancer metastasis is unknown. Here we show discontinuation of anti-VEGF treatment creates a time-window of profound structural changes of liver sinusoidal vasculatures, exhibiting hyper-permeability and enlarged open-pore sizes of the fenestrated endothelium and loss of VE-cadherin. The drug cessation caused highly leaky hepatic vasculatures permit tumour cell intravasation and extravasation. Discontinuation of an anti-VEGF antibody-based drug and sunitinib markedly promotes liver metastasis. Mechanistically, host hepatocyte, but not tumour cell-derived vascular endothelial growth factor (VEGF), is responsible for cancer metastasis. Deletion of hepatocyte VEGF markedly ablates the ‘off-drug'-induced metastasis. These findings provide mechanistic insights on anti-VEGF cessation-induced metastasis and raise a new challenge for uninterrupted and sustained antiangiogenic therapy for treatment of human cancers.

Abstract LB-006: Macrophage-dependent activation of Notch1 signaling regulates breast tumor cell intravasation

Abstract 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.

Abstract 4579: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased cell signaling and tumor cell intravasation

Abstract G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which new signaling entities can be created with respect to agonist binding. Preliminary data from our lab indicates that agonist-bound GPCR C-X-C Chemokine Receptor 4 (CXCR4) and agonist-bound cannabinoid receptor 2 (CB2R) interact at the cell membrane and that receptor interaction mitigates the effects of CXCL12-mediated CXCR4 signaling. RhoA, Rac1 and Cdc42 are small GTP-binding proteins of the Rho family that are activated downstream of CXCR4 signaling. The effects of RhoA, Rac1 and Cdc42 on the actin cytoskeleton have led to established roles for Rho proteins in cancer cell motility, metastasis and intravasation. Here we report the novel finding that CXCR4/CB2R heterodimerization decreases Rho signaling, resulting in decreased intravasation. To test the effect of receptor heterodimerization on RhoA, Rac1, and Cdc42 activation, PC3 cells were treated with the CXCR4 agonist SDF1α, the CB2R agonist AM1241 or with a combination of SDF1α and AM1241 for 1 minute. Cell lysates were collected and immunoprecipitation experiments were performed to detect RhoA, Rac1 and Cdc42. To test the effects of CXCR4/CB2R heterodimerization on intravsation, we performed a zymography assay for the detection of metalloproteinase-2 (MMP2) and metalloproteinase-9 (MMP9), which are commonly secreted by CXCR4 signaling. Immunopreciptation results indicated an increase in RhoA and Rac1 protein levels after 1 minute in PC3 cells compared to the untreated control. However, when treated with SDF1α and AM1241 for 1 minute, RhoA and Rac1 levels decreased compared to SDF1α independent treatment; there was no observable change in Cdc42 protein levels. The results from the zymography experiments demonstrated that co-treatment with SDF1α and AM1241, which induces CXCR4/CB2R heterodimerization, led to decreased MMP-2 and MMP-9 activity, as compared to the SDF1α-independent treatment. Preliminary results provide novel evidence that CXCR4 and CB2R heterodimerize at the cell membrane, which may result in decreased signaling through RhoA and Rac1, but not through Cdc42. Furthermore, CXCR4/CB2R heterodimerization results in decreased MMP activation, thus abrogating intravsation. Citation Format: Imani N. Fennell, Kisha A. Scarlett, Christopher J. Coke. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased cell signaling and 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 4579.

Abstract 4572: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 leads to decreased Rho-induced prostate cancer metastasis

