Transendothelial Migration Of Tumor Cells Research Articles

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.

Abstract A63: Macrophages expressing fibroblast activation protein-alpha are a tumor-educated polarization phenotype which facilitates metastasis

Abstract Tumor associated macrophages (TAMs) are one of the most abundant stromal cell types in cancer, and promote tumor progression by supporting angiogenesis, immunosuppression, and tumor cell migration. However, there is increasing evidence to suggest that the functions of these cells could be refined to specific subsets. A subset of TAMs identified by the cell surface marker fibroblast activation protein-alpha (FAP) has previously been demonstrated to facilitate immune suppression in an ectopic Lewis lung carcinoma model through its expression of the heme degrading enzyme heme oxygenase-1 (HO-1) (Arnold et al, 2014). Little is known about the origin of these cells, and whether they represent an autonomous population or a polarization phenotype. Here we show in orthotopic mammary 4T1 adenocarcinomas that FAP+ TAMs are a tumor-educated polarization phenotype which is functionally distinct from IL-4/-13 polarized alternatively activated macrophages. In contrast, IL-6 and integrin β1 signaling contribute to FAP+ TAM polarization. In addition to the previously described role of these cells in immune suppression we show that FAP+ TAMs facilitate pulmonary metastasis. FAP+ TAMs are the primary tumoral source of HO-1 and pharmacological inhibition of HO activity reduces pulmonary metastasis in both orthotopic 4T1 adenocarcinomas and aggressive autochthonous murine mammary adenocarcinomas (MMTV-PyMT). Using an in vitro transwell assay, we demonstrate that HO-1 activity by macrophages enhances the transendothelial migration of tumor cells. In vivo HO inhibition does not affect seeding of tumor cells in the lung once these cells have entered the circulation, suggesting that FAP+ TAMs facilitate the intravasation event. Together, these results demonstrate FAP+ TAMs to be a tumor-educated polarization phenotype which facilitates tumor cell metastasis by its expression of HO-1. Arnold, J.N., et al., Tumoral immune suppression by macrophages expressing fibroblast activation protein-α and heme oxygenase-1. Cancer Immunol Res, 2014. 2(2): p. 121-6 Citation Format: Tamara de Koning, Joy M. Burchell, Sandra S. Diebold, James N. Arnold. Macrophages expressing fibroblast activation protein-alpha are a tumor-educated polarization phenotype which facilitates metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A63.

Tamoxifen Directly Inhibits Platelet Activation, Angiogenic Potential and Platelet-Mediated Metastasis

Objective - Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in tumor metastasis and neovascularization. Metastatic disease is the cause of roughly 90% of all cancer-related deaths and understanding the mechanisms leading to dissemination of tumor cells to distant sites remains one of the main challenges of cancer research. Platelets carry a plethora of potent angiogenic and metastatic mediators within their alpha-granules and exposure to breast tumor cells induces platelet activation, leading to release of these mediators. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment and prevention of breast cancer; its use is associated with a 50% reduction in the risk of invasive and noninvasive breast cancer in women who utilized the drug for at least 5 years. Interestingly, tamoxifen has demonstrated anti-cancer efficacy in estrogen negative breast cancers suggesting that this drug has additional mechanisms of action. Previously tamoxifen and its metabolites have been shown to directly impact platelet function. Because platelets are critical for metastatic spread, we posited that tamoxifen or its metabolites may exert anti-tumor effects through direct platelet inhibition. To test this hypothesis, we examined the impact of tamoxifen on platelet activation, angiogenic potential and metastasis both ex vivoand in breast cancer patients utilizing tamoxifen therapy.Approach and Results - We found that ex vivo pretreatment with tamoxifen or its metabolite 4-hydroxytamoxifen (4-OH) lead to a significant inhibition of platelet activation in response to TRAP, ADP or the breast tumor cell lines MDA-MB-231 and MCF-7 in platelets from healthy human donors. Platelets, known to promote tumor angiogenesis, are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. Activated platelets exposed to tamoxifen or 4-OH released significantly lower amounts of the pro-angiogenic regulator VEGF in while anti-angiogenic endostatin release is unaffected, thus shifting the balance of angiogenic regulators that are released. To examine the impact of this alteration in angiogenic protein release, we performed functional angiogenesis assays using releasates generated from tamoxifen or 4-OH treated platelets. These in vitroangiogenesis assays confirmed that tamoxifen pretreatment led to dramatically diminished capillary tube formation and decreased endothelial migration. Tamoxifen also significantly inhibited the ability of platelets to promote metastasis in vitro, causing a dramatic decrease in breast tumor cell invasion and transendothelial migration. Next, we utilized membrane-based cytokine arrays to further interrogate the effect of tamoxifen on the release of stored platelet factors. Using this method, we identified several key proteins known to be associated with metastasis that were lower in releasate from tamoxifen treated platelets including TGF-b, IL-6 and IGF-1 while anti-angiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished VEGF release and lower angiogenic potential.Conclusions - Overall we demonstrate that tamoxifen directly influences the release of specific stored platelet factors leading to decreased tumor cell support. The mechanism is directly linked to tamoxifen's inhibitory role in platelet activation, causing altered of release of key platelet-derived angiogenic and metastatic factors including VEGF, angiopoietin-1, TGF-b, IL-6 and IGF-1 during tumor cell and platelet crosstalk. Furthermore, translational studies confirmed that platelet activation and angiogenic potential are significantly suppressed in breast cancer patients utilizing tamoxifen therapy. Our work stresses the importance of platelets for successful angiogenesis and metastatic spread and, ultimately, supports the idea of utilizing targeted platelet therapies to inhibit the platelet's role in malignancy. DisclosuresNo relevant conflicts of interest to declare.

Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/MenaINV-initiated invadopodium formation.

The process of intravasation involving transendothelial migration is a key step in metastatic spread. How the triple cell complex composed of a macrophage, Mena over-expressing tumor cell and endothelial cell, called the tumor microenvironment of metastasis (TMEM), facilitates tumor cell transendothelial migration is not completely understood. Previous work has shown that the physical contact between a macrophage and tumor cell results in the formation of invadopodia, actin-rich matrix degrading protrusions, important for tumor cell invasion and transendothelial migration and tumor cell dissemination. Herein, we show that the macrophage-induced invadopodium is formed through a Notch1/MenaINV signaling pathway in the tumor cell upon macrophage contact. This heterotypic tumor cell – macrophage interaction results in the upregulation of MenaINV through the activation of MENA transcription. Notch1 and MenaINV expression are required for tumor cell transendothelial migration, a necessary step during intravasation. Inhibition of the Notch signaling pathway blocked macrophage-induced invadopodium formation in vitro and the dissemination of tumor cells from the primary tumor in vivo. Our findings indicate a novel role for Notch1 signaling in the regulation of MenaINV expression and transendothelial migration and provide mechanistic information essential to the use of therapeutic inhibitors of metastasis.

Monocyte Induction of E-Selectin-Mediated Endothelial Activation Releases VE-Cadherin Junctions to Promote Tumor Cell Extravasation in the Metastasis Cascade.

Tumor cells interact with blood constituents and these interactions promote metastasis. Selectins are vascular receptors facilitating interactions of tumor cells with platelets, leukocytes, and endothelium, but the role of endothelial E-selectin remains unclear. Here we show that E-selectin is a major receptor for monocyte recruitment to tumor cell-activated endothelium. Experimental and spontaneous lung metastasis using murine tumor cells, without E-selectin ligands, were attenuated in E-selectin-deficient mice. Tumor cell-derived CCL2 promoted endothelial activation, resulting in enhanced endothelial E-selectin expression. The recruitment of inflammatory monocytes to metastasizing tumor cells was dependent on the local endothelial activation and the presence of E-selectin. Monocytes promoted transendothelial migration of tumor cells through the induction of E-selectin-dependent endothelial retractions and a subsequent modulation of tight junctions through dephosphorylation of VE-cadherin. Thus, endothelial E-selectin shapes the tumor microenvironment through the recruitment, adhesion, and activation of monocytes that facilitate tumor cell extravasation and thereby metastasis. These findings provide evidence that endothelial E-selectin is a novel factor contributing to endothelial retraction required for efficient lung metastasis. Cancer Res; 76(18); 5302-12. ©2016 AACR.

