Tumor Microenvironment Of Metastasis Function Research Articles

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in naïve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

Abstract P2-05-01: Chemotherapy-induced metastasis: Mechanisms and translational opportunities

Abstract 1 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, NY, USA 2 Integrated Imaging Program, Albert Einstein College of Medicine, NY, USA 3 Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, NY, USA 4 Department of Pathology, Albert Einstein College of Medicine, NY, USA Background: Chemotherapy has become essential in the care of patients with advanced breast cancer. However, as we have previously discovered, chemotherapy may induce pro-metastatic changes in the breast cancer microenvironment by promoting the assembly and function of cancer cell intravasation sites called tumor microenvironment of metastasis (TMEM), and by increasing the proportion of the highly-invasive MenaINV-HI tumor cells that utilize TMEM sites for hematogenous dissemination. Consequently, chemotherapy-treated animals demonstrate increased numbers of circulating-tumor cells and micrometastases. Since the formation of metastases depends on both the presence of functional doorways for dissemination (TMEM) and cancer cells capable of using these doorways (MenaINV-HI tumor cells), we investigated the cellular and molecular contexts required for chemotherapy-mediated induction of TMEM function and MenaINV expression. Since TMEM function depends on Tie2 expressing macrophages and MenaINV expression is inducible by cancer cell -macrophage contact, we focused on inhibiting Tie2 receptors and chemotherapy-induced macrophage influx to inhibit chemotherapy-induced metastasis. Materials and Methods: We used spontaneous models of mouse breast carcinoma (MMTV-PyMT) and patient-derived xenografts treated with chemotherapy with or without co-treatment with either Tie2 inhibitor (rebastinib), inhibitors of Cxcr4+ macrophage chemotaxis (Cxcl12 inhibitors) or macrophage depletion agents (clodronate). Pro-metastatic endpoints were measured by intravital imaging, multichannel immunofluorescence and standard metastasis dissemination assays. Results: We demonstrated, using multiple methods of macrophage suppression, that chemotherapy-mediated induction of MenaINV-HI tumor cells depends on the presence of macrophages. In particular, depletion of either the entire macrophage lineage using clodronate liposomes or the use of specific inhibitors of the Cxcl12/Cxcr4 chemotactic pathway, resulted in a significant suppression of the MenaINV-hi cancer cell subpopulation in all mammary tumors examined. Moreover, inhibition of Tie2 by rebastinib blocked TMEM function and decreased the number of circulating tumor cells and metastatic foci, despite the chemotherapy-mediated induction of MenaINV-HI tumor cells. Conclusions: Our data indicate that both the MenaINV-HI disseminating cancer cell population and the TMEM doorways are necessary but not individually sufficient for metastasis. As such, suppression of either the MenaINV-HI population or TMEM function can suppress chemotherapy-induced metastasis, thus providing a target to improve clinical care and eliminate non-beneficial effects of chemotherapy. Citation Format: Rivera Sanchez LG, Karagiannis GS, Wang Y, Sharma VP, Burt J, Entenberg D, Oktay M, Condeelis JS. Chemotherapy-induced metastasis: Mechanisms and translational opportunities [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-05-01.

Abstract P6-18-22: Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2-negative metastatic breast cancer (MBC)

