Transendothelial Migration Of Tumor Cells Research Articles

Unraveling the Action Mechanism of Tubeimoside-1 against Tumor Microvessels via Network Pharmacology and Experimental Validation.

Objective: Tubeimoside-1 (TBMS1) is a plant-derived triterpenoid saponin that exhibits pharmacological properties and anti-tumor effects, but the anti-tumor microvessels of action of TBMS1 remains to be completely elucidated. This study aims to verify the effect of TBMS1 on tumor microvessels and its underlying mechanism. Methods: A SKOV3 xenografted mouse model were constructed to evaluate the anti-tumor microvessels of TBMS1 in vivo, followed by function assays to verify the effects of TBMS1 on the proliferation, cell cycle, migration, and tubule formation of vascular endothelial cells in vitro. Next, based on network pharmacology, the drug/disease-target protein-protein interaction (PPI) networks, biological functions and gene enrichment analyses were performed to predict the underlying mechanism. Finally, molecules and pathways associated with tumor trans-endothelial migration were identified. Results: TBMS1 treatment effectively reduced tumor microvessel density in ovarian cancer model and inhibited the proliferation, cell cycle, migration, and induced apoptosis of vascular endothelial cells in vitro. Network pharmacological data suggested that tumor cell adhesion and trans-endothelial migration may participate in antiangiogenic effects of TBMS1. By endothelial adhesion and permeability assay, we identified that tumor adhesion and the permeability of endothelial monolayers were reduced by TBMS1. Furthermore, adhesion protein (VCAM-1and ICAM-1) and tight junction (TJ) proteins (VE-cadhsion, ZO-1 and claudin-5) were found to be regulated. Finally, Akt, Erk1/2, Stat3 and NF-κB signaling were decreased by TBMS1 treatment. Conclusion: To sum up, our findings strongly suggest that clinical application of TBSM1 may serve as a vasoactive drug treatment to suppress tumor progression.

Platelet Angiopoietin-1 Protects Against Experimental Lung Metastasis By Limiting Tumor Cell Extravasation

In addition to their fundamental roles in hemostasis, platelets can contribute to both tumor growth and metastasis. However, despite being a reservoir for pro-angiogenic and metastatic cytokines, platelets also harbor negative regulators of tumor progression. One such regulator is angiopoietin-1 (Angpt1), an angiogenic cytokine that preserves vascular integrity by promoting cell-cell endothelial interactions downstream of its receptor, the receptor tyrosine kinase Tie-2. In addition, Angpt1 also protects against tumor cell metastasis through an unidentified mechanism. Although platelets are believed to be the primary source of Angpt1 in circulation, the contributions of platelet-derived Angpt1 to tumor growth and metastasis have not been investigated. In this study, we demonstrate that platelet Angpt1 is selectively upregulated in the spontaneous PyMT breast cancer mouse model, but not pre-clinical models of melanoma or non-malignant inflammation (colitis), suggesting a distinct contribution of platelet Angpt1 in breast cancer. To test whether deletion of platelet Angpt1 affects tumor growth or metastasis, we generated mice selectively lacking Angpt1 in megakaryocytes and their platelet progeny (Angpt1 fl/flPF4 Cre, referred to as Angpt1 Plt KO). Plasma Angpt1 was undetectable in Angpt1 Plt KO mice, comparable to mice deficient in the megakaryocyte differentiation factor thrombopoietin, which display approximately 10% of platelet counts. Taken together, these findings establish that platelets are the principal source of circulating Angpt1. In tumor implantation studies, orthotopic tumor growth and vascularization were unaffected in Angpt1 Plt KO mice in pre-clinical models of breast cancer and melanoma. However, Angpt1 Plt KO mice had increased experimental metastasis during both early (3-24 hours) and late (10 days, Figure 1) stages of tumor colonization in both breast cancer and melanoma models, revealing an important role for platelet Angpt1 in restraining the latter stages of tumor metastasis following tumor engraftment. Next, we sought to mechanistically determine how platelet Angpt1 limits tumor cell metastasis. Static adhesion assays demonstrated that platelets lacking Angpt1 did not affect tumor cell attachment to endothelial cells when compared to platelets containing Angpt1. However, Angpt1 Plt KO mice had increased tumor cell retention and extravasation in their lungs following experimental metastasis (3-24 hours), suggesting that platelet Angpt1 limits endothelial permeabilization and the trans-endothelial migration of tumor cells out of blood vessels. Most importantly, all intravascular tumor cells were found within platelet-rich microthrombi immediately after sequestration in the lungs. We therefore conclude that localized Angpt1 released by activated platelets at the site of tumor cell arrest likely reprograms the surrounding endothelium to limit successful tumor cell extravasation. These findings reveal an unexpected role for platelets in restricting tumor cell metastasis.