A Novel Three-Dimensional Platform to Investigate Neoangiogenesis, Transendothelial Migration, and Metastasis of MDAMB-231 Breast Cancer Cells.

A crucial step in the progression of cancer involves the transendothelial migration of tumor cells into the bloodstream and invasion at distant sites. Most in vitro models of malignant cell behavior do not account for the presence of and interaction with vascular cells. Three-dimensional platforms to further explore the factors responsible for metastatic cellular behavior are under intensive investigation. Hydrogels with encapsulated MDAMB-231 breast cancer cells were fabricated with a central microchannel. The microchannel was lined with a co-culture of human umbilical vein endothelial cells and human aortic smooth muscle cells. For comparison, co-culture-seeded microchannels without breast cancer cells (MDAMB-negative) were fabricated. After 7 and 14 days, the endoluminal lining of encapsulated MDAMB-231 co-culture-seeded microchannels demonstrated aberrant endothelial cell and smooth muscle cell organization and breast cancer cell transendothelial migration. MDAMB-231 cells performed matrix remodeling, forming tumor aggregates within the bulk, migrating preferentially toward the hydrogel "neovessel." In contrast, MDAMB-negative constructs demonstrated maintenance of an intact endoluminal lining composed of endothelial cells and smooth muscle cells that organized into discrete layers. Furthermore, the thicknesses of the endoluminal lining of MDAMB-negative constructs were significantly greater than encapsulated MDAMB-231 co-culture-seeded constructs after 7 and 14 days (p = 0.012 and p < 0.001, respectively). The authors have created a powerful tool that may have tremendous impact on furthering our understanding of cancer recurrence and metastasis, shedding light on these poorly understood phenomena.

GM-CSF Mediates Mesenchymal-Epithelial Cross-talk in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by a dense stroma consisting of a prevalence of activated fibroblasts whose functional contributions to pancreatic tumorigenesis remain incompletely understood. In this study, we provide the first identification and characterization of mesenchymal stem cells (MSC) within the human PDA microenvironment, highlighting the heterogeneity of the fibroblast population. Primary patient PDA samples and low-passage human pancreatic cancer-associated fibroblast cultures were found to contain a unique population of cancer-associated MSCs (CA-MSC). CA-MSCs markedly enhanced the growth, invasion, and metastatic potential of PDA cancer cells. CA-MSCs secreted the cytokine GM-CSF that was required for tumor cell proliferation, invasion, and transendothelial migration. Depletion of GM-CSF in CA-MSCs inhibited the ability of these cells to promote tumor cell growth and metastasis. Together, these data identify a population of MSCs within the tumor microenvironment that possesses a unique ability, through GM-CSF signaling, to promote PDA survival and metastasis. The role of stroma in pancreatic cancer is controversial. Here, we provide the first characterization of MSCs within the human PDA microenvironment and demonstrate that CA-MSCs promote tumorigenesis through the production of GM-CSF. These data identify a novel cytokine pathway that mediates mesenchymal-epithelial cross-talk and is amenable to therapeutic intervention. Cancer Discov; 6(8); 886-99. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

Abstract 902: A small molecule glycomimetic antagonist of E-selectin and CXCR4 (GMI-1359) delays pancreatic tumor metastasis and significantly alters the pancreatic tumor microenvironment

