Tumor Cell Intravasation Research Articles

Abstract B20: The molecular function of ASPSCR1-TFE3 in alveolar soft part sarcoma

Abstract Alveolar soft part sarcoma (ASPS) is a soft-tissue sarcoma that affects predominantly adolescents and young adults with lower legs and buttocks as frequent primary sites. ASPS is characterized by alveolar structure of tumor cells, abundant vascularization, and high frequency of hematogenous metastasis. As for gene abnormality, ASPSCR1-TFE3 fusion gene is observed in all ASPS cases. To understand the exact mechanisms of ASPSCR1-TFE3 and tumorigenesis of ASPS, we have developed an ex vivo mouse model for human ASPS (Tanaka et al., Cancer Res 2017;77(4). In mouse ASPS, the tumor cells frequently showed tumor intravasation, with the intravascular tumor cells presenting as organoid structures covered with hemangiopericytes, which is also observed in human ASPS. Moreover, culture supernatant of mouse ASPS cells strongly induced hemangiopericyte migration in vitro. The search for hemangiopericyte-inducing factors which are released from ASPS cells is in progress. We identified Gpnmb that plays an important role in intravasation of ASPS tumor cells as a direct target of the ASPSCR1-TFE3 fusion transcription factor. The ChIP-seq analysis detected an ASPSCR1-TFE3 binding peak at the Gpnmb promoter, and Gpnmb expression was significantly upregulated in ASPS tumors. Enhanced localization of GPNMB at the front of tumor invasion and intravasation in both mouse and human ASPS. Knockdown of Gpnmb suppressed transendothelial migration of ASPS tumor cells, indicating that Gpnmb is required for intravasation. TFE3 is a member of the TFE/MITF family, which has been found involved in human cancer such as renal cell carcinoma and malignant melanoma. The present model enabled us to evaluate tumorigenic activity of TFE/MITF family proteins. To examine the oncogenic activity of ASPSCR1 fusion genes, artificial chimeric genes were generated consisting of the 5 ASPSCR1 and 3 TFE/MITF sequences. The ASPSCR1-TFE3 and ASPSCR1-TFEB but not ASPSCR1-TFEC and ASPSCR1-MITF induced sarcoma. The results suggest the presence of TFE3/TFEB-specific cofactors and mechanisms for tumorigenesis. In order to identify such novel cofactors and functional motifs, human ASPS cell-nuclear extract was incubated with TFE3 or MITF proteins and TFE3- or MITF-bound fractions were analyzed by mass spectrometry. The TFE3-specific interaction partners were identified and their roles in tumorigenesis, angiogenesis, and/or metastasis are subjected to the subsequent analysis. These studies will provide valuable information for our understanding of the biology of ASPS and functions of TFE/MITF family transcription factors. Citation Format: Miwa Tanaka, Takuro Nakamura. The molecular function of ASPSCR1-TFE3 in alveolar soft part sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B20.

Chemotherapy-induced metastasis: mechanisms and translational opportunities.

Tumors often overcome the cytotoxic effects of chemotherapy through either acquired or environment-mediated drug resistance. In addition, signals from the microenvironment obfuscate the beneficial effects of chemotherapy and may facilitate progression and metastatic dissemination. Seminal mediators in chemotherapy-induced metastasis appear to be a wide range of hematopoietic, mesenchymal and immune progenitor cells, originating from the bone marrow. The actual purpose of these cells is to orchestrate the repair response to the cytotoxic damage of chemotherapy. However, these repair responses are exploited by tumor cells at every step of the metastatic cascade, ranging from tumor cell invasion, intravasation and hematogenous dissemination to extravasation and effective colonization at the metastatic site. A better understanding of the mechanistic underpinnings of chemotherapy-induced metastasis will allow us to better predict which patients are more likely to exhibit pro-metastatic responses to chemotherapy and will help develop new therapeutic strategies to neutralize chemotherapy-driven prometastatic changes.