Interleukin 4 Controls the Pro-Tumoral Role of Macrophages in Mammary Cancer Pulmonary Metastasis in Mice.

Simple SummaryMetastasis is the main cause of death from breast cancer. In mouse models of breast cancer lung metastasis, macrophages enhance metastasis by promoting tumor cell seeding and persistent growth. Here, we show that interleukin-4 (IL4) is required for this process as IL4 receptor (IL4rα)-null mice develop fewer and smaller lung metastases. This deficiency is partially rescued by adoptive transfer of wild-type monocytes. IL4 signaling in macrophages upregulates the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. In addition, expression of several other genes already causally associated with lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1 are upregulated in macrophages. High-resolution intravital imaging at the time of metastatic seeding showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages, showing the dependence on IL4. We conclude that IL4 signaling in monocytes and macrophages is important during seeding and growth of breast metastasis in the lung.Metastasis is the systemic manifestation of cancer and the main cause of death from breast cancer. In mouse models of lung metastases, recruitment of classical monocytes from blood to the lung and their differentiation to metastasis-associated macrophages (MAMs) facilitate cancer cell extravasation, survival and growth. Ablation of MAMs or their monocytic progenitors inhibits metastasis. We hypothesized that factors controlling macrophage polarization modulate tumor cell extravasation in the lung. We evaluated whether signaling by Th1 or Th2 cytokines in macrophages affected transendothelial migration of tumor cells in vitro. Interferon gamma and LPS inhibited macrophage-dependent tumor cell extravasation while the Th2 cytokine interleukin-4 (IL4) enhanced this process. We demonstrated that IL4 receptor (IL4rα)-null mice developed fewer and smaller lung metastasis in E0771-LG mammary cancer models of this disease. Adoptive transfer of wild-type monocytes to IL4rα-deficient mice partially rescued this phenotype. IL4 signaling in macrophages controlled the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. Furthermore, IL4 signaling in macrophages regulated the transcript abundance of several other genes already causally associated with mammary cancer lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1. The central role of IL4 signaling in MAMs was confirmed by high-resolution intravital imaging of the lung in mice at the time of metastatic seeding, which showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages. This interaction with wild-type MAMs enhanced tumor cell survival and seeding, which was lost in the IL4rα mice. These data indicate that IL4 signaling in monocytes and macrophages is key during seeding and growth of breast metastasis in the lung, as it regulates pro-tumoral paracrine signaling between cancer cells and macrophages.

Hyperglycemia-induced oxidative stress promotes tumor metastasis by upregulating vWF expression in endothelial cells through the transcription factor GATA1.

