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Introduction Brain tumors cause morbidity and mortality in part through peritumoral brain edema. The current main treatment for peritumoral brain edema are corticosteroids. Due to the increased recognition of their side-effect profile, there is growing interest in finding alternatives to steroids but there is little formal study of animal models of peritumoral brain edema. This study aims to summarize the available literature. Methods A systematic search was undertaken of 5 literature databases (Medline, Embase, CINAHL, PubMed and the Cochrane Library). The generic strategy was to search for various terms associated with “brain tumors”, “brain edema” and “animal models”. Results We identified 603 reports, of which 112 were identified as relevant for full text analysis that studied 114 peritumoral brain edema animal models. We found significant heterogeneity in the species and strain of tumor-bearing animals, tumor implantation method and edema assessment. Most models did not produce appreciable brain edema and did not test for observable manifestations thereof. Conclusion No animal model currently exists that enable the investigation of novel candidates for the treatment of peritumoral brain edema. With current interest in alternative treatments for peritumoral brain edema, there is an unmet need for clinically relevant animal models.

Table containing all brain metastatic cell lines in the BrMPanel

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood–brain barrier, blood–tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model in vivo. These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.

Contains experimental therapies tested on brain metastatic cell lines and associated clinical trials

Supporting information is available online at https://aasldpubs.onlinelibrary.wiley.com/doi/full/10.1002/hep.31983#support-information-section . From 2023 Hepatology is published by Wolters Kluwer Health on behalf of the American Association for the Study of Liver Diseases. Background and Aims Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive type of liver cancer in urgent need of treatment options. Aberrant activation of c-Jun N-terminal kinase (JNK) pathway is a key feature in ICC and an attractive candidate target for its treatment. However, the mechanisms by which constitutive JNK activation promotes ICC growth, and thus the key downstream effectors of this pathway remain unknown for their applicability as therapeutic targets. Our aim was to obtain a better mechanistic understanding of the role of JNK signalling in ICC that could open new therapeutic opportunities. Approach and Results Using loss- and gain-of-function studies in vitro and in vivo, we show that activation of the JNK pathway promotes ICC cell proliferation by affecting the protein stability of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a key driver of tumorigenesis. PIN1 is highly expressed in ICC primary tumours, and its expression positively correlates with active JNK. Mechanistically, the JNK kinases directly bind to and phosphorylate PIN1 at Ser115, and this phosphorylation prevents PIN1 mono-ubiquitination at Lys117 and its proteasomal degradation. Moreover, pharmacological inhibition of PIN1 via all-trans retinoic acid (ATRA), an FDA-approved drug, impairs the growth of both cultured and xenografted ICC cells. Conclusions Our findings implicate the JNK-PIN1 regulatory axis as a functionally important determinant for ICC growth, and provide a rationale for therapeutic targeting of JNK activation via PIN1 inhibition. AMMF - The Cholangiocarcinoma Charity. Grant Number: 2014; Foundation for Liver Research. Grant Number: Start-up 2010; Guts UK. Grant Number: DGO2019_02; Rosetrees Trust. Grant Number: M894; Brunel University London. Grant Number: BRIEF LBL348; Blood Cancer UK. Grant Number: 17014; Faculty of Medicine and Health, University of Leeds. Grant Number: 250 ; Great Minds University Academic Fellowship 2016.

Background Brain metastases (BrM) develop in 20–40% of cancer patients and represent an unmet clinical need. Limited access of drugs into the brain because of the blood-brain barrier is at least partially responsible for therapeutic failure, necessitating improved drug delivery systems. Methods Green fluorescent protein (GFP)-transduced murine and nontransduced human hematopoietic stem cells (HSCs) were administered into mice (n = 10 and 3). The HSC progeny in mouse BrM and in patient-derived BrM tissue (n = 6) was characterized by flow cytometry and immunofluorescence. Promoters driving gene expression, specifically within the BrM-infiltrating HSC progeny, were identified through differential gene-expression analysis and subsequent validation of a series of promoter-green fluorescent protein-reporter constructs in mice (n = 5). One of the promoters was used to deliver tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) to BrM in mice (n = 17/21 for TRAIL vs control group). Results HSC progeny (consisting mostly of macrophages) efficiently homed to macrometastases (mean [SD] = 37.6% [7.2%] of all infiltrating cells for murine HSC progeny; 27.9% mean [SD] = 27.9% [4.9%] of infiltrating CD45+ hematopoietic cells for human HSC progeny) and micrometastases in mice (19.3–53.3% of all macrophages for murine HSCs). Macrophages were also abundant in patient-derived BrM tissue (mean [SD] = 8.8% [7.8%]). Collectively, this provided a rationale to optimize the delivery of gene therapy to BrM within myeloid cells. MMP14 promoter emerged as the strongest promoter construct capable of limiting gene expression to BrM-infiltrating myeloid cells in mice. TRAIL delivered under MMP14 promoter statistically significantly prolonged survival in mice (mean [SD] = 19.0 [3.4] vs mean [SD] = 15.0 [2.0] days for TRAIL vs control group; two-sided P = .006), demonstrating therapeutic and translational potential of our approach. Conclusions Our study establishes HSC gene therapy using a myeloid cell–specific promoter as a new strategy to target BrM. This approach, with strong translational value, has potential to overcome the blood-brain barrier, target micrometastases, and control multifocal lesions.