Tumor Metastasis in the Microcirculation.

Tumor cell metastasis through blood circulation is a complex process and is one of the great challenges in cancer research as metastatic spread is responsible for ∼90% of cancer-related mortality. Tumor cell intravasation into, arrest and adhesion at, and extravasation from the microvessel walls are critical steps in metastatic spread. Understanding these steps may lead to new therapeutic concepts for tumor metastasis. Vascular endothelium forming the microvessel wall and the glycocalyx layer at its surface are the principal barriers to and regulators of the material exchange between circulating blood and body tissues. The cleft between adjacent endothelial cells is the principal pathway for water and solute transport through the microvessel wall in health. Recently, this cleft has been found to be the location for tumor cell adhesion and extravasation. The blood-flow-induced hydrodynamic factors such as shear rates and stresses, shear rate and stress gradients, as well as vorticities, especially at the branches and turns of microvasculatures, also play important roles in tumor cell arrest and adhesion. This chapter therefore reports the current advances from in vivo animal studies and in vitro culture cell studies to demonstrate how the endothelial integrity or microvascular permeability, hydrodynamic factors, microvascular geometry, cell adhesion molecules, and surrounding extracellular matrix affect critical steps of tumor metastasis in the microcirculation.

Abstract B030: Neoadjuvant chemotherapy promotes macrophage-induced MenaINV-dependent pro-metastatic changes in breast tumors

Abstract Neoadjuvant chemotherapy (NAC) is used for treatment of localized breast cancer to decrease tumor size and improve surgical outcome. However, our recent study has uncovered a previously unrecognized side effect of NAC, which involves the induction of pro-metastatic changes in the primary breast cancer microenvironment. In particular, NAC promotes the assembly of structures that serve as doorways for intravasation of tumor cells called tumor microenvironment of metastasis (TMEM), and increases the proportion of the highly invasive and migratory MenaINV-hi/Mena11alo (MenaCalc+) tumor cells, which utilize the TMEM sites for hematogenous dissemination (1). MenaINV expression in particular is associated with decreased sensitivity of cancer cells to receptor tyrosine-kinase (RTK) and tyrosine-kinase (TK) inhibitors and with dramatically increased TMEM-dependent intravasation. The mechanism behind these pro-metastatic changes was not known. Here we report that NAC induces increased levels of Tie2Hi macrophages in the tumors, resulting in increased contact between macrophages and tumor cells leading to MenaINV expression in the cancer cells, and MenaINV-dependent TMEM assembly and tumor cell intravasation. Furthermore, we demonstrate that the Tie2 switch control inhibitor rebastinib sufficiently suppresses Tie2 signaling in macrophages, thereby inhibiting increased levels of Tie2Hi macrophages in the tumor, resulting in decreased MenaINV expression in the cancer cells and decreased TMEM-associated tumor cell intravasation. Our work has uncovered a previously unrecognized mechanism behind pro-metastatic changes in response to cytotoxic chemotherapy, the markers that predict these changes (TMEM, MenaCalc and MenaINV), and shows that this mechanism could be addressed by combining chemotherapy with rebastinib. Reference: 1. Karagiannis GS, et al. Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism. Sci Transl Med 2017;9:aeen0026. Citation Format: George S. Karagiannis, Ved P. Sharma, Sara Brizio, Joseph Burt, Yarong Wang, David Entenberg, Bryan Smith, Daniel Flynn, Maja H. Oktay, John Condeelis. Neoadjuvant chemotherapy promotes macrophage-induced MenaINV-dependent pro-metastatic changes in breast tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B030.

