Promote Tumor Cell Extravasation Research Articles

Metastasis and the Microbiome: The Impact of Bacteria in Disseminated Colorectal Cancer.

Metastasis remains a leading cause of mortality for patients with solid tumors. An expanding body of literature suggests interplay between the host, gut, and tumoral microbiomes may play a role in cancer initiation and distant dissemination. These associations have been particularly well-studied in colorectal cancer, where gut dysbiosis and an endotoxin-induced inflammatory milieu foster premalignant polyp formation, setting the stage for carcinogenesis. Subsequent violation of the gut vascular barrier enables dissemination of bacterial agents to sites such as the liver, where they contribute to establishment of pre-metastatic niches, which promote tumor cell extravasation and metastatic outgrowth. Intriguingly, breakdown of this vascular barrier has been shown to be aided by the presence of tumoral bacteria. The presence of similar species, including Fusobacterium nucleatum and Escherichia Coli, in both primary and metastatic colorectal tumors, supports this hypothesis and their presence is associated with chemotherapy resistance and an overall poor prognosis. Specific gut microbial populations are also associated with differential response to immunotherapy, which has a growing role in microsatellite unstable colorectal cancers. Recent work suggests that modulation of gut microbiome using dietary modification, targeted antibiotics, or fecal microbiota transplantation may improve response to immunotherapy and oncologic outcomes. Elucidation of the precise mechanistic links between the microbiome and cancer dissemination will open the doors to additional therapeutic possibilities.

Macrophages Promote Tumor Cell Extravasation across an Endothelial Barrier through Thin Membranous Connections.

Macrophages are important players involved in the progression of breast cancer, including in seeding the metastatic niche. However, the mechanism by which macrophages in the lung parenchyma interact with tumor cells in the vasculature to promote tumor cell extravasation at metastatic sites is not clear. To mimic macrophage-driven tumor cell extravasation, we used an in vitro assay (eTEM) in which an endothelial monolayer and a matrigel-coated filter separated tumor cells and macrophages from each other. The presence of macrophages promoted tumor cell extravasation, while macrophage conditioned media was insufficient to stimulate tumor cell extravasation in vitro. This finding is consistent with a requirement for direct contact between macrophages and tumor cells. We observed the presence of Thin Membranous Connections (TMCs) resembling similar structures formed between macrophages and tumor cells called tunneling nanotubes, which we previously demonstrated to be important in tumor cell invasion in vitro and in vivo. To determine if TMCs are important for tumor cell extravasation, we used macrophages with reduced levels of endogenous M-Sec (TNFAIP2), which causes a defect in tunneling nanotube formation. As predicted, these macrophages showed reduced macrophage-tumor cell TMCs. In both, human and murine breast cancer cell lines, there was also a concomitant reduction in tumor cell extravasation in vitro when co-cultured with M-Sec deficient macrophages compared to control macrophages. We also detected TMCs formed between macrophages and tumor cells through the endothelial layer in the eTEM assay. Furthermore, tumor cells were more frequently found in pores under the endothelium that contain macrophage protrusions. To determine the role of macrophage-tumor cell TMCs in vivo, we generated an M-Sec deficient mouse. Using an in vivo model of experimental metastasis, we detected a significant reduction in the number of metastatic lesions in M-Sec deficient mice compared to wild type mice. There was no difference in the size of the metastases, consistent with a defect specific to tumor cell extravasation and not metastatic outgrowth. Additionally, with an examination of time-lapse intravital-imaging (IVI) data sets of breast cancer cell extravasation in the lungs, we could detect the presence of TMCs between extravascular macrophages and vascular tumor cells. Overall, our data indicate that macrophage TMCs play an important role in promoting the extravasation of circulating tumor cells in the lungs.

Galectin-3: A factotum in carcinogenesis bestowing an archery for prevention.