T cell receptor (TCR) triggering by antigen results in metabolic reprogramming that, in turn, facilitates the exit of T cells from quiescence. The increased nutrient requirements of activated lymphocytes are met, in part, by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids, and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine is a nonessential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular asparagine and ASNS activity in CD8+ T cell activation. At early time points of activation in vitro, CD8+ T cells expressed little or no ASNS, and, as a consequence, viability and TCR-stimulated growth, activation, and metabolic reprogramming were substantially impaired under conditions of asparagine deprivation. At later time points (more than 24 hours of activation), TCR-induced mTOR-dependent signals resulted in ASNS upregulation that endowed CD8+ T cells with the capacity to function independently of extracellular asparagine. Thus, our data suggest that the coordinated upregulation of ASNS expression and uptake of extracellular asparagine is involved in optimal T cell effector responses.

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood–brain barrier, blood–tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model in vivo. These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.

BACKGROUND Up to 60% of melanoma patients develop brain metastases (BrM). These patients have a poor prognosis and limited treatment options. Immune checkpoint inhibitors (ICI) targeting Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and Programmed cell death protein-1 (PD-1) have revolutionized the treatment of melanoma and their efficacy has been also demonstrated in melanoma BrM. Our group previously demonstrated that ICI (combined α-PD-1 and α-CTLA-4) enhances chemokine-dependent infiltration of cytotoxic T lymphocytes (CTLs) into melanoma BrMs in preclinical models, accompanied by upregulated expression of T cell attracting chemokines in tumours. Notably, CTLs infiltrating BrM expressed only some of the chemokine receptors (CRs) interacting with ICI-induced chemokines in BrM, providing a rationale to over-express the “missing” CRs in T cells to enhance their homing to tumours in the context of adoptive T cell therapy (ACT). MATERIALS AND METHODS OT-I cells were isolated from OT-I mice and differentiated ex vivo into effector (TEF) and memory (TCM) CD8+ T cells. Tumour infiltrating lymphocytes (TILs) from B16 tumour-bearing mice treated with ICI were isolated using magnetic beads, activated and expanded ex vivo. Expression of CRs and activation markers in ex vivo cultured T cells were quantified by qPCR and/or flow cytometry. The migration of human blood CD8+ T cells towards chemokines of interest were measured in ex vivo migration assays. RESULTS The same CRs that were missing on BrM-infiltrating CTLs in vivo models were also absent from OT-I TEF (CCR7low/CD44high/CD62Llow) and TCM (CCR7high/CD44low/CD62Lhigh) cells, as well as from TILs expanded ex vivo for use in ACT. Furthermore, we observed no increase in migration of human T cells towards chemokines interacting with the “missing” CRs in comparison to the baseline migration, suggesting that these CRs are also absent from human T cells. CONCLUSION Ex vivo expanded T cells that are used in ACT are missing several CRs that are interacting with chemokines upregulated in BrM. We hypothesise that the use of genetically engineered T cells expressing the “missing” CRs in ACT has the potential to enhance ACT efficacy in combination with ICI.

Recent evidence suggests that the ion channel TRPA1 is implicated in lung adenocarcinoma (LUAD), where its role and mechanism of action remain unknown. We have previously established that the membrane receptor FGFR2 drives LUAD progression through aberrant protein–protein interactions mediated via its C-terminal proline-rich motif. Here we report that the N-terminal ankyrin repeats of TRPA1 directly bind to the C-terminal proline-rich motif of FGFR2 inducing the constitutive activation of the receptor, thereby prompting LUAD progression and metastasis. Furthermore, we show that upon metastasis to the brain, TRPA1 gets depleted, an effect triggered by the transfer of TRPA1-targeting exosomal microRNA (miRNA-142-3p) from brain astrocytes to cancer cells. This downregulation, in turn, inhibits TRPA1-mediated activation of FGFR2, hindering the metastatic process. Our study reveals a direct binding event and characterizes the role of TRPA1 ankyrin repeats in regulating FGFR2-driven oncogenic process; a mechanism that is hindered by miRNA-142-3p., TRPA1 has been reported to contribute lung cancer adenocarcinoma (LUAD), but the mechanisms are unclear. Here the authors propose that TRPA1/FGFR2 interaction is functional in LUAD and show that astrocytes oppose brain metastasis by mediating the downregulation of TRPA1 through exosome-delivered miRNA-142-3p.