Neutrophils: Homing in on the myeloid mechanisms of metastasis

The metastasis cascade is complex and comprises several stages including local invasion into surrounding tissue, intravasation and survival of tumour cells in the circulation, and extravasation and colonisation of a distant site. It is increasingly clear that these processes are driven not only by signals within the tumour cells, but are also profoundly influenced by stromal cells and signals in the tumour microenvironment. Amongst the many cell types within the tumour microenvironment, immune cells such as lymphocytes, macrophages and neutrophils play a prominent role in tumour development and progression. Neutrophils, however, have only recently emerged as important players, particularly in metastasis. Here we review the current evidence suggesting a multi-faceted role for neutrophils in the metastatic cascade.

Mechanism of action and efficacy of LY2109761, a TGF-β receptor inhibitor, targeting tumor microenvironment in liver cancer after TACE.

TACE (transcatheter arterial chemoembolization) has been recognized as an effective palliative treatment option for patients with HCC, however, the medium-long term efficacy of it remains modest. LY2109761, a TGF-β receptor inhibitor, was confirmed to reduce tumor cell growth, intravasation, and metastatic dissemination of HCC cells through different molecular mechanisms. This study aims to investigate the treatment effect of combining TACE therapy with LY2109761- a TGF-β receptor I kinase inhibitor on suppressing tumor growth and metastasis in a rabbit VX2 tumor model. The molecular mechanisms underlying the biological activities of LY2109761 was also evaluated through an in vitro model. And we found that LY2109761 could inhibit cell proliferation by down-regulating the phosphorylation of Smad-2 as well as improved the therapeutic effect of TACE in a VX2 hepatocellular carcinoma model. And we further found that LY2109761 may play a modulating role in the process of T cell transformation. Hence, based on those obsevations in our research, we concluded that combing LY2109761 with TACE for the treatment of VX2 rabbit liver cancer can help inhibit tumor growth as well as increase the tumor cell necrosis after TACE.

Mitosis-Mediated Intravasation in a Tissue-Engineered Tumor-Microvessel Platform.

Intravasation involves the migration of tumor cells across the local endothelium and escape into vessel flow. Although tumor cell invasiveness has been correlated to increased intravasation, the details of transendothelial migration and detachment into circulation are still unclear. Here, we analyzed the intravasation of invasive human breast cancer cells within a tissue-engineered microvessel model of the tumor microenvironment. Using live-cell fluorescence microscopy, we captured 2,330 hours of tumor cell interactions with functional microvessels and provide evidence for a mitosis-mediated mechanism where tumor cells located along the vessel periphery are able to disrupt the vessel endothelium through cell division and detach into circulation. This model provides a framework for understanding the physical and biological parameters of the tumor microenvironment that mediate intravasation of tumor cells across an intact endothelium. Cancer Res; 77(22); 6453-61. ©2017 AACR.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2Hi Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors.

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486-501. ©2017 AACR.

Highlights of This Issue

Cytotoxic chemotherapy is known to induce pro-metastatic changes resulting in poor survival. Harney, Karagiannis and colleagues show that the TIE2 kinase inhibitor rebastinib reduces primary tumor growth and metastasis in a preclinical breast cancer model, and is synergistic in combination with taxane chemotherapy. They demonstrate that taxane chemotherapy is associated with metastasis through recruitment of tumor-infiltrating TIE2+ macrophages that promote angiogenesis, TMEM-dependent tumor cell intravasation and lung metastasis, events reversed by rebastinib. In an adjuvant study, rebastinib in combination with eribulin led to a significant improvement in survival compared to single agent eribulin. Thus, rebastinib is a promising therapy for inhibiting pro-metastatic microenvironments in cancer patients.Epithelial-mesenchymal transition (EMT) plays an important role in cancer progression, relapse, and metastasis. By using the myxobacterial natural compound Archazolid A as a tool, Merk and colleagues discovered the V-ATPase, a multimeric proton pump that regulates lysosomal acidification, as a crucial player in EMT and Archazolid A as a promising strategy to block EMT through the inhibition of VATPase. Mechanistic studies found that Archazolid A disrupts the process of EMT, the generation of tumor-initiating cells and the turnover of E-cadherin. These evidences suggest that Archazolid A is a promising cancer therapeutic agent that is worth further investigation.The extracellular matrix limits the efficacy of chemotherapy in pancreatic ductal adenocarcinoma (PDA) in part by active cell signaling. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds fibrillar collagens and is expressed by PDA tumor cells. Aguilera, Huang and colleagues demonstrate that pharmacologic inhibition of DDR1 with the small molecule 7rh substantially improves the efficacy of standard chemotherapy in robust preclinical models of PDA in the absence of additional normal tissue toxicity. These results suggest that inhibition of collagen signaling with 7rh has the potential to improve efficacy of conventional chemotherapy in PDA patients.High tumor mutational burden (TMB) predicts response to checkpoint blockade in lung cancer and melanoma. Our study demonstrates that TMB can be used to predict response to checkpoint blockade independent of tumor histology. Higher TMB was independently and linearly associated with improved response rates and progression-free survival on multivariate analysis. Response rates were 67% with very high TMB (>50 mutations/mb). Further, the median TMB was significantly higher for patients who responded to anti-PD-1/PD-L1 monotherapy compared to those who did not. Interestingly, benefit from dual checkpoint blockade did not show a similarly strong dependence on TMB.

