Secreted Gaussia Luciferase as a Biomarker for Monitoring Tumor Progression and Treatment Response of Systemic Metastases

Background

Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses.

Methodology/Principal Findings

We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R² = 0.84) and urine (R² = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2.

Conclusion/Significance

We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments.     

NM23 and metastasis suppressor genes: update

Metastatic dissemination represents a leading cause of death in cancer patients. Elucidating the mechanisms of the metastatic process is therefore essential to control it. Since 1988, when the NME (NM23) gene was discovered, several genes specifically suppressing the metastatic potential of tumor cells, have been identified. These metastasis suppressor genes, which exhibit a reduced expression in metastatic tumor cells, are defined by their capacity to suppress metastatic dissemination in vivo without inhibiting primary tumor growth when transfected into metastatic cell lines and injected into experimental animals. Their decreased expression in a subset of human tumor cohorts is associated with a high metastatic potential, thus confirming the data obtained in experimental models. Most of these genes affect key signal transduction pathways, including mitogen-activated protein kinases, Rho-GTPases and G-protein-coupled receptors. These signaling categories control cell-cell and cell-matrix interactions, which are important in monitoring adhesion, invasion and migration properties of metastatic tumor cells. Reduced expression of metastasis suppressor genes is most often due to epigenetic mechanisms, suggesting that their re-expression could constitute a new anti-metastatic therapy. In this paper, we review the literature on metastasis suppressor genes, with a particular focus on NM23.     

Cyclooxygenase inhibitors modulate NK activities that control metastatic disease

Cyclooxygenase (COX) inhibitors have demonstrated efficacy in models of human cancer but the relevant mechanisms have not all been elucidated. Both Cox-dependent as well as Cox-independent mechanisms have been implicated. Using a syngeneic model of metastatic breast cancer, we have investigated the effect of Cox inhibitors on NK functions that are critical to the control of metastatic disease. NK recognition of target cells is governed by a balance of activating and inhibiting receptors that bind ligands including MHC class I. We now show that treatment of tumor cells with the nonselective COX-1/COX-2 inhibitor indomethacin or the selective COX-2 inhibitor celecoxib leads to decreased expression of the MHC class I molecules L^d and K^d . Downregulated class I expression is associated with concomitant increased sensitivity to NK cell-mediated lysis. Both COX inhibitors limit tumor metastasis and this therapeutic effect is dependent on NK but not T cell function. Antimetastatic activity is also lost in the absence of interferon- (IFN-). Both COX inhibitors also suppress local tumor growth of subcutaneously implanted mammary tumor cells in immune competent Balb/cByJ mice. This therapeutic activity is lost in the absence of either CD4+ or CD8+ T cells, but is not compromised by the loss of NK activity. Thus, the mechanism of tumor inhibition differs in the context of local versus metastatic disease. Taken together, these findings are consistent with a mechanism not previously described, whereby COX inhibitors may relieve MHC-mediated inhibition of NK cytotoxicity leading to recognition and lysis of metastatic tumor cells.     

A Distinct Macrophage Population Mediates Metastatic Breast Cancer Cell Extravasation,Establishment and Growth

Background

The stromal microenvironment and particularly the macrophage component of primary tumors influence their malignant potential. However, at the metastatic site the role of these cells and their mechanism of actions for establishment and growth of metastases remain largely unknown.

Methodology/Principal Findings

Using animal models of breast cancer metastasis, we show that a population of host macrophages displaying a distinct phenotype is recruited to extravasating pulmonary metastatic cells regardless of species of origin. Ablation of this macrophage population through three independent means (genetic and chemical) showed that these macrophages are required for efficient metastatic seeding and growth. Importantly, even after metastatic growth is established, ablation of this macrophage population inhibited subsequent growth. Furthermore, imaging of intact lungs revealed that macrophages are required for efficient tumor cell extravasation.

Conclusion/Significance

These data indicate a direct enhancement of metastatic growth by macrophages through their effects on tumor cell extravasation, survival and subsequent growth and identifies these cells as a new therapeutic target for treatment of metastatic disease.     

