Basement Membrane-Rich Organoids with Functional Human Blood Vessels Are Permissive Niches for Human Breast Cancer Metastasis

Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention.     

Imaging Circulating Tumor Cells in Freely Moving Awake Small Animals Using a Miniaturized Intravital Microscope

Metastasis, the cause for 90% of cancer mortality, is a complex and poorly understood process involving the invasion of circulating tumor cells (CTCs) into blood vessels. These cells have potential prognostic value as biomarkers for early metastatic risk. But their rarity and the lack of specificity and sensitivity in measuring them render their interrogation by current techniques very challenging. How and when these cells are circulating in the blood, on their way to potentially give rise to metastasis, is a question that remains largely unanswered. In order to provide an insight into this "black box" using non-invasive imaging, we developed a novel miniature intravital microscopy (mIVM) strategy capable of real-time long-term monitoring of CTCs in awake small animals. We established an experimental 4T1-GL mouse model of metastatic breast cancer, in which tumor cells express both fluorescent and bioluminescent reporter genes to enable both single cell and whole body tumor imaging. Using mIVM, we monitored blood vessels of different diameters in awake mice in an experimental model of metastasis. Using an in-house software algorithm we developed, we demonstrated in vivo CTC enumeration and computation of CTC trajectory and speed. These data represent the first reported use we know of for a miniature mountable intravital microscopy setup for in vivo imaging of CTCs in awake animals.     

Modelling Circulating Tumour Cells for Personalised Survival Prediction in Metastatic Breast Cancer

Ductal carcinoma is one of the most common cancers among women, and the main cause of death is the formation of metastases. The development of metastases is caused by cancer cells that migrate from the primary tumour site (the mammary duct) through the blood vessels and extravasating they initiate metastasis. Here, we propose a multi-compartment model which mimics the dynamics of tumoural cells in the mammary duct, in the circulatory system and in the bone. Through a branching process model, we describe the relation between the survival times and the four markers mainly involved in metastatic breast cancer (EPCAM, CD47, CD44 and MET). In particular, the model takes into account the gene expression profile of circulating tumour cells to predict personalised survival probability. We also include the administration of drugs as bisphosphonates, which reduce the formation of circulating tumour cells and their survival in the blood vessels, in order to analyse the dynamic changes induced by the therapy.We analyse the effects of circulating tumour cells on the progression of the disease providing a quantitative measure of the cell driver mutations needed for invading the bone tissue. Our model allows to design intervention scenarios that alter the patient-specific survival probability by modifying the populations of circulating tumour cells and it could be extended to other cancer metastasis dynamics.     

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.     

Three-dimensional microfluidic tumor–macrophage system for breast cancer cell invasion

The recrudescence of breast cancer can partly be attributed to poor understanding of the early steps and the mechanisms involved in breast cancer metastasis, especially how tumor inflammatory cells including tumor-associated macrophages (TAM) affect invasion process. However, invasion-related biological studies in traditional in vitro assays or in vivo models are challenging due to the arduousness in establishing models that precisely reproduce the tumor invasion environment. To this end, we proposed a juxtaposed dual-layer cell-loaded hydrogels biomimetic microfluidic system and formed monolayer size-selective permeable vascular endothelial barriers besides the dual layer to mimic mammalian blood vessels. We clarified that in this system, TAM promoted the invasion of breast cancer cells, whereas breast cancer cells maintained the phenotype of TAM cells and promoted the differentiation of U937 cells into TAM. It formed a tumor–macrophage bidirectional crosstalk system. This system could be used for drug screening. So finally, through the calculation of the survival rate of breast cancer cells when cocultured with different macrophages under paclitaxel treatment, we analyzed the antagonism of tumor–macrophage bidirectional crosstalk on anticancer drugs.     

Tumor cells caught in the act of invading: their strategy for enhanced cell motility

Invasion of neighboring extracellular matrix tissue, the lymphatic system and blood vessels is a key element of tumor cell metastasis in many epithelial tumors. Understanding the cell motility pathways that contribute to invasion can provide new approaches and targets for anticancer therapy. The recent convergence of technologies for expression profiling and intravital imaging has revealed the identities of some of the genes that contribute to motility and chemotaxis of cancer cells in tumors. In particular, the genes encoding a minimum motility machine are coordinately upregulated in tumor cells collected by an in vivo invasion assay. These results support a "tumor microenvironment invasion model" and provide new target opportunities for cancer therapy.     

Hypoxia-induced metastasis model in embryonic zebrafish

Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent DiI-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average experimental time for this model is 7 d. Our protocol offers a remarkable opportunity to study molecular mechanisms of hypoxia-induced cancer metastasis.     

