Brain Metastatic Cells Research Articles

Discordance of Epidermal Growth Factor Receptor Mutation between Brain Metastasis and Primary Non-Small Cell Lung Cancer.

BackgroundThe aim of this study was to compare epidermal growth factor receptor (EGFR) mutations between non-small cell lung cancer (NSCLC) and corresponding brain metastases (BMs) in Korea society.MethodsFrom 2011 to 2016, a total of 74 patients underwent surgical resection of a metastatic brain tumor from NSCLC. Among them, we performed retrospective analysis for 46 patients who underwent EGFR sequencing of primary NSCLC tissues.ResultsAmong these 46 cases, 18 (39.1%) cases showed EGFR mutation in primary lung cancer. Detected mutation sites were exon 19 (8 cases), exon 21 (6 cases), exon 18 (1 cases), and multiple mutations (3 cases). In 18 cases of BM, EGFR mutation studies were done. Among them, 8 (25.6%) cases showed mutation on exon 19 (5 cases) or exon 21 (3 cases). To compare EGFR mutation status between primary lung cancer and BM, 18 paired tissues from both NSCLC and matched BM were collected. Four (22.5%) patients were discordant for the status of EGFR between primary and metastatic sites.ConclusionEGFR mutations were significantly discordant between primary tumors and corresponding metastases in a significant portion of NSCLC. In treatment of BM of EGFR mutant metastatic NSCLC, due to possibility of discordance, pathologic confirming through brain biopsy is recommended.

Circular RNA profile of breast cancer brain metastasis: identification of potential biomarkers and therapeutic targets.

To explore the circular RNA (circRNA) profile of breast cancer brain metastasis (BCBM). RNA-seq was performed to identify the circRNA expression profile of brain metastatic breast cancer cell line 231-BR, in comparison with its parental nonspecific metastatic cell line MDA-MB-231. A total of 215 upregulated and 191 downregulated circRNAs were identified. The expression levels of ten randomly selected majorly altered circRNAs were re-examined by real-time quantitative PCR and agarose gel electrophoresis, and the following alterations were verified: upregulation of hsa_circ_0001944, hsa_circ_0001481, hsa_circ_0000646, hsa_circ_0001006 and hsa_circ_0000732, and downregulation of hsa_circ_0001910, hsa_circ_0008285 and hsa_circ_0000002. CircRNA/miRNA analysis revealed that hsa_circ_0001944 may be involved in BCBM through sponging up miR-509 and interfering with its binding to the downstream targets. This study provides a leading and fundamental circRNA profile of BCBM.

STEM-03. NOVEL METASTATIC BRAIN TUMOR TARGETS ISOLATED THROUGH PHAGE DISPLAY BIOPANNING AGAINST BRAIN METASTASIS-INITIATING CELLS

Despite treatment advances for primary cancers that may result in brain metastases, the treatment for brain metastases has lagged behind, partly due to the unique environment created by the central nervous system. Recent work has highlighted that metastatic brain tumors demonstrate significant genotypic deviation from their parental tumors, which highlights the need to focus on metastatic brain tumors for the development of more effective targeted therapeutics. The epithelial-mesenchymal transition suggests the existence of brain metastasis-initiating cells (BMICs), which may possess stem cell-like phenotypes that play a pivotal role in metastasis. To properly isolate this subpopulation of tumor cells, we compared patient derived metastatic brain tumor cells against brain metastasis cell lines. We found that both types of cells demonstrated similar morphology when grown in standard serum media conditions, but when grown in serum-free media, both demonstrated a tumor sphere morphology. Gene expression analysis showed increased expression of stem cell markers CD133 and EYA1 when grown in serum-free media. To isolate therapeutic targets specific to BMICs, we performed in vitro and in vivo phage display biopanning against BMICs. Analysis of the recovered peptides derived several potential targets. Among them were angiomotin (AMOT) and bromodomain testis-specific factor (BRDT). Gene expression analysis confirmed significant upregulation of these gene in metastatic brain tumors cells when compared to non-tumor neural cells and primary lung cancer cell lines. Tissue microarray analysis of eleven matched brain metastasis and primary lung cancer patient tissue from Moffitt Cancer Center demonstrated an increased expression in brain metastasis over primary lung tissue. Analysis through NIH GEO database demonstrated decreased survival with increased gene expression. Our results show that we can use phage display biopanning to isolate novel targets that may be specific to metastatic brain tumors by isolating the proper growth conditions that support the BMIC phenotype.

