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Tumor cell intravasation is an essential step in the metastatic cascade, but its exact mechanism is not completely understood. We have previously shown that the direct physical association of a tumor cell over-expressing Mena, a Tie2hi/Vegfhi macrophage and an endothelial cell, creates a micro-anatomic doorway called “tumor microenvironment of metastasis” (TMEM). TMEM are responsible for cancer cell intravasation and dissemination to distant sites. The density of TMEM doorways is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that TMEM doorways create increased localized vascular permeability which cancer cells utilize to intravasate, the precise molecular mechanisms relating TMEM-doorway function and intravasation has not been elucidated. Active TMEM doorways are found in pre-invasive and invasive ductal breast carcinoma as well as in metastatic foci in lymph nodes and lungs, indicating that TMEM-mediated cancer cell dissemination occurs not only at the primary tumor site but also at metastatic sites, which may perpetuate metastatic dissemination even after removal of the primary tumor. Thus it is essential to understand the exact molecular mechanism of TMEM-doorway function so that specific targeted therapies can be developed to intercept systemic cancer cell dissemination. We outline here the exact molecular mechanism of TMEM-doorway functions. TMEM doorway endothelial cell-secreted Ang2 (a Tie2 ligand) stimulates VEGF expression and production by the Tie2hi TMEM macrophage. Subsequently, the TMEM doorway tumor cell- secreted CSF1 stimulates local secretion of VEGF from the Tie2hi TMEM macrophages, leading to dissociation of endothelial adherens and tight junctions near TMEM and cancer cell intravasation. In addition, we show that acute blockage of CSF1R and Tie2-Ang2 signaling by inhibitors and blocking antibodies both in vitro and in mammary tumors leads to decreased macrophage VEGF production and secretion, decreased tumor cell trans-endothelial migration, and decreased TMEM-dependent vascular permeability, tumor cell dissemination and circulating tumor cells. This is the first description of the molecular mechanisms regulating TMEM doorway function and thus represents a major step in defining new biomarkers and targets for the treatment of metastatic tumors. Citation Format: Chinmay Surve, Allison S. Harney, Mary Chen, Yarong Wang, Xianjun Ye, Yu Lin, Ved Sharma, Richard Stanley, Maja H. Oktay, John S. Condeelis. Regulation of breast tumor metastasis by the dynamic interaction between the TMEM doorway macrophage, tumor and endothelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-10.

Background: Cancer stem cells (CSCs) play an important role during metastatic progression of breast cancer. However, little is known, at the single cell level, about the process of stemness induction in non-stem cells or the dynamic behavior of CSCs during hematogenous dissemination. Methods: Here, we employed high-resolution intravital multiphoton microscopy with a SOX2/OCT4 responsive fluorescent biosensor for stemness to directly observe the induction of stemness in single non-stem cells and their evolution through the metastatic cascade in living animals using orthotopic breast cancer xenograft model. We confirmed our findings in vitro using tumor cell-macrophage co-culture assays. Results: We report that, both in vitro and in vivo, direct physical contact with macrophages induces stemness in non-stem cancer cells via juxtacrine Notch-Jagged1 signaling. In vivo, macrophage depletion with clodronate treatment showed a significant decrease in stem cells. In vitro, using either the fate mapping of non-stem cells with or without macrophage contact, or the origin-mapping of stem cells to find whether they originated from non-stem cells or pre-existing stem cells, we found that there was four-fold increase in new CSC induction after direct macrophage contact. In contrast, we did not see any role of macrophages in the expansion of pre-existing CSCs, both in vivo and in vitro, indicating that macrophage contact-dependent stem induction is the primary mechanism of CSC generation. Using immunohistochemical staining in fixed tissue and live imaging of primary tumors and lungs in mice using optical windows, we found that during the course of dissemination of tumor cells from the primary site, CSCs become progressively enriched in the tumor cell population as they approach dissemination doorways (known as TMEM, Tumor MicroEnvironment of Metastasis), intravasate, circulate and arrive at the lung. Association with and passage through TMEM doorways is the step that generates the greatest enrichment in CSCs (~ 60-fold). On arrival in the lung, CSCs represent more than 75% of the disseminated tumor cell population, greatly enriched compared with their representation in the bulk primary tumor of ~ 1%. Conclusion: Overall, these data indicate, for the first time, that macrophages associated with TMEM induce CSCs and promote TMEM-mediated CSC intravasation and early metastatic seeding. Our results are consistent with the dramatic enrichment of cancer stem cell markers in association with TMEM in breast cancer patients (Kim et al 2020 AACR abstract) and support a strategy for anti-metastatic therapy. Citation Format: Ved P. Sharma, Yarong Wang, Binwu Tang, George S. Karagiannis, Emily A. Xue, David Entenberg, Lucia Borriello, Anouchka Coste, Joan G. Jones, Chinmay R. Surve, Dominic Esposito, Maja H. Oktay, Lalage M. Wakefield, John S. Condeelis. Macrophage contact-dependent stemness induction and progressive CSC enrichment during metastatic dissemination in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 372.

