Brain Metastatic Breast Cancer Cells Research Articles

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling.

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also showthat brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

Abstract 3157: The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis

Abstract Brain metastasis (BM) affects approximately 10% of cancer patients with metastatic disease, representing a major cause of morbidity and mortality. BM is a complex multi-step process involving various immune checkpoint proteins. Mitogen-activated protein kinase (MAPK), particularly extracellular signal-regulated kinases 1/2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3) are implicated in tumorigenesis and are critical upstream regulators of Programmed Death Ligand 1 (PD-L1) expression, a common immunotherapy target. Tumor suppressor p53 is shown to regulate STAT3 and ERK1/2 signaling, which are dysregulated in various malignancies. We hypothesize that p53 along with STAT3 and ERK1/2 play a critical role in the initiation and maintenance of BM. Twenty-six BM tumors with various primary sites of origin were used following IRB guidelines. pSTAT3Tyr705, pERK1/2Thr202/Tyr204, mutant p53 (p53mt), and PD-L1 expression was determined by immunohistochemistry. Gene expression analysis of BM was performed on a cDNA microarray. Human brain metastatic breast cancer cells (MDA-MB-231) were treated with STAT3 inhibitor (NSC74859) or MAPK inhibitor (U0126) in regular or astrocytic media to determine cell proliferation and migration by MTT assay and scratch-wound, respectively. ERK1/2 signaling pathway status was determined by Western blotting. Results demonstrated: 1) pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were expressed profusely at tumor margins, while p53mt was uniformly expressed throughout. 81% of BM expressed p53mt while 86% and 63% expressed pERK1/2Thr202/Tyr204 and pSTAT3Tyr705, respectively. Venn analysis displayed significant overlap between expression, with 62% overlap between p53mt and pERK1/2Thr202/Tyr204 and a 50% overlap between both pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 with p53mt. Expression of PD-L1 was seen in a majority of BM tumors. 2) Gene expression analysis demonstrated alterations in 32 STAT3- and MAPK-associated genes; these included upregulation of stathmin-1 and stathmin-like 3, which control microtubule dynamics in STAT3-related cell migration, and changes in cytokine-related genes, suggesting altered immune responses. 3) Cell proliferation showed augmented growth in astrocytic media (p≤0.05) and was significantly decreased following treatment with NSC74850 or U0126 (p≤0.01). While migration showed a trend towards significance in reduction following NSC74859 (p=0.08), migration stagnated following U0126 treatment in astrocytic media (p≤0.01). 4) pERK1/2Thr202/Tyr204 expression was reduced following U0126 or NSC74859 treatment. These findings suggest activation of STAT3 and ERK1/2 promote BM and suppression of these pathways may lead to a reduction in cell viability and motility. Therefore, this provides compelling evidence for the use of STAT3 and ERK1/2 along with p53 as potential targets for immunotherapy in brain metastatic disease. Citation Format: Sabrina L. Zeller, Eris Spirollari, John V. Wainwright, Simon J. Hanft, Chirag D. Gandhi, Meena Jhanwar-Uniyal. The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3157.

O-Glycome Profiling of Breast Cancer Cell Lines to Understand Breast Cancer Brain Metastasis.

Breast cancer is the second leading cause of cancer-related death among women and a major source of brain metastases. Despite the increasing incidence of brain metastasis from breast cancer, the underlying mechanisms remain poorly understood. Altered glycosylation is known to play a role in various diseases including cancer metastasis. However, profiling studies of O-glycans and their isomers in breast cancer brain metastasis (BCBM) are scarce. This study analyzed the expression of O-glycans and their isomers in human breast cancer cell lines (MDA-MB-231, MDA-MB-361, HTB131, and HTB22), a brain cancer cell line (CRL-1620), and a brain metastatic breast cancer cell line (MDA-MB-231BR) using nanoLC-MS/MS, identifying 27 O-glycan compositions. We observed significant upregulation in the expression of HexNAc1Hex1NeuAc2 and HexNAc2Hex3, whereas the expression of HexNAc1Hex1NeuAc1 was downregulated in MDA-MB-231BR compared to other cell lines. In our isomeric analysis, we observed notable alterations in the isomeric forms of the O-glycan structure HexNAc1Hex1NeuAc1 in a comparison of different cell lines. Our analysis of O-glycans and their isomers in cancer cells demonstrated that changes in their distribution can be related to the metastatic process. We believe that our investigation will contribute to an enhanced comprehension of the significance of O-glycans and their isomers in BCBM.

