The clustering status of detached gastric cancer cells inhibits anoikis-induced ferroptosis to promote metastatic colonization.

BACKGROUND AND PURPOSE: Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation. Its role in cancer metastasis remains unclear. In this study, we aimed to investigate the potential involvement of ferroptosis in gastric cancer (GC) metastasis. METHODS: GC cells (AGS, MKN45, HGC27) were used to explore the role of ferroptosis in single and clustered cells with extracellular matrix (ECM) detachment in vitro. We overexpressed glutathione peroxidase 4 (GPX4) to inhibit ferroptosis and assessed the changes in cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Then tumor tissues from 54 GC patients with and without lymphatic metastasis were collected for immunohistochemical staining to investigate the expression of ferroptosis and EMT markers. Finally, Kaplan-Meier survival curves were used to investigate the relationship between overall survival and expression of GPX4 in 178 GC patients. RESULTS: Detached single cells had lower viability than adherent cells, but cell clustering improved their survival under matrix-detached conditions. Detached single cells exhibited an induction of iron-dependent reactive oxygen species (ROS) accumulation, glutathione (GSH) depletion, lipid peroxidation, upregulation of ACSL4, TFRC and HO-1, increased iron levels, and changes in mitochondrial morphology. Opposite effects were observed in detached clustered cells, including the upregulation of the ferroptosis suppressors GPX4 and SLC7A11. Overexpression of GPX4 inhibited ferroptosis and promoted GC cell proliferation, migration, invasion, and EMT. Immunohistochemical analysis of tumor tissues from GC patients indicated that lymphatic metastasis was associated with higher potential for ferroptosis inhibition and EMT induction. Finally, Kaplan-Meier survival curves demonstrated a significant decrease in overall survival among GC patients with high GPX4 expression. CONCLUSIONS: Our study provides the first evidence that inhibition of ferroptosis is a crucial mechanism promoting GC metastasis. GPX4 may be a valuable prognostic factor for GC patients. These findings suggest that targeting ferroptosis inhibition may be a promising strategy for GC patients with metastatic potential. Trial registration The ethical approval code of this study in Institutional Review Board of Peking Union Medical College Hospital is No: K1447.

An emerging paradigm of CXCL12 involvement in the metastatic cascade.

The chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF1), has emerged as a pivotal regulator in the intricate molecular networks driving cancer progression. As an influential factor in the tumor microenvironment, CXCL12 plays a multifaceted role that spans beyond its traditional role as a chemokine inducing invasion and metastasis. Indeed, CXCL12 has been assigned functions related to epithelial-to-mesenchymal transition, cancer cell stemness, angiogenesis, and immunosuppression, all of which are currently viewed as specialized biological programs contributing to the "metastatic cascade" among other cancer hallmarks. Its interaction with its cognate receptor, CXCR4, initiates a cascade of events that not only shapes the metastatic potential of tumor cells but also defines the niches within the secondary organs that support metastatic colonization. Given the profound implications of CXCL12 in the metastatic cascade, understanding its mechanistic underpinnings is of paramount importance for the targeted elimination of rate-limiting steps in the metastatic process. This review aims to provide a comprehensive overview of the current knowledge surrounding the role of CXCL12 in cancer metastasis, especially its molecular interactions rationalizing its potential as a therapeutic target.

[The Role and Significance of Hepatic Environmental Cells in Tumor Metastatic Colonization to Liver].

Metastasis, a main cause of death in tumor patients, is a complicated process that involves multiple steps, presenting a major clinical challenge. Tumor cells break the physical boundaries of a primary tumor, intravasate into the lumina of blood vessels, travel around through blood circulation, extravasate into distant organs, colonize the host organs, and eventually develop into the foci of metastatic cancer. The metastasis of tumor cells exhibits organ-tropism, i.e., tumor cells preferentially spread to specific organs. Liver is a common site for metastasis. The pattern of metastasis in uveal melanoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma shows organ-tropism for liver. The anatomical structure of liver determines its hemodynamic characteristics, e.g., low pressure and slow blood flow, which tend to facilitate the stasis and colonization of tumor cells in the liver. Besides the hemodynamic features, the metastatic colonization of liver depends largely on the interaction between tumor cells and the hepatic microenvironment (especially liver-resident cellular components). Resident cells of the hepatic microenvironment include hepatocytes, liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), Kupffer cells (KCs), etc. Herein, we discussed the role and significance of liver-resident cells in the metastatic colonization of tumor in the liver.