Cancer metastasis and therapy resistance are the foremost hurdles in oncology at the moment. This review aims to pinpoint the functional aspects of a unique multifaceted glycosylated molecule in both intracellular and extracellular compartments of a cell namely galectin-3 along with its metastatic potential in different types of cancer. All materials reviewed here were collected through the search engines PubMed, Scopus, and Google scholar. Among the 15 galectins identified, the chimeric gal-3 plays an indispensable role in the differentiation, transformation, and multi-step process of tumor metastasis. It has been implicated in the molecular mechanisms that allow the cancer cells to survive in the intravascular milieu and promote tumor cell extravasation, ultimately leading to metastasis. Gal-3 has also been found to have a pivotal role in immune surveillance and pro-angiogenesis and several studies have pointed out the importance of gal-3 in establishing a resistant phenotype, particularly through the epithelial-mesenchymal transition process. Additionally, some recent findings suggest the use of gal-3 inhibitors in overcoming therapeutic resistance. All these reports suggest that the deregulation of these specific lectins at the cellular level could inhibit cancer progression and metastasis. A more systematic study of glycosylation in clinical samples along with the development of selective gal-3 antagonists inhibiting the activity of these molecules at the cellular level offers an innovative strategy for primary cancer prevention.

TAK1 regulates endothelial cell necroptosis and tumor metastasis.

Formation of metastases is the major cause of death in patients diagnosed with cancer. It is a complex multistep process, including tumor cell migration, intravasation, survival in the circulation, and extravasation. Previously it was shown that tumor cell-induced endothelial necroptosis promotes tumor cell extravasation and metastasis. Here, we identified endothelial TGF-β-activated kinase 1 (TAK1) as a critical regulator of endothelial necroptosis and metastasis. Human and murine endothelial cells lacking TAK1 exhibit higher levels of necroptosis both in vitro and in vivo, and mice with endothelial cell-specific loss of TAK1 are more prone to form metastases. Endothelial RIPK3, a key component of the necroptotic machinery, was upregulated in mice with endothelial TAK1-deficiency, and endothelial knockout of RIPK3 reverted the effects of TAK1-deficiency. Moreover, altered expression levels of TAK1 and RIPK3 in pulmonary endothelial cells of mice bearing primary tumors correlated with increased endothelial necroptosis and metastasis. Together, our data suggest an important protective role for endothelial TAK1 in tumor progression by keeping endothelial necroptosis in check.

Inflamed neutrophils sequestered at entrapped tumor cells via chemotactic confinement promote tumor cell extravasation

Systemic inflammation occurring around the course of tumor progression and treatment are often correlated with adverse oncological outcomes. As such, it is suspected that neutrophils, the first line of defense against infection, may play important roles in linking inflammation and metastatic seeding. To decipher the dynamic roles of inflamed neutrophils during hematogenous dissemination, we employ a multiplexed microfluidic model of the human microvasculature enabling physiologically relevant transport of circulating cells combined with real-time, high spatial resolution observation of heterotypic cell-cell interactions. LPS-stimulated neutrophils (PMNs) and tumor cells (TCs) form heterotypic aggregates under flow, and arrest due to both mechanical trapping and neutrophil-endothelial adhesions. Surprisingly, PMNs are not static following aggregation, but exhibit a confined migration pattern near TC-PMN clusters. We discover that PMNs are chemotactically confined by self-secreted IL-8 and tumor-derived CXCL-1, which are immobilized by the endothelial glycocalyx. This results in significant neutrophil sequestration with arrested tumor cells, leading to the spatial localization of neutrophil-derived IL-8, which also contributes to increasing the extravasation potential of adjacent tumor cells through modulation of the endothelial barrier. Strikingly similar migration patterns and extravasation behaviors were also observed in an in vivo zebrafish model upon PMN-tumor cell coinjection into the embryo vasculature. These insights into the temporal dynamics of intravascular tumor-PMN interactions elucidate the mechanisms through which inflamed neutrophils can exert proextravasation effects at the distant metastatic site.

Abstract 4620: Signaling cascade of metastasis-associated macrophages determines breast cancer distal metastasis