Our laboratory has identified a number of small molecules that bind to G protein βγ subunits (Gβγ) by competing for peptide binding to the Gβγ "hot spot." M119/Gallein were identified as inhibitors of Gβγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on Gβγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γ activation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca(2+) release, and activated other downstream targets of Gβγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases Gβγ in vitro from Gαi1β1γ2 heterotrimers by causing its dissociation from GαGDP without inducing nucleotide exchange in the Gα subunit. We used this novel probe to examine the hypothesis that Gβγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free Gβγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner.

Cancer stem cells (CSCs) play an important role during metastatic progression of breast cancer. However, the in vivo properties and dynamic behavior of CSCs are not well understood. Here, we employed high-resolution intravital multiphoton microscopy using a SOX2/OCT4 responsive fluorescent stem cell biosensor to directly observe CSC dynamics in the living animal using an orthotopic breast cancer xenograft model. We report that CSCs constitute a minority population (1-3%) in the primary tumors, and display the slow-migratory, invasive phenotype that is specifically associated with disseminating tumor cell population. We also report, for the first time, that CSCs are preferentially localized in direct contact with macrophages near and in tumor microenvironment of metastasis (TMEM) sites, the macrophage-containing intravasation doorway for tumor cells and that CSCs metastasize to lung and are strikingly enriched in early lung metastatic colonies. This is explained by our observation that, in vitro and in vivo, direct physical contact with macrophages induces stemness in non-stem cancer cells via juxtacrine Notch-Jagged1 signaling. These data indicate for the first time that macrophages play an actively inductive role in the CSC niche and promote TMEM-mediated CSC intravasation and early metastatic seeding. Citation Format: Ved P. Sharma, Yarong Wang, Binwu Tang, George S. Karagiannis, Emily A. Xue, David Entenberg, Lucia Borriello, Anouchka Coste, Chinmay R. Surve, Dominic Esposito, Maja H. Oktay, Lalage M. Wakefield, John S. Condeelis. Direct observation in living tumors shows macrophage-dependent induction and dissemination of cancer stem cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 972.

Tumor cell intravasation is an essential step in the metastatic cascade, but its exact mechanism is not completely understood. We have previously shown that the direct physical association of a tumor cell over-expressing Mena, a perivascular Tie2hi/Vegfhi macrophage and an endothelial cell, forming a cell triad termed “tumor microenvironment of metastasis” (TMEM), increases vascular permeability, facilitating intravasation of tumor cells. It is only at the TMEM site that intravasation occurs leading to breast tumor metastasis. TMEM density is a clinically validated prognostic marker of distant metastasis in breast cancer patients. The precise molecular mechanisms relating TMEM function had not been elucidated. Here we describe the molecular mechanism. We show here that TMEM function involves the three cells in TMEM: firstly endothelial cell-secreted Ang2 stimulates VEGF build up in the TMEM macrophage, secondly a tumor cell secretes CSF1 which, third, stimulates the TMEM macrophage VEGF secretion, leading to vascular opening and metastasis. In addition, we show that acute blockage of CSF1R and Tie2-Ang2 signaling by inhibitors and blocking antibodies both in vitro and in mammary tumors leads to decreased macrophage VEGF production and secretion, decreased trans-endothelial migration of tumor cells, and decreased TMEM-dependent vascular permeability, circulating tumor cells and lung metastases. We conclude that dynamic interaction between the cells associated with TMEM leads to Ang2 and CSF1-mediated stimulation of macrophage VEGF expression and secretion leading to vascular opening, resulting in tumor cell intravasation. This is the first description of the molecular mechanism behind the predictive power of the clinically used prognostic marker TMEM and represents a major step in defining new biomarkers and targets for the treatment of metastatic tumors. Citation Format: Chinmay R. Surve, Allison Harney, Yarong Wang, Xiaoming Chen, Ved Sharma, Richard Stanley, Maja Oktay, John Condeelis. Regulation of breast tumor metastasis by the dynamic interaction between the TMEM macrophage, tumor, and endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 976.