The metastatic microenvironment: Melanoma-microglia cross-talk promotes the malignant phenotype of melanoma cells.

Melanoma has the highest propensity to metastasize to the brain compared to other cancers, as brain metastases are found frequently high in patients who have prolonged survival with visceral metastasis. Once disseminated in the brain, melanoma cells communicate with brain resident cells that include astrocytes and microglia. Microglia cells are the resident macrophages of the brain and are the main immunological cells in the CNS involved in neuroinflammation. Data on the interactions between brain metastatic melanoma cells and microglia and on the role of microglia-mediated neuroinflammation in facilitating melanoma brain metastasis are lacking. To elucidate the role of microglia in melanoma brain metastasis progression, we examined the bidirectional interactions between microglia and melanoma cells in the tumor microenvironment. We identified the molecular and functional modifications occurring in brain-metastasizing melanoma cells and microglia cells after the treatment of each cell type with supernatants of the counter cell type. Both cells induced alteration in gene expression programs, cell signaling, and cytokine secretion in the counter cell type. Moreover, melanoma cells exerted significant morphological changes on microglia cells, enhanced proliferation, induced matrix metalloproteinase-2 (MMP-2) activation, and cell migration. Microglia cells induced phenotypic changes in melanoma cells increasing their malignant phenotype: increased melanoma proliferation, MMP-2 activity, cell migration, brain endothelial penetration, and tumor cells ability to grow as spheroids in 3D cultures. Our work provides a novel insight into the bidirectional interactions between melanoma and micoglia cells, suggesting the contribution of microglia to melanoma brain metastasis formation.

Heterogeneous distribution of trastuzumab in HER2-positive xenografts and metastases: role of the tumor microenvironment

Most HER2-positive metastatic breast cancer patients continue to relapse. Incomplete access to all target HER2-positive cells in metastases and tumor tissues is a potential mechanism of resistance to trastuzumab. The location of locally bound trastuzumab was evaluated in HER2-positive tissues in vivo and as in vivo xenografts or metastases models in mice. Microenvironmental elements of tumors were related to bound trastuzumab using immunohistochemical staining and include tight junctions, vasculature, vascular maturity, vessel patency, hypoxia and HER2 to look for correlations. Trastuzumab was evaluated alone and in combination with bevacizumab. Dynamic contrast-enhanced magnetic resonance imaging parameters of overall vascular function, perfusion and apparent permeability were compared with matched histological images of trastuzumab distribution and vascular patency. Trastuzumab distribution is highly heterogeneous in all models examined, including avascular micrometastases of the brain and lung. Trastuzumab distributes well through the extravascular compartment even in conditions of high HER2 expression and poor convective flow in vivo. Microregional patterns of trastuzumab distribution in vivo do not consistently correlate with vascular density, patency, function or maturity; areas of poor trastuzumab access are not necessarily those with poor vascular supply. The number of vessels with perivascular trastuzumab increases with time and higher doses and dramatically decreases when pre-treated with bevacizumab. Areas of HER2-positive tissue without bound trastuzumab persist in all conditions. These data directly demonstrate tissue- and vessel-level barriers to trastuzumab distribution in vivo that can effectively limit access of the drug to target cells in brain metastases and elsewhere.

Expression of LC3B and FIP200/Atg17 in brain metastases of breast cancer.