Abstract Macrophages are abundantly found in the tumor microenvironment and enhance malignancy. At distal metastatic sites, our previous studies identified a distinct population of metastasis-associated macrophages (MAMs) that promote tumor cell extravasation, seeding and persistent growth. These macrophages were derived from inflammatory monocytes recruited by CCL2/CCR2 chemokine signaling and directly promote tumour cell extravasation through VEGF production. Our recent studies identified two subsequent signaling pathways that mediate the retention and function of MAMs after recruitment that are critical for tumour cell survival and persistent growth. Specifically, activation of CCR2 signaling turns on CCL3 expression in MAMs. Genetic deletion of CCL3 or its receptor CCR1 in macrophages reduces lung metastasis, and MAM accumulation in tumor-challenged lung. Adoptive transfer of wild type monocytes restores lung metastasis in Ccl3 deficient mice. Mechanistically, Ccr1 deficiency prevents MAM retention in the lung by reducing direct MAM-tumour cell interactions. On the other hand, MAMs turn on cell surface expression of FMS-like tyrosine kinase 1 (FLT1) upon differentiation from monocytes. Using several genetic models of Flt1 deficiency, we show that macrophage specific FLT1 signaling is critical for breast tumor distal metastatic potential. FLT1 specific inhibitory antibody significantly inhibits the metastatic seeding and persistent growth but does not affect the recruitment or retention of MAMs. Furthermore, we identified that FLT1 signaling regulates a set of downstream inflammatory response genes including Colony Stimulating Factor 1 (CSF1) a central regulator of macrophage biology. Using a genetic gain-of-function approach we show that CSF1 mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity in MAMs even when FLT1 is inhibited. Together, our data indicates CCL3/CCR1 and FLT1 signaling pathways are critical in regulating MAMs and promoting breast cancer distal metastasis, and suggests the therapeutic potential of targeting these pathways in treating metastatic disease. Note: This abstract was not presented at the meeting. Citation Format: Bin-Zhi Qian. Signaling cascade of metastasis-associated macrophages determines breast cancer distal metastasis [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 4620. doi:10.1158/1538-7445.AM2017-4620

Death Receptor 6–Mediated Endothelial Necroptosis Drives Metastasis

Abstract Tumor cell–induced endothelial cell necroptosis promotes tumor cell extravasation and 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.

Abstract A13: Macrophage FLT1 mediated inflammatory response determines breast cancer distal metastasis

Abstract Macrophages are abundantly found in the tumor microenvironment and enhance malignancy. At distal metastatic sites, our previous studies identified a distinct population of metastasis associated macrophages (MAMs) that promotes tumor cell extravasation, seeding and persistent growth. These macrophages were derived from circulating inflammatory monocytes recruited by CCL2/CCR2 chemokine signaling and directly promote tumor cell extravasation and metastatic seeding in vivo through VEGF production. Our recent studies identified that MAMs express high levels of cell surface FMS-like tyrosine kinase 1 (FLT1, also known as VEGFR1) after their recruitment. Blockade of FLT1 signaling using specific inhibitory antibodies significantly inhibited the metastatic seeding and persistent growth. Using several genetic models of Flt1 deficiency, we show that macrophage specific FLT1 signaling is critical for breast tumor distal metastatic potential. FLT1 is not expressed by other hematopoietic cells and its inhibition did not affect the recruitment of MAMs, which indicated that specific FLT1 signaling in MAMs are important for their metastasis promoting functions. Indeed, we identified that FLT1 regulates a set of inflammatory response genes including Colony Stimulating Factor 1 (CSF1) a central regulator of macrophage biology. Using a genetic gain-of-function approach we show that CSF1 mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity in MAMs even when FLT1 has been inhibited. Together, our data established a link between inflammation and cancer metastasis and suggested the therapeutic potential of targeting these pathways in treating metastatic disease. Citation Format: Bin-Zhi Qian, Hui Zhang, Jiufeng Li, Eun-Jin Yeo, Neil O. Carragher, Anne R. Bresnick, Richard A. Lang, Jeffrey W. Pollard. Macrophage FLT1 mediated inflammatory response determines breast cancer distal metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A13.

Abstract 1531: Macrophage FLT1 signaling activates an inflammatory response that promotes breast cancer distal metastasis.