Central nervous system (CNS), notably brain, metastases are most prevalent in lung cancer (20–56% of patients), breast cancer (5–20%) and melanoma (7–16%). Lesions occur in both the brain parenchyma and the meninges. To mechanistically understand CNS metastasis formation and develop preventive and therapeutic strategies, it is essential to use model systems that, as much as possible, faithfully recapitulate the clinical disease process. Furthermore, the complexities of brain metastases dictate that studies should utilize multiple model systems in various stages of brain metastases progression. To facilitate brain metastasis research, 19 laboratories around the world have compiled comprehensive information on their brain metastasis mouse models. Each lab has provided details on the cell lines that they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. This Brain Metastasis Cell Lines Panel (BrMPanel, https://apps.cnio.es/app/BrainMetastasis/CellLines) represents the first of its class and includes information about each cell line, how tropism to the brain was established, and the behavior of each model in vivo. The BrMPanel is composed of 60 cell lines, derived from patients (32 cell lines, 53%), mouse (27, 45%) or rat (1, 2%), and represent the three main cancer types that result in brain metastasis: breast cancer (38 cell lines, 63%), lung cancer (8, 13%) and melanoma (14, 23%). This resource is intended to assist investigators in choosing the most suitable model for research on brain metastasis, and is available to the entire scientific community. The ultimate goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources. We invite other collaborators to contribute their models to the BrMPanel to grow this resource.

Immune checkpoints restrain the immune system following its activation and their inhibition unleashes anti-tumor immune responses. Immune checkpoint inhibitors revolutionized the treatment of several cancer types, including melanoma, and immune checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies is becoming a frontline therapy in metastatic melanoma. Notably, up to 60% of metastatic melanoma patients develop metastases in the brain. Brain metastases (BrM) are also very common in patients with lung and breast cancer, and occur in ∼20-40% of patients across different cancer types. Metastases in the brain are associated with poor prognosis due to the lack of efficient therapies. In the past, patients with BrM used to be excluded from immune-based clinical trials due to the assumption that such therapies may not work in the context of "immune-specialized" environment in the brain, or may cause harm. However, recent trials in patients with BrM demonstrated safety and intracranial activity of anti-PD-1 and anti-CTLA-4 therapy. We here discuss how immune checkpoint therapy works in BrM, with focus on T cells and the cross-talk between BrM, the immune system, and tumors growing outside the brain. We discuss major open questions in our understanding of what is required for an effective immune checkpoint inhibitor therapy in BrM.

Patients with glioblastoma (GBM) have a poor prognosis, and inefficient delivery of drugs to tumors represents a major therapeutic hurdle. Hematopoietic stem cell (HSC)-derived myeloid cells efficiently home to GBM and constitute up to 50% of intratumoral cells, making them highly appropriate therapeutic delivery vehicles. Because myeloid cells are ubiquitously present in the body, we recently established a lentiviral vector containing matrix metalloproteinase 14 (MMP14) promoter, which is active specifically in tumor-infiltrating myeloid cells as opposed to myeloid cells in other tissues, and resulted in a specific delivery of transgenes to brain metastases in HSC gene therapy. Here, we used this novel approach to target transforming growth factor beta (TGFβ) as a key tumor-promoting factor in GBM. Transplantation of HSCs transduced with lentiviral vector expressing green fluorescent protein (GFP) into lethally irradiated recipient mice was followed by intracranial implantation of GBM cells. Tumor-infiltrating HSC progeny was characterized by flow cytometry. In therapy studies, mice were transplanted with HSCs transduced with lentiviral vector expressing soluble TGFβ receptor II–Fc fusion protein under MMP14 promoter. This TGFβ-blocking therapy was compared with the targeted tumor irradiation, the combination of the two therapies, and control. Tumor growth and survival were quantified (statistical significance determined by t-test and log-rank test). T cell memory response was probed through a repeated tumor challenge. Myeloid cells were the most abundant HSC-derived population infiltrating GBM. TGFβ-blocking HSC gene therapy in combination with irradiation significantly reduced tumor burden as compared with monotherapies and the control, and significantly prolonged survival as compared with the control and TGFβ-blocking monotherapy. Long-term protection from GBM was achieved only with the combination treatment (25% of the mice) and was accompanied by a significant increase in CD8+ T cells at the tumor implantation site following tumor rechallenge. We demonstrated a preclinical proof-of-principle for tumor myeloid cell-specific HSC gene therapy in GBM. In the clinic, HSC gene therapy is being successfully used in non-cancerous brain disorders and the feasibility of HSC gene therapy in patients with glioma has been demonstrated in the context of bone marrow protection. This indicates an opportunity for clinical translation of our therapeutic approach.