Macroautophagy/autophagy is considered to play key roles in tumor cell evasion of therapy and establishment of metastases in breast cancer. High expression of LC3, a residual autophagy marker, in primary breast tumors has been associated with metastatic disease and poor outcome. FIP200/Atg17, a multi-functional pro-survival molecule required for autophagy, has been implicated in brain metastases in experimental models. However, expression of these proteins has not been examined in brain metastases from patients with breast cancer. In this retrospective study, specimens from 44 patients with brain metastases of infiltrating ductal carcinoma of the breast (IDC), unpaired samples from 52 patients with primary IDC (primary-BC) and 16 matched-paired samples were analyzed for LC3 puncta, expression of FIP200/Atg17, and p62 staining. LC3-puncta+ tumor cells and FIP200/Atg17 expression were detected in greater than 90% of brain metastases but there were considerable intra- and inter-tumor differences in expression levels. High numbers of LC3-puncta+ tumor cells in brain metastases correlated with a significantly shorter survival time in triple-negative breast cancer. FIP200/Atg17 protein levels were significantly higher in metastases that subsequently recurred following therapy. The percentages of LC3 puncta+ tumor cells and FIP200/Atg17 protein expression levels, but not mRNA levels, were significantly higher in metastases than primary-BC. Meta-analysis of gene expression datasets revealed a significant correlation between higher FIP200(RB1CC1)/Atg17 mRNA levels in primary-BC tumors and shorter disease-free survival. These results support assessments of precision medicine-guided targeting of autophagy in treatment of brain metastases in breast cancer patients.

A novel calix[4]pyrrole derivative as a potential anticancer agent that forms genotoxic adducts with DNA

meso-(p-acetamidophenyl)-calix[4]pyrrole 3 was found to exhibit remarkable cytotoxicity towards A549 cancer cells. A comparative study including the isomer of 3meso-(m-acetamidophenyl)-calix[4]pyrrole 5, as well as molecules containing ‘fragments’ of these structures, demonstrated that both the calix[4]pyrrole and the acetamidophenyl units are essential for high cytotoxicity. Although calix[4]pyrroles and other anion-complexing ionophores have recently been reported to induce apoptosis by perturbing cellular chloride concentrations, in our study an alternative mechanism has emerged, as proven by the isolation of covalent DNA adducts revealed by the 32P postlabelling technique. Preliminary pharmacokinetic studies indicate that 3 is able to cross the Blood-Brain-Barrier, therefore being a potential drug that could kill primary and brain metastatic cancer cells simultaneously.

Abstract 3027: Single-cell RNA sequencing defines regulatory networks in ER+ breast cancer organ-specific metastases

Abstract Patients with estrogen receptor (ER)+ breast cancer are at chronic and prolonged risk of metastatic recurrences, which are usually fatal. The most frequent sites of ER+ breast cancer metastasis are bone, liver, lung, and brain, with individual tumors preferring one or more sites. The underlying mechanisms of site-specific metastasis are poorly understood and are critical for prevention and treatment of metastases to more lethal (brain, liver, lung) vs. static (bone) sites. The purpose of this study is to use single-cell RNA sequencing (sc RNA-seq) of ER+ breast cancer cells dispersed to breast tropic sites to define unique molecular determinants of organ-specific metastasis. Our group has developed ER+ patient-derived xenografts (PDX) at the University of Colorado Denver (UCD) labeled with constitutive luciferase-GFP. Our ER+ PDX fall into two general categories-pure “luminal” tumors with high ER plus/minus progesterone receptor (PR) (>50% of cells) and an absence of cytokeratin 5 (CK5)+ cells, and “luminobasal” tumors with a low percent of ER+PR+ cells (5-10%) and high CK5+ cells (15-90%). Using intracardiac (ic) dispersal as a measure of late-stage metastasis, we have defined the organ homing preference of several of these lines. Luminal PDX frequently colonize bone, with occasional secondary lesions to brain and liver. Luminobasal PDX ubiquitously colonize liver, with secondary lesions mostly to lung. We performed sc RNA-seq of luminal UCD65 brain metastatic and luminobasal UCD46 liver metastatic cells compared to their cognate primary tumors grown in the mammary fat pad. UCD65 brain metastatic vs. primary tumor cells have elevated expression of PR and carbonic anhydrase 2 (CA2), a cellular guard against intracellular pH changes, and a prospective PR-regulated gene. UCD46 liver metastatic cells have activated TGFbeta signaling, and increased cancer stem cell markers. We conclude that ER+ breast cancer PDX retain innate preference for specific breast-tropic organs, that this may relate to their ER+ subtype (luminal vs. luminobasal), and that sc RNA-seq may identify key molecular signatures of ER+ breast cancer metastases. We are profiling additional tumors and sites to make further comparisons and generate testable hypotheses. Furthermore, our models allow for functional studies to test innovative interventions and therapeutics that target site-specific metastases. This work was supported by grants from the Breast Cancer Research Foundation (16-072, CAS, KBH, PK), the NIH (CA140985, CAS) and the UCD RNA Biosciences Initiative (AEG, CAS, PK). Citation Format: Nuria Padilla Just, Jessica Finlay-Schultz, Austin E. Gillen, Diana M. Cittelly, Elizabeth A. Wellberg, Kathryn B. Horwitz, Peter Kabos, Carol A. Sartorius. Single-cell RNA sequencing defines regulatory networks in ER+ breast cancer organ-specific metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3027.