Dissection of a metastatic gene expression signature into distinct components

Background

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.

Results

For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed.

Conclusion

Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.     

Green fluorescent protein (GFP)-expressing tumor model derived from a spontaneous osteosarcoma in a vascular endothelial growth factor (VEGF)-GFP transgenic mouse

Vascular endothelial growth factor (VEGF) mediates tumor angiogenesis, growth, and metastasis. Murine models of metastatic tumors in which green fluorescent protein (GFP) expression is driven by the VEGF promoter can be imaged both intravitally and externally and thus offer many possibilities for real-time studies of tumor angiogenesis, metastasis, and treatment in vivo. In our defined-flora animal facility, an 11-month-old female transgenic mouse with a C3H background (VEGF(P)-GFP/C3H) developed a spontaneous tumor that expressed GFP under the control of VEGF. Necropsy and histopathologic examination revealed an osteosarcoma with lung metastases. Fresh tumor fragments were transplanted successfully into other VEGF(P)-GFP/C3H transgenic mice. During the first five generations, the tumor "take rate" was 100% (25 of 25 animals), with a latent period of 8 days and an average tumor volume of 1500 mm3 at 36 days. Transplanted tumors have maintained their original histopathologic characteristics and metastatic behavior. In addition, the tumor grows in wild-type C3H mice with an 83% take rate (10 of 12 animals) and as monolayer cells in vitro. GFP was expressed strongly in tumor tissue, lung metastatic foci, and cultured tumor cells. Real-time growth of tumors grown in dorsal skin chambers in C3H mice could be visualized using GFP fluorescence. In addition, GFP fluorescence of metastatic lesions in lungs of C3H mice was clearly visible by multiphoton laser scanning microscopy. This in vitro and in vivo transplantable and metastatic osteosarcoma (Os-P0107) is an attractive model for further study of tumor pathophysiology and treatment efficiency affecting VEGF expression.     

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.Download video file.^{(52M, mov)}     

Role of platelets and breast cancer stem cells in metastasis

The high mortality rate of breast cancer is mainly caused by the metastatic ability of cancer cells, resistance to chemotherapy and radiotherapy, and tumor regression capacity. In recent years, it has been shown that the presence of breast cancer stem cells is closely associated with the migration and metastatic ability of cancer cells, as well as with their resistance to chemotherapy and radiotherapy. The tumor microenvironment is one of the main molecular factors involved in cancer and metastatic processes development, in this sense it is interesting to study the role of platelets, one of the main communicator cells in the human body which are activated by the signals they receive from the microenvironment and can generate more than one response. Platelets can ingest and release RNA, proteins, cytokines and growth factors. After the platelets interact with the tumor microenvironment, they are called "tumor-educated platelets." Tumor-educated platelets transport material from the tumor microenvironment to sites adjacent to the tumor, thus helping to create microenvironments conducive for the development of primary and metastatic tumors. It has been observed that the clone capable of carrying out the metastatic process is a cancer cell with stem cell characteristics. Cancer stem cells go through a series of processes, including epithelial-mesenchymal transition, intravasation into blood vessels, movement through blood vessels, extravasation at the site of the establishment of a metastatic focus, and site colonization. Tumor-educated platelets support all these processes.     

A Novel 3-D Mineralized Tumor Model to Study Breast Cancer Bone Metastasis

Background

Metastatic bone disease is a frequent cause of morbidity in patients with advanced breast cancer, but the role of the bone mineral hydroxyapatite (HA) in this process remains unclear. We have developed a novel mineralized 3-D tumor model and have employed this culture system to systematically investigate the pro-metastatic role of HA under physiologically relevant conditions in vitro.