失巢凋亡及其在肿瘤侵袭、转移中的调控

作为肿瘤转移的屏障, 细胞与邻近细胞或者细胞外基质(Extracellular matrix, ECM)失去联系后将遭受凋亡, 这种细胞死亡方式称为“失巢凋亡”。正常上皮细胞或不具备转移性质的实体瘤细胞从原位脱落进入血液循环后就会引发失巢凋亡, 失巢凋亡的意义在于防止这些脱落的细胞种植并生长于其他不适宜的地方。而肿瘤细胞, 尤其是一些容易发生远距离转移的恶性肿瘤细胞, 具有极强的抗失巢凋亡特性, 便于转移侵袭。研究发现肿瘤细胞能通过多种方式抵抗失巢凋亡, 比如细胞自分泌生长因子或者由邻近细胞旁分泌, 激活促存活信号通路; 细胞改变整合蛋白的表达模式, 使之能够接收新环境的生存信号; 活性氧(Reactive oxygen species, ROS)通过不依赖配体的方式激活生长因子受体, 从而逃逸凋亡; 上皮间质转化(Epithelial-mesenchymal transition, EMT)激活等。这些方式导致细胞存活信号激活和凋亡途径抑制, 最终使肿瘤细胞抗失巢凋亡, 促进转移。文章综述了当前研究的肿瘤转移的关键机制, 这些策略也将成为肿瘤治疗的重要靶点。     

黏着斑激酶与肿瘤微环境

黏着斑激酶（focal adhesion kinase, FAK）是一种胞质非受体酪氨酸激酶。FAK和肿瘤密切相关,在多种癌细胞中高表达,促进癌细胞的发生、生长、存活、增殖、粘附、转移和侵袭以及血管生成等过程。肿瘤微环境包括肿瘤细胞、周围血管、免疫细胞、纤维母细胞、内皮细胞、信号分子和细胞外基质,它对癌症的发展和恶化具有重要作用。肿瘤细胞可以通过分泌细胞外信号影响微环境,使其有利于肿瘤生存和发展|肿瘤微环境中的基质细胞能通过产生趋化因子、基质降解酶和生长因子促进肿瘤侵袭和转移。本文综述肿瘤微环境在癌症发生发展过程中的作用及FAK在肿瘤微环境中的调控作用,为肿瘤疾病的治疗提供新思路。     

Patterns of blood-vessel invasion by mammary tumor cells

Rat mammary tumor TMT-081 was employed as a model for blood vessel invasion because its mode of metastasis resembles that of human tumors. The invasive mechanism was studied with two methods of serial transplantation: transfer of enzymatically dispersed solid tumors, and transfer of buffy coat containing circulating tumor cells. The latter method produced greater invasion of blood vessels, including larger veins and occasionally arteries, perhaps by obviating damage to tumor cells during enzyme treatment. The course of migration was traced by three-dimensional examination in the high voltage electron microscope, as well as the light microscope. Two broad patterns were found for the course of invasion of small and large vessels respectively.     

Breakdown of the endothelial barrier function in tumor cell transmigration

The ability of tumor cells to metastasize is associated with a poor prognosis for cancer. During the process of metastasis, tumor cells circulating in the blood or lymph vessels can adhere to, and potentially transmigrate through, the endothelium and invade the connective tissue. We studied the effectiveness of the endothelium as a barrier against the invasion of 51 tumor cell lines into a three-dimensional collagen matrix. Only nine tumor cell lines showed attenuated invasion in the presence of an endothelial cell monolayer, whereas 17 cell lines became invasive or showed a significantly increased invasion. Endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-β. Expression of the IL-8 and Gro-β receptor, CXCR2, was upregulated in invasive tumor cells. Addition of IL-8 or Gro-β increased tumor cell invasiveness by more than twofold. Tumor cell variants selected for high CXCR2 expression were fourfold more invasive in the presence of an endothelial cell layer, whereas CXCR2 siRNA knock-down cells were fivefold less invasive. We demonstrate that Gro-β and IL-8 secreted by endothelial cells, together with CXCR2 receptor expression on invasive tumor cells, contribute to the breakdown of the endothelial barrier by enhancing tumor cell force generation and cytoskeletal remodeling dynamics.     

Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model

The metastatic invasion of cancer cells from the primary lesion into the adjacent stroma is a key step in cancer progression, and is associated with poor outcome. The principles of cancer invasion have been experimentally addressed in various in vitro models; however, key steps and mechanisms in vivo remain unclear. Here, we establish a modified skin-fold chamber model for orthotopic implantation, growth and invasion of human HT-1080 fibrosarcoma cells, dynamically reconstructed by epifluorescence and multiphoton microscopy. This strategy allows repeated imaging of tumor growth, tumor-induced angiogenesis and invasion, as either individual cells, or collective strands and cell masses that move along collagen-rich extracellular matrix and coopt host tissue including striated muscle strands and lymph vessels. This modified window model will be suited to address mechanisms of cancer invasion and metastasis, and related experimental therapy.     

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.     