In contrast to primary tumors, the understanding of macrophages within metastases is very limited. In order to compare macrophage phenotypes between different metastatic sites, we established a pre-clinical mouse model of intracranial breast cancer metastasis in which cancer lesions develop simultaneously within the brain parenchyma and the dura. This mimics a situation that is commonly occurring in the clinic. Flow cytometry analysis revealed significant differences in the activation state of metastasis-associated macrophages (MAMs) at the two locations. Concurrently, gene expression analysis identified significant differences in molecular profiles of cancer cells that have metastasized to the brain parenchyma as compared to the dura. This included differences in inflammation-related pathways, NF-kB1 activity and cytokine profiles. The most significantly upregulated cytokine in brain parenchyma- versus dura-derived cancer cells was Lymphotoxin β and a gain-of-function approach demonstrated a direct involvement of this factor in the M2 polarization of parenchymal MAMs. This established a link between metastatic site-specific properties of cancer cells and the MAM activation state.

Pharmacological inhibition of uncontrolled cell growth with small-molecule inhibitors is a potential strategy for treating glioblastoma multiforme (GBM), the most malignant primary brain cancer. We showed that the synthetic small-molecule KHS101 promoted tumor cell death in diverse GBM cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM.

Brain metastases are an unmet clinical need with high frequency in melanoma patients. With immune checkpoint inhibitors targeting programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) becoming a frontline therapy in melanoma, it is critical to understand how this therapy works in the “immune-specialized” brain microenvironment. Our study shows that in the absence of extracranial tumor, melanoma tumors growing in the brain escape anti–PD-1/anti–CTLA-4 therapy. A synergy between immune checkpoint inhibition and extracranial tumor is required to put a break on brain metastases by enhancing CD8+ T cell recruitment to the brain via peripheral expansion of effector cells and upregulation of T cell entry receptors on tumor blood vessels. Augmentation of these processes could be explored to enhance the efficacy of immunotherapy in brain metastases.

Inhibition of immune checkpoints programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) on T cells results in durable antitumor activity in melanoma patients. Despite high frequency of melanoma brain metastases (BrM) and associated poor prognosis, the activity and mechanisms of immune checkpoint inhibitors (ICI) in metastatic tumors that develop within the “immune specialized” brain microenvironment, remain elusive. We established a melanoma tumor transplantation model with intracranial plus extracranial (subcutaneous) tumor, mimicking the clinically observed coexistence of metastases inside and outside the brain. Strikingly, intracranial ICI efficacy was observed only when extracranial tumor was present. Extracranial tumor was also required for ICI-induced increase in CD8 + T cells, macrophages, and microglia in brain tumors, and for up-regulation of immune-regulatory genes. Combined PD-1/CTLA-4 blockade had a superior intracranial efficacy over the two monotherapies. Cell depletion studies revealed that NK cells and CD8 + T cells were required for intracranial anti–PD-1/anti–CTLA-4 efficacy. Rather than enhancing CD8 + T cell activation and expansion within intracranial tumors, PD-1/CTLA-4 blockade dramatically (∼14-fold) increased the trafficking of CD8 + T cells to the brain. This was mainly through the peripheral expansion of homing-competent effector CD8 + T cells and potentially further enhanced through up-regulation of T cell entry receptors intercellular adhesion molecule 1 and vascular adhesion molecule 1 on tumor vasculature. Our study indicates that extracranial activation/release of CD8 + T cells from PD-1/CTLA-4 inhibition and potentiation of their recruitment to the brain are paramount to the intracranial anti–PD-1/anti–CTLA-4 activity, suggesting augmentation of these processes as an immune therapy-enhancing strategy in metastatic brain cancer.

Pharmacological inhibition of uncontrolled cell growth with small molecule inhibitors is a potential strategy against glioblastoma multiforme (GBM), the most malignant primary brain cancer. Phenotypic profiling of the neurogenic small molecule KHS101 revealed the chemical induction of lethal cellular degradation in molecularly-diverse GBM cells, independent of their tumor subtype, whereas non-cancerous brain cells remained viable. Mechanism-of-action (MOA) studies showed that KHS101 specifically bound and inhibited the mitochondrial chaperone HSPD1. In GBM but not non-cancerous brain cells, KHS101 elicited the aggregation of an enzymatic network that regulates energy metabolism. Compromised glycolysis and oxidative phosphorylation (OXPHOS) resulted in the metabolic