Brain Metastases Are Regulated by Immuno-inflammatory Signaling Pathways Governed by STAT3, MAPK and Tumor Suppressor p53 Status: Possible Therapeutic Targets.

Brain metastasis (BM) is a complex multi-step process involving various immune checkpoint proteins. Mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1/2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3) are implicated in tumorigenesis and are critical upstream regulators of Programmed Death Ligand 1 (PD-L1), an immunotherapy target. Tumor suppressor p53, dysregulated in cancers, regulates STAT3 and ERK1/2 signaling. This study examined the roles of STAT3, MAPK and p53 status in BM initiation and maintenance. Twenty-six BM, with various primary malignancies, were used (IRB-approved) to determine mutant p53 (p53mt), pSTAT3Tyr705, pERK1/2Thr202/Tyr204, and PD-L1 expression using immunohistochemistry. cDNA microarray was used for gene expression analysis. Brain-metastatic breast cancer cells (MDA-MB-231) were treated with STAT3 (NSC74859) or MAPK/ERK1/2 (U0126) inhibitors in regular or astrocytic media. ERK1/2 pathway was assessed using western blotting, and cell proliferation and migration were determined using MTT and scratch-wound assays, respectively. pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were expressed at tumor margins, whereas p53mt and PD-L1 were uniformly expressed, with significant overlap between expression of these proteins. Gene expression analysis identified alterations in 18 p53- and 32 STAT3- or MAPK-associated genes contributing to dysregulated immune responses and cell cycle regulation. U0126 and NSC74859 reduced pERK1/2Thr202/Tyr204 expression. Cell proliferation decreased following each treatment (p≤0.01). Migration stagnated following U0126 treatment in astrocytic media (p≤0.01). Activation of STAT3 and ERK1/2 promotes BM and provides compelling evidence for use of STAT3, ERK1/2 and p53 status as potential immunotherapeutic targets in BM.

Varying the RGD concentration on a hyaluronic acid hydrogel influences dormancy versus proliferation in brain metastatic breast cancer cells.

A majority of breast cancer deaths occur due to metastasis of cancer cells to distant organs. In particular, brain metastasis is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of dormant cancer cells. However, how these cues influence dormancy remains poorly understood. Herein, we employed hyaluronic acid (HA) hydrogels with a stiffness of ~0.4 kPa as an in vitro biomimetic platform to investigate the impact of biochemical cues, specifically alterations in RGD concentration, on dormancy versus proliferation in MDA-MB-231Br brain metastatic breast cancer cells. We applied varying concentrations of RGD peptide (0, 1, 2, or 4 mg/mL) to HA hydrogel surfaces and confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA-MB-231Br cells were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded to spindle-like morphology as well as an increase in cell spreading area. Also, an increase in RGD concentration resulted in an increase in cell proliferation. Cellular dormancy was assessed using the ratio of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK) to phosphorylated p38 (p-p38) positivity, which was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to hydrogels functionalized with higher RGD concentration. We also demonstrated that the HA hydrogel-induced cellular dormancy was reversible. Finally, we demonstrated the involvement of β1 integrin in mediating cell phenotype in our hydrogel platform. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.