Webinar | Deciphering cancer: Understanding tumor invasion and the metastatic microenvironment

Tumor metastasis is a multistep process that includes stimulation of angiogenesis, intravasation of tumor cells into blood or lymphatic vessels, and subsequent engraftment and extravasation to peripheral tissues where secondary tumors are seeded. It is known that metastatic progression is driven by mutational and epigenetic changes in tumor cells, but more recent efforts have focused on the evolving interplay between the tumor cell and the tumor microenvironment (TME) during invasion and transition from micrometastasis to macrometastasis. Whether the tumor cell or the subverted vascular niche/premetastatic niche is the driver of TME evolution remains the subject of debate. This webinar examines the role of tumor-secreted factors, including exosomes and tumor vascular niche—derived protumorigenic factors in tumor—environment interactions, and their implications for development of therapeutic interventions to arrest metastasis.

MDA-MB-231 Cell Beads Surrounded by Human Umbilical Vein Endothelial Cells for the In Vitro Study of Tumor Cell Intravasation

MDA-MB-231 Cell Beads Surrounded by Human Umbilical Vein Endothelial Cells for the<i> In Vitro</i> Study of Tumor Cell Intravasation

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

Abstract 2968: Examining the role of breast cancer Neuregulin1 and macrophage ErbB3 in intravasation

Abstract The purpose of our studies is to investigate the role of Neuregulin1 (NRG1) and ErbB3 signaling between breast cancer cells and macrophages in facilitating tumor cell intravasation. The interaction of breast cancer cells with other cell types within the tumor microenvironment plays an important role in metastasis. These interactions are thought to influence tumor cell invasion and intravasation, two important steps in the process of metastasis. Our studies are specifically interested in examining the signaling occurring between tumor cells and macrophages. Previous studies have established the presence of paracrine signaling between breast cancer cells and macrophages, where colony stimulating factor 1 (CSF-1) produced by the tumor cells stimulates the production of epidermal growth factor (EGF) by macrophages, leading to chemotactic invasion of the tumor cells. In addition to this paracrine loop signaling between tumor cells and macrophages, it has been seen that macrophage expression of ErbB3, a member of the EGFR family of receptor tyrosine kinases, may play a role in facilitating tumor cell invasion. In order to examine the effects of signaling between tumor cells and macrophages in intravasation, we utilize an in vitro transendothelial migration (iTEM) assay. This assay uses transwells coated with matrigel and endothelial cells in order to mimic the entry of tumor cells into blood vessels. We show that using an ErbB3 blocking antibody results in a significant reduction of macrophage-induced transendothelial migration of breast cancer cells. Additionally, reduction of expression of the ErbB3 receptor ligand Neuregulin1 in tumor cells yields a similar result. Stimulation of macrophages with NRG1 leads to increased expression of Jagged1 (JAG1), a ligand of the Notch receptor. Activation of the Notch receptor pathway has been shown to be involved in tumor cell invasion. Overall our studies look to further examine the interaction between tumor cells and macrophages, and these observations indicate that ErbB3, NRG1, and JAG1 could all serve as novel targets in metastasis and the tumor microenvironment. Citation Format: Ramon Cabrera, Serena Chiang, Jeffrey Segall. Examining the role of breast cancer Neuregulin1 and macrophage ErbB3 in intravasation [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 2968. doi:10.1158/1538-7445.AM2017-2968