Abstract 4376: Tumor treating fields (TTFields) decrease proliferation of patient-derived lung cancer brain metastasis cells in vitro

Abstract Up to 40% of all non-small cell lung cancer (NSCLC) patients will develop brain metastases during the course of their disease, with ~20-25% of patients presenting with brain metastases at the time of their initial lung cancer diagnosis. For the latter patient population, median survival is a dismal 8 months. Systemic therapies are typically ineffective at treating the brain metastases, even in cases when the extracranial disease responds to the therapy. Clinical trials utilizing TTFields therapy in patients with lung cancer brain metastases are underway. For these trials, TTFields are delivered at 150 kHz, as that is the frequency that has been determined for the treatment of primary lung cancer. In contrast, for the FDA-approved treatment of the most common malignant primary brain tumor, glioblastoma, TTFields are delivered at 200 kHz. Therefore, in this study, a patient-derived lung cancer brain metastatic cell line was assessed to compare the response to in vitro TTFields application at either 150 or 200 kHz compared to untreated control cells. Methods: Use of patient tumor tissue for this study was approved by the Wayne State University Institutional Review Board and written informed consent was obtained from the patient. An adenocarcinoma lung cancer brain metastasis (from a patient that presented with the brain metastasis at the time of diagnosis) was collected immediately following microsurgical resection. A single-cell suspension from the tumor tissue was prepared by enzymatic and mechanical disruption. After 7 passages in vitro, cells were plated on plastic coverslips (10,000 each) in DMEM/F12 media supplemented with 10% fetal bovine serum. TTFields were applied at ~1.6 V/cm at 150 or 200 kHz (groups n=4-5) with one control group. Culture media was replaced every day. After 10 days of in vitro TTFields application, cell proliferation was assessed with the XTT assay. Control vs. TTFields-treated groups were compared with two-tailed t-tests. Results: The 10-day TTFields treatment resulted in significantly reduced cellular proliferation in the cells that received 150 kHz TTFields (0.220±0.006; mean±SEM) compared to the control cells (0.250±0.009; p=0.0237) with no statistical difference between the control and the cells that received 200 kHz TTFields (0.230±0.008; p>0.05). Conclusions: Application of TTFields in vitro at 150 kHz reduced cellular proliferation in patient-derived lung cancer brain metastasis cells, with no reduction of proliferation after application of TTFields at 200 kHz. This is the first report on the in vitro effects of TTFields in a patient-derived NSCLC brain metastasis cell line. Citation Format: Sharon K. Michelhaugh, Sam Kiousis, Neil V. Klinger, Sandeep Mittal. Tumor treating fields (TTFields) decrease proliferation of patient-derived lung cancer brain metastasis cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4376.

Abstract LB-359: Role of metabolic adaptation to the brain microenvironment in facilitating brain metastasis progression