Microglia increase CEMIP expression and promote brain metastasis in breast cancer through the JAK2/STAT3 signaling pathway

Abstract Objectives Brain metastasis is the most lethal metastatic site for patients with breast cancer, and the incidence of brain metastasis is increasing every year. Microglia act a pivotal part in promoting the proliferation and metastasis of breast cancer cells in the brain. Therefore, understanding the biological process of brain metastasis in breast cancer is important to improve therapeutic outcomes and prolong the survival of patients. Materials and Methods The role of microglia on the prognosis of patients with breast cancer with brain metastasis was verified by immunohistochemistry and the Kaplan–Meier curve. Cell experiments in vitro were used to analyze the effect of microglia on cell proliferation, migration and invasion. Knockdown of cell migration-inducing hyaluronan-binding protein (CEMIP) expression and co-culture experiments were carried out to study the mechanism of microglia on the progression of brain metastasis of breast cancer. Results We found that microglia may shorten the survival time of patients with breast cancer by regulating the expression of CEMIP in brain metastatic tumors. Co-culture experiments in vitro indicated that microglia enhance the proliferation, migration, and invasion abilities of brain metastatic breast cancer cells; however, the knockdown of CEMIP expression suppresses this effect. In addition, we also found that CEMIP expression, increased by microglia, activates the JAK2/STAT3 pathway in brain metastatic breast cancer cells, which induces the secretion of CCL2, IL-6, TGF-β, and VEGF. CCL2 recruits microglia to gather around brain metastases, whereas IL-6, TGF-β, and VEGF induce high CEMIP expression, triggering a positive feedback loop between microglia and brain metastatic breast cancer cells. Conclusions Our study proposes a possible mechanism of microglia promoting brain metastasis of breast cancer, indicating that both microglia and CEMIP may be valuable therapeutic targets for patients with breast cancer with brain metastasis.

CNSC-02. GLUTAMATERGIC SYNAPTIC INPUT DRIVES BRAIN METASTASIS OF BREAST CANCER AND MELANOMA

Abstract The nervous system is implicated in pathology of both cranial and extracranial cancers. In glioblastoma, direct excitatory glutamatergic synapses between neurons and cancer cells were discovered. Importantly, these neuron-cancer contacts drive glioblastoma proliferation and invasion. However, such synapses have not yet been documented in other types of brain cancer. In the context of brain metastases, indirect, peri-synaptic contacts between breast cancer cells and neurons were observed, which promote brain metastatic growth through glutamatergic signaling via NMDA receptors on the neoplastic cells. Recently, two studies have demonstrated that neuronal activity supports the growth of both the primary tumor and brain metastases from neuroendocrine carcinoma small cell lung cancer. In the work presented here, we demonstrate direct neuron-cancer synapses in early-stage brain metastases originating from breast cancer and melanoma. These synapses exhibited the typical structural features observed in synaptic connections and induced postsynaptic currents in the cancer cells through the activation of AMPA subtype glutamate receptors. Importantly, genetic and pharmacological perturbation of these receptors reduces the number of brain metastases and the overall metastatic burden in preclinical models of breast cancer and melanoma. These results uncover a previously unknown form of direct communication between neurons and brain-metastatic breast cancer and melanoma tumor cells. This neuronal-tumor synaptic interaction demonstrates the significance of the neuronal microenvironment in the early stages of the brain metastatic cascade. Furthermore, it unveils a potential therapeutic vulnerability associated with the neuronal microenvironment, suggesting that targeting the synaptic communication between neurons and cancer cells could be a promising strategy to impede the progression of brain metastases.