Diabetes mellitus (DM) characterized by hyperglycemia is a chronic metabolic disorder that leads to many symptoms and vascular complications. Despite the close association between DM and cancer progression, the response and role of endothelial cells (ECs) under diabetic conditions in tumor metastasis remain to be elucidated. In this study, we sought to determine whether and how ECs under diabetic conditions contribute to tumor metastasis. We have taken advantage of syngeneic mouse tumor models of Lewis lung carcinoma (LLC) and melanoma (B16F10) cells and a streptozotocin (STZ)-induced hyperglycemia model. We demonstrated that hyperglycemia increased the metastasis of LLC and B16F10 cells in an experimental metastasis model with an intravenous injection of the tumor cells. We also found that hyperglycemia promoted lung metastasis of tumor cells by increasing the adhesiveness of ECs to facilitate the adhesion of tumor cells to ECs rather than affecting the metastatic behavior of tumor cells themselves. From the analysis of gene expression in primary lung ECs from STZ-treated mice, we identified that vWF promoted the adhesion of tumor cells to ECs and the transendothelial migration of tumor cells. Mechanistically, hyperglycemia-induced oxidative stress in ECs, and increased oxidative stress enhanced vWF expression in ECs through an increase in the transcription factor GATA1. These results provide evidence for the role of vWF in ECs in promoting hyperglycemia-induced tumor metastasis and potential therapeutic targets for the regulation of vWF expression in ECs and hyperglycemia-induced tumor metastasis.

CADM1 promotes adhesion to vascular endothelial cells and transendothelial migration in cultured GIST cells.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the human gastrointestinal tract. Small intestinal GISTs appear to be associated with poorer prognosis and higher metastasis rate than gastric GISTs of the same size and mitotic index. Recently, we reported that cell adhesion molecule 1 (CADM1) is expressed specifically in most small intestinal GISTs, but not in most gastric GISTs, suggesting that this difference in CADM1 expression between gastric GISTs and small intestinal GISTs might influence the difference in clinical behavior between them. The aim of the present study was to examine whether high CADM1 expression affected proliferation, migration, invasion, adhesion to endothelial cells and transendothelial migration of cultured GIST cells by comparing original GIST-T1 cells with very low CADM1 expression with GIST-T1 cells with high CADM1 expression induced by CADM1 cDNA transfection (GIST-T1-CAD cells). GIST-T1-CAD cells had reduced ability to proliferate, migrate and invade compared with the original GIST-T1 cells, but showed significantly higher ability to adhere to human umbilical vein endothelial cells and migrate through endothelial cell monolayers. Thus, CADM1 may contribute to higher metastasis rates in small intestinal GISTs facilitating tumor cell adhesion to vascular endothelial cell and transendothelial migration of tumor cells. CADM1 might serve as a potential target for inhibition of metastasis in small intestinal GISTs.

ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC.

Abstract PO015: JAGGED-1 as a novel metastatic extravasation promoter of triple negative breast cancer cells

Abstract Though survival is improving for breast cancer patients, clinical outcomes remain poor for patients with aggressive molecular subtypes. Triple negative breast cancer (TNBC) is considered the most aggressive breast cancer subtype, and despite having a 90% five-year relative survival rate for localized disease, that number drops to as low as 11% for patients with distant TNBC metastasis. The poor clinical outcomes for TNBC patients are largely due to lack of clinically actionable molecular targets and pathways compared to other breast cancer subtypes. Given the lack of effective treatment strategies for advanced TNBC, there is an urgent and unmet need to understand the means through which tumor cells disseminate and metastasize to distant tissues in hopes of identifying new targets for therapeutic intervention. For tumor cells to metastasize, they acquire complex series of pathophysiological phenotypes, starting with invasion into surrounding tissue and intravasation into systemic lymphatic or blood vascular systems. Only a small fraction of tumor cells can survive in circulation, with a smaller fraction able to extravasate from vessels and establish distant metastases. While some cancer cells partially coopt the mechanisms that leukocytes use to extravasate, circulating TNBC cells also use distinct mechanisms to breach an endothelial barrier. However, the mechanism(s) of TNBC extravasation remains largely unknown, underlying the need to study further. High expression of Notch ligand JAGGED-1 (JAG1) is associated with increased metastasis and mortality in patients with breast cancer. While JAG1 enhances tumor angiogenesis and tumor growth, the means through which JAG1 promotes tumor cell dissemination is not clear. Our preliminary data supports a role for JAG1 as a key regulator of tumor cell interactions with the endothelium, controlling tumor cell adhesion, transendothelial migration (TEM), and transcription of critical paracrine-acting extravasation proteins, such as SPARC and ICAM1. Using both JAG-specific ligand traps as well as CRISPR/Cas9 mediated deletion of JAG1, we show that JAG1 promotes TEM in static transwell models, promotes primary tumor growth, and directs a tumor cell transcriptional program that upregulates cell surface interaction pathways distinct from global Notch inhibition. It is our central hypothesis that JAG1 promotes TNBC migration across the vascular endothelium, thereby allowing disseminated cancer cells to exit from circulation and enhancing TNBC metastasis. To define the JAG1-mediated elements of extravasation, we will examine five CRISPR/Cas9 JAG1 knockout lines in a cutting-edge flow system that recapitulates the vascular shear stress microenvironments where extravasation is thought to occur in vivo. We will evaluate multiple steps of extravasation including tumor cell binding, rolling, and TEM under various flow parameters. Moreover, we will study in vivo lung extravasation using a murine tail vein injection model and interrogate transcriptional candidates downstream of JAG1 as identified by our RNAseq data. Citation Format: Benjamin Gordon, Reyhaan A. Chaudhri, Tim Sargis, L.A. Naiche, Jan K. Kitajewski. JAGGED-1 as a novel metastatic extravasation promoter of triple negative breast cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO015.