SEC22B inhibition attenuates colorectal cancer aggressiveness and autophagic flux under unfavorable environment.

Autophagy has bidirectional functions in cancer by facilitating cell survival and death in a context-dependent manner. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a large family of proteins essential for numerous biological processes, including autophagy; nevertheless, their potential function in cancer malignancy remains unclear. Here, we explored the gene expression patterns of SNAREs in tissues of patients with colorectal cancer (CRC) and discovered that SEC22B expression, a vesicle SNARE, was higher in tumor tissues than in normal tissues, with a more significant increase in metastatic tissues. Interestingly, SEC22B knockdown dramatically decreased CRC cell survival and growth, especially under stressful conditions, such as hypoxia and serum starvation, and decreased the number of stress-induced autophagic vacuoles. Moreover, SEC22B knockdown successfully attenuated liver metastasis in a CRC cell xenograft mouse model, with histological signs of decreased autophagic flux and proliferation within cancer cells. Together, this study posits that SEC22B plays a crucial role in enhancing the aggressiveness of CRC cells, suggesting that SEC22B might be an attractive therapeutic target for CRC.

Distinct tumor architectures for metastatic colonization of the brain.

Brain metastasis is a dismal cancer complication, hinging on the initial survival and outgrowth of disseminated cancer cells. To understand these crucial early stages of colonization, we investigated two prevalent sources of cerebral relapse, triple-negative (TNBC) and HER2+ breast cancer (HER2BC). We show that these tumor types colonize the brain aggressively, yet with distinct tumor architectures, stromal interfaces, and autocrine growth programs. TNBC forms perivascular sheaths with diffusive contact with astrocytes and microglia. In contrast, HER2BC forms compact spheroids prompted by autonomous extracellular matrix components and segregating stromal cells to their periphery. Single-cell transcriptomic dissection reveals canonical Alzheimer's disease-associated microglia (DAM) responses. Differential engagement of tumor-DAM signaling through the receptor AXL suggests specific pro-metastatic functions of the tumor architecture in both TNBC perivascular and HER2BC spheroidal colonies. The distinct spatial features of these two highly efficient modes of brain colonization have relevance for leveraging the stroma to treat brain metastasis.

Immune surveillance of brain metastatic cancer cells is mediated by IFITM1.

Brain metastasis, most commonly originating from lung cancer, increases cancer morbidity and mortality. Although metastatic colonization is the rate-limiting and most complex step of the metastatic cascade, the underlying mechanisms are poorly understood. Here, in vivo genome-wide CRISPR-Cas9 screening revealed that loss of interferon-induced transmembrane protein 1 (IFITM1) promotes brain colonization of human lung cancer cells. Incipient brain metastatic cancer cells with high expression of IFITM1 secrete microglia-activating complement component 3 and enhance the cytolytic activity of CD8+ T cells by increasing the expression and membrane localization of major histocompatibility complex class I. After activation, microglia (of the innate immune system) and cytotoxic CD8+ T lymphocytes (of the adaptive immune system) were found to jointly eliminate cancer cells by releasing interferon-gamma and inducing phagocytosis and T-cell-mediated killing. In human cancer clinical trials, immune checkpoint blockade therapy response was significantly correlated with IFITM1 expression, and IFITM1 enhanced the brain metastasis suppression efficacy of PD-1 blockade in mice. Our results exemplify a novel mechanism through which metastatic cancer cells overcome the innate and adaptive immune responses to colonize the brain, and suggest that a combination therapy increasing IFITM1 expression in metastatic cells with PD-1 blockade may be a promising strategy to reduce metastasis.

Arginase-1 inhibition reduces migration ability and metastatic colonization of colon cancer cells.