Abstract BACKGROUND: The binding of the homeostatic chemokine SDF-1 to the G -protein-coupled receptor C-X-C Chemokine Receptor 4 (CXCR4) induces cell migration via heterotrimeric G-proteins of the Gi family. Additionally, activated CXCR4 can stimulate the small GTPase RhoA through Gα13 which is required for directional cell migration, contributing to the metastatic potential of cancer cells. Recently, GPCR hetero-oligomerization has emerged as a means by which new signaling entities can be created, yet how receptor heteromers affect receptor pharmacology remains largely unknown. Here we report that agonist-bound CXCR4 is able to form a heteromer with agonist-bound cannabinoid receptor 2 (CB2) at the cell membrane of prostate cancer (PCa) cells and that receptor interaction inhibits the activation of the cytoskeleton regulator RhoA. EXPERIMENTAL METHODS: To confirm CXCR4-CB2 interactions on the cell membrane, we utilized an in situ proximity ligation assay to detect heterodimer formation on the surface PC-3 cells treated with SDF-1 alone or, simultaneous treatment with SDF-1 and the CB2 agonist AM1241. In order to establish that RhoA activity decreased upon heterodimerization of CXCR4 and CB2, we performed RhoA-GTP pulldown assays in PC-3 cells. We then tested the effects of CXCR4/CB2 heterodimerization on actin cytoskeletal reorganization by performing immunocytochemistry using an anti-Phalloidin antibody to detect F-actin. The ROCK kinase inhibitor Y-27632 was used as positive control of RhoA inhibition in these studies. Next, we assessed the effects of receptor heterodimerization on cellular adhesion to extracellular matrix components as the ability of cancer cells to adhere to the endothelium is critical of transendothelial migration during tumor cell intravasation and extravasation. Intuitively, alterations in adhesion capabilities should affect the ability of PCa cells to migrate through an endothelial cell monolayer. Thus we performed transendothelial migration assays. RESULTS: The proximity ligation assay revealed that CXCR4 and CB2 physically interacted on the cell surface of prostate cancer cells. Furthermore, receptor heterodimerization diminished SDF-1 induced actin cytoskeleton reorganization as characterized by membrane ruffling in a similar manner to the ROCK kinase inhibitor Y-27632. Heterodimer formation, stimulated by simultaneous treatment of cells with SDF-1 and AM1241 also attenuated PCa adhesion and transendothelial migration. CONCLUSION The attenuation of RhoA has multiple functional consequences leading to a less metastatic phenotype. Thus induction of CXCR4-CB2 heterodimerization is a viable pharmacological approach to targeting the CXCR4-RhoA axis as a means of preventing the metastatic dissemination of PCa cells. Citation Format: Kisha A. Scarlett, Christopher J. Coke, Imani Fennell, Cimona V. Hinton. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 leads to decreased Rho-induced prostate cancer metastasis. [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 4572.

Abstract 244: Paclitaxel increases the assembly and function of the tumor microenvironment of metastasis in breast cancer

Abstract Chemotherapy induces influx of bone marrow-derived progenitors such as mesenchymal stem cells, endothelial progenitors and proangiogenic monocytes into the primary tumor to promote angiogenesis. Thus it is feared that chemotherapy may potentiate tumor cell invasion and metastasis. Here, we show that paclitaxel delays tumor growth in several mammary carcinoma mouse and human breast cancer models, yet it significantly increases the density of microanatomical sites called “tumor microenvironment of metastasis” (TMEM) that are responsible for tumor cell intravasation and dissemination of breast cancer. The TMEM site consists of a Mena-overexpressing cancer cell in direct contact with a Tie2hi/VEGFhi macrophage and an underlying endothelial cell. Mice treated with paclitaxel have significantly more circulating tumor cells (CTCs) and metastatic foci when compared to vehicle-treated animals indicating that the chemotherapy-induced TMEM are active in assisting tumor cell intravasation. Moreover, syngeneic transplantation of Dendra2+/PyMT tumors into FVB recipients showed significantly higher incidence of Dendra2+ cells in the lung, following paclitaxel administration. In parallel experiments, paclitaxel induced the influx of macrophages and intravasation of cancer cells as observed using intravital imaging of MMTV-PyMT-Dendra2/Cfms-CFP mice, in which blood vessels were visualized with Quantum dots. Furthermore, paclitaxel treatment in experimental mice caused a significant increase in the expression of Mena at the gene and protein levels. PCR assays for total Mena (PanMena) or specific Mena isoforms (MenaINV, Mena11a) revealed that this increase was particularly attributed to the invasive Mena isoforms [i.e. MenaINV and MenaCalc (Menacalc = PanMena - Mena11a)]. These pre-clinical data are supported by the findings from a cohort of 10 breast cancer patients who received neoadjuvant dose-dense paclitaxel followed by doxorubicin/ cyclophosphamide. Of these tumors, 7/10 patients had more than 2-fold increase in TMEM density following neoadjuvant chemotherapy regimen. Moreover, chemotherapy produced an acute increase of up to 150-fold in MenaINV expression in 3/7 and up to 5.5-fold in MenaCalc in 3/4 patients who underwent serial fine needle aspiration (FNA) biopsy before and after 1-2 doses of either neoadjuvant paclitaxel or doxorubicin-cyclophosphamide. This is provocative because an increase in either MenaCalc score or TMEM density are independently associated with increased risk of distant recurrence in breast cancer patients. In conclusion, our data indicate that paclitaxel treatment induces intravasation-mediated dissemination of breast cancer cells in rodents and in certain clinical scenarios in humans by promoting increases in MenaCalc expression and TMEM intravasation sites. Citation Format: George S. Karagiannis, Allison H. Harney, Yarong Wang, Jessica Pastoriza, Jeanine Pignatelli, Jesus Anampa, Joseph A. Sparano, Joan G. Jones, David Entenberg, John S. Condeelis, Maja H. Oktay. Paclitaxel increases the assembly and function of the tumor microenvironment of metastasis in breast cancer. [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 244.