Abstract BACKGROUND: TMEM (Tumor Microenvironment of Metastasis) are the portal for tumor cell intravasation into the circulation and subsequent metastasis (Harney et al Cancer Discov 2015). The potent Tie2 kinase inhibitor rebastinib inhibits intravasation at TMEM sites, reduces circulating tumor cell (CTC) burden, increases angiopoietin (Ang) 1/2 levels, prevents distant metastases, and improves survival in breast cancer animal models when added to either P or E (Harney et al MCT 2017), and circumvent chemotherapy-induced pro-metastatic changes in the tumor microenvironment mediated by TMEM (Karagiannis et al STM 2017). We sought to determine the safety of rebastinib combined with antitubulin therapy (P or E) in patients with HER2- MBC. We also hypothesized that addition of rebastinib would reduce CTC burden and increase Ang levels by blocking Ang-mediated stimulation of VEGF release from TMEM-associated macrophages. METHODS: We aimed to determine the safety and recommended phase 2 dose (RP2D) of rebastinib (2 dose levels: 50 mg or 100 mg PO BID) in combination with P (80 mg/m2 x 12 weeks) or E (1.4 mg/m2 on day 1 & 8 q 21 days) using a standard 3+3 design (1 cycle = 21 days). Secondary objectives included evaluating the effect of the P/E + rebastinib combination on CTCs (TelomeScan) and Ang levels. Dose limiting toxicity (DLT) was defined as grade 3-4 febrile neutropenia, thrombocytopenia, and non-hematologic toxicity during the first 6 weeks of therapy. Eligibility included HER2- MBC, ECOG PS 0-1, CDK4/6 inhibitor progression if ER+. Patients with ≤ 2 prior non-taxane chemotherapy regimens received P+ rebastinib, whereas those with ≥ 2 chemo regimens (including a taxane) received E+ rebastinib. RESULTS: Of 11 treated patients, 6 received rebastinib + P and 5 received rebastinib + E (2 non-evaluable due to rapid disease progression and non-compliance). Among 11 patients who received 60 treatment cycles, only 1 patient (treated with eribulin) had grade 3 events (anemia and neuropathy after week 6) potentially related to treatment. When combined with P, the RP2D of rebastinib was 100 mg PO BID, with DLT occurring in 0/6 patients. When combined with E, 0/3 evaluable patients had a DLT at 50 mg BID of rebastinib (accrual ongoing for 100mg BID). Best response included partial response/stable disease in 4(2PR/2SD) of 6 treated with P+ rebastinib, and 1(1PR) of 5 treated with E+ rebastinib. CTCs decreased during therapy (median decrease 99.7 %) and 4/8 patients converted from CTC+ to CTC-. Ang1 levels increased during therapy in 8 patients (0.2-7.0 fold), while Ang2 levels were also increased in 8 patients (0.2-1.4 fold). CONCLUSIONS: When combined with P x 12 weeks, the RP2D of rebastinib is 100 mg PO BID. When combined with E, the RP2D of rebastinib is at least 50mg PO BID; however, the 100 mg PO BID dose level is still accruing patients. The P/E + rebastinib combinations are associated with antitumor activity and exhibit pharmacodynamic evidence indicating blockade of Tie2 (increased Ang) and TMEM function (reduced CTCs) We plan to further evaluate the P+ rebastinib combination as neoadjuvant therapy in the I-SPY program, and continue further evaluation of P/E + rebastinib combinations in MBC. Citation Format: Anampa JD, Xue X, Oh S-y, Kornblum N, Sadan S, Oktay MH, Condeelis J, Sparano JA. Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2-negative metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-22.

Abstract P2-08-18: Tumor microenvironment of metastasis (TMEM) score in residual breast carcinoma post-neoadjuvant chemotherapy as an independent prognosticator of distant recurrence