Biomimetic Nanocomposites Camouflaged with Hybrid Cell Membranes for Accurate Therapy of Early‐Stage Glioma

AbstractGlioma features high fatality rate and short survival time of patients due to its fast growth speed and high invasiveness, hence timely treatment of early‐stage glioma is extremely important. However, the blood brain barrier (BBB) severely prevents therapeutic agents from entering the brain; meanwhile, the non‐targeted distribution of agents always causes side effects to vulnerable cerebral tissues. Therefore, delivery systems that possess both BBB penetrability and precise glioma targeting ability are keenly desired. We herein proposed a hybrid cell membrane (HM) camouflage strategy to construct therapeutic nanocomposites, in which HM consisting of brain metastatic breast cancer cell membrane and glioma cell membrane was prepared with a simple membrane fusion pathway. By coating HM onto drug‐loaded nanoparticles, the as‐obtained biomimetic therapeutic agent (termed HMGINPs) inherited satisfying BBB penetrability and homologous glioma targeting ability simultaneously from the two source cells. HMGINPs exhibited good biocompatibility and superior therapeutic efficacy towards early‐stage glioma.

Biomimetic Nanocomposites Camouflaged with Hybrid Cell Membranes for Accurate Therapy of Early-Stage Glioma.

Glioma features high fatality rate and short survival time of patients due to its fast growth speed and high invasiveness, hence timely treatment of early-stage glioma is extremely important. However, the blood brain barrier (BBB) severely prevents therapeutic agents from entering the brain; meanwhile, the non-targeted distribution of agents always causes side effects to vulnerable cerebral tissues. Therefore, delivery systems that possess both BBB penetrability and precise glioma targeting ability are keenly desired. We herein proposed a hybrid cell membrane (HM) camouflage strategy to construct therapeutic nanocomposites, in which HM consisting of brain metastatic breast cancer cell membrane and glioma cell membrane was prepared with a simple membrane fusion pathway. By coating HM onto drug-loaded nanoparticles, the as-obtained biomimetic therapeutic agent (termed HMGINPs) inherited satisfying BBB penetrability and homologous glioma targeting ability simultaneously from the two source cells. HMGINPs exhibited good biocompatibility and superior therapeutic efficacy towards early-stage glioma.

DH5α Outer Membrane-Coated Biomimetic Nanocapsules Deliver Drugs to Brain Metastases but not Normal Brain Cells via Targeting GRP94.

Receptor-mediated vesicular transport has been extensively developed to penetrate the blood-brain barrier (BBB) and has emerged as a class of powerful brain-targeting delivery technologies. However, commonly used BBB receptors such as transferrin receptor and low-density lipoprotein receptor-related protein 1, are also expressed in normal brain parenchymal cells and can cause drug distribution in normal brain tissues and subsequent neuroinflammation and cognitive impairment. Here, the endoplasmic reticulum residing protein GRP94 is found upregulated and relocated to the cell membrane of both BBB endothelial cells and brain metastatic breast cancer cells (BMBCCs) by preclinical and clinical investigations. Inspired by that Escherichia coli penetrates the BBB via the binding of its outer membrane proteins with GRP94, avirulent DH5α outer membrane protein-coated nanocapsules (Omp@NCs) are developed to cross the BBB, avert normal brain cells, and target BMBCCs via recognizing GRP94. Embelin (EMB)-loaded Omp@EMB specifically reduce neuroserpin in BMBCCs, which inhibits vascular cooption growth and induces apoptosis of BMBCCs by restoring plasmin. Omp@EMB plus anti-angiogenic therapy prolongs the survival of mice with brain metastases. This platform holds the translational potential to maximize therapeutic effects on GRP94-positive brain diseases.

MiR-623 Targets Metalloproteinase-1 and Attenuates Extravasation of Brain Metastatic Triple-Negative Breast Cancer Cells.