Abstract The cellular and molecular composition of the pancreatic tumor microenvironment includes a complex matrix that encases tumor cells and presents a conundrum in respect to therapy. The dense desmoplasia that accompanies tumor growth reduces blood flow to tumors and creates an environment that compromises delivery of therapeutics. As well, elimination of certain stromal elements enhances the aggressiveness and metastatic potential of pancreatic cancer cells. Recent studies attempt to target stroma in an effort to enhance delivery and efficacy of therapeutic agents and to block metastasis. In this context we have investigated the activity of GMI-1359, a potent dual antagonist that targets both E-selectin and CXCR4. Adhesion protein E-selectin is crucial for regulating vascular and lymphatic endothelial cell (EC) interactions with tumor cells during transmigration. The CXCL12-CXCR4 chemokine axis contributes to the formation of the tumor microenvironment including fibroblast and immune cell recruitment, lymph- and angiogenesis, tumor cell proliferation/survival, and tumor stem cell mobilization. Our in vitro results demonstrate that pancreatic fibroblasts secrete significant amounts of CXCL12 which promotes tumor cell and lymphatic and vascular EC directional migration; this migration toward CXCL12-secreting fibroblasts was completely blocked by GMI-1359. Additionally, CXCL12-stimulated ECs facilitated increased transendothelial migration (TEM) by pancreatic tumor cells. Dual antagonist GMI-1359 inhibited this CXCL12-dependent increase in pancreatic tumor cell TEM and was more effective than an independent small molecule E-selectin only inhibitor. Using an in vivo orthotopic model of pancreatic cancer in athymic mice, we evaluated the ability of GMI-1359 (with and without co-administration Gemcitabine (Gem)) to suppress tumor progression, modulate tumor microenvironment composition, and prolong survival. Our work demonstrated GMI-1359 slightly inhibited tumor growth when used alone or in combination with Gem but did not prolong survival in this immune compromised model. However, GMI-1359 inhibited tumor metastasis to the spleen, liver, and lungs. Interestingly, GMI-1359 significantly modulated the cellular composition of the tumor microenvironment. Immunohistochemical analysis revealed mice treated with GMI-1359 (with or without Gem administration) had drastically reduced desmoplasia and reduced lymphatic and blood vessel densities compared to mice treated with vehicle control or Gem alone. The E-selectin only inhibitor had no effect on tumor microenvironment composition. Further studies of GMI-1359, particularly in immune competent models, are warranted to understand its potential for disrupting the pancreatic tumor microenvironment, inhibiting dissemination, and enhancing anti-tumor immune responses. Citation Format: Maria M. Steele, William E. Fogler, John L. Magnani, Michael A. Hollingsworth. A small molecule glycomimetic antagonist of E-selectin and CXCR4 (GMI-1359) delays pancreatic tumor metastasis and significantly alters the pancreatic tumor microenvironment. [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 902.

Targeting of CCL2-CCR2-Glycosaminoglycan Axis Using a CCL2 Decoy Protein Attenuates Metastasis through Inhibition of Tumor Cell Seeding

The CCL2-CCR2 chemokine axis has an important role in cancer progression where it contributes to metastatic dissemination of several cancer types (e.g., colon, breast, prostate). Tumor cell–derived CCL2 was shown to promote the recruitment of CCR2+/Ly6Chi monocytes and to induce vascular permeability of CCR2+ endothelial cells in the lungs. Here we describe a novel decoy protein consisting of a CCL2 mutant protein fused to human serum albumin (dnCCL2-HSA chimera) with enhanced binding affinity to glycosaminoglycans that was tested in vivo. The monocyte-mediated tumor cell transendothelial migration was strongly reduced upon unfused dnCCL2 mutant treatment in vitro. dnCCL2-HSA chimera had an extended serum half-life and thus a prolonged exposure in vivo compared with the dnCCL2 mutant. dnCCL2-HSA chimera bound to the lung vasculature but caused minimal alterations in the leukocyte recruitment to the lungs. However, dnCCL2-HSA chimera treatment strongly reduced both lung vascular permeability and tumor cell seeding. Metastasis of MC-38GFP, 3LL, and LLC1 cells was significantly attenuated upon dnCCL2-HSA chimera treatment. Tumor cell seeding to the lungs resulted in enhanced expression of a proteoglycan syndecan-4 by endothelial cells that correlated with accumulation of the dnCCL2-HSA chimera in the vicinity of tumor cells. These findings demonstrate that the CCL2-based decoy protein effectively binds to the activated endothelium in lungs and blocks tumor cell extravasation through inhibition of vascular permeability.