CX3CL1 Induces Vertebral Microvascular Barrier Dysfunction via the Src/P115-RhoGEF/ROCK Signaling Pathway.

Trans-endothelial migration (TEM) of cancer cells is a critical step in metastasis. Micro-vascular barrier disruptions of distant organs play important roles in tumor cells TEM. The spine is a preferred site for multiple cancer cell metastases. Our previous study found that vertebral spongy bone was rich in CX3CL1 and that CX3CL1 can attract fractalkine receptor-expressing tumor cells to the spine. In the present study, we determined whether CX3CL1 was involved in vertebral micro-vascular barrier disruption and promoted tumor cell TEM after circulating tumor cells were arrested in the vertebral micro-vasculature. We examined the role of CX3CL1 in the barrier function of vertebral micro-vascular endothelial cells (VMECs) and explored the molecular mechanisms of CX3CL1-induced VMEC barrier disruption. Our results demonstrated that CX3CL1 led to F-actin formation and ZO-1 disruption in VMECs and induced the vertebral micro-vascular barrier disruption. Importantly, we found that the activation of the Src/P115-RhoGEF/ROCK signaling pathway plays an important role in CX3CL1-induced VMEC stress fiber formation, ZO-1 disruption and then vertebral micro-vascular barrier hyper-permeability. Inhibiting Src/P115-RhoGEF/ROCK signaling in VMECs effectively blocked CX3CL1-induced vertebral vascular endothelial dysfunction and subsequent tumor cell TEM. The results of this study and our previous study indicate that in addition to its chemotaxis, CX3CL1 plays a critical role in regulating vertebral micro-vascular barrier function and tumor cell TEM. CX3CL1 induced VMECs stress fiber formation, ZO-1 disruption and then vascular endothelial hyperpermeability via activation of the Src/P115-RhoGEF/ROCK signaling pathway. The inhibition of the Src/P115-RhoGEF/ROCK signaling pathway in VMECs effectively blocked tumor cells TEMs in vertebral spongy bone and maybe a potential therapeutic strategy for spine metastases in the future.

Abstract PD8-10: Regulation of breast tumor metastasis by the dynamic interaction between the TMEM doorway macrophage, tumor and endothelial cells

Abstract 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.

Engineered fluidic systems to understand lymphatic cancer metastasis.

The majority of all cancers metastasize initially through the lymphatic system. Despite this, the mechanisms of lymphogenous metastasis remain poorly understood and understudied compared to hematogenous metastasis. Over the past few decades, microfluidic devices have been used to model pathophysiological processes and drug interactions in numerous contexts. These devices carry many advantages over traditional 2D in vitro systems, allowing for better replication of in vivo microenvironments. This review highlights prominent fluidic devices used to model the stages of cancer metastasis via the lymphatic system, specifically within lymphangiogenesis, vessel permeability, tumor cell chemotaxis, transendothelial migration, lymphatic circulation, and micrometastases within the lymph nodes. In addition, we present perspectives for the future roles that microfluidics might play within these settings and beyond.