Tissue-engineered 3D microvessel and capillary network models for the study of vascular phenomena.

Advances in tissue engineering, cell biology, microfabrication, and microfluidics have led to the development of a wide range of vascular models. Here, we review platforms based on templated microvessel fabrication to generate increasingly complex vascular models of (i) the tumor microenvironment, (ii) occluded microvessels, and (iii) perfused capillary networks. We outline fabrication guidelines and demonstrate a number of experimental methods for probing vascular function such as permeability measurements, tumor cell intravasation, flow characterization, and endothelial cell morphology and proliferation.

Abstract SY31-02: Macrophages–evildoers in cancer

Abstract Tumor evolution to malignancy requires manipulation of its tissue microenvironment to be promoting. This is particularly true for the immune infiltrate that is biased away from responding to the tumor to effect control and instead actively promotes progression. The tumor immune response thus downregulates cytotoxic T-cell responses and promotes tissue repair and morphogenic activities of the infiltrating immune cells (1). Thus the environment tends to be dominated by innate immune cells, particularly macrophages and neutrophils, while cytotoxic T cells are often excluded. Our interest has been in macrophages as these, in many different mouse models of cancer, promote tumor progression and enhance metastasis. In fact, macrophages appear to be involved in every step of tumor progression. They stimulate tumor initiation, enhance angiogenesis, promote tumor cell migration and intravasation, increase stem cell viability, suppress immune responses, and, at the metastatic site, promote extravasation and persistent growth. Consequently, macrophage ablation results in inhibition of tumor progression and metastasis (2). These macrophage biologic activities are induced through a dynamic interplay with tumor cells that often involves reciprocal signaling. We have been particularly interested in the involvement of macrophages in enhancing metastasis since it is metastatic disease that is responsible for most cancer deaths. We have demonstrated a chemokine-signaling cascade that results in the recruitment of the progenitor monocytes and their retention in the tissue (3, 4). This results in differentiation of what we have termed metastasis-associated macrophages (MAMs) that confer survival signals and growth advantage to metastatic cells. These MAMs in term respond to local signals to upregulate an inflammatory gene signature through the tyrosine kinase transmembrane receptors, vascular endothelial growth factor receptor 1 (VEGFR1 or FLT1) and colony stimulating factor 1 receptor (CSF1R) (5). Furthermore, monocytes appear to be preadapted by the primary tumor to promote metastasis by the generation of preferred sites know as premetastatic niches (6). Thus, understanding monocyte biology, the mechanisms of their recruitment, and differentiation is of central importance to the fundamental appreciation of the role of macrophages in the tumor. My talk will focus on these questions.

Abstract 3963: Neoadjuvant chemotherapy promotes prometastatic changes in the primary breast tumor microenvironment in mice and humans