Methodology/Principal Findings

MDA-MB231 breast cancer cells were cultured within non-mineralized or mineralized polymeric scaffolds fabricated by a gas foaming-particulate leaching technique. Tumor cell adhesion, proliferation, and secretion of pro-osteoclastic interleukin-8 (IL-8) was increased in mineralized tumor models as compared to non-mineralized tumor models, and IL-8 secretion was more pronounced for bone-specific MDA-MB231 subpopulations relative to lung-specific breast cancer cells. These differences were pathologically significant as conditioned media collected from mineralized tumor models promoted osteoclastogenesis in an IL-8 dependent manner. Finally, drug testing and signaling studies with transforming growth factor beta (TGFβ) confirmed the clinical relevance of our culture system and revealed that breast cancer cell behavior is broadly affected by HA.

Conclusions/Significance

Our results indicate that HA promotes features associated with the neoplastic and metastatic growth of breast carcinoma cells in bone and that IL-8 may play an important role in this process. The developed mineralized tumor models may help to reveal the underlying cellular and molecular mechanisms that may ultimately enable more efficacious therapy of patients with advanced breast cancer.     

间质-上皮转化在肿瘤转移中的作用

肿瘤转移是一个多步骤、多因素参与的复杂过程,是目前临床上绝大多数肿瘤患者的致死因素.上皮-间质转化(epithelial-mesenchymal transition, EMT)过程已被证实可促使肿瘤细胞发生转移.近年来许多研究表明,间质-上皮转化(mesenchymal-epithelial transition, MET)即EMT的逆过程,与肿瘤也密切相关,特别是肿瘤转移即形成继发性的肿瘤转移灶.深入研究肿瘤MET有望为肿瘤转移的预防和诊治提供新思路.     

Metastatic tumor cells – genotypes and phenotypes

Background

Metastasis is the primary cause of mortality in cancer patients. Therefore, elucidating the genetics and epigenetics of metastatic tumor cells and the mechanisms by which tumor cells acquire metastatic properties constitute significant challenges in cancer research.

Objective

To summarize the current understandings of the specific genotype and phenotype of the metastatic tumor cells.

Method and Result

In-depth genetic analysis of tumor cells, especially with advances in the next-generation sequencing, have revealed insights of the genotypes of metastatic tumor cells. Also, studies have shown that the cancer stem cell (CSC) and epithelial to mesenchymal transition (EMT) phenotypes are associated with the metastatic cascade.

Conclusion

In this review, we will discuss recent advances in the field by focusing on the genomic instability and phenotypic dynamics of metastatic tumor cells.

     

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.     

Early origin of cancer metastases: Dissemination and evolution of premalignant cells

Metastasis is the main cause of death by cancer. Hence, establishing predictive markers constitutes a major clinical objective. The capacity for a tumor cell to migrate and survive from a primary tumor is often described as the ultimate step of Darwinian selection. These metastatic cells are believed to emerge from a subpopulation of cells present in a primary tumor. In line with this hypothesis, various gene "signatures" associated with poor prognosis and/or with tumors displaying high metastatic potential have been promoted. However, over the last few years, a growing body of evidence supports the idea that metastatic cells disseminate early from the primordial tumor and evolve independently of it. Herein, we propose to review to the data favoring this alternative model and discuss the interplay between metastatic mechanisms and failsafe mechanism pathways.     

Invasion of human embryonic fibroblast cell aggregates by rat tumor cells of different metastatic capacities

Aggregates of normal human Wi38 cells are used as a three dimensional substrate to test in vitro the behavior of rat tumor cells which exhibit different invasive and metastatic capabilities in vivo. The invasive but non metastatic tumor cells colonize the Wi38 cell aggregates, invade and destroy them within three days. The non invasive but highly metastatic tumor cells settle in a limited number on the aggregates but show no further activities. Co-cultivation of these tumor cells with cell suspension of single Wi38 cells under aggregation conditions does not hinder the Wi38 cells in forming aggregates. The results show that the invasive process in the metastatic cascade needs more specific reaction partners and host environment than local tumor growth. The conditions of the first process cannot be mimicked by a simple model.     