Triclosan Potentiates Epithelial-To-Mesenchymal Transition in Anoikis-Resistant Human Lung Cancer Cells

Alteration of cancer cell toward mesenchymal phenotype has been shown to potentiate tumor aggressiveness by increasing cancer cell metastasis. Herein, we report the effect of triclosan, a widely used antibacterial agent found in many daily products, in enhancing the epithelial-to-mesenchymal transition (EMT) in aggressive anoikis resistant human H460 lung cancer cells. EMT has been long known to increase abilities of the cells to increase migration, invasion, and survival in circulating system. The present study reveals that treatment of the cancer cells with triclosan at the physiologically related concentrations significantly increased the colony number of the cancer cells assessed by tumor formation assay. Also, the mesenchymal-like morphology and decrease in cell-to-cell adhesion were observed in triclosan-treated cells. Importantly, western blot analysis revealed that triclosan-treated cells exhibited decreased E-cadherin, while the levels of EMT markers, namely N-cadherin, vimentin, snail and slug were found to be significantly up-regulated. Furthermore, EMT induced by triclosan treatment was accompanied by the activation of focal adhesion kinase/ATP dependent tyrosine kinase (FAK/Akt) and Ras-related C3 botulinum toxin substrate 1 (Rac1), which enhanced the ability of the cells to migrate and invade. In conclusion, we demonstrated for the first time that triclosan may potentiate cancer cells survival in detached condition and motility via the process of EMT. As mentioned capabilities are required for success in metastasis, the present study provides the novel toxicological information and encourages the awareness of triclosan use in cancer patients.     

Cancer cell invasion is enhanced by applied mechanical stimulation

Metastatic cells migrate from the site of the primary tumor, through the stroma, into the blood and lymphatic vessels, finally colonizing various other tissues to form secondary tumors. Numerous studies have been done to identify the stimuli that drive the metastatic cascade. This has led to the identification of multiple biochemical signals that promote metastasis. However, information on the role of mechanical factors in cancer metastasis has been limited to the affect of compliance. Interestingly, the tumor microenvironment is rich in many cell types including highly contractile cells that are responsible for extensive remodeling and production of the dense extracellular matrix surrounding the cancerous tissue. We hypothesize that the mechanical forces produced by remodeling activities of cells in the tumor microenvironment contribute to the invasion efficiency of metastatic cells. We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments. Furthermore, this mechanically enhanced invasion is dependent upon substrate protein composition, and influenced by topography. Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion. We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro. We further propose that in vivo, non-cancerous cells located within the tumor micro-environment may be capable of providing the necessary mechanical stimulus during the remodeling of the extracellular matrix surrounding the tumor.     

Circulation times of prostate cancer and hepatocellular carcinoma cells by in vivo flow cytometry

In metastasis, the cancer cells that travel through the body are capable of establishing new tumors in locations remote from the site of the original disease. To metastasize, a cancer cell must break away from its tumor and invade either the circulatory or lymphatic system, which will carry it to a new location, and establish itself in the new site. Once in the blood stream, the cancer cells now have access to every portion of the body. Here, we have used the "in vivo flow cytometer" to study if there is any relationship between metastatic potential and depletion kinetics of circulating tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labelled cells in vivo. We have improved the counting algorithm and measured the depletion kinetics of cancer cells with different metastatic potential. Interestingly, more invasive PC-3 prostate cancer cells are depleted faster from the circulation than LNCaP cells. In addition, we have measured the depletion kinetics of two related human hepatocellular carcinoma (liver cancer) cell lines, high-metastatic HCCLM3 cells, and low-metastatic HepG2 cells. More than 60% HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.     

The countercurrent principle in invasion and metastasis of cancer cells. Recent insights on the roles of chemokines

Chemokine production by cancer cells constitutes a duality. Leukocyte recruitment under the pressure of chemokines may be beneficial for the host or for the tumor. Here, the emphasis will be on the detrimental effects of chemokines in tumor biology. A decade ago, the countercurrent principle of tumor-derived chemokine and peritumoral protease production was formulated to explain chemokine expression as a selective advantage for specific tumors and as a phenotype of invasive and metastasizing cancer cells. Chemoattracted leukocytes may provide trophic factors and produce invasion and metastasis-promoting proteinases. On the basis of the consensus sequence glutamic acid-leucine-arginine (ELR) preceding the canonical cysteine-any amino acid-cysteine (CXC), ELR-positive CXC chemokines, such as interleukin-8 and granulocyte chemotactic protein-2, are angiogenic and thus instruct the host to feed the tumor and bring the vessels into closer contact with the tumor cells. These mechanisms may enhance lymphogenic and hematogenic metastasis. Recent research and proofs of this countercurrent concept are here reviewed and compared. In addition, we discuss how alterations in chemokine ligand and receptor expression profiles may contribute to tumor growth, invasion, metastasis and immune evasion. These comparisons imply practical consequences for future cancer diagnosis and therapy. The implications include methods to diminish metastasis by inhibiting angiogenic CXC chemokine ligands and receptors, therapeutic combinations of chemokine overexpression with antigenic stimuli and co-treatment with angiostatic chemokines and tumor antigens.     

Macrophages: obligate partners for tumor cell migration, invasion, and metastasis

Macrophages within the tumor microenvironment facilitate angiogenesis and extracellular-matrix breakdown and remodeling and promote tumor cell motility. Recent studies reveal that direct communication between macrophages and tumor cells leads to invasion and egress of tumor cells into the blood vessels (intravasation). Thus, macrophages are at the center of the invasion microenvironment and are an important drug target for cancer therapy.