Abstract 397: Rebastinib potently inhibits function of perivascular TIE2 expressing macrophages in vitro and in vivo

Abstract Introduction Significant cross-talk between tumor cells and the surrounding stromal tissue are essential for tumor vascularization, survival, immunotolerance, invasion, and metastasis. The angiopoietin (ANG)/TIE2 kinase signaling pathway is a pivotal cross talk axis in the tumor microenvironment. It has been demonstrated that TIE2-expressing macrophages (TEMs) mediate invasion and metastasis in the PyMT syngeneic breast cancer model, and that TIE2 expression correlates with poor overall survival and high risk of metastasis in breast cancer patients. In this study, rebastinib was evaluated as a TIE2 inhibitor in in vitro and in vivo models, and was shown to have profound effects on the structure and function of perivascular TEMs. Procedures TIE2 kinase assays used a standard PK/LDH coupled spectrophotometric continuous assay. CHO cells were transiently transfected to express human TIE2 for cellular studies. CHOs, HUVECs and EA.hy926 cells were used to evaluate rebastinib for inhibition of ANG1-stimulated TIE2 phosphorylation. In vitro intravasation studies were performed in a transwell transendothelial migration chamber wherein TIE2HI macrophages interact with breast tumor cells to cause transendothelial migration of tumor cells across a sealed HUVEC endothelial monolayer. In vivo evaluations of rebastinib were performed using intravital high-resolution two-photon microscopy in the murine PyMT breast cancer model to evaluate effects on tumor vascular permeability and tumor cell intravasation. Results Rebastinib is a potent inhibitor of TIE2 kinase (IC50 = 0.63 nM). Rebastinib slowly dissociated from TIE2 (koff = 0.0012 minutes−1; T1/2 = 10 hr). In HUVECs or EA.hy926 cells, rebastinib inhibited ANG1-stimulated TIE2 kinase activity (IC50s of 0.018 and 0.091 nM, respectively). In TIE2 CHO cells, rebastinib inhibited TIE2 phosphorylation (IC50 2.0 nM), and demonstrated a prolonged off-rate (&amp;gt; 24 hr) against TIE2 after inhibitor washout. Rebastinib exhibited an IC50 &amp;lt; 5 nM for inhibiting macrophage-inducedbreast tumor cell intravasation in the in vitro transwell transendothelial migration assay. Rebastinib was evaluated in vivo in the PyMT syngeneic breast cancer model. Rebastinib dosed at 10 mg/kg orally twice weekly impaired tumoral perivascular TEMs, resulting in a significant reduction in vascular permeability and in tumor cell intravasation as quantified by CTCs. Conclusion Rebastinib is a potent inhibitor of TIE2 kinase and exhibits durable cellular inhibition in endothelial cells and in TIE2 macrophages. Oral dosing of rebastinib resulted in a significant reduction in TIE2-macrophage mediated tumor vascular permeability and in the intravasation of tumor cells into the circulation. Rebastinib is currently in Phase 1 clinical evaluation in solid tumors. Citation Format: Allison Harney, Jeanine Pignatelli, Edison Leung, Maja Oktay, Yarong Wang, Bryan D. Smith, Daniel L. Flynn, John S. Condeelis. Rebastinib potently inhibits function of perivascular TIE2 expressing macrophages in vitro and in vivo. [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 397. doi:10.1158/1538-7445.AM2015-397

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

Endothelial Rac1 is essential for hematogenous metastasis to the lung.

A variety of vasoactive stimuli induce endothelial permeability through Rac1, a membrane of Rho small GTPases. Here, we determine whether tumor-secreted vasoactive stimulant through Rac1 inducing permeability contributes to hematogenous metastasis. Activation of Rac1 was assayed in human umbilical vein endothelial cells (HUVEC), transendothelial passages were measured by Transwell chambers, and hematogenously metastatic mouse model was generated by intravenous injection with Lewis lung carcinoma cells (LLC). LLC secreted abundant vascular endothelial growth factor (VEGF) in the culture media and sera of mice bearing LLC xenografts or metastatic LLC, and VEGF activated Rac1 through VEGF receptors/PI3Kβ signaling cascade, resulting in hyperoxidative stress and consequent hyperpermeability in HUVEC. Moreover, in co-culture of LLC and HUVEC, significant increases in endothelial permeability and transendothelial migration of LLC were robustly attenuated by either anti-VEGF neutralizing antibody or Rac1 knockdown in HUVEC. Finally, in metastatic mouse model, deletion of one copy of Rac1 in endothelium not only significantly attenuated LLC-induced vascular permeability, but robustly reduced the metastasis of LLC to lungs. This study supports that tumor-secreted vasoactive stimuli activate Rac1 to induce permeability and consequent transendothelial migration of tumor cells, and that loss of Rac1 function in endothelium is an effective therapeutic intervention for hematogenous metastasis.