Abstract Background: Tumor microenvironment of metastasis (TMEM) is a microanatomical structure composed by 3 cells in direct contact, including a tumor cell expressing the actin-regulatory protein Mammalian-enabled (Mena), a perivascular Tie2hi/Vegfhi-expressing macrophage, and an endothelial cell. TMEM are intravasation sites that function as doorways for hematogenous tumor cell dissemination and metastases (Harney et al. Cancer Discovery 2015). TMEM may be identified and enumerated by triple immunohistochemistry in mouse and human mammary carcinomas. High TMEM score is associated with increased risk of distant metastasis in early stage breast cancer, and provides complementary prognostic information to IHC4 (Rohan et al. JNCI 2014) and Oncotype DX Recurrence Score in ER+, HER2-negative breast cancer (Sparano et al. NPJ Breast Cancer, 2017). Neoadjuvant chemotherapy (NAC) increases TMEM score in breast carcinoma in animal models and humans, indicating a previously unrecognized mechanism of resistance to cytotoxic therapy (Karagiannis et al. Science Trans Med 2017). Intravasation at TMEM sites may be inhibited using agents that block release of VEGF from TMEM-associated TIE2-hi, VEGF-hi macrophages (Harney et al. Mol Cancer Ther, 2017). Here we investigated whether TMEM score in post-NAC treated breast carcinoma is prognostic of distant recurrence in localized breast cancer after NAC, and thus provides a foundation for testing agents that block TMEM function in combination with NAC. Methods: We determined TMEM score in 80 evaluable patients' post-NAC specimens with residual invasive ductal carcinomas of at least 0.5 cm. Approximately 60% of patients had ER+/HER2-negative, 28% had triple negative and 12% had HER2+ disease. Most of the patients received doxorubicin/cyclophosphamide + taxane and an anti-HER2 therapy if applicable. Tissue sections from residual tumors were stained for TMEM using triple immunohistochemistry for Mena-expressing cancer cells, CD31-expressing endothelial cells and CD68-expressing macrophages. The stained slides were scanned, and the images were analyzed by three pathologists, blinded to outcome, who independently determined the tissue areas appropriate for TMEM scoring. TMEM was scored within these areas using an automated algorithm. Results: TMEM score was significantly higher in patients with distant recurrence (average TMEM=106), compared to patients without distant recurrence (average TMEM=71) (p<0.01, two-sided t-test). Moreover, in a Cox proportional hazards model that included TMEM score (upper tertile vs. lower 2 tertiles), age (>50 yrs. vs. <50), race (black vs non-black), tumor stage (T 1-3), estrogen receptor (ER) status (+ vs -), high TMEM score was associated with a increased risk of distant recurrence (HR=2.2, 95% CI=1.0 to 4.9, p=0.05) Conclusion: TMEM score may provide independent prognostic information for distant recurrence in patients with residual invasive carcinoma after NAC. These results support the use of agents that block TMEM function in combination with NAC, as planned in the I-SPY2 trial. Citation Format: Oktay MH, D'Alfonso T, Ginter P, Lanjewar S, Entenberg D, Pastoriza JM, Wang Y, Lin Y, Karagiannnis GS, Lin J, Ye X, Anampa J, Xue X, Rohan TE, Sparano JA, Condeelis JS. Tumor microenvironment of metastasis (TMEM) score in residual breast carcinoma post-neoadjuvant chemotherapy as an independent prognosticator of distant recurrence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-18.

Abstract CT021: Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2 negative metastatic breast cancer (MBC)

Abstract BACKGROUND: Metastasis is the primary cause of death in breast cancer, yet no current therapies specifically inhibit metastasis. TMEM (Tumor Microenvironment of Metastasis) are the portal for tumor cell intravasation into the circulation and subsequent metastasis (Harney et al Cancer Discov 2015 [PMCID:4560669). The potent Tie2 kinase inhibitor rebastinib 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 E (Karagiannis et al STM 2017 [PMCID:5592784]; Harney et al MCT 2017 [PMCID:28838996]). We hypothesized that the addition of rebastinib to antitubulin therapy (P or E) in patients with HER2- MBC would be safe, and by inhibiting TMEM function, would also reduce CTC burden by blocking hematogenous dissemination. METHODS: The primary objective was to determine the safety and recommended phase 2 dose (RP2D) of rebastinib (2 dose levels: 50 mg or 100 mg PO BID) in combination with P (80 mg/m2 x 12 weeks) or E (1.4 mg/m2 on day 1 & 8 q 21 days) using a standard 3+3 design (1 cycle = 21 days). Secondary objectives included evaluating the effect of the P/E + rebastinib combination on CTCs (TelomeScan). Dose limiting toxicity (DLT) was defined as grade 3-4 febrile neutropenia, thrombocytopenia, and non-hematologic toxicity during the first 6 weeks of therapy. Eligibility included HER2- MBC, ECOG PS 0-1, progression after a CDK4/6 inhibitor if ER+. Patients with ≤ 2 prior non-taxane chemotherapy regimens received P+ rebastinib, whereas those with ≥ 2 chemo regimens (including a taxane) received E+ rebastinib. RESULTS: Of 10 treated patients, 6 received rebastinib + P and 4 received rebastinib + E (2 non-evaluable due to rapid disease progression [n=1] and non-compliance [n=1]). Among 10 patients who received 48 treatment cycles, only 1 patient (treated with eribulin) had grade 3 events (anemia and neuropathy after week 6) potentially related to treatment. When combined with P, the RP2D of rebastinib was 100 mg PO BID, with DLT occurring in 0/6 patients. When combined with E, 0/2 evaluable patients had a DLT at 50 mg BID of rebastinib (accrual ongoing). Best response included partial response/stable disease in 4(2PR/2SD) of 5 treated with P+ rebastinib (1 too early), and 2(1PR/1SD) of 4 treated with E+ rebastinib. CTCs decreased during therapy (baseline vs. day 43, median decrease 99.7 %) and 4/8 patients converted from CTC+ to CTC-. CONCLUSIONS: When combined with P x 12 weeks, the RP2D of rebastinib is 100 mg PO BID. The P/E + rebastinib combinations are associated with antitumor activity and exhibit pharmacodynamic evidence suggesting TMEM function blockade. We plan to further evaluate the P+ rebastinib combination as neoadjuvant therapy in the I-SPY program, and continue further evaluation of P/E + rebastinib combinations in MBC. Citation Format: Jesus D. Anampa, Xiaonan Xue, Sun-young Oh, Noah Kornblum, Sara Sadan, Maja Oktay, John Condeelis, Joseph A. Sparano. Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2 negative metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT021.