BackgroundMost breast cancer-related deaths result from metastasis. Understanding the molecular basis of metastasis is needed for the development of effective targeted and preventive strategies. Matrix metalloproteinase-1 (MMP1) plays an important role in brain metastasis (BM) of triple-negative breast cancer (TNBC) by promoting extravasation of cancer cells across the brain endothelium (BE). MMP1 expression is controlled by endogenous microRNAs. Preliminary bioinformatics analysis has revealed that miR-623, known to target the 3ʹUTR of MMP1, is significantly downregulated in brain metastatic tumors compared to primary BC tumors. However, the involvement of miR-623 in MMP1 upregulation in breast cancer brain metastatic cells (BCBMC) remains unexplored. Here, we investigated the role of miR-623 in MMP1 regulation and its impact on the extravasation of TNBC cells through the BE in vitro.Materials and MethodsA loss-and-gain of function method was employed to address the effect of miR-623 modulation on MMP1 expression. MMP1 regulation by miR-623 was investigated by real-time PCR, western blot, luciferase and transwell migration assays using an in vitro human BE model.ResultsOur results confirmed that brain metastatic TNBC cells express lower levels of miR-623 compared with cells having low propensity to spread toward the brain. miR-623 binds to the 3′-untranslated region of MMP1 transcript and downregulates its expression. Restoring miR-623 expression significantly decreased MMP1 expression, preserved the endothelial barrier integrity, and attenuated transmigration of BCBMC through the BE.ConclusionOur study elucidates, for the first time, the crucial role of miR-623 as MMP1 direct regulator in BCBMC and sheds light on miR-623 as a novel therapeutic target that can be exploited to predict and prevent brain metastasis in TNBC. Importantly, the presents study helps in unraveling a brain metastasis-specific microRNA signature in TNBC that can be used as a guide to personalized metastasis prediction and preventive approach with better therapeutic outcome.

OGT/CDK5/ACSS2 Axis Regulates Breast Cancer Brain Metastasis Growth

Onset of brain metastases (BMs) in breast cancer patients is considered an end‐stage event, with no effective drug treatment and a median survival after diagnosis measured in months. Thus, there is an urgent need to develop novel treatment strategies. Metastatic breast cancer cells colonizing the brain encounter adverse ‘nutritional environment’, as the levels of key metabolic fuels such as glucose are much lower, in part because they are avidly consumed by neurons. To overcome this challenge, brain‐metastatic cancer cells rely on acetate as a fuel source and convert it into acetyl‐CoA via upregulation of acetyl‐CoA synthetase 2 (ACSS2) enzyme. We have previously shown that brain tumors increase the nutrient sensing post‐translational modification O‐GlcNAcylation and O‐GlcNAc transferase (OGT) levels to regulate acetate metabolism into acetyl‐CoA via increased phosphorylation of ACSS2 on Ser‐267 in a cyclin dependent kinase 5 (CDK5)‐dependent manner, increasing brain tumor growth. Here, we show that human breast cancer cells selected to metastasize to the brain contain increased levels of O‐GlcNAc, OGT and ACSS2‐Ser267 phosphorylation compared to parental cells and show that human breast cancer brain metastatic patient samples contain elevated ACSS2‐Ser‐267 levels. Moreover, overexpression of OGT or ACSS2‐S267D phospho‐mimetic mutant confer a growth advantage on brain metastatic breast cancer cells in an in vivo intracranial xenograft model, may be due to an increase in lipogenic proteins. Additionally, we show that pharmacologically targeting CDK5 and ACSS2 with small molecule inhibitors reduces tumor growth in a novel orthotopic ex vivo brain slice model. These results suggest a crucial role for OGT/CDK5/ACSS2 signaling in transducing nutritional state to regulate acetate metabolism in breast cancer BM cells and identify CDK5 and ACSS2 as novel therapeutic targets for treatment of breast cancer brain metastasis.

Combinatorial targeting of microRNA-26b and microRNA-101 exerts a synergistic inhibition on cyclooxygenase-2 in brain metastatic triple-negative breast cancer cells.

Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells. Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE. We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA. These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.

Multifunctional Liposomes Enable Active Targeting and Twinfilin 1 Silencing to Reverse Paclitaxel Resistance in Brain Metastatic Breast Cancer.