Myeloid heme oxygenase-1 promotes metastatic tumor colonization in mice.

Heme oxygenase-1 (HO-1) is a heme degradation enzyme with antioxidant and immune-modulatory functions. HO-1 promotes tumorigenesis by enhancing tumor cell proliferation and invasion. Whether HO-1 has an effect on cancer progression through stromal compartments is less clear. Here we show that the growth of tumor engrafted subcutaneously in syngeneic mice was not affected by host HO-1 expression. However, lung metastasis arisen from subcutaneous tumor or circulating tumor cells was significantly reduced in HO-1+/− mice comparing to wild type (WT) mice. The reduced lung metastasis was also observed in B6 mice bearing HO-1+/− bone marrow as comparing to WT chimeras, indicating that HO-1 expression in hematopoietic cells impacts tumor colonization at the metastatic site. Further experiments demonstrated that the numbers of myeloid cells recruited to pulmonary premetastatic niches and metastatic loci were significantly lower in HO-1+/− mice than in WT mice. Likewise, the extents of tumor cell extravasation and colonization at the metastatic loci in the early phase of metastasis were significantly lower in HO-1+/− mice. Mechanistic studies revealed that HO-1 impacted chemoattractant-induced myeloid cell migration by modulating p38 kinase signaling. Moreover, myeloid HO-1-induced expressions of vascular endothelial growth factor and interleukin-10 promoted tumor cell transendothelial migration and STAT3 activation in vitro. These data support a pathological role of myeloid HO-1 in metastasis and suggest a possibility of targeting myeloid HO-1 for cancer treatment.

A microfluidic platform for quantitative analysis of cancer angiogenesis and intravasation.

Understanding the mechanism behind cancer metastasis is a major challenge in cancer biology. Several in vitro models have been developed to mimic a cancer microenvironment by engineering cancer-endothelial cell (EC) and cancer-stromal cell interactions. It has been challenging to realistically mimic angiogenesis, intravasation, and extravasation using macro-scale approaches but recent progress in microfluidics technology has begun to yield promising results. We present a metastasis chip that produce microvessels, where EC and stromal cells can be patterned in close proximity to tumor cells. The vessels are formed following a natural morphogenic process and have smooth boundaries with proper cell-cell junctions. The engineered microvessels are perfusable and have well-defined openings toward inlet and outlet channels. The ability to introduce cancer cells into different locations bordering to the microvessel wall allowed generation and maintenance of appropriate spatial gradients of growth factors and attractants. Cancer angiogenesis and its inhibition by anti-vascular endothelial growth factor (bevacizumab) treatment were successfully reproduced in the metastasis chip. Cancer intravasation and its modulation by treatment of tumor necrosis factor-α were also modeled. Compared to other models, the unique design of the metastasis chip that engineers a clear EC-cancer interface allows precise imaging and quantification of angiogenic response as well as tumor cell trans-endothelial migration. The metastasis chip presented here has potential applications in the investigation of fundamental cancer biology as well as in drug screening.

Macrophage contact induces RhoA GTPase signaling to trigger tumor cell intravasation

Most cancer patients die as a result of metastasis, thus it is important to understand the molecular mechanisms of dissemination, including intra- and extravasation. Although the mechanisms of extravasation have been vastly studied in vitro and in vivo, the process of intravasation is still unclear. Furthermore, how cells in the tumor microenvironment facilitate tumor cell intravasation is still unknown. Using high-resolution imaging, we found that macrophages enhance tumor cell intravasation upon physical contact. Macrophage and tumor cell contact induce RhoA activity in tumor cells, triggering the formation of actin-rich degradative protrusions called invadopodia, enabling tumor cells to degrade and break through matrix barriers during tumor cell transendothelial migration. Interestingly, we show that macrophage-induced invadopodium formation and tumor cell intravasation also occur in patient-derived tumor cells and in vivo models, revealing a conserved mechanism of tumor cell intravasation. Our results illustrate a novel heterotypic cell contact mediated signaling role for RhoA, as well as yield mechanistic insight into the ability of cells within the tumor microenvironment to facilitate steps of the metastatic cascade.

