Tumor cells frequently disseminate through the lymphatic system during metastatic spread of breast cancer and many other types of cancer. Yet it is not clear how tumor cells make their way into the ...lymphatic system and how they choose between lymphatic and blood vessels for migration. Here we report that mammary tumor cells undergoing epithelial-mesenchymal transition (EMT) in response to transforming growth factor-β (TGF-β1) become activated for targeted migration through the lymphatic system, similar to dendritic cells (DCs) during inflammation. EMT cells preferentially migrated toward lymphatic vessels compared with blood vessels, both in vivo and in 3D cultures. A mechanism of this targeted migration was traced to the capacity of TGF-β1 to promote CCR7/CCL21-mediated crosstalk between tumor cells and lymphatic endothelial cells. On one hand, TGF-β1 promoted CCR7 expression in EMT cells through p38 MAP kinase-mediated activation of the JunB transcription factor. Blockade of CCR7, or treatment with a p38 MAP kinase inhibitor, reduced lymphatic dissemination of EMT cells in syngeneic mice. On the other hand, TGF-β1 promoted CCL21 expression in lymphatic endothelial cells. CCL21 acted in a paracrine fashion to mediate chemotactic migration of EMT cells toward lymphatic endothelial cells. The results identify TGF-β1-induced EMT as a mechanism, which activates tumor cells for targeted, DC-like migration through the lymphatic system. Furthermore, it suggests that p38 MAP kinase inhibition may be a useful strategy to inhibit EMT and lymphogenic spread of tumor cells.
During breast cancer progression, transforming growth factor-beta (TGF-β) switches from acting as a growth inhibitor to become a major promoter of epithelial-mesenchymal transition (EMT), invasion ...and metastasis. However, the mechanisms involved in this switch are not clear. We found that loss of CCAAT-enhancer binding protein beta (C/EBPβ), a differentiation factor for the mammary epithelium, was associated with signs of EMT in triple-negative human breast cancer, and in invasive areas of mammary tumors in MMTV-PyMT mice. Using an established model of TGF-β-induced EMT in mouse mammary gland epithelial cells, we discovered that C/EBPβ was repressed during EMT by miR-155, an oncomiR in breast cancer. Depletion of C/EBPβ potentiated the TGF-β response towards EMT, and contributed to evasion of the growth inhibitory response to TGF-β. Furthermore, loss of C/EBPβ enhanced invasion and metastatic dissemination of the mouse mammary tumor cells to the lungs after subcutaneous injection into mice. The mechanism by which loss of C/EBPβ promoted the TGF-β response towards EMT, invasion and metastasis, was traced to a previously uncharacterized role of C/EBPβ as a transcriptional activator of genes encoding the epithelial junction proteins E-cadherin and coxsackie virus and adenovirus receptor. The results identify miR-155-mediated loss of C/EBPβ as a mechanism, which promotes breast cancer progression by shifting the TGF-β response from growth inhibition to EMT, invasion and metastasis.
Therapy-induced resistance remains a significant hurdle to achieve long-lasting responses and cures in cancer patients. We investigated the long-term consequences of genetically impaired angiogenesis ...by engineering multiple tumor models deprived of endoglin, a co-receptor for TGF-β in endothelial cells actively engaged in angiogenesis. Tumors from endoglin-deficient mice adapted to the weakened angiogenic response, and refractoriness to diminished endoglin signaling was accompanied by increased metastatic capability. Mechanistic studies in multiple mouse models of cancer revealed that deficiency for endoglin resulted in a tumor vasculature that displayed hallmarks of endothelial-to-mesenchymal transition, a process of previously unknown significance in cancer biology, but shown by us to be associated with a reduced capacity of the vasculature to avert tumor cell intra- and extravasation. Nevertheless, tumors deprived of endoglin exhibited a delayed onset of resistance to anti-VEGF (vascular endothelial growth factor) agents, illustrating the therapeutic utility of combinatorial targeting of multiple angiogenic pathways for the treatment of cancer.
Blood vessel leakiness is an early, transient event in acute inflammation but can also persist as vessels undergo remodeling in sustained inflammation. Angiopoietin/Tie2 signaling can reduce the ...leakiness through changes in endothelial cells. The role of pericytes in this action has been unknown. We used the selective PDGF-B-blocking oligonucleotide aptamer AX102 to determine whether disruption of pericyte-endothelial crosstalk alters vascular leakiness or remodeling in the airways of mice under four different conditions: i) baseline, ii) acute inflammation induced by bradykinin, iii) sustained inflammation after 7-day infection by the respiratory pathogen Mycoplasma pulmonis , or iv) leakage after bradykinin challenge in the presence of vascular stabilization by the angiopoietin-1 (Ang1) mimic COMP-Ang1 for 7 days. AX102 reduced pericyte coverage but did not alter the leakage of microspheres from tracheal blood vessels at baseline or after bradykinin; however, AX102 exaggerated leakage at 7 days after M. pulmonis infection and increased vascular remodeling and disease severity at 14 days. AX102 also abolished the antileakage effect of COMP-Ang1 at 7 days. Together, these findings show that pericyte contributions to endothelial stability have greater dependence on PDGF-B during the development of sustained inflammation, when pericyte dynamics accompany vascular remodeling, than under baseline conditions or in acute inflammation. The findings also show that the antileakage action of Ang1 requires PDGF-dependent actions of pericytes in maintaining endothelial stability.