Abstract Breast cancer is the most commonly diagnosed cancer among American women, with a 1 in 8 lifetime risk of occurrence. The efficacy of treatment of primary tumors is dramatically improving, resulting in prolonged life span; however, given this increase in patient survival, the incidence of metastases is increasing as patients live long enough for metastases to develop. When breast cancer metastasizes to the brain or central nervous system, expected 5-year survival decreases dramatically. Where 5- year survival is 99% for patients with localized disease, it is only 25% for patients with CNS metastases. Further, brain metastases now account for 30% of breast cancer mortality. In breast cancer brain metastasis, interactions between tumor cells and glial cells (the main cell type present in the brain) appear vital to tumor growth and survival under the sub-ideal conditions presented by the unique brain microenvironment. However, the mechanisms through which glial cells support and alter tumor cells, particularly with respect to metabolism are poorly understood. One mechanism by which cells have been found to adapt to metabolic stress is through the transfer of mitochondria from stromal cells. Indeed, mitochondrial transfer has been observed in the central nervous system where glial cells have been found to transfer mitochondria to neurons following ischemic stroke to support neuronal cell viability and recovery. We hypothesize that interactions between tumor cells and glial cells promotes metabolic adaptation to the brain microenvironment. Further, we theorize that this may result from the acquisition of mitochondria derived from glial cells by tumor cells, which may confer a proliferative advantage to the tumor cells. To study this phenomenon, we are utilizing an in vitro system where breast cancer cells are co-cultured with primary murine glial cells. Our initial findings show that that breast cancer cells have decreased proliferation and survival in low glucose/low glutamine growth conditions, which are more representative of the brain environment. Interestingly, the presence of glia provides a survival advantage, and allows the breast cancer cells to survive longer in this condition. We have further found that glia are able to rapidly transfer mitochondria to breast cancer cells. Together, our findings suggest that glial cells play an important role in the progression of breast cancer brain metastases and that mitochondrial transfer may be a novel mechanism by which glial cells protect brain metastatic breast cancer cells. We have planned further experiments to specifically explore the link between the survival phenotype evidenced with the presence of glia to the transfer of mitochondria. Citation Format: Treasa K. O'Tighearnaigh, Erin N. Howe, Patricia M. Schnepp, Siyuan Zhang. Role of metabolic adaptation to the brain microenvironment in facilitating brain metastasis progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-359.

Abstract 1971: A novel approach to overcome resistance to HER2 targeted therapy in a new syngeneic model of breast cancer brain metastasis

Abstract The human epidermal growth factor receptor-2 (HER2) subtype of breast cancer is aggressive and associated with a high propensity to metastasize to the brain. Whilst the introduction of HER2-targeted therapies has significantly improved survival outcomes in affected patients, effective treatment of brain metastasis remains a challenge due to acquired resistance and limited drug permeability across the blood brain barrier. Moreover, progress in identifying more effective treatments, particularly against therapy-resistant brain-metastatic HER2 breast cancer, has been hampered by the lack of robust and clinically relevant preclinical models. Here, we introduce a new Balb/c syngeneic model of spontaneous breast cancer brain metastasis that naturally expresses HER2 (RCSI-Her2). In vivo metastasis assays confirmed that this model gives rise to high incidence of spontaneous brain metastases from mammary tumors or experimental brain metastases following intra-cardiac inoculation. RT-PCR, western blotting and immunohistochemical analyses of RCSI-Her2 cells, tumors or brain metastases confirmed the lack of hormone receptor expression but high levels of HER2. Accordingly, RCSI-Her2 cell proliferation is inhibited by HER2 inhibitors, lapatinib or neratinib, but not by anti-estrogens, indicating phenotypic and functional similarities to human HER2 breast cancer. Importantly, short term in vitro proliferation assays show that the combination of neratinib with αvβ3 integrin inhibitors (Cilengitide or DisBa-01) potently and synergistically inhibits the proliferation of RCSI-HER2 or brain-metastatic triple negative breast cancer cells (4T1Br4). Moreover, treatment of neratinib-resistant RCSI-Her2 variants with Cilengitide or DisBa-01 restores sensitivity to neratinib. Preliminary in vivo experiments indicate that neratinib is well-tolerated in immune-competent mice, inhibits RCSI-HER2 primary tumour growth and extends survival. Transcriptomic analysis (RNAseq) of neratinib-sensitive and resistant variants treated or not with neratinib to identify signaling pathways implicated in resistance is underway. The results from this analysis will be presented. To our knowledge, RCSI-Her2 is the only model that fully recapitulates the spontaneous spread of HER2 breast cancer to the brain in immune-competent hosts. This model provides a unique tool to validate the efficacy of neratinib/αvβ3 integrin inhibitors in vivo against advanced brain-metastatic and therapy-resistant HER2 breast cancer. Citation Format: Normand Pouliot, Aadya Nagpal, Miriam Fuentes, Effie Mouhtouris, Francesca Avogadri-Connors, Alshad S. Lalani, Heloisa Sobreiro Selistre de Araujo. A novel approach to overcome resistance to HER2 targeted therapy in a new syngeneic model of breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1971.