Abstract Chemotherapy induces influx of bone marrow-derived proangiogenic Tie2hi monocytes in primary tumors. Tie2hi perivascular macrophages specifically induce the prometastatic Mena isoforms in tumor cells and can assemble specialized microanatomical sites called “tumor microenvironment of metastasis” (TMEM), structures that may serve as doorways for intravasation of tumor cells in mammary tumors. Both TMEM and MenaINV are required for tumor cell intravasation and dissemination. Thus, we hypothesized that chemotherapy may increase the density of TMEM sites and MenaINV-expressing, intravasation-competent tumor cells, resulting in increased tumor cell invasion and metastasis. We studied these potential pro-metastatic effects of chemotherapy in a neoadjuvant setting (NAC) by either administering paclitaxel or a combination of doxorubicin and cyclophosphamide in several mammary carcinoma mouse and human breast cancer models. As expected, chemotherapy delayed tumor growth, yet it significantly increased the recruitment of TMEM-forming, perivascular Tie2hi/Vegfhi macrophages and TMEM density. Using high-resolution multiphoton intravital imaging in live tumor-bearing mice, we observed that paclitaxel also increased the activity of TMEM sites, visualized as endothelial cell tight-junction disruption around TMEM and subsequent intravasation of the migratory cancer cell subpopulation. Indeed, paclitaxel-treated mice have higher numbers of circulating tumor cells, single cell seeding in lungs and incidence and number of micrometastatic foci, all associated with increased TMEM activity, as demonstrated by high-resolution imaging techniques. Tie2 inhibitors reversed paclitaxel-induced pro-metastatic phenotypes without affecting the assembly of TMEM, indicating that Tie2-mediated signaling is required for paclitaxel-mediated cancer cell dissemination via TMEM. Paclitaxel also caused a significant increase in the expression of MenaINV at both the gene and protein levels. Furthermore, paclitaxel treatment in Mena-/- breast tumor-bearing mice resulted in failure to assemble TMEM and to increase circulating-tumor cells and cancer cell metastasis despite the fact that Tie2hi macrophages are attracted to perivascular niches as a result of paclitaxel treatment. This indicated that Mena is involved in the paclitaxel-mediated increase in cancer cell dissemination but not required for Tie2hi macrophage recruitment. These pre-clinical data are further supported by findings from a cohort (N=20) of breast cancer patients, who received pre-operative paclitaxel-based chemotherapy and demonstrated significant increases in TMEM density and MenaINV expression. Together, our data provide solid evidence that NAC leads to metastasis in rodents via TMEM/ MenaINV-mediated mechanisms, and to cancer cell dissemination in certain clinical scenarios in humans. Citation Format: George S. Karagiannis, Jessica Pastoriza, Jeanine Pignatelli, Yarong Wang, Allison S. Harney, David Entenberg, Ved P. Sharma, Emily Xue, Esther Cheng, Timothy M. D'Alfonso, Joan G. Jones, Jesus Anampa, Thomas E. Rohan, Joseph A. Sparano, John S. Condeelis, Maja H. Oktay. Neoadjuvant chemotherapy promotes prometastatic changes in the primary breast tumor microenvironment in mice and humans [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 3963. doi:10.1158/1538-7445.AM2017-3963

Abstract 878: A new SOX2/OCT4 stem cell biosensor reveals the mechanism of cancer stem cell dissemination in human breast cancer