Vascular remodeling is a feature of chronic inflammation during which capillaries transform into venules that expand the region of the vasculature in which leakage and leukocyte emigration both ...occur. Recently, we found that angiopoietin/Tie2 receptor signaling drives the transformation of capillaries into venules at an early stage of the sustained inflammatory response in the airways of mice infected with Mycoplasma pulmonis . However, the precise contributions of both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are not clear. In this study, we sought to determine the contribution of Ang2 to this vascular remodeling. Ang2 mRNA expression levels increased and phosphorylated Tie2 immunoreactivity in mucosal blood vessels decreased, indicative of diminished receptor signaling after infection. Selective inhibition of Ang2 throughout the infection by administration of either of two distinct function-blocking antibodies reduced the suppression of Tie2 phosphorylation and decreased the remodeling of mucosal capillaries into venules, the amount of leukocyte influx, and disease severity. These findings are consistent with Ang2 acting as an antagonist of Tie2 receptors and the reduction of Tie2 phosphorylation in endothelial cells rendering the vasculature more responsive to cytokines that promote both vascular remodeling and the consequences of inflammation after M. pulmonis infection. By blocking such changes, Ang2 inhibitors may prove beneficial in the treatment of sustained inflammation in which vascular remodeling, leakage, and leukocyte influx contribute to its pathophysiology.
Tumor cells frequently disseminate through the lymphatic system during metastatic spread of breast cancer and many other types of cancer. Yet it is not clear how tumor cells make their way into the ...lymphatic system and how they choose between lymphatic and blood vessels for migration. Here we report that mammary tumor cells undergoing epithelial-mesenchymal transition (EMT) in response to transforming growth factor- beta (TGF- beta 1) become activated for targeted migration through the lymphatic system, similar to dendritic cells (DCs) during inflammation. EMT cells preferentially migrated toward lymphatic vessels compared with blood vessels, both in vivo and in 3D cultures. A mechanism of this targeted migration was traced to the capacity of TGF- beta 1 to promote CCR7/CCL21-mediated crosstalk between tumor cells and lymphatic endothelial cells. On one hand, TGF- beta 1 promoted CCR7 expression in EMT cells through p38 MAP kinase-mediated activation of the JunB transcription factor. Blockade of CCR7, or treatment with a p38 MAP kinase inhibitor, reduced lymphatic dissemination of EMT cells in syngeneic mice. On the other hand, TGF- beta 1 promoted CCL21 expression in lymphatic endothelial cells. CCL21 acted in a paracrine fashion to mediate chemotactic migration of EMT cells toward lymphatic endothelial cells. The results identify TGF- beta 1-induced EMT as a mechanism, which activates tumor cells for targeted, DC-like migration through the lymphatic system. Furthermore, it suggests that p38 MAP kinase inhibition may be a useful strategy to inhibit EMT and lymphogenic spread of tumor cells.
It was already in the 18th century when the French surgeon LeDran first noted that breast cancer patients with spread of tumor cells to their axillary lymph nodes had a drastically worse prognosis ...than patients without spread (LeDran et al., ). Since then, metastatic spread of cancer cells to regional lymph nodes has been established as the most important prognostic factor in many types of cancer (Carter et al., ; Elston and Ellis, ). However, despite its clinical importance, lymph metastasis remains an underexplored area of tumor biology. Fundamental questions, such as when, how, and perhaps most importantly, why tumor cells disseminate through the lymphatic system, remain largely unanswered. Accordingly, no treatment strategies exist that specifically target lymph metastasis. The identification of epithelial‐mesenchymal transition (EMT) as a mechanism, which allows cancer cells to dedifferentiate and acquire enhanced migratory and invasive properties, has been a game changer in cancer research. Conceptually, EMT provides an explanation for why epithelial cancers with poor differentiation status are generally more aggressive and prone to metastasize than more differentiated cancers. Inflammatory cytokines, such as TGF‐β, which are produced and secreted by tumor‐infiltrating immune cells, are potent inducers of EMT. Thus, reactivation of EMT also links cancer‐related inflammation to invasive and metastatic disease. Recently, we found that breast cancer cells undergoing TGF‐β‐induced EMT acquire properties of immune cells allowing them to disseminate in a targeted fashion through the lymphatic system similar to activated dendritic cells during inflammation. Here, we review our current understanding of the mechanisms by which cancer cells spread through the lymphatic system and the links to inflammation and the immune system. We also emphasize how imaging techniques have the potential to further expand our knowledge of the mechanisms of lymph metastasis, and how lymph nodes serve as an interface between cancer and the immune system.