BACKGROUND: Arginase-1 (ARG1), a urea cycle-related enzyme, catalyzes the hydrolysis of arginine to urea and ornithine, which regulates the proliferation, differentiation, and function of various cells. However, it is unclear whether ARG1 controls the progression and malignant alterations of colon cancer. METHODS: We established metastatic colonization mouse model and ARG1 overexpressing murine colon cancer CT26 cells to investigate whether activation of ARG1 was related to malignancy of colon cancer cells in vivo. Living cell numbers and migration ability of CT26 cells were evaluated in the presence of ARG inhibitor in vitro. RESULTS: Inhibition of arginase activity significantly suppressed the proliferation and migration ability of CT26 murine colon cancer cells in vitro. Overexpression of ARG1 in CT26 cells reduced intracellular L-arginine levels, enhanced cell migration, and promoted epithelial-mesenchymal transition. Metastatic colonization of CT26 cells in lung and liver tissues was significantly augmented by ARG1 overexpression in vivo. ARG1 gene expression was higher in the tumor tissues of liver metastasis than those of primary tumor, and arginase inhibition suppressed the migration ability of HCT116 human colon cancer cells. CONCLUSION: Activation of ARG1 is related to the migration ability and metastatic colonization of colon cancer cells, and blockade of this process may be a novel strategy for controlling cancer malignancy.

Exosomal microRNA-4535 of Melanoma Stem Cells Promotes Metastasis by Inhibiting Autophagy Pathway.

High mortality rate and poor survival in melanoma are associated with efficient metastatic colonization. The underlying mechanisms remain elusive. Elucidating the role of exosomes in mediating the interactions between cancer cells and the metastatic microenvironment has been focused on cancer cell derived exosomes in modulating the functions of stromal cells. Whether cancer stem cells (CSCs) can modify the metastatic properties of non-CSC cells, and whether exosomal crosstalk plays a role have not been demonstrated prior to this report. In this study, a paired M14 melanoma derivative cell line, i.e., melanoma parental cell (MPC) and its CSC derivative cell line melanoma stem cell (MSC) were employed. We demonstrated that exosomal crosstalk betwen MSCs and non-CSC MPCs is a new mechanism that underlies melanoma metastasis. Low metastatic melanoma cells (MPCs) can acquire the "metastatic power" from highly metastatic melanoma CSCs (MSCs). We illustrated an uncharacterized microRNA, miR-4535 in mediating such exosomal crosstalk. MSCs deliver its exosomal miR-4535 to the targeted MPCs. Upon entering MPCs, miR-4535 augments metastatic colonization of MPCs by inactivating the autophagy pathway.

Metastasis Model to Test the Role of Notch Signaling in Prostate Cancer.

Distant metastasis is the main cause of death in prostate cancer patients. Notch signaling plays an important role in driving prostate cancer aggressiveness and metastasis. In this chapter, we describe a protocol to measure prostate cancer metastatic colonization, incidences of metastasis, accurately quantify the burden of metastasis, and test the role of NOTCH1 receptor on prostate cancer metastatic colonization and homing to distant sites. The metastasis model presented here is established by intracardiac injection of control human prostate cancer cells and NOTCH1 downregulated cells. The cells are engineered to express both red fluorescent protein (RFP) and luciferase. In this model, whole body bioluminescence imaging, high-resolution, and quantitative fluorescence imaging are utilized for quantitative assessment of metastatic colonization and metastasis burden. Further, histopathology analyses of diverse metastatic organs are performed. This model is a powerful and versatile tool to investigate the mechanisms underlying the function of NOTCH receptors in metastatic colonization in prostate cancer.

Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer.

Tumor-resident intracellular microbiota is an emerging tumor component that has been documented for a variety of cancer types with unclear biological functions. Here, we explored the functional significance of these intratumor bacteria, primarily using a murine spontaneous breast-tumor model MMTV-PyMT. We found that depletion of intratumor bacteria significantly reduced lung metastasis without affecting primary tumor growth. During metastatic colonization, intratumor bacteria carried by circulating tumor cells promoted host-cell survival by enhancing resistance to fluid shear stress by reorganizing actin cytoskeleton. We further showed that intratumor administration of selected bacteria strains isolated from tumor-resident microbiota promoted metastasis in two murine tumor models with significantly different levels of metastasis potential. Our findings suggest that tumor-resident microbiota, albeit at low biomass, play an important role in promoting cancer metastasis, intervention of which might therefore be worth exploring for advancing oncology care.