Abstract OT2-06-04: Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel or eribulin in patients with metastatic breast cancer

Abstract Background: Metastasis is the primary cause of death in breast cancer, yet no specific therapies are available that inhibit the metastatic process. 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. High TMEM score in the primary tumor is associated with higher risk of recurrence in ER+, HER2- early breast cancer. Paclitaxel induces the formation of TMEM in the primary tumors of patients treated with neoadjuvant chemotherapy, and in the primary tumor and distant metastases in the PyMT/PDX models. Tumor cell intravasation is mediated by release of VEGF that promotes focal vascular leakiness specifically at TMEM sites, and is derived from TMEM-associated Tie2HI/VEGFHI macrophages that release VEGF upon binding of the Tie2 receptor to angiopoietin2 (ANG2), which is elaborated by TMEM-associated endothelial cells. Moreover, ANG2-stimulated release of IL-10 by tumor-associated macrophages suppresses T cell proliferation, increases the ratio of CD4+T cells to CD8+ T cells, and promotes the expansion of CD4+CD25highFOXP3+ cells. The Tie2 inhibitor rebastinib 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 paclitaxel or eribulin. We therefore hypothesize that the addition of a potent Tie2 inhibitor (rebastinib) to antitubulin therapy in patients with HER2 negative metastatic breast cancer (MBC) will prevent hematogenous dissemination and distant metastasis by inhibition of TMEM function, reduction in CTC burden, and inhibition of immune-system suppression resulting in improvement in breast clinical outcomes Methods: Primary objective of this phase Ib study (NCT02824575) is to evaluate safety and tolerability of rebastinib in two dose levels (DL) (50mg or 100mg po BID) combined with paclitaxel IV 80mg/m2 (day 1, 8 and 15) or eribulin IV 1.4mg/m2 (day1 and 8) for four 21-day cycles. Key eligibility includes histologically confirmed HER2 negative MBC. ≤ 2 non-taxane chemotherapy regimens are allowed for rebastinib plus paclitaxel arm, while ≥ 2 chemotherapy regimens (including a taxane) are required for eribulin plus rebastinib arm. ≥ 2 endocrine regimens, including an approved CDK4/6 inhibitor, is required for ER+ disease. Patients require ECOG PS 0 or 1 and normal organ and marrow function. Exclusion criteria include significant ocular disease, significant history of cardiac disease or concomitant use drugs that prolong QTc interval. Pharmacodynamic biomarkers to be measured during cycle 1-3 include CTCs, ANG 1/2 levels and Tie-2 expressing monocytes. Tissue biopsy after two treatment cycles in 6 patients who have accessible tumors 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 three patients assigned paclitaxel arm (DL1) and one patient in eribulin arm(DL1). Citation Format: Anampa JD, Xue X, Oktay M, Condeelis J, Sparano JA. Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel or eribulin in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT2-06-04.

A permanent window for the murine lung enables high-resolution imaging of cancer metastasis.

Stable, high-resolution intravital imaging of the lung has become possible through the utilization of vacuum-stabilized imaging windows. However, this technique is extremely invasive and limited to only hours in duration. Here we describe a minimally invasive, permanently implantable window for high-resolution intravital imaging of the murine lung that allows the mouse to survive surgery, recover from anesthesia, and breathe independently. Compared to vacuum-stabilized windows, this window produces the same high-quality images without vacuum-induced artifacts; it is also less invasive, which allows imaging of the same lung tissue over a period of weeks. We further adapt the technique of microcartography for reliable relocalization of the same cells longitudinally. Using commonly employed experimental, as well as more clinically relevant, spontaneous metastasis models, we visualize all stages of metastatic seeding, including: tumor cell arrival; extravasation; growth and progression to micrometastases; as well as tumor microenvironment of metastasis function, the hallmark of hematogenous dissemination of tumor cells.