Paclitaxel (PTX) is a first-line chemotherapeutic drug for breast cancer, but PTX resistance often occurs in metastatic breast cancer. In addition, due to the poor targeting of chemotherapeutic drugs and the presence of the blood-brain barrier (BBB), it is hard to effectively treat brain metastatic breast cancer using paclitaxel. Thus, it is urgent to develop an effective drug delivery system for the treatment of brain metastatic breast cancer. The current study found that TWF1 gene, an epithelial-mesenchymal transition-associated gene, was overexpressed in brain metastatic breast cancer (231-BR) cells and was associated with the PTX resistance of 231-BR cells. Knockdown of TWF1 by small interference RNA (siRNA) in 231-BR cells could effectively increase the sensitivity of brain metastatic breast cancer cells to paclitaxel. Then, a liposome-based drug delivery system was developed for PTX delivery across BBB, enhancing PTX sensitivity and brain metastases targeting via BRBP1 peptide modification. The results showed that BRBP1-modified liposomes could effectively cross the BBB, specifically accumulate in brain metastases, and effectively interfere TWF1 gene expression in vitro and in vivo, and thus they enhanced proliferation inhibition, cell cycle arrest, and apoptosis induction, thereby inhibiting the formation and growth of brain metastases. In summary, our results indicated that BRBP1-modified and PTX- and TWF1 siRNA-loaded liposomes have the potential for the treatment of brain metastatic breast cancer, which lays the foundation for the development of a new targeted drug delivery system.

Comparing the fate of brain metastatic breast cancer cells in different immune compromised mice with cellular magnetic resonance imaging.

Metastasis is the leading cause of mortality in breast cancer patients, with brain metastases becoming increasingly prevalent. Studying this disease is challenging due to the limited experimental models and methods available. Here, we used iron-based cellular MRI to track the fate of a mammary carcinoma cell line (MDA-MB-231-BR) in vivo to characterize the growth of brain metastases in the nude and severely immune-compromised NOD/SCID/ILIIrg-/- (NSG) mouse. Nude and NSG mice received injections of iron-labeled MDA-MB-231-BR cells. Images were acquired with a 3T MR system and assessed for signal voids and metastases. The percentage of signal voids and the number and volume of metastases were quantified. Ex vivo imaging of the liver, histology, and immunofluorescence labeling was performed. Brain metastases grew more rapidly in NSG mice. At day 21 post cell injection, the average number of brain tumors in NSG mice was approximately four times greater than in nude mice. The persistence of iron-labeled cells, visualized as signal voids by MRI, was also examined. The percentage of voids decreased significantly over time for both nude and NSG mice. Body images revealed that the NSG mice also had metastases in the liver, lungs, and lymph nodes while tumors were only detected in the brains of nude mice. This work demonstrates the advantages of using the highly immune-compromised NSG mouse to study breast cancer metastasis, treatments aimed at inhibiting metastasis and outgrowth of breast cancer metastases in multiple organs, and the role that imaging can play toward credentialing these models that cannot be done with other in vitro or histopathologic methods alone.

Exosomes Derived from Brain Metastatic Breast Cancer Cells Destroy the Blood-Brain Barrier by Carrying lncRNA GS1-600G8.5.

Brain metastasis is a major cause of death in breast cancer patients. The greatest event for brain metastasis is the breaching of the blood-brain barrier (BBB) by cancer cells. The role of exosomes in cancer metastasis is clear, whereas the role of exosomes in the integrity of the BBB is unknown. Here, we established a highly brain metastatic breast cancer cell line by three cycles of in vivo selection. The effect of exosomes on the BBB was evaluated in vitro by tracking, transepithelial/transendothelial electrical resistance (TEER), and permeability assays. BBB-associated exosomal long noncoding RNA (lncRNA) was selected from the GEO dataset and verified by real-time PCR, TEER, permeability, and Transwell assays. The cells obtained by the in vivo selection showed higher brain metastatic capacity in vivo and higher migration and invasion in vitro compared to the parental cells. Exosomes from the highly brain metastatic cells were internalized by brain microvascular endothelial cells (BMECs), which reduced TEER and increased permeability of BBB. The exosomes derived from the highly metastatic cells promoted invasion of the breast cancer cells in the BBB model. lncRNA GS1-600G8.5 was highly expressed in the highly brain metastatic cells and their exosomes, as compared to the samples with reduced metastatic behavior. Silencing of GS1-600G8.5 significantly abrogated the BBB destructive effect of exosomes. GS1-600G8.5-deficient exosomes failed to promote the infiltration of cancer cells through the BBB. Furthermore, BMECs treated with GS1-600G8.5-deprived exosomes expressed higher tight junction proteins than those treated with the control exosomes. These data suggest the exosomes derived from highly brain metastatic breast cancer cells might destroy the BBB system and promote the passage of cancer cells across the BBB, by transferring lncRNA GS1-600G8.5.