STAT3 labels a subpopulation of reactive astrocytes required for brain metastasis

The brain microenvironment imposes a particularly intense selective pressure on metastasis-initiating cells, but successful metastases bypass this control through mechanisms that are poorly understood. Reactive astrocytes are key components of this microenvironment that confine brain metastasis without infiltrating the lesion. Here, we describe that brain metastatic cells induce and maintain the co-option of a pro-metastatic program driven by signal transducer and activator of transcription 3 (STAT3) in a subpopulation of reactive astrocytes surrounding metastatic lesions. These reactive astrocytes benefit metastatic cells by their modulatory effect on the innate and acquired immune system. In patients, active STAT3 in reactive astrocytes correlates with reduced survival from diagnosis of intracranial metastases. Blocking STAT3 signaling in reactive astrocytes reduces experimental brain metastasis from different primary tumor sources, even at advanced stages of colonization. We also show that a safe and orally bioavailable treatment that inhibits STAT3 exhibits significant antitumor effects in patients with advanced systemic disease that included brain metastasis. Responses to this therapy were notable in the central nervous system, where several complete responses were achieved. Given that brain metastasis causes substantial morbidity and mortality, our results identify a novel treatment for increasing survival in patients with secondary brain tumors.

The influence of matrix stiffness on the behavior of brain metastatic breast cancer cells in a biomimetic hyaluronic acid hydrogel platform.

Breast cancer brain metastasis marks the most advanced stage of breast cancer no longer considered curable with a median survival period of ∼4-16 months. Apart from the genetic susceptibility (subtype) of breast tumors, brain metastasis is also dictated by the biophysical/chemical interactions of tumor cells with native brain microenvironment, which remain obscure, primarily due to the lack of tunable biomimetic in vitro models. To address this need, we utilized a biomimetic hyaluronic acid (HA) hydrogel platform to elucidate the impact of matrix stiffness on the behavior of MDA-MB-231Br cells, a brain metastasizing variant of the triple negative breast cancer line MDA-MB-231. We prepared HA hydrogels of varying stiffness (0.2-4.5 kPa) bracketing the brain relevant stiffness range to recapitulate the biophysical cues provided by brain extracellular matrix. In this system, we observed that the MDA-MB-231Br cell adhesion, spreading, proliferation, and migration significantly increased with the hydrogel stiffness. We also demonstrated that the stiffness based responses of these cells were mediated, in part, through the focal adhesion kinase-phosphoinositide-3 kinase pathway. This biomimetic material system with tunable stiffness provides an ideal platform to further the understanding of mechanoregulation associated with brain metastatic breast cancer cells. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1832-1841, 2018.

CD74 regulates complexity of tumor cell HLA class II peptidome in brain metastasis and is a positive prognostic marker for patient survival

Despite multidisciplinary local and systemic therapeutic approaches, the prognosis for most patients with brain metastases is still dismal. The role of adaptive and innate anti-tumor response including the Human Leukocyte Antigen (HLA) machinery of antigen presentation is still unclear. We present data on the HLA class II-chaperone molecule CD74 in brain metastases and its impact on the HLA peptidome complexity.We analyzed CD74 and HLA class II expression on tumor cells in a subset of 236 human brain metastases, primary tumors and peripheral metastases of different entities in association with clinical data including overall survival. Additionally, we assessed whole DNA methylome profiles including CD74 promoter methylation and differential methylation in 21 brain metastases. We analyzed the effects of a siRNA mediated CD74 knockdown on HLA-expression and HLA peptidome composition in a brain metastatic melanoma cell line.We observed that CD74 expression on tumor cells is a strong positive prognostic marker in brain metastasis patients and positively associated with tumor-infiltrating T-lymphocytes (TILs). Whole DNA methylome analysis suggested that CD74 tumor cell expression might be regulated epigenetically via CD74 promoter methylation. CD74high and TILhigh tumors displayed a differential DNA methylation pattern with highest enrichment scores for antigen processing and presentation. Furthermore, CD74 knockdown in vitro lead to a reduction of HLA class II peptidome complexity, while HLA class I peptidome remained unaffected.In summary, our results demonstrate that a functional HLA class II processing machinery in brain metastatic tumor cells, reflected by a high expression of CD74 and a complex tumor cell HLA peptidome, seems to be crucial for better patient prognosis.

Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.

TMIC-07. HIGH ALDEHYDE DEHYDROGENASE ACTIVITY IDENTIFIES BRAIN METASTASIS-INITIATING CELLS IN BREAST CANCER

Brain metastases (BM) from breast cancer (BC) are associated with significant morbidity and mortality. However, the mechanism of how BC cells initiate and propagate BM tumors remains elusive. In this study, we established a set of patient-derived models of circulating tumor cells (CTCs) from either hormone receptor positive (HR+) or negative (HR-) cells, and subsequent BM cells in vivo. Our brain-primed BC derivative lines exhibited a trend to form metastatic tumors in the brain more frequently than in the bones or lungs. In contrast, the naive BC cells rarely formed BM tumors. Micro-MRI of mouse brains after cardiac injection of brain-primed BC derivatives detected the persistent attachment and subsequent colonization of these circuiting cells, whereas naive BC cells, when injected into arterial circulation, were quickly eradicated from the brain. Transcriptomic expression analysis showed that ALDH isoforms including ALDH1A3 are highly upregulated in BM cells in contrast to BC cells. Silencing of ALDH1A3 by lentiviral shRNA infection specifically attenuated in vivo BM formation from the BC derivatives through arterial circulation, while it did not affect formation of the metastases in the lungs and bones. RNA sequencing detected that the expression of tissue transglutaminase (TG2), constitutive activator of transcription factor-kB (NF-kB), is higher in BM but not in lung and bone metastasis and its expression was attenuated by reducing ALDH1A3. Clinically, matched pairs of human primary BC and BM tissue samples showed the elevation of ALDH1A3 expression in BM tumors. In addition, ALDH1A3 expression was significantly correlated with poor prognosis. To develop therapeutics for ALDH1A3, we designed and synthesized a small molecule ALDH1A3 inhibitor, termed GA11. Systemic treatment of the CTC-BM mouse models with GA11 yielded a prominent inhibitory effect on BM formation. Taken together, our results suggest that ALDH1A3 might be a promising therapeutic target for BM tumors.

CMET-19. THE ROLE OF BRAIN SPECIFIC FATTY ACID BINDING PROTEIN 7 IN BREAST CANCER METASTASES TO THE BRAIN

HER2 overexpression in breast cancer increases the incidence of brain metastasis. Brain metastases are established after extravasation of cancer cells in the glia followed by invasion in the brain microenvironment. To identify critical genes for establishment of brain metastases, we conducted an In silico screening of publicly available datasets. Our screening identified FABP7 to be significantly overexpressed in brain metastatic patients as compared to patients with systemic metastases. In the NKI dataset overexpression of FABP7 was also correlated with poor prognosis. FABP7 is a brain-specific gene responsible for brain development and lipid metabolism. To establish the role of FABP7 in the brain metastatic process, FABP7 shRNA knockdown studies were conducted in BT-474BrM3 cells. Our results demonstrated a reduction in invasion ability of FABP7 knockdown cells. This result was accompanied with a significant decreased expression of genes that play a key role in the metastatic process such as OCT4, NANOG, ZEB2 or Carbonic anhydrase IX (Ca9). Moreover, phosphorylation of the p38 MAPK and ATF-2, another key signaling pathway regulating metastasis, was abrogated by FABP7 knockdown, suggesting that FABP7 is required for the kinase activity of p38 MAPK. In lieu of the fact that brain is normally under hypoxic environment, we simulated hypoxia in cell culture models. Interestingly under hypoxic conditions, FABP7 knockdown cells failed to induce angiogenic and invasive markers, such as VEGFA or PTPRZ1. Altogether, our results indicate that the expression of FABP7 by brain metastatic breast cancer cells allows the breast cancer cells to invade within the central nervous system.