Abstract 898: Elucidation of the molecular mechanism of MenaINV expression, invadopodium maturation and tumor cell intravasation during breast cancer dissemination by TMEM

Abstract 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

Abstract CT040: Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel or eribulin in patients with metastatic breast cancer

Abstract Background: Metastasis is the primary cause of death in breast cancer, yet no specific therapies are available that inhibit the metastatic process. 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. High TMEM score in the primary tumor is associated with higher risk of recurrence in ER+, HER2- early breast cancer. Paclitaxel 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 rodent model and patient-derived xenograft (PDX) models. Intravasation of tumor cells is mediated by release of vascular endothelial growth factor (VEGF) that promotes focal vascular leakiness specifically at TMEM sites, and is derived from TMEM-associated Tie2HI/VEGFHI macrophages that release VEGF upon binding of the Tie2 receptor to angiopoietin, which is elaborated by TMEM-associated endothelial cells. The Tie2 inhibitor rebastinib 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 paclitaxel or eribulin. We therefore hypothesize that the addition of a potent Tie2 inhibitor (rebastinib) to antitubulin therapy with paclitaxel or eribulin in patients with HER2-negative metastatic breast cancer (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 rebastinib (Tie2 inhibitor) in two dose levels (DL) (50mg or 100mg po BID) combined with paclitaxel IV 80mg/m2 (day 1, 8 and 15) or eribulin IV 1.4mg/m2 (day1 and 8) for four 21-day cycles. Key eligibility includes histologically confirmed HER2 negative MBC or not amenable to curative surgery. Up to two prior non-taxane chemotherapy regimens are allowed for rebastinib plus paclitaxel arm, while at least two chemotherapy regimens (including a taxane) are required for eribulin plus rebastinib arm. ER positive patients must have failed at least two lines of endocrine therapy including an approved CDK4/6 inhibitor. Patients require ECOG PS 0 or 1 and normal organ and marrow function. Exclusion criteria include significant ocular disease (retinal neovascularization, macular edema or macular degeneration), significant history of cardiac disease or concomitant use drugs that prolong QTc interval. Pharmacodynamic biomarkers to be measured during cycle 1(day1 and 15), cycle 2(day1 and 15) and cycle 3(day1) include CTCs, angiopoietin 1/2 levels and Tie-2 expressing monocytes. Tissue biopsy after two treatment cycles in 6 patients who have accessible tumors 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 paclitaxel arm combined with rebastinib 50mg BID. Citation Format: Jesus D. Anampa, Xiaonan Xue, Maja oktay, John Condeelis, Joseph A. Sparano. Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel or eribulin in patients with metastatic breast cancer [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 CT040. doi:10.1158/1538-7445.AM2017-CT040

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.

Abstract PD5-02: Paclitaxel induced mena- and TMEM-mediated pro-metastatic changes in the breast cancer microenvironment