Tumor cell intravasation.

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.

When the endothelium scores an own goal: endothelial cells actively augment metastatic extravasation through endothelial-mesenchymal transition.

Endothelial-mesenchymal transition (EndMT) is an important mechanism during organ development and in certain pathological conditions. For example, EndMT contributes to myofibroblast formation during organ fibrosis, and it has been identified as an important source of cancer-associated fibroblasts, facilitating tumor progression. Recently, EndMT was proposed to modulate endothelial function during intravasation and extravasation of metastatic tumor cells. Evidence suggests that endothelial cells are not passive actors during transendothelial migration (TEM) of cancer cells, as there are profound changes in endothelial junctional protein expression, signaling, permeability, and contractility. This review describes these alterations in endothelial characteristics during TEM of metastatic tumor cells and discusses them in the context of EndMT. EndMT could play an important role during metastatic intravasation and extravasation, a novel hypothesis that may lead to new therapeutic approaches to tackle metastatic disease.

Chorioallantoic Membrane Microtumor Model to Study the Mechanisms of Tumor Angiogenesis, Vascular Permeability, and Tumor Cell Intravasation.

The mechanisms governing the development of angiogenic blood vessels, which not only deliver the nutrients to growing tumors but also provide the conduits for tumor cell dissemination, are still not fully resolved. The model systems based on the grafting of human tumor cells onto the chorioallantoic membrane (CAM) of the chick embryo offer several advantages to study complex processes underlying tumor angiogenesis and tumor cell dissemination. In particular, the CAM model described here allows for investigation of multiple microtumors as independent entities, thereby greatly facilitating quantification and statistical analyses of tumor neovascularization and cancer spreading. This CAM microtumor system was designed specifically to measure the level of tumor cell intravasation in combination with quantitative analyses of the microarchitecture and permeability of the intratumoral angiogenic blood vessels. By using this newly established microtumor model we have demonstrated the functional involvement of tumor matrix metalloproteinase-1 (MMP-1) and epidermal growth factor receptor (EGFR) in regulating the development of a distinct angiogenic vasculature capable of sustaining tumor cell intravasation and metastasis.

Isolation of Mouse and Human Tumor-Associated Macrophages.

The tumor microenvironment is a complex network of cells that support tumor progression and malignancy. It has been demonstrated that tumor cells can educate the immune system to promote a tumor-friendly environment. Among all these immune cells, tumor-associated macrophages (TAMs) are well represented and their presence in mouse models has been shown to promote tumor progression and metastasis. These effects are through the stimulation of angiogenesis, enhancement of tumor cell invasion and intravasation, immunosuppression, and at the metastatic site tumor cell extravasation and growth. However, the precise mechanisms are not fully understood. Furthermore there is limited information on TAMs derived from human cancers. For this reason it is important to be able to extract TAMs from tumors in order to compare their phenotypes, functions, and transcriptomes with normal resident tissue macrophages. Isolation of these cells is challenging due to the lack of markers and standardized protocols. Here we show an optimized protocol for the efficient isolation and extraction of resident macrophages and TAMs from human and mouse tissues by using multicolor flow cytometry. These protocols allow for the extraction of thousands of macrophages in less than 5 h from tissues as small as half a gram. The isolated macrophages can then be used for both "omics" and in vitro studies.

Immunosuppressive cells in tumor immune escape and metastasis.