The contribution of platelets to intravascular arrest, extravasation, and outgrowth of disseminated tumor cells.

Platelets are primarily known for their contribution to hemostasis and subsequent wound healing. In addition to these functions, platelets play a role in the process of metastasis. Since the first study that suggested a metastasis-promoting function for platelets was published in 1968, various mechanisms have been proposed to explain how platelets contribute to the metastatic process. These include roles in the intravascular arrest of tumor cells, in tumor cell transendothelial migration, in the degradation of basement membrane barriers, in migration and invasion at the metastatic site, and in the proliferation of disseminated tumor cells. Nevertheless, conflicting observations about the role of platelets in these processes have also been reported. Here, we review the in vivo evidence that supports a role for platelets in metastasis formation, propose several scenarios for the contribution of platelets to tumor cell arrest and transendothelial migration, and discuss the evidence that platelets contribute to metastatic invasion and outgrowth.

Ionizing radiation increases the endothelial permeability and the transendothelial migration of tumor cells through ADAM10-activation and subsequent degradation of VE-cadherin

BackgroundWe analyzed the changes in permeability of endothelial cell layers after photon irradiation, with a focus on the metalloproteases ADAM10 and ADAM17, and on VE-cadherin, components crucial for the integrity of endothelial intercellular junctions, and their roles in the transmigration of cancer cells through endothelial cell monolayers.MethodsPrimary HUVEC were irradiated with 2 or 4 Gy photons at a dose rate of 5 Gy/min. The permeability of an irradiated endothelial monolayer for macromolecules and tumor cells was analyzed in the presence or absence of the ADAM10/17 inhibitors GI254023X and GW280264X. Expression of ADAM10, ADAM17 and VE-Cadherin in endothelial cells was quantified by immunoblotting and qRT. VE-Cadherin was additionally analyzed by immunofluorescence microscopy and ELISA.ResultsIonizing radiation increased the permeability of endothelial monolayers and the transendothelial migration of tumor cells. This was effectively blocked by a selective inhibition (GI254023X) of ADAM10. Irradiation increased both, the expression and activity of ADAM10, which led to increased degradation of VE-cadherin, but also led to higher rates of VE-cadherin internalization. Increased degradation of VE-cadherin was also observed when endothelial monolayers were exposed to tumor-cell conditioned medium, similar to when exposed to recombinant VEGF.ConclusionsOur results suggest a mechanism of irradiation-induced increased permeability and transendothelial migration of tumor cells based on the activation of ADAM10 and the subsequent change of endothelial permeability through the degradation and internalization of VE-cadherin.

Abstract 976: Regulation of breast tumor metastasis by the dynamic interaction between the TMEM macrophage, tumor, and endothelial cells

Abstract 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.

The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen.

The vacuolar-type H+-ATPase (v-ATPase) is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells (ECs), in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.

A novel neuregulin \u2013 jagged1 paracrine loop in breast cancer transendothelial migration