Modelling metastatic colonization of cholangiocarcinoma organoids in decellularized lung and lymph nodes.

Cholangiocarcinoma (CCA) is a type of liver cancer with an aggressive phenotype and dismal outcome in patients. The metastasis of CCA cancer cells to distant organs, commonly lung and lymph nodes, drastically reduces overall survival. However, mechanistic insight how CCA invades these metastatic sites is still lacking. This is partly because currently available models fail to mimic the complexity of tissue-specific environments for metastatic CCA. To create an in vitro model in which interactions between epithelial tumor cells and their surrounding extracellular matrix (ECM) can be studied in a metastatic setting, we combined patient-derived CCA organoids (CCAOs) (n=3) with decellularized human lung (n=3) and decellularized human lymph node (n=13). Decellularization resulted in removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin. Proteomic analyses showed a tissue-specific ECM protein signature reflecting tissue functioning aspects. The macro and micro-scale mechanical properties, as determined by rheology and micro-indentation, revealed the local heterogeneity of the ECM. When growing CCAOs in decellularized lung and lymph nodes genes related to metastatic processes, including epithelial-to-mesenchymal transition and cancer stem cell plasticity, were significantly influenced by the ECM in an organ-specific manner. Furthermore, CCAOs exhibit significant differences in migration and proliferation dynamics dependent on the original patient tumor and donor of the target organ. In conclusion, CCA metastatic outgrowth is dictated both by the tumor itself as well as by the ECM of the target organ. Convergence of CCAOs with the ECM of its metastatic organs provide a new platform for mechanistic study of cancer metastasis.

The EMT transcription factor ZEB1 blocks osteoblastic differentiation in bone development and osteosarcoma.

Osteosarcoma is an often-fatal mesenchyme-derived malignancy in children and young adults. Overexpression of EMT-transcription factors (EMT-TFs) has been associated with poor clinical outcome. Here, we demonstrated that the EMT-TF ZEB1 is able to block osteoblastic differentiation in normal bone development as well as in osteosarcoma cells. Consequently, overexpression of ZEB1 in osteosarcoma characterizes poorly differentiated, highly metastatic subgroups and its depletion induces differentiation of osteosarcoma cells. Overexpression of ZEB1 in osteosarcoma is frequently associated with silencing of the imprinted DLK-DIO3 locus, which encodes for microRNAs targeting ZEB1. Epigenetic reactivation of this locus in osteosarcoma cells reduces ZEB1 expression, induces differentiation, and sensitizes to standard treatment, thus indicating therapeutic options for ZEB1-driven osteosarcomas. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Advances in the study of cancer metastasis and calcium signaling as potential therapeutic targets.

Metastasis is still the primary cause of cancer-related mortality. However, the underlying mechanisms of cancer metastasis are not yet fully understood. Currently, the epithelial-mesenchymal transition, metabolic remodeling, cancer cell intercommunication and the tumor microenvironment including diverse stromal cells, are reported to affect the metastatic process of cancer cells. Calcium ions (Ca2+) are ubiquitous second messengers that manipulate cancer metastasis by affecting signaling pathways. Diverse transporter/pump/channel-mediated Ca2+ currents form Ca2+ oscillations that can be decoded by Ca2+-binding proteins, which are promising prognostic biomarkers and therapeutic targets of cancer metastasis. This paper presents a review of the advances in research on the mechanisms underlying cancer metastasis and the roles of Ca2+-related signals in these events.

Long non-coding RNA TMPO-AS1 promotes tumor progression via sponging miR-140-5p in breast cancer.