Abstract Macrophages are abundantly found in the tumor microenvironment enhance malignancy. At distal metastatic sites a distinct population of metastasis associated macrophages (MAMs) promotes tumor cell extravasation, seeding and persistent growth. Compared with lung resident macrophages, MAMs associated with breast cancer lung metastasis in pre-clinical models express high levels of cell surface FMS-like tyrosine kinase 1 (Flt1). Blockade of FLT1 signaling using specific inhibitory antibodies significantly inhibited the metastatic seeding and persistent growth of both human and murine breast tumor cell. This was confirmed using a genetic model of Flt1 tyrosine kinase domain (TK) ablation. Using bone marrow transplantation techniques, we further showed that this metastasis inhibitory effect was due to the lack of FLT1 signaling in hematopoietic cells. Furthermore, macrophage specific ablation of the tyrosine kinase domain of Flt1 by crossing Flt1 flox/flox mice with macrophage restricted Cre mice (Csf1r-Cre) also significantly inhibited tumor cell metastatic potential. Since Flt1 is not expressed by other hematopoietic cells and FLT1 inhibition did not affect the recruitment of MAMs, it indicated that specific FLT1 signaling in MAMs are important for their metastasis promoting functions. FLT1 regulated genes were identified by comparing gene expression profiles using microarray techniques of FACS sorted MAMs from mice treated with control and anti-FLT1 inhibitory antibody. Bioinformatics analysis showed that inflammatory response genes were highly enriched in these genes. Together, our studies indicated that FLT1 signaling is required for the activation of metastasis associated macrophages and regulates a set of inflammatory genes that promote breast tumor distal metastasis. Citation Format: Bin-Zhi Qian, Richard A. Lang, Jeffrey W. Pollard. Macrophage FLT1 signaling activates an inflammatory response that promotes breast cancer distal metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1531. doi:10.1158/1538-7445.AM2013-1531

The Primacy of β1 Integrin Activation in the Metastatic Cascade

After neoplastic cells leave the primary tumor and circulate, they may extravasate from the vasculature and colonize tissues to form metastases. β1 integrins play diverse roles in tumorigenesis and tumor progression, including extravasation. In blood cells, activation of β1 integrins can be regulated by “inside-out” signals leading to extravasation from the circulation into tissues. However, a role for inside-out β1 activation in tumor cell metastasis is uncertain. Here we show that β1 integrin activation promotes tumor metastasis and that activated β1 integrin may serve as a biomarker of metastatic human melanoma. To determine whether β1 integrin activation can influence tumor cell metastasis, the β1 integrin subunit in melanoma and breast cancer cell lines was stably knocked down with shRNA and replaced with wild-type or constitutively-active β1. When tumor cells expressing constitutively-active β1 integrins were injected intravenously into chick embryos or mice, they demonstrated increased colonization of the liver when compared to cells expressing wild-type β1 integrins. Rescue expression with mutant β1 integrins revealed that tumor cell extravasation and hepatic colonization required extracellular ligand binding to β1 as well as β1 interaction with talin, an intracellular mediator of integrin activation by the Rap1 GTPase. Furthermore, shRNA-mediated knock down of talin reduced hepatic colonization by tumor cells expressing wild-type β1, but not constitutively-active β1. Overexpression in tumor cells of the tumor suppressor, Rap1GAP, inhibited Rap1 and β1 integrin activation as well as hepatic colonization. Using an antibody that detects activated β1 integrin, we found higher levels of activated β1 integrins in human metastatic melanomas compared to primary melanomas, suggesting that activated β1 integrin may serve as a biomarker of invasive tumor cells. Altogether, these studies establish that inside-out activation of β1 integrins promotes tumor cell extravasation and colonization, suggesting diagnostic and therapeutic approaches for targeting of β1 integrin signaling in neoplasia.

Abstract 2967: CCL3 autocrine signaling regulate retention of metastasis associated macrophages and promote breast cancer metastasis

Abstract Macrophages abundantly found in the tumor microenvironment enhance malignancy. At metastatic sites a distinct population of metastasis associated macrophages (MAMs) promotes tumor cell extravasation, seeding and persistent growth. These macrophages are derived from circulating inflammatory monocytes recruited by CCL2/CCR2 chemokine signaling. Our current study identified a distinct gene expression signature of the MAMs in comparison with resident macrophages in lung and spleen. Majority of these genes encodes cell surface proteins and secreted factors which mediate MAM's interaction with the metastatic microenvironment. Interestingly, we identified another chemokine, CCL3, is highly expressed by MAMs in a CCL2/CCR2 dependent manner. This indicates that CCL2 not only regulate MAM recruitment but also its function. RNA interference mediated CCL3 knockdown significantly reduced their potential in promoting tumor cell extravasation in vitro. Anti-CCL3 antibody and genetic ablation of CCL3 significantly reduced tumor cell metastatic potential in vivo. Importantly, MAM also express high level of CCR1 and CCR5, receptors for CCL3, upon differentiation. Anti-CCL3 inhibitory antibody significantly reduced MAM retention in pulmonary metastasis. Together, our data indicates an autocrine signaling via CCL3 regulated by CCL2/CCR2 controls MAM retention and promote breast tumor lung metastasis which identified a novel therapeutic target for treating metastatic breast disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2967. doi:1538-7445.AM2012-2967