Interaction of tumor cells and immune system in the metastatic process

The metastatic process is rather complicated and relatively inefficient. Millions of tumor cells are constantly shedding from the primary tumor into the blood stream, but very few of them are able to form metastatic tumors in the different organs or tissues of the host. It is widely accepted that metastatic cells have to possess a complex array of various properties that allow them to complete the metastatic cascade. The realization of the metastatic potential largely depends on the ability of tumor cells to evade host defense mechanisms. The potential role of specific and nonspecific immune mechanisms in the control of metastatic spread and growth is the subject of the present review. A better understanding of the mechanisms of antimetastatic defense is of prime importance for development of efficient immunotherapeutic methods for the treatment and eradication of disseminated tumor metastases.     

Arresting supporters: targeting neutrophils in metastasis

It is becoming increasingly clear that leukocytes dynamically regulate cancer progression and metastasis, and among leukocytes, granulocytic cells abundantly accumulate in metastatic organs; however, their function in metastasis remains controversial. In a recent report in Nature, Wculek and Malanchi clarify the role of mature neutrophils as mediators of metastatic initiation and provide a targeted approach to prevent the pro-metastatic activity of neutrophils in breast cancer models.Metastasis remains the primary cause of death in solid tumors. Metastatic tumors often respond poorly to standard therapies, underscoring the need for new and effective drugs targeting metastasis¹. Several factors are thought to determine the survival and growth of disseminated cancer cells in a distant microenvironment. These include cell-autonomous processes such as tumor-reinitiating capacity at the metastatic site and cell-extrinsic interactions with non-cancer cells in the tumor microenvironment.Research over the past decades has demonstrated that leukocytes dynamically regulate cancer progression and metastasis². Some of these paracrine interactions are tumor suppressive (NK or CD8⁺ T cells), while many others (macrophage subsets or platelets) support metastatic progression³. Among leukocytes, neutrophils represent the most abundant class of circulating immune cells. Although their role as the first responders in infection is well characterized, their function in metastasis is poorly understood and remains controversial. Some studies have demonstrated a tumor-promoting effect of neutrophils during metastatic progression^4,5,6, whereas others have found a contrasting, anti-tumor function of these cells⁷. Other close cousins of neutrophils, granulocytic myeloid-derived suppressor cells (G-MDSCs) and Ly6G⁺Ly6C⁺ granulocytes, have been shown to promote metastatic progression^8,9,10. It is currently unclear whether subpopulations of granulocytic cells with such distinct functions coexist in metastatic tissues, whether they are localized differentially within the metastatic organ or regulated temporally in metastasis. In a recent paper in Nature, Wculek and Malanchi¹¹ clarified some of these open questions and defined the role of neutrophils as mediators of metastatic initiation.In line with previous findings⁶, Wculek and Malanchi confirmed that CD11b⁺Ly6G⁺ neutrophils accumulated in the pre-metastatic lung before the arrival of tumor cells in the genetically engineered MMTV-PyMT mammary cancer mouse model, and the number of these neutrophils subsequently increased with metastatic progression¹¹. Since granulocyte-colony stimulating factor (G-CSF) regulates neutrophil production and mobilization from the bone marrow, neutrophil accumulation and lung metastasis were expectedly impaired in Gcsf-null mice. Surprisingly, G-CSF deficiency in MMTV-PyMT tumor cells did not alter either lung neutrophil accumulation or metastasis, although these tumor cells are known to secrete G-CSF¹⁰. However, lung metastasis was reduced when neutrophils were depleted in mice that were genetically engineered with an inducible, neutrophil-targeted diphtheria toxin. As a complementary strategy, the authors showed that depletion of neutrophils using anti-Ly6G blocking antibody in the pre-metastatic phase decreased spontaneous metastasis. Using labeled MMTV-PyMT⁺ tumor cells, the authors showed reduced lung colonization