Abstract Background: Breast cancer cell intravasation and dissemination occurs specifically at microanatomical structures that we call tumor-microenvironment of metastasis (TMEM), representing direct physical contact between a tumor cell expressing the actin-regulatory protein Mammalian-enabled (Mena), a perivascular Tie2hi/Vegfhi-expressing macrophage, and an endothelial cell (Harney et al. Cancer Discovery 2015). TMEM sites have been identified in mouse and human mammary carcinomas, and both TMEM density (Rohan et al. JNCI 2014) and invasive Mena isoform expression (Agarwal et al. Breast Cancer Res, 2012; Forse et al. BMC Cancer, 2015]) correlates with metastasis in early stage breast cancer. Since cytotoxic agents such as PTX induce influx of bone marrow-derived progenitors that differentiate into Tie2hi/VEGFhi macrophages in the primary tumor, we hypothesized that PTX may potentiate tumor cell invasion and metastasis by inducing the formation of TMEM sites and/or function. Methods and Results in humans: We analyzed the effect of chemotherapy on TMEM and invasive Mena isoforms in 10 patients with localized breast cancer who had residual disease after neoadjuvant chemotherapy (NAC: weekly paclitaxel followed by dose-dense doxorubicin-cyclophosphamide [AC]), of whom 7 had more than 2-fold increase in TMEM density in residual disease compared with pretreatment. In a separate cohort of 5 patients, NAC produced an acute increase of up to 150-fold in invasive Mena isoforms after 1-2 doses of NAC. Methods and Results in mice: After our preliminary data in humans, we evaluated effects of PTX in 4 different models, including 2 mouse models (PyMT-spontaneous & transplantation) and 2 patient-derived xenograft (PDX) triple negative models (HT17, HT33). Although PTX delayed primary tumor growth, tumors in PTX-treated mice had significantly more TMEM sites, circulating tumor cells (CTCs) and metastatic foci when compared to vehicle-treated animals. Using intravital imaging of MMTV-PyMT-Dendra2/Cfms-CFP mice, PTX induced influx of macrophages into primary tumors and intravasation of cancer cells at TMEM sites. Furthermore, PTX treatment significantly increased expression of Mena at the gene and protein levels, including invasive Mena isoforms. Deletion of the Mena gene completely abolished dissemination and metastasis in all cases, including those treated with PTX. Conclusions: We show in mammary carcinoma mouse models and PDX models that although PTX delays tumor growth, it induces invasive Mena isoform expression and significantly increases the density of TMEM sites that are responsible for cancer cell intravasation, dissemination and metastasis. Thus, our data indicate that PTX paradoxically induces dissemination of breast cancer cells by promoting invasive Mena isoforms and TMEM-mediated cancer cell intravasation, suggesting that blockade of TMEM assembly and/or function could enhance the effectiveness of PTX and possibly other cytotoxic agents commonly used to treat early and advanced stage breast cancer. Citation Format: Karagiannis GS, Pastoriza JM, Wang Y, Harney AS, Entenberg D, Pignatelli J, Jones JG, Anampa J, Sparano JA, Rohan TE, Condeelis JS, Oktay MH. Paclitaxel induced mena- and TMEM-mediated pro-metastatic changes in the breast cancer microenvironment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD5-02.

Signatures of Breast Cancer Metastasis: aMENAble to Interpretation?

MenaINV, an isoform of the motility regulator protein Mena, contributes to prometastatic phenotypes. Tumor microenvironment of metastasis (TMEM), a three-cell structure associated with intravasation, contains a stationary Mena-expressing tumor cell. TMEM density and MenaINV expression both correlate with poor clinical outcome in breast cancer patients. However, is MenaINV involved in TMEM assembly and function?

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 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 4034: Macrophage-dependent activation of a non-canonical NOTCH-RhoA signaling pathway regulates tumor cell intravasation

Abstract Intravital imaging of rodent mammary tumors has shown that direct contacts between a Mena expressing tumor cell, a perivascular macrophage and an endothelial cell forms a microanatomical structure named TMEM (Tumor Microenvironment of Metastasis). Clinical studies have shown that the number of TMEM is correlated with an 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 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. Using high resolution FRET imaging, we further found that macrophage-induced invadopodium formation is dependent on RhoA activation in the tumor cell. However, what remained to be determined was the signaling pathway that regulated this heterotypic cell contact-mediated phenomenon. We explored the role of NOTCH, a known receptor involved in heterotypic cell contact signaling, in stimulating macrophage-dependent tumor cell invadopodium formation and transendothelial migration. We found that, in the absence of Notch signaling, macrophage-induced invadopodium formation and tumor cell intravasation are abolished. Moreover, RhoA is no longer activated in tumor cells upon macrophage contact when Notch signaling is perturbed. These results show that Notch signaling regulates heterotypic cell contact mediated invadopodium formation through RhoA activation, and reveals a novel non-canonical NOTCH/RhoA pathway as a molecular target to prevent TMEM function and therefore metastasis. Citation Format: Jose Javier Bravo Cordero, Minna Roh-Johnson, Jeanine Pignatelli, Maja Oktay, Louis Hodgson, John Condeelis. Macrophage-dependent activation of a non-canonical NOTCH-RhoA signaling pathway regulates 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 4034. doi:10.1158/1538-7445.AM2015-4034