This article presents a review on the role of EMT in cancer metastasis through the lymphatic system. The emphasis is on the role of TGF‐beta‐induced EMT as a link between cancer and inflammation, and how EMT cells acquire properties of immune cells.
CCAAT-enhancer binding protein β (C/EBPβ) is a transcription factor that has a critical role in mammary gland development and breast cancer progression. Loss of C/EBPβ increases metastatic ...dissemination of mouse mammary tumor cells. However, the mechanism by which C/EBPβ expression affects metastasis formation remains unknown. This study aims at determining the relationship between C/EBPβ and survival of breast cancer patients, and elucidating C/EBPβ's link with metastasis formation. C/EBPβ expression was evaluated in 137 cases of human breast cancer, and the correlation with overall survival was estimated by Kaplan-Meier analysis. Additionally, the mouse 4T1 tumor model was used for in vivo studies. Decreased C/EBPβ expression was found to be associated with shorter overall survival of breast cancer patients. In the murine 4T1 model, loss of C/EBPβ affects tumor growth, morphology and promotes metastatic spread to the lungs. Immunohistochemical analyses showed that C/EBPβ inhibition leads to increased major histocompatibility complex II (MHCII) expression, followed by the accumulation of CD45-, CD3- and CD4-positive (CD4+) lymphocytes in the tumors. Inflammation involvement in C/EBPβ-mediated metastasis formation was confirmed by DNA microarray and by experiments on CD4+ cell-deprived nude mice. Additionally, anti-CD3 and anti-CD4 treatments of C/EBPβ-silenced tumor-bearing mice resulted in reverting the C/EBPβ effect on tumor growth and metastasis. Altogether, C/EBPβ is a predictor of overall survival in breast cancer patients, and affects tumor growth, morphology and lung metastasis formation in murine 4T1 model. The mechanism of metastasis formation involves immunologic response depending on C/EBPβ-mediated activation of MHCII and accumulation of CD4+ lymphocytes in the tumor.
Tumors are composed of multiple cell types besides the tumor cells themselves, including innate immune cells such as macrophages. Tumor-associated macrophages (TAMs) are a heterogeneous population of ...myeloid cells present in the tumor microenvironment (TME). Here, they contribute to immunosuppression, enabling the establishment and persistence of solid tumors as well as metastatic dissemination. We have found that the pattern recognition scavenger receptor MARCO defines a subtype of suppressive TAMs and is linked to clinical outcome. An anti-MARCO monoclonal antibody was developed, which induces anti-tumor activity in breast and colon carcinoma, as well as in melanoma models through reprogramming TAM populations to a pro-inflammatory phenotype and increasing tumor immunogenicity. This anti-tumor activity is dependent on the inhibitory Fc-receptor, FcγRIIB, and also enhances the efficacy of checkpoint therapy. These results demonstrate that immunotherapies using antibodies designed to modify myeloid cells of the TME represent a promising mode of cancer treatment.
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•Scavenger receptor MARCO is expressed by suppressive tumor-associated macrophages•Antibody targeting of MARCO-expressing TAMs blocks tumor growth and metastasis•Anti-MARCO enhances the effect of checkpoint therapy in melanoma and colon carcinoma•MARCO is expressed on TAMs in human breast cancer and metastatic melanoma
Georgoudaki et al. show that tumor-associated macrophages can be targeted using an antibody toward the pattern recognition receptor MARCO. This results in altered macrophage polarization and a reduction in tumor growth and metastasis.
Tumor cells undergoing the epithelial-mesenchymal transition (EMT) acquire the capacity to migrate, invade the stroma and metastasize. EMT cells also acquire stem cell characteristics suggesting ...crosstalk between EMT and stem cell pathways and contribution of the EMT process to the generation of cancer stem cells. Indeed, transforming growth factor-beta (TGF-β), a major inducer of EMT, cooperates with stem cell pathways like Wnt, Ras, Hedgehog and Notch to induce EMT. A molecular basis for this cooperative signaling is indicated by recent data showing that many EMT associated transcription factors like Snail1, Zeb1/2, Twist, β-catenin, Lef/TCF, Foxc2 and AP-1 interact with Smads and form EMT promoting Smad complexes (EPSC) engaged in both repressing epithelial genes and activating mesenchymal genes. Thus, formation and activation of EPSC seems to represent a point of convergence between EMT and stem cell pathways. Here, we review our current understanding of the mechanisms involved in the transcriptional crosstalk between TGF-β and stem cell pathways and discuss how a fundament for the activation of these mechanisms may lead to the induction of EMT in tumors.
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