Tumor immune escape and the initiation of metastasis are critical steps in malignant progression of tumors and have been implicated in the failure of some clinical cancer immunotherapy. Tumors develop numerous strategies to escape immune surveillance or metastasize: Tumors not only modulate the recruitment and expansion of immunosuppressive cell populations to develop the tumor microenvironment or pre-metastatic niche but also switch the phenotype and function of normal immune cells from a potentially tumor-reactive state to a tumor-promoting state. Immunosuppressive cells facilitate tumor immune escape by inhibiting antitumor immune responses and furthermore promote tumor metastasis by inducing immunosuppression, promoting tumor cell invasion and intravasation, establishing a pre-metastatic niche, facilitating epithelial-mesenchymal transition, and inducing angiogenesis at primary tumor or metastatic sites. Numerous translational studies indicate that it is possible to inhibit tumor immune escape and prevent tumor metastasis by blocking immunosuppressive cells and eliminating immunosuppressive mechanisms that are induced by either immunosuppressive cells or tumor cells. Furthermore, many clinical trials targeting immunosuppressive cells have also achieved good outcome. In this review, we focus on the underlying mechanisms of immunosuppressive cells in promoting tumor immune escape and metastasis, discuss our current understanding of the interactions between immunosuppressive cells and tumor cells in the tumor microenvironment, and suggest future research directions as well as potential clinical strategies in cancer immunotherapy.

Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA.

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 TIE2(hi) 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. Tumor vasculature is abnormal with increased permeability. Here, we show that VEGFA signaling from TIE2(hi) 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.

Abstract 5125: Imaging the tumor microenvironment of metastasis reveals the mechanism of transient blood vessel permeability and tumor cell intravasation

Abstract Sites of direct contact between a macrophage, a tumor cell and endothelial cell [Tumor MicroEnvironment of Metastasis (TMEM)], correlates with metastasis in breast cancer patients independently of other clinical prognostic indicators suggesting a direct role for TMEM function in hematogenous dissemination. Here we show, using intravital high-resolution two-photon microscopy, that tumor cell intravasation occurs only at TMEM. Tumor cell intravasation occurs concurrently with transient, local vascular permeability at TMEM in an autochthonous mouse mammary carcinoma model and a human patient-derived xenograft model. Ablation of the presence or activity of the TMEM associated macrophages blocks tumor cell intravasation at TMEM demonstrating an essential role of perivascular macrophages in TMEM function. A subset of TMEM macrophages are identified as Tie2-expressing macrophages that are characterized by F4/80+/CD11b+/CD68+/MRC1+/Tie2Hi/VEGFAHi/CD11c-. VEGFA signaling from Tie2Hi TMEM-associated macrophages causes the local loss of vascular junctions resulting in transient vascular permeability and tumor cell intravasation at TMEM. Macrophage-specific ablation of VEGFA results in increased vascular junction stability and inhibition of intravasation, demonstrating that vascular junction dissolution at VEGFAHi/Tie2Hi TMEM-associated macrophages leads to vascular permeability and tumor cell intravasation. Inhibition of Tie2 with the first in class small molecular inhibitor rebastinib impairs TMEM function leading to a reduction in vascular permeability, tumor cell dissemination and metastasis. Further, rebastinib inhibition of Tie2 blocks tumor cell extravasation and metastatic growth in the lungs. Together, the findings that TMEM macrophages mediate vascular permeability and tumor cell intravasation demonstrate an essential role for TMEM within the primary mammary tumor as sites of tumor cell dissemination. These data reveal the mechanism of tumor cell intravasation in breast cancer, explain the prognostic value of TMEM density in predicting distant metastatic recurrence in breast cancer patients and document a strategy for inhibition of dissemination. This research is supported by the Department of Defense Breast Cancer Research Program under award number BC120227 (ASH), NIH CA100324 (JSC) and the Integrated Imaging Program. Citation Format: Allison S. Harney, Esther N. Arwert, David Entenberg, Yarong Wang, Peng Guo, Bin-Zhi Qian, Bryan D. Smith, Jeffrey W. Pollard, Joan G. Jones, Daniel L. Flynn, John S. Condeelis. Imaging the tumor microenvironment of metastasis reveals the mechanism of transient blood vessel permeability and tumor cell intravasation. [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 5125. doi:10.1158/1538-7445.AM2015-5125

Abstract 4019: Examining mechanisms by which macrophages enhance intravasation in breast cancer

Abstract 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<10-4) in iTEM. Together, these results suggest that cellular signaling through macrophage CXCR2 is critical to tumor cell intravasation. 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