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., United States. National Institutes of Health (CA150344), United States. National Institutes of Health (CA100324)

Activation of the Gi family of heterotrimeric guanine nucleotide–binding proteins (G proteins) releases βγ subunits, which are the major transducers of chemotactic G protein–coupled receptor (GPCR)–dependent cell migration. The small molecule 12155 binds directly to Gβγ and activates Gβγ signaling without activating the Gαi subunit in the Gi heterotrimer. We used 12155 to examine the relative roles of Gαi and Gβγ activation in the migration of neutrophils on surfaces coated with the integrin ligand intercellular adhesion molecule–1 (ICAM-1). We found that 12155 suppressed basal migration by inhibiting the polarization of neutrophils and increasing their adhesion to ICAM-1–coated surfaces. GPCR-independent activation of endogenous Gαi and Gβγ with the mastoparan analog Mas7 resulted in normal migration. Furthermore, 12155-treated cells expressing a constitutively active form of Gαi1 became polarized and migrated. The extent and duration of signaling by the second messenger cyclic adenosine monophosphate (cAMP) were enhanced by 12155. Inhibiting the activity of cAMP-dependent protein kinase (PKA) restored the polarity of 12155-treated cells but did not decrease their adhesion to ICAM-1 and failed to restore migration. Together, these data provide evidence for a direct role of activated Gαi in promoting cell polarization through a cAMP-dependent mechanism and in inhibiting adhesion through a cAMP-independent mechanism.

Numerous clinical studies have identified Tumor MicroEnvironment of Metastasis (TMEM) and MenaCalc as distinct but functionally interrelated prognostic indicators of distant metastasis in breast cancer patients. TMEM sites are the only sites where tumor cells intravasate in mammary tumors. TMEM score is calculated histologically as the density of tripartite microanatomical structures involving a perivascular macrophage, a Mena-expressing tumor cell and an endothelial cell, all three in direct physical contact. On the other hand, MenaCalc represents the pattern of Mena splice-isoforms present in a tumor sample. MenaINV is the key metastasis-promoting Mena splice-isoform driving tumor cell migration toward blood vessels, intravasation and dissemination. However, the precise molecular mechanisms relating TMEM formation and function and MenaINV expression in these critical steps of the metastatic cascade have not been elucidated. Here we show that MenaINV promotes invadopodium-based proteolysis, which is required for tumor cell invasion and transendothelial migration, by preventing the localization of the phosphatase PTP1B to invadopodia. Interestingly, PTP1B regulates invadopodium maturation by limiting cortactin phosphorylation at a key residue (Y421) that is necessary for actin polymerization during invadopodium maturation. Additionally, we demonstrate that MenaINV expression, invadopodium activity, and subsequent transendothelial migration are induced in tumor cells via Notch1-mediated signaling induced by contact of tumor cells with macrophages. Knock-down of MenaINV expression in tumor cells leads to a proportional decrease in mature invadopodium formation. Complete knock-out of Mena in mouse mammary tumors (PyMT-MMTV) abolishes TMEM assembly and TMEM functions, including TMEM-dependent vascular permeability, circulating tumor cells and lung metastases. In summary, our work shows that macrophage contact of TMEM-associated tumor cells during TMEM assembly stimulates Notch1 to drive expression of MenaINV in tumor cells. MenaINV expression then inhibits PTP1B at invadopodia to promote cortactin phosphorylation at invadopodium precursors, driving invadopodium maturation and promoting transendothelial migration. These findings provide, for the first time, an integrative molecular mechanism for two clinically validated prognostic indicators of metastatic risk, TMEM and MenaCalc, and identify new drug targets for limiting the metastatic spread of breast cancer. Citation Format: Maxwell D. Weidmann, Chinmay R. Surve, Jeanine Pignatelli, Javier J. Bravo-Cordero, George S. Karagiannis, Maja H. Oktay, John S. Condeelis. Elucidation of the molecular mechanism of MenaINV expression, invadopodium maturation and tumor cell intravasation during breast cancer dissemination by TMEM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 898. doi:10.1158/1538-7445.AM2017-898

Synergistic activation by heterotrimeric guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110beta from other class IA phosphoinositide 3 kinases (PI3Ks). Activation of p110beta is specifically implicated in various physiological and pathophysiological processes such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110beta dependent functions we identified the site in p110beta that binds to the Gbetagamma subunit of G proteins. Mutation of this site eliminated Gbetagamma dependent activation of PI3Kbeta (a dimer of p110beta and the p85 regulatory subunit) in vitro and in cells without affecting basal activity or phosphotyrosine peptide mediated activation. Disrupting the p110beta Gbetagamma interaction by mutation or with a cell permeable peptide inhibitor blocked the transforming capacity of PI3Kbeta in fibroblasts and reduced the proliferation chemotaxis and invasiveness of PTEN null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kbeta could provide a therapeutic approach for tumors that depend on p110beta for growth and metastasis.