Abstract There is increasing consensus that cancer stem cells (CSC) play an important role during metastatic progression of breast cancer. Previous studies have shown that stem cells display a pro-invasive phenotype in breast cancer. However, the phenotypes specific to breast cancer stem cells have not been evaluated at single cell resolution in vivo. Here, we employ high-resolution intravital two-photon microscopy in both orthotopic xenograft tumors, and their metastases, formed from human breast cancer cell lines expressing a previously characterized SOX2/OCT4 transcription-based fluorescent stem cell biosensor (SORE6). Using this high resolution imaging technology we found that SORE6+ stem cells: (a) constitute a minority population of the primary mammary tumors, (b) move approximately ten times slower than non-stem breast cancer cells (0.1 vs 1.1 µm/min, respectively), (c) are migratory toward blood vessels and, (d) compared to non-stem cells, they are enriched for invasive cellular protrusions called invadopodia. This is important because we have shown that these phenotypes are specifically associated with the disseminating population of tumor cells in the primary tumor site and, in addition, invadopodia are required for transendothelial migration during intravasation. Stem cells also have a three-fold higher incidence of direct contact with macrophages compared to non-stem cells. In fact, stem cells were frequently seen to be part of the tripartite macrophage-tumor cell- endothelial cell complex called TMEM, which was previously shown to be the doorway for intravasation of tumor cells in primary mammary tumors and is validated as a prognostic of metastasis in human breast cancer patients. Furthermore, we followed breast cancer stem cell dissemination to the lung by using a novel lung window for high resolution imaging of the lung (WHRIL), which allows visualization of the same lung tissue in a single mouse, serially, over days to weeks. Using WHRIL, in combination with intravital multiphoton microscopy and the above biosensor, we visualized the arrival, extravasation and outgrowth of spontaneously arriving breast tumor cells in the lung. We report here for the first time, the kinetics and stemness state of the spontaneously metastasizing tumor cells and, the record at single cell resolution, of their fate and metastatic outgrowth in the lung over time. Citation Format: Ved P. Sharma, Sonia Voiculescu, Yarong Wang, George S. Karagiannis, Binwu Tang, Lalage Wakefield, David Entenberg, Sumanta Goswami, Maja Oktay, John Condeelis. A new SOX2/OCT4 stem cell biosensor reveals the mechanism of cancer stem cell dissemination in human 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 878. doi:10.1158/1538-7445.AM2017-878

Cancer metastasis through the prism of epithelial-to-mesenchymal transition in circulating tumor cells.

Metastasis of epithelial cancer cells to distant sites is a particularly critical stage of cancer progression that typically marks the incurability of the disease. It is governed by a complex series of events including invasion and intravasation of tumor cells into the stroma and blood, respectively. Epithelial‐to‐mesenchymal transition (EMT), a phenotypic change marked by the loss of epithelial characteristics and the acquisition of invasive mesenchymal properties, is implicated in the dissemination of tumor cells. Circulating tumor cells (CTCs), the precursors of metastasis, can be used to interrogate the contribution of EMT in metastasis and therapeutic responses. The analysis of these CTCs and in particular the presence of inter‐ and intrapatient heterogeneity for markers of EMT has provided new insights into the metastatic process. This review will focus on epithelial–mesenchymal plasticity in CTCs and its potential clinical implications.

Platelet "first responders" in wound response, cancer, and metastasis.

Platelets serve as "first responders" during normal wounding and homeostasis. Arising from bone marrow stem cell lineage megakaryocytes, anucleate platelets can influence inflammation and immune regulation. Biophysically, platelets are optimized due to size and discoid morphology to distribute near vessel walls, monitor vascular integrity, and initiate quick responses to vascular lesions. Adhesion receptors linked to a highly reactive filopodia-generating cytoskeleton maximizes their vascular surface contact allowing rapid response capabilities. Functionally, platelets normally initiate rapid clotting, vasoconstriction, inflammation, and wound biology that leads to sterilization, tissue repair, and resolution. Platelets also are among the first to sense, phagocytize, decorate, or react to pathogens in the circulation. These platelet first responder properties are commandeered during chronic inflammation, cancer progression, and metastasis. Leaky or inflammatory reaction blood vessel genesis during carcinogenesis provides opportunities for platelet invasion into tumors. Cancer is thought of as a non-healing or chronic wound that can be actively aided by platelet mitogenic properties to stimulate tumor growth. This growth ultimately outstrips circulatory support leads to angiogenesis and intravasation of tumor cells into the blood stream. Circulating tumor cells reengage additional platelets, which facilitates tumor cell adhesion, arrest and extravasation, and metastasis. This process, along with the hypercoagulable states associated with malignancy, is amplified by IL6 production in tumors that stimulate liver thrombopoietin production and elevates circulating platelet numbers by thrombopoiesis in the bone marrow. These complex interactions and the "first responder" role of platelets during diverse physiologic stresses provide a useful therapeutic target that deserves further exploration.