Long non-coding RNAs (lncRNAs) are involved in carcinogenesis and tumor suppression, and are novel biological tumor regulators. However, the functional roles of lncRNAs and their underlying dysregulation mechanisms in breast cancer are not completely understood. The aim of the present study was to investigate the clinical significance and biological functions of lncRNA TMPO antisense RNA 1 (TMPO-AS1) in breast cancer. TMPO-AS1 levels were measured in human cancer tissues and breast cancer cell lines, and the functional roles of TMPO-AS1 in breast cancer cells were investigated by performing in vitro and in vivo assays. Additionally, luciferase reporter assays were conducted to detect the association between microRNA (miR)-140-5p and TMPO-AS1. TMPO-AS1 expression levels were significantly increased in breast cancer tissues and cell lines compared with adjacent non-cancerous tissues and MCF-10A cells, respectively. In vitro and in vivo studies indicated that TMPO-AS1 knockdown significantly suppressed breast cancer cell viability at 48 and 72 h compared with the small interfering (si)RNA negative control group (NC; siNC). TMPO-AS1 knockdown in vitro inhibited MCF-7 and T47D cell migration and invasion compared with the siNC group. TMPO-AS1 knockdown in metastatic breast cancer cells also decreased metastatic colonization in the mouse lung compared with the short hairpin RNA NC group. Mechanistically, TMPO-AS1 promoted cellular viability and migration as a competing endogenous RNA by sponging miR-140-5p. The results suggested that TMPO-AS1 may serve as a potential therapeutic target in patients with breast cancer.

How does fascin promote cancer metastasis?

Fascin is an F-actin-bundling protein that cross-links individual actin filaments into straight and stiff bundles. Fascin overexpression in cancer is strongly associated with poor prognosis and metastatic progression across different cancer types. It is well established that fascin plays a causative role in promoting metastatic progression. We will review the recent progress in our understanding of mechanisms underlying fascin-mediated cancer metastasis. This review will cover the biochemical basis for fascin-bundling activity, the mechanisms by which cancer cells upregulate fascin expression and the mechanism underlying fascin-mediated cancer cell migration, invasion, and metastatic colonization. We propose that fascin has broad roles in both metastatic dissemination and metastatic colonization. Understanding these mechanisms will be crucial to the development of anti-metastasis therapeutics targeting fascin.

NLGP Attenuates Murine Melanoma and Carcinoma Metastasis by Modulating Cytotoxic CD8+ T Cells.

Neem leaf glycoprotein (NLGP), a natural immunomodulator, attenuates murine carcinoma and melanoma metastasis, independent of primary tumor growth and alterations in basic cellular properties (cell proliferation, cytokine secretion, etc.). Colonization event of invasion-metastasis cascade was primarily inhibited by NLGP, with no effect on metastasis-related invasion, migration, and extravasation. High infiltration of interferon γ (IFN-γ)-secreting cytotoxic CD8+ T cells [CD44+, CD69+, GranB+, IFN-γ+, and interleukin 2+] was documented in the metastatic site of NLGP-treated mice. Systemic CD8+ T cell depletion abolished NLGP-mediated metastasis inhibition and reappeared upon adoptive transfer of NLGP-activated CD8+ T cells. Interferon γ-secreting from CD8+ T cells inhibit the expression of angiogenesis regulatory vascular endothelial growth factor and transforming growth factor ß and have an impact on the prevention of colonization. Neem leaf glycoprotein modulates dendritic cells (DCs) for proper antigen presentation by its DC surface binding and upregulation of MHC-I/II, CD86, and CCR7. Neem leaf glycoprotein-treated DCs specifically imprint CXCR3 and CCR4 homing receptors on activated CD8+ T cells, which helps to infiltrate into metastatic sites to restrain colonization. Such NLGP's effect on DCs is translation dependent and transcription independent. Studies using ovalbumin, OVA257-264, and crude B16F10 antigen indicate MHC-I upregulation depends on the quantity of proteasome degradable peptide and only stimulates CD8+ T cells in the presence of antigen. Overall data suggest NLGP inhibits metastasis, in conjunction with tumor growth restriction, and thus might appear as a promising next-generation cancer immunotherapeutic.

LEF1 supports metastatic brain colonization by regulating glutathione metabolism and increasing ROS resistance in breast cancer.