Gene expression profiling of brain metastatic cell from triple negative breast cancer: Understanding the molecular events

Brain metastatic triple negative breast cancer (BM-TNBC) is afflicted with unfavorable prognosis. However, the molecular events underlying BM-TNBC remain largely unknown. In the present study, we conducted gene expression microarray analysis using the triple negative breast cancer cell line MDA-MB-231 and its brain metastatic derivative (MDA-MB-231Brm). Results of microarray analysis showed that a total of 4296 genes were differentially expressed, of which 2433 genes were up-regulated and 1863 genes were down-regulated. Gene Ontology (GO), KEGG pathway and protein-protein interaction (PPI) analyses indicated differentially expressed genes functionally categorized as genes of signal transduction, multicellular organismal development, ion transport, nervous system development, plasma membrane, extracellular region, calcium ion binding, GTP binding neuroactive ligand-receptor interaction. The validity of the microarray results was verified by quantitative real-time PCR analysis of twelve representative genes. The present findings revealed molecular basis and events associated with brain metastasis in TNBC, which will potentially contribute to the understanding of underlying mechanism and develop therapeutic targets.

Effects of FSTL1 on cell proliferation in breast cancer cell line MDA‑MB‑231 and its brain metastatic variant MDA‑MB‑231‑BR.

In the past decades, altered Follistatin-like 1 (FSTL1) expression has been documented in a variety of cancers, while its functional roles are poorly understood. Particularly in breast cancer, the expression of FSTL1 and its signaling pathway remain to be determined. In the present study, an elevated FSTL1 expression and a supressed cell proliferation were detected in a specific brain metastatic cell line MDA-MB-231-BR (231-BR), compared with its parental cell line MDA-MB-231. However, this protein was hardly detected in the other three breast cancer cell lines. Next, lentiviral vectors encoding FSTL1 or FSTL1 specific shRNAs were used to overexpress or knock down FSTL1 in MDA-MB-231 or 231-BR, respectively (MDA-MB-231FSTL1 or 231-BRsh FSTL1). Results showed that overexpression of FSTL1 inhibited MDA-MB-231 cell proliferation, while knockdown of FSTL1 in 231-BR cells promotes cell proliferation, compared with their corresponding control groups. These results were further confirmed in nude mouse xenografts. The tumor volume in 231-BR cell-bearing mice was significantly smaller than that of MDA-MB-231 group, and reduction of tumor volume was detected in MDA-MB-231FSTL1 cell-bearing mice compared with the control group. Previous studies revealed that TGF-β-Smad2/3 signaling pathway was activated in 231-BR and MDA-MB-231FSTL1 cells, which may contribute to the inhibited cell proliferation. In addition, Smad3 knockdown could restore the inhibition of cell proliferation induced by FSTL1 overexpression in MDA-MB-231FSTL1 cells, indicating that the anti-proliferative effect of FSTL1 overexpression may be associated with Smad3 involved TGF-β signaling pathway regulation. This study identified FSTL1 as an inhibitor of cell proliferation in MDA-MB-231 and 231-BR cell lines, which may provide new insights into the development and management of breast cancer.

CD271 determines migratory properties of melanoma cells

Melanoma cell expression of the nerve growth factor receptor CD271 is associated with stem-like properties. However, the contributing role of the receptor in melanoma cell migration is elusive. Here, we explored extracranial (skin, soft tissue, lymph node and liver, n = 13) and matched brain metastases (BM, n = 12) and observed a heterogeneous distribution of phenotypically distinct subsets of CD271+ cells. In addition, we observed that CD271 expression gradually rises along with melanoma progression and metastasis by exploration of publicly available expression data of nevi, primary melanoma (n = 31) and melanoma metastases (n = 54). Furthermore, we observed highest levels of CD271 in BM. Sub-clustering identified 99 genes differentially expressed among CD271high and CD271low (p < 0.05) BM-subgroups. Comparative analysis of subsets revealed increased ( ≥ 1.5fold, log2) expression of migration-associated genes and enrichment of CD271-responsible genes involved in DNA-repair and stemness. Live cell-imaging based scratch-wound assays of melanoma cells with stable knock-down of CD271 revealed a significantly reduced cell migration (3.9fold, p = 1.2E-04) and a reduced expression of FGF13, CSPG4, HMGA2 and AKT3 major candidate regulatory genes of melanoma cell migration. In summary, we provide new insights in melanoma cell migration and suggest that CD271 serves as a candidate regulator, sufficient to determine cellular properties of melanoma brain metastatic cells.