Platelet-Derived Nucleotides Promote Tumor-Cell Transendothelial Migration and Metastasis via P2Y2 Receptor

Tumor cells can activate platelets, which in turn facilitate tumor cell survival and dissemination. The exact mechanisms by which platelets promote metastasis have remained unclear. Here, we show that adenine nucleotides released from tumor cell-activated platelets induce opening of the endothelial barrier to allow transendothelial migration of tumor cells and thereby promote cancer cell extravasation. We identified the endothelial P2Y2 receptor, which is activated by ATP, as the primary mediator of this effect. Mice deficient in P2Y2 or lacking ATP secretion from platelets show strongly reduced tumor cell metastasis. These findings demonstrate a mechanism by which platelets promote cancer cell metastasis and suggest the P2Y2 receptor and its endothelial downstream signaling mechanisms as a target for antimetastatic therapies.

Integrin-Associated CD151 Drives ErbB2-Evoked Mammary Tumor Onset and Metastasis

ErbB2+ human breast cancer is a major clinical problem. Prior results have suggested that tetraspanin CD151 might contribute to ErbB2-driven breast cancer growth, survival, and metastasis. In other cancer types, CD151 sometimes supports tumor growth and metastasis. However, a definitive test of CD151 effects on de novo breast cancer initiation, growth, and metastasis has not previously been done. We used CD151 gene-deleted mice expressing the MMTV-ErbB2 transgene to show that CD151 strongly supports ErbB2+ mammary tumor initiation and metastasis. Delayed tumor onset (by 70–100 days) in the absence of CD151 was accompanied by reduced survival of mammary epithelial cells and impaired activation of FAK- and MAPK-dependent pathways. Both primary tumors and metastatic nodules showed smooth, regular borders, consistent with a less invasive phenotype. Furthermore, consistent with impaired oncogenesis and decreased metastasis, CD151-targeted MCF-10A/ErbB2 cells showed substantial decreases in three-dimensional colony formation, EGF-stimulated tumor cell motility, invasion, and transendothelial migration. These CD151-dependent functions were largely mediated through α6β4 integrin. Moreover, CD151 ablation substantially prevented PKC- and EGFR/ERK-dependent α6β4 integrin phosphorylation, consistent with retention of epithelial cell polarity and intermediate filament cytoskeletal connections, which helps to explain diminished metastasis. Finally, clinical data analyses revealed a strong correlation between CD151 and ErbB2 expression and metastasis-free survival of breast cancer patients. In conclusion, we provide strong evidence that CD151 collaborates with LB integrins (particularly α6β4) and ErbB2 (and EGFR) receptors to regulate multiple signaling pathways, thereby driving mammary tumor onset, survival, and metastasis. Consequently, CD151 is a useful therapeutic target in malignant ErbB2+ breast cancer.

Inhibition of Prostate Cancer Bone Metastasis by Synthetic TF Antigen Mimic/Galectin-3 Inhibitor Lactulose-l-Leucine

Currently incurable, prostate cancer metastasis has a remarkable ability to spread to the skeleton. Previous studies demonstrated that interactions mediated by the cancer-associated Thomsen-Friedenreich glycoantigen (TF-Ag) and the carbohydrate-binding protein galectin-3 play an important role in several rate-limiting steps of cancer metastasis such as metastatic cell adhesion to bone marrow endothelium, homotypic tumor cell aggregation, and clonogenic survival and growth. This study investigated the ability of a synthetic small-molecular-weight nontoxic carbohydrate-based TF-Ag mimic lactulose-L-leucine (Lac-L-Leu) to inhibit these processes in vitro and, ultimately, prostate cancer bone metastasis in vivo. Using an in vivo mouse model, based on intracardiac injection of human PC-3 prostate carcinoma cells stably expressing luciferase, we investigated the ability of Lac-L-Leu to impede the establishment and growth of bone metastasis. Parallel-flow chamber assay, homotypic aggregation assay, modified Boyden chamber assay, and clonogenic growth assay were used to assess the effects of Lac-L-Leu on tumor cell adhesion to the endothelium, homotypic tumor cell aggregation, transendothelial migration, and clonogenic survival and growth, respectively. We report that daily intraperitoneal administration of Lac-L-Leu resulted in a three-fold (P < .05) decrease in metastatic tumor burden compared with the untreated control. Mechanistically, the effect of Lac-L-Leu, which binds and inhibits galectins by mimicking essential structural features of the TF-Ag, was associated with a dose-dependent inhibition of prostate cancer cell adhesion to bone marrow endothelium, homotypic aggregation, transendothelial migration, and clonogenic growth. We conclude that small-molecular-weight carbohydrate-based compounds targeting β-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton.