More than half of all brain metastases show infiltrating rather than displacing growth at the macro-metastasis/organ parenchyma interface (MMPI), a finding associated with shorter survival. The lymphoid enhancer-binding factor-1 (LEF1) is an epithelial-mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain-colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the antioxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma.

Fascin Controls Metastatic Colonization and Mitochondrial Oxidative Phosphorylation by Remodeling Mitochondrial Actin Filaments.

The deregulation of the actin cytoskeleton has been extensively studied in metastatic dissemination. However, the post-dissemination role of the actin cytoskeleton dysregulation is poorly understood. Here, we report that fascin, an actin-bundling protein, promotes lung cancer metastatic colonization by augmenting metabolic stress resistance and mitochondrial oxidative phosphorylation (OXPHOS). Fascin is directly recruited to mitochondria under metabolic stress to stabilize mitochondrial actin filaments (mtF-actin). Using unbiased metabolomics and proteomics approaches, we discovered that fascin-mediated mtF-actin remodeling promotes mitochondrial OXPHOS by increasing the biogenesis of respiratory Complex I. Mechanistically, fascin and mtF-actin control the homeostasis of mtDNA to promote mitochondrial OXPHOS. The disruption of mtF-actin abrogates fascin-mediated lung cancer metastasis. Conversely, restoration of mitochondrial respiration by using yeast NDI1 in fascin-depleted cancer cells is able to rescue lung metastasis. Our findings indicate that the dysregulated actin cytoskeleton in metastatic lung cancer could be targeted to rewire mitochondrial metabolism and to prevent metastatic recurrence.

Targeting neddylation inhibits intravascular survival and extravasation of cancer cells to prevent lung-cancer metastasis.

Metastasis is the leading cause of tumor-related death from lung cancer. However, limited success has been achieved in the treatment of lung cancer metastasis due to the lack of understanding of the mechanisms that underlie the metastatic process. In this study, Lewis lung carcinoma (LLC) cells which expressed green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm were used to record metastatic process in real-time via a whole-mouse imaging system. Using this system, we show the neddylation inhibitor MLN4924 inhibits multiple steps of the metastatic process, including intravascular survival, extravasation, and formation of metastatic colonies, thus finally suppressing tumor metastasis. Mechanistically, MLN4924 efficiently inhibits the expression of MMP2, MMP9, and vimentin and disrupts the actin cytoskeleton at an early stage to impair invasive potential and subsequently causes a DNA damage response, cell cycle arrest, and apoptosis upon long exposure to MLN4924. Furthermore, MMP2 and MMP9 are overexpressed in patient lung adenocarcinoma, which conferred a worse overall survival. Together, targeting the neddylation pathway via MLN4924 suppresses multiple steps of the metastatic process, highlighting the potential therapeutic value of MLN4924 for the treatment of metastatic lung cancer.

Human bone perivascular niche-on-a-chip for studying metastatic colonization.

Eight out of 10 breast cancer patients die within 5 years after the primary tumor has spread to the bones. Tumor cells disseminated from the breast roam the vasculature, colonizing perivascular niches around blood capillaries. Slow flows support the niche maintenance by driving the oxygen, nutrients, and signaling factors from the blood into the interstitial tissue, while extracellular matrix, endothelial cells, and mesenchymal stem cells regulate metastatic homing. Here, we show the feasibility of developing a perfused bone perivascular niche-on-a-chip to investigate the progression and drug resistance of breast cancer cells colonizing the bone. The model is a functional human triculture with stable vascular networks within a 3D native bone matrix cultured on a microfluidic chip. Providing the niche-on-a-chip with controlled flow velocities, shear stresses, and oxygen gradients, we established a long-lasting, self-assembled vascular network without supplementation of angiogenic factors. We further show that human bone marrow-derived mesenchymal stem cells, which have undergone phenotypical transition toward perivascular cell lineages, support the formation of capillary-like structures lining the vascular lumen. Finally, breast cancer cells exposed to interstitial flow within the bone perivascular niche-on-a-chip persist in a slow-proliferative state associated with increased drug resistance. We propose that the bone perivascular niche-on-a-chip with interstitial flow promotes the formation of stable vasculature and mediates cancer cell colonization.