of these tumor cells upon neutrophil depletion and further delineated that the neutrophils support metastatic initiation. One of the strengths of this study lies in the demonstration of the role of neutrophils in metastasis using multiple experimental strategies and models.One question that arises is how neutrophils support metastatic initiation. Using the MMTV-PyMT model, the authors showed that neutrophil-derived signals promote metastatic initiation by increasing the number of metastatic initiating cancer cells (MICs). Among the neutrophil-derived factors, the authors identified high levels of leukotrienes (LTs). LTs are eicosanoids that belong to a family of molecules that are derived from arachidonic acid. LTs mediate inflammatory processes and increase vasodilation and vascular permeability¹². Interestingly, LT stimulation of mouse MMTV-PyMT⁺ breast cancer cells enhanced tumor initiation potential of these cancer cells both in vitro and in vivo. In addition, the authors showed that the LT receptors (LTRs; BLT2 and CysLT2) are enriched among the MICs in mouse and human breast cancer cell lines. LTs are products of the arachidonate 5-lipoxygenase (Alox5) enzyme. Therefore, to address the role of the neutrophil Alox5-LT pathway in metastasis, the authors generated bone marrow chimeric mice using the Alox5-null mice, implanted MMTV-PyMT mammary cancer cells and quantified lung metastasis. Alox5 inhibition decreased spontaneous lung metastasis.LTs are important mediators of inflammatory and allergic responses¹². As such, Alox5 inhibitors such as Zileuton (Zil) are in use for treating asthma. Using Zil, this study revealed a therapeutic strategy to block the pro-metastatic activity of neutrophils in multiple mouse models of breast cancer. The authors showed that lung metastasis was reduced with Zil treatment; however, the primary tumor load or lung neutrophil accumulation remained unaffected. It needs to be explored how LT inhibition selectively affects pro-metastatic neutrophils and whether there are any side effects of long-term LT inhibition in preclinical cancer models. From a translational aspect, combination therapy with standard care will further determine the efficacy of this asthma drug as an anti-metastatic agent in breast cancer.A corollary question is how LTs produced by neutrophils benefit the MICs. The authors showed that LT treatment increased MIC proliferation through ERK/MAPK activation. Interestingly, activation of the MAPK pathway in cancer cells is a common feature of several paracrine inputs from granulocytic cells (Figure 1). Granulocytic myeloid derived suppressor cells (G-MDSC) recruited by CXCL1/2 chemokines secrete pro-survival, S100A8/9 factors that activate MAPK signaling in metastatic cancer cells⁹. Metastatic tumors secrete G-CSF that mobilizes Ly6G⁺Ly6C⁺ granulocytes to the lungs¹⁰. Ly6G⁺Ly6C⁺ cells produce Bv8, a small protein belonging to the prokineticin family, which binds to cancer cell Bv8/prokineticin receptor and activates the MAPK signaling pathway, thereby promoting cancer cell migration. Therefore, different granulocyte-derived factors ranging from lipid eicosanoids to Bv8 and S100A8/9 proteins trigger MAPK pathway activation in metastatic cancer cells, which subsequently promotes their proliferation, migration and survival in metastatic organs (Figure 1).Open in a separate windowFigure 1Diverse paracrine inputs from granulocytic cells activate the MAPK signaling pathway in cancer cells, which increases their metastatic fitness. Drugs that inhibit these paracrine inputs and reduce metastasis in mouse models are also shown.Based on these encouraging experimental results, it is necessary to evaluate the clinical importance of these findings. In this regard, Wculek and Malanchi showed that LTR expression was detected in a cohort of human breast primary and lymph node metastasis. Further investigation is warranted to explore how the neutrophil paracrine LT-LTR-MAPK pathway might be linked to lung metastasis and survival in breast cancer patients. Validation of these experimental findings from mouse models in patient samples would pave the way towards clinical translation of these important findings. In summary, the study by Wculek and Malanchi addresses the functional contribution of a key player, neutrophils, in the metastatic microenvironment, and provides a targeted approach to prevent pro-metastatic activity of these cells in breast cancer models.     