Abstract PL03-01: Macrophage diversity promotes tumor progression and metastasis

Abstract The tumor microenvironment represents a complex and changing ecology of many different cell types whose interactions determines the overall malignancy of the tumor. This environment consists of tumor cells harboring oncogenic and tumor suppressor mutations together with normal cells that include resident ones and those recruited from the bone marrow. In this latter class myeloid cells and especially macrophages, the subject of our studies, are particularly well represented. Associative clinical studies have indicated that the abundance of macrophages or overexpression of their signaling pathways is often an indicator of poor prognosis in a wide variety of cancers. These data suggests a pro-tumoral role for tumor-associated macrophages. This hypothesis that macrophages promoted malignancy is supported by a significant number of studies using genetic or pharmacological ablation of macrophages or their signaling pathways. In our studies, we used genetic ablation of macrophages in the model of breast cancer caused by the mammary epithelial expression of the Polyoma Middle T oncoprotein (PyMT). This ablation resulted in a slowing of tumor progression to malignancy and an inhibition of metastasis. We and our collaborators have identified several traits that macrophages confer in the primary tumor that account for their enhancement of malignancy. These are in the promotion of angiogenesis, stimulation of tumor cell invasion, migration and intravasation. At the metastatic site we have also shown that macrophages promote tumor cell extravasation and subsequent growth. Macrophage isolation and characterization from PyMT tumors and their metastatic sites suggest that each of these activities is regulated by a distinct subsets of macrophages that nevertheless express canonical macrophage markers. Each of these macrophage populations have defining cell surface markers and unique transcriptional profiles that suggest individual functions (Condeelis and Pollard, 2006; Qian and Pollard, 2010). Metastasis is the major cause of the failure of therapy and therefore death in cancer patients. Therefore we have focused our recent research on this process. We showed that macrophages are rapidly recruited to tumor cells as they metastasize to the lung. Ablation of these metastasis-associated macrophages (MAMs) using either genetic or pharmacological means results in an inhibition of tumor cell seeding and subsequent growth. Importantly ablation of these macrophages after the tumor cells have become established also inhibited subsequent metastatic growth suggesting that these cells represent therapeutic targets. Using ex vivo imaging methods we showed that a major action of these macrophages was on the promotion of tumor cell extravasation with subsequent effects on viability. This effect of macrophage on tumor cells could be recapitulated in an ex vivo extravasation assay thus enabling the interrogation of specific signaling molecules that may have function in promoting extravasation. To analyze the source of these MAMs, we performed adoptive transfers of the two different populations of monocytes that are defined by the presence or absence of the surface antigen Ly6C. Interestingly there was preferential recruitment of the Ly6C+ population to the metastatic sites whether in an experimental model of metastasis or into spontaneous metastases from PyMT tumors. Tracking of these monocytes showed that they rapidly differentiated into MAMs in the lung. A similar monocytic population (CD14+CD16-) circulating in human peripheral blood was recruited to human breast cancer metastases following adoptive transfer into immunocompromised mice. Furthermore, in mice we found that the Ly6C+ monocytes are preferentially recruited to bone metastases. In contrast to these data, Ly6C negative monocytes are recruited to the spontaneous tumors and to liver metastases there is a reduction in the recruitment of both populations of monocytes. Inhibition of the recruitment of these Ly6C+ monocytes inhibited tumor cell extravasation and thus metastases in bone and lung but not liver. These data suggest that alternative and tissue-specific mechanisms are operative at different metastatic sites. The dynamic acquisition of monocytic populations to primary and metastatic tumors and their differentiation into distinct macrophage populations represents the potential to target aspects of the tumor microenvironment. Such novel therapeutics in combination with conventional chemotherapeutic and/or radiation therapy may provide synergistic benefits to the patient. 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 PL03-01. doi:10.1158/1538-7445.AM2011-PL03-01