BackgroundThe interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis. Invasion and intravasation, two critical steps in the metastatic process, are influenced by these interactions. Macrophages are of particular interest when it comes to studying tumor cell invasiveness. Previous studies have shown that there is paracrine loop signaling between breast cancer cells and macrophages involving colony stimulating factor 1 (CSF-1) produced by tumor cells and epidermal growth factor (EGF) production by macrophages. In this paper, we identify a novel paracrine loop between tumor cells and macrophages involving neuregulin (NRG1) and notch signaling.MethodsThe aim of this study was to determine the role of NRG1, a ligand of the ErbB3 receptor, in macrophage stimulation of tumor cell transendothelial migration and intravasation. We used fluorescence-activated cell sorting (FACS) and western blot to determine ErbB3 and NRG1 expression, respectively. An in vitro transendothelial migration (iTEM) assay was used to examine the effects of short hairpin (sh)RNA targeting NRG1 in tumor cells and clustered regularly interspaced short palindromic repeats (CRISPR) knockout of jagged 1 (JAG1) in macrophages. Orthotopic xenograft injections in mice were used to confirm results in vivo.ResultsIn our system, macrophages were the primary cells showing expression of ErbB3, and a blocking antibody against ErbB3 resulted in a significant decrease in macrophage-induced transendothelial migration of breast cancer cells. Stimulation of macrophages with NRG1 upregulated mRNA and protein expression of JAG1, a ligand of the Notch receptor, and JAG1 production by macrophages was important for transendothelial migration of tumor cells.ConclusionsThis study demonstrates that stimulation of macrophages by tumor cell NRG1 can enhance transendothelial migration and intravasation. We also demonstrate that this effect is due to induction of macrophage JAG1, an important ligand of the Notch signaling pathway.

Tumor-derived exosomes enhance invasion and metastasis of salivary adenoid cystic carcinoma cells.

Tumor-derived exosomes (TDE) have been shown to participate in different steps of the dissemination of cancer cells. However, the role of salivary adenoid cystic carcinoma-derived (SACC-derived) exosomes had not been documented in SACC. The study aims to explore the functions of SACC-derived TDE in SACC progression and investigate potential mechanisms. Salivary adenoid cystic carcinoma cell line SACC-83 was used to generate TDE. Afterward, SACC-83 or HUVECs were cocultured with or without TDE. Tumor migration, tumor invasion, and endothelial permeability were examined by wound healing assay, tumor invasion assay, endothelial permeability assay, and tumor cell transendothelial migration assay, respectively. Moreover, the expression levels of cell junction-related proteins were examined by qRT-PCR and Western blot. Salivary adenoid cystic carcinoma -83-derived exosomes were taken up by their host cells. Meanwhile, TDE increased migration and invasion capacity of SACC-83 cells and enhanced endothelial cell permeability. Furthermore, we demonstrated that the expression of cell junction-related proteins (Claudins and ZO-1) was downregulated, whichis presumablyinvolved in the TDE-mediated promotion of migration, invasion, and metastasis. The results suggested that SACC cell-derived exosomes were loaded with individual components that could enhance invasiveness and induce microenvironment changes, thus promoting SACC aggression.

Endothelial Rab7 GTPase mediates tumor growth and metastasis in lysosomal acid lipase–deficient mice

Tumors depend on their microenvironment for sustained growth, invasion, and metastasis. In this environment, endothelial cells (ECs) are an important stromal cell type interacting with malignant cells to facilitate tumor angiogenesis and cancer cell extravasation. Of note, lysosomal acid lipase (LAL) deficiency facilitates melanoma growth and metastasis. ECs from LAL-deficient (lal-/-) mice possess enhanced proliferation, migration, and permeability of inflammatory cells by activating the mammalian target of rapamycin (mTOR) pathway. Here we report that lal-/- ECs facilitated in vivo tumor angiogenesis, growth, and metastasis, largely by stimulating tumor cell proliferation, migration, adhesion, and transendothelial migration via increased expression of IL-6 and monocyte chemoattractant protein 1 (MCP-1). This prompted us to look for lysosomal proteins that are involved in lal-/- EC dysfunctions. We found that lal-/- ECs displayed increased expression of Rab7, a late endosome/lysosome-associated small GTPase. Moreover, Rab7 and mTOR were co-increased and co-localized to lysosomes and physically interacted in lal-/- ECs. Rab7 inhibition reversed lal-/- EC dysfunctions, including decreasing their enhanced migration and permeability of tumor-stimulatory myeloid cells, and suppressed EC-mediated stimulation of in vitro tumor cell transmigration, proliferation, and migration and in vivo tumor growth and metastasis. Finally, Rab7 inhibition reduced overproduction of reactive oxygen species and increased IL-6 and MCP-1 secretion in lal-/- ECs. Our results indicate that metabolic reprogramming resulting from LAL deficiency enhances the ability of ECs to stimulate tumor cell proliferation and metastasis through stimulation of lysosome-anchored Rab7 activity.