The tumor microenvironment: An irreplaceable element of tumor budding and epithelial-mesenchymal transition-mediated cancer metastasis

Tumor budding occurs at the invasive front of cancer; the tumor cells involved have metastatic and stemness features, indicating a poor prognosis. Tumor budding is partly responsible for cancer metastasis, and its initiation is based on the epithelial-mesenchymal transition (EMT) process. The EMT process involves the conversion of epithelial cells into migratory and invasive cells, and is a profound event in tumorigenesis. The EMT, associated with the formation of cancer stem cells (CSCs) and resistance to therapy, results from a combination of gene mutation, epigenetic regulation, and microenvironmental control. Tumor budding can be taken to represent the EMT in vivo. The EMT process is under the influence of the tumor microenvironment as well as tumor cells themselves. Here, we demonstrate that the tumor microenvironment dominates EMT development and impacts cancer metastasis, as well as promotes CSC formation and mediates drug resistance. In this review, we mainly discuss components of the microenvironment, such as the extracellular matrix (ECM), inflammatory cytokines, metabolic products, and hypoxia, that are involved in and impact on the acquisition of tumor-cell motility and dissemination, the EMT, metastatic tumor-cell formation, tumor budding and CSCs, and cancer metastasis, including subsequent chemo-resistance. From our point of view, the tumor microenvironment now constitutes a promising target for cancer therapy.     

IL-6 promotes head and neck tumor metastasis by inducing epithelial-mesenchymal transition via the JAK-STAT3-SNAIL signaling pathway

Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions and cell polarity, resulting in the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process in head and neck cancers are poorly understood. Increasing evidence suggests that tumor microenvironment plays an important role in promoting EMT in tumor cells. We have previously shown that head and neck tumors exhibit significantly higher Bcl-2 expression in tumor-associated endothelial cells and overexpression of Bcl-2 alone in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a severe combined immunodeficient (SCID) mouse model. In this study, we show that endothelial cells expressing Bcl-2 (EC-Bcl-2), when cocultured with head and neck tumor cells (CAL27), significantly enhance EMT-related changes in tumor cells predominantly by the secretion of IL-6. Treatment with recombinant IL-6 or stable IL-6 overexpression in CAL27 cells or immortalized oral epithelial cells (IOE) significantly induced the expression of mesenchymal marker, vimentin, while repressing E-cadherin expression via the JAK/STAT3/Snail signaling pathway. These EMT-related changes were further associated with enhanced tumor and IOE cell scattering and motility. STAT3 knockdown significantly reversed IL-6-mediated tumor and IOE cell motility by inhibiting FAK activation. Furthermore, tumor cells overexpressing IL-6 showed marked increase in lymph node and lung metastasis in a SCID mouse xenograft model. Taken together, these results show a novel function for IL-6 in mediating EMT in head and neck tumor cells and increasing their metastatic potential.     

Receptor for advanced glycation end products (RAGE) functions as receptor for specific sulfated glycosaminoglycans, and anti-RAGE antibody or sulfated glycosaminoglycans delivered in vivo inhibit pulmonary metastasis of tumor cells

Altered expression of chondroitin sulfate (CS) and heparan sulfate (HS) at the surfaces of tumor cells plays a key role in malignant transformation and tumor metastasis. Previously we demonstrated that a Lewis lung carcinoma (LLC)-derived tumor cell line with high metastatic potential had a higher proportion of E-disaccharide units, GlcUA-GalNAc(4,6-O-disulfate), in CS chains than low metastatic LLC cells and that such CS chains are involved in the metastatic process. The metastasis was markedly inhibited by the pre-administration of CS-E from squid cartilage rich in E units or by preincubation with a phage display antibody specific for CS-E. However, the molecular mechanism of the inhibition remains to be investigated. In this study the receptor molecule for CS chains containing E-disaccharides expressed on LLC cells was revealed to be receptor for advanced glycation end products (RAGE), which is a member of the immunoglobulin superfamily predominantly expressed in the lung. Interestingly, RAGE bound strongly to not only E-disaccharide, but also HS-expressing LLC cells. Furthermore, the colonization of the lungs by LLC cells was effectively inhibited by the blocking of CS or HS chains at the tumor cell surface with an anti-RAGE antibody through intravenous injections in a dose-dependent manner. These results provide the clear evidence that RAGE is at least one of the critical receptors for CS and HS chains expressed at the tumor cell surface and involved in experimental lung metastasis and that CS/HS and RAGE are potential molecular targets in the treatment of pulmonary metastasis.