The novel chemokine receptor CXCR7 regulates trans-endothelial migration of cancer cells

BackgroundMigration of metastatic tumor cells from the bloodstream into lymph nodes is thought to be facilitated by expression of the chemokine receptors CCR7, CXCR4 and, for B cell-derived tumors, CXCR5. Expression of their respective chemokine ligands (CCL19, CCL21, CXCL12 and CXCL13) by endothelial cells inside the lymph nodes facilitates the trans-endothelial migration (TEM) of these cells through high endothelial venules into the lymph node parenchyma. It is known that CXCR7, a second CXCL12 receptor, regulates TEM of CXCR4+CXCR7+ tumor cells towards a CXCL12 source. In this study, we set out to assess the potential stimulation by CXCL12 of tumor cell TEM towards other chemokines and whether CXCR7 might be able to regulate such effects.MethodsThe human Burkitt's lymphoma cell line NC-37, which expresses CXCR4, CXCR5, CXCR7 and CCR7, was selected as a model system. TEM of these cells through a human HUVEC endothelial cell monolayer was used as the main model system for these studies. Regulation of their TEM behavior by various concentrations of the various cognate chemokines for the above-mentioned receptors, placed in either the source or target wells of modified Boyden chamber migration plates, was assessed by quantifying the number of cells migrated under each experimental condition.ResultsExposure of CXCR4+CXCR7+ cancer cells to CXCL12 greatly potentiated their TEM towards the chemokines CCL19 and CXCL13. This CXCL12-potentiated TEM was inhibited by the second CXCR7 chemokine ligand, CXCL11, as well as CXCR7-specific small molecule antagonists and antibodies. In contrast, the CXCR4 antagonist AMD3100 was less effective at inhibiting CXCL12-potentiated TEM. Thus, CXCR7 antagonists may be effective therapeutic agents for blocking CXCL12-mediated migration of CXCR4+CXCR7+ tumor cells into lymph nodes, regardless of whether the cancer cells follow a CXCL12 gradient or whether serum CXCL12 stimulates their migration towards CCR7 and CXCR5 chemokines in the lymph nodes.

Abstract 2842: CCL2 recruits inflammatory monocytes to facilitate breast tumor metastasis

Abstract Macrophages are abundant in the tumor microenvironment and enhance malignancy through their promotion of angiogenesis and invasion at the primary tumor site as well as extravasation, target organ seeding and persistent growth at distant metastatic sites. To determine the origin and recruitment of tumor associated macrophages, different populations of monocytes were FACS sorted and adoptively transferred into mice bearing MMTV-PyMT induced mouse mammary tumors with or without spontaneous pulmonary metastases. Ly6C+ inflammatory monocytes were preferentially recruited to pulmonary metastases but not primary tumors in a CCL2 (also known as MCP1) dependent manner. Human inflammatory monocytes were also preferentially recruited to pulmonary metastases of human breast cancer cells in immuno-compromised mice in a CCL2 dependent manner. These inflammatory monocytes promote tumor cell trans-endothelial migration in vitro, a process that is abrogated by an anti-CCL2 neutralizing antibody. Neutralizing CCL2 in vivo blocks the recruitment of metastasis associated macrophages and their direct interaction with metastasizing tumor cells, leading to inhibition of tumor cell extravasation and metastatic seeding. Inhibition of tumor cell-derived CCL2 inhibits their metastatic seeding. Secretory factors from inflammatory monocytes were identified to mediate their metastasis-promoting function in vitro and in vivo. Both CCL2 expression and macrophage infiltration are correlated with poor prognosis in human breast cancer. Our data strongly suggest that recruitment of Ly6C+ inflammatory monocytes is the mechanistic link between these two clinical associations and suggests new therapeutic targets for treating metastatic breast disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2842. doi:10.1158/1538-7445.AM2011-2842