Decreasing the survival ability of brain metastatic breast cancer cell by targeting glucose transporter 3

Breast cancer brain metastases (BBMs) are found in 15–20 % of breast cancer patients. And it results in a high mortality once the brain metastases are diagnosed. Therefore, making it more comprehensive about the mechanisms of BBMs is emergently necessary. Several studies have demonstrated that tumor microenvironment has potent roles in the tumor progression and metastasis. Besides, according to the former studies, cells in different organs using glucose might be various; as well as the dominant glucose transporters used are distinct. Here, we analyzed the brain metastatic breast cancer (BR) cells collected from intracardiac injection of nude mice and found the utilization of glucose transporters in the BR cells were different from the parental breast cancer cells; the elevated expressions of glucose transporter 3 were observed. In addition, the higher anaerobic glycolysis ability was found in BR cells. And after GLUT3 knockdown, the glucose reprogramming in BR cells was significantly decreased as well as the cancer malignant behaviors both in vitro and in vivo. Besides manipulating GLUT3 expressions, we also provide a potential mechanism that CREB could be a potent regulator of GLUT3 in BR cells by chromatin immunoprecipitation (ChIP) and knockdown assay. After decreasing the expressions of CREB in BR cells, the expressions of GLUT3 were also significantly reduced. In sum, our results reveal that GLUT3 plays an important role in provoking breast cancer cells surviving in brain by mediating glucose utilizations. And these results could provide a potential therapeutic way to target BBMs.Support or Funding Information1. Ministry of Science and Technology, R.O.C 2. Molecular Medicine Taiwan International Graduate Program, Academia Sinica

An in vitro hyaluronic acid hydrogel based platform to model dormancy in brain metastatic breast cancer cells

Breast cancer cells (BCCs) can remain dormant at the metastatic site, which when revoked leads to formation of metastasis several years after the treatment of primary tumor. Particularly, awakening of dormant BCCs in the brain results in breast cancer brain metastasis (BCBrM) which marks the most advanced stage of the disease with a median survival period of ~4–16 months. However, our understanding of dormancy associated with BCBrM remains obscure, in part, due to the lack of relevant in vitro platforms to model dormancy associated with BCBrM. To address this need, we developed an in vitro hyaluronic acid (HA) hydrogel platform to model dormancy in brain metastatic BCCs via exploiting the bio-physical cues provided by HA hydrogels while bracketing the normal brain and metastatic brain malignancy relevant stiffness range. In this system, we observed that MDA-MB-231Br and BT474Br3 brain metastatic BCCs exhibited a dormant phenotype when cultured on soft (0.4 kPa) HA hydrogel compared to stiff (4.5 kPa) HA hydrogel as characterized by significantly lower EdU and Ki67 positivity. Further, we demonstrated the nuclear localization of p21 and p27 (markers associated with dormancy) in dormant MDA-MB-231Br cells contrary to their cytoplasmic localization in the proliferative population. We also demonstrated that the stiffness-based dormancy in MDA-MB-231Br cells was reversible and was, in part, mediated by focal adhesion kinases and the initial cell seeding density. Finally, RNA sequencing confirmed the dormant phenotype in MDA-MB-231Br cells. This platform could further our understanding of dormancy in BCBrM and could be adapted for anti-metastatic drug screening. Statement of SignificanceOur understanding of dormancy associated with BCBrM remains obscure, in part, due to the lack of relevant in vitro platforms to model dormancy associated with BCBrM. Herein, we present a HA hydrogel-based platform to model dormancy in brain metastatic BCCs while recapitulating key aspects of brain microenvironment. We demonstrated that the biophysical cues provided the HA hydrogel mediates dormancy in brain metastatic BCCs by assessing both proliferation and cell cycle arrest markers. We also established the role of focal adhesion kinases and initial cell seeding density in the stiffness-mediated dormancy in brain metastatic BCCs. Further, RNA-seq. confirmed the dormant phenotype in brain metastatic BCCs. This platform could be utilized to further our understanding of microenvironmental regulation of dormancy in BCBrM.