19P - An endothelial premetastatic-like niche is promoted by tumor-secreted factors derived from highly metastatic breast cancer cells in vitro

Background: It is well established that primary tumour can modify secondary organs long before the circulating tumor cells reach their metastatic targets. These modifications are mainly exerted through tumor-secreted factors and can act on several cell types to form what is called the pre-metastatic niche. Pre-metastatic endothelial niche play a key role in the extravasation process during metastasis. Here we describe the effect of the mixture of secreted factors by tumor cells over endothelial changes that facilitates tumor cell transendothelial migration. Methods: Adhesion assay Human umbilical vein endothelial cells (HUVEC) cells were stimulated or not with TNF-α [10ng/ml] or Tumor Secreted Factors (TSFs) [10 µg/ml] from MDA-MB-231 or MCF-7 cell lines. HUVEC monolayers were co-cultured with a U937(3H) cells. After 3h firm attached U937(3H) were lysed and radioactivity was measured. Vascular permeability assay HUVEC monolayers were cultured in Boyden chambers. Cells were stimulated or not with TNF-α [10ng/ml] or TSFs [10 µg/ml] and incubated for 12h. Afterwards a dextran-FITC solution was added for 20 min and the bottom well fluorescence was quantified. Transendothelial migration assay HUVEC monolayers were cultured in Boyden chambers. Cells were stimulated or not with TNF-α [10ng/ml] or TSFs [10 µg/ml] and incubated for 10h. Fluorescent labeled MDA-MB-231 cell suspension 2x104 was added (24h) and the migrant cells were counted under an epifluorescence microscope. Results: Adhesion assay revealed that HUVEC stimulated with MDA-231 TSFs attached U937 cells 6-fold comparing to the MCF-7 TSFs or control, in a similar way as TNF-α did. The MDA-TSFs were able to increase the HUVEC monolayer permeability exceeded about 30% of the TNF-α induced permeability. A 1.5-fold increase of transendothelial migration cells was observed in HUVEC stimulated with MDA-MB-231 TSFs. Conclusions: Tumor secreted factors derived from highly metastatic cell line MDA-MB-231 are capable to induce a premetastatic-like endothelial state, increasing the tumor cell transendothelial migration, adhesion to the HUVEC monolayer as well as vascular permeability enhancement. Clinical trial identification: 11-62-2014 Legal entity responsible for the study: Instituto de Investigaciones Biomédicas, UNAM Funding: Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), fellowship 509589 from CONACYT. Disclosure: All authors have declared no conflicts of interest.

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

VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells

During metastasis, breakdown of the endothelial barrier is critical for tumor cell extravasation through blood vessel walls and is mediated by a combination of tumor secreted soluble factors and receptor-ligand interactions. However, a complete mechanism governing tumor cell transendothelial migration remains unclear. Here, we investigate the roles of tumor-associated signals in regulating endothelial cell contractility and adherens junction disassembly leading to endothelial barrier breakdown. We show that Src mediates VE-cadherin disassembly in response to metastatic melanoma cells. Through the use of pharmacological inhibitors of cytoskeletal contractility we find that endothelial cell contractility is responsive to interactions with metastatic cancer cells and that reducing endothelial cell contractility abrogates migration of melanoma cells across endothelial monolayers. Furthermore, we find that a combination of tumor secreted soluble factors and receptor-ligand interactions mediate activation of Src within endothelial cells that is necessary for phosphorylation of VE-cadherin and for breakdown of the endothelial barrier. Together, these results provide insight into how tumor cell signals act in concert to modulate cytoskeletal contractility and adherens junctions disassembly during extravasation and may aid in identification of therapeutic targets to block metastasis.