Abstract P3-03-04: Role of ABL kinase signaling in metastatic breast cancer colonization of the brain

Abstract Breast cancer brain metastasis (BCBM) occurs in approximately 50% of late-stage Human Epidermal Growth Factor Receptor 2-positive (HER2+) breast cancer patients. BCBM may result in cognitive decline and cranial neuropathies and is associated with a median survival rate of nine months. Current therapies for BCBM, such as whole brain radiation therapy, can enhance cognitive impairment while targeted therapies and chemotherapy are ineffective. Despite the increased patient survival due to HER2 targeted therapies, patients often relapse due to brain metastases. Recently, our laboratory has discovered a role for Abelson (Abl) family of tyrosine kinases, ABL1 and ABL2, in breast cancer metastasis. Evaluation of 279 HER2+ breast cancer patients demonstrates a correlation between high expression of ABL1 and decreased distant metastasis free survival. Expression of ABL1 and ABL2 increase in brain metastatic breast cancer cells compared to parental cells, suggesting a role for the Abl kinases in breast cancer brain metastasis. I have found that treatment with the allosteric Abl kinase inhibitor GNF5 decreases colonization of the brain by two distinct human HER2+ breast cancer cell lines. Increasing evidence suggests that HER2 is upregulated in breast cancer cells colonizing the brain and may interact with the brain microenvironment to promote outgrowth. Both pharmacologic and genetic inhibition of the Abl kinases decreases HER2 protein expression in brain metastatic HER2+ breast cancer cells, potentially ablating the cell’s ability to proliferate in the brain. These data support a role for Abl kinases in promoting colonization of the brain by breast cancer cells. Due to its blood-brain barrier penetrance and efficacy in pre-clinical mouse models, Abl kinase-specific allosteric inhibitors may provide a novel therapeutic strategy for HER2+ breast cancer brain metastasis. Citation Format: Courtney Michelle McKernan, Ann M Pendergast. Role of ABL kinase signaling in metastatic breast cancer colonization of the brain [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-04.

Glucose Transporter 3 is Essential for the Survival of Breast Cancer Cells in the Brain.

Breast cancer brain metastasis commonly occurs in one-fourth of breast cancer patients and is associated with poor prognosis. Abnormal glucose metabolism is found to promote cancer metastasis. Moreover, the tumor microenvironment is crucial and plays an active role in the metabolic adaptations and survival of cancer cells. Glucose transporters are overexpressed in cancer cells to increase glucose uptake. The glucose transporter 3 (GLUT3) is a high-affinity glucose transporter that is highly expressed in mammalian neurons. GLUT3 is also overexpressed in several malignant brain tumors. However, the role of GLUT3 in breast cancer brain metastasis remains unknown. The results of the present study demonstrated that GLUT3 is highly overexpressed in brain metastatic breast cancers and mediates glucose metabolic reprogramming. Furthermore, knockdown of cAMP-response element binding protein (CREB) could directly regulate GLUT3 expression in brain metastatic breast cancer cells. Notably, we verified and provided a novel role of GLUT3 in mediating glucose metabolism and assisting breast cancer cells to survive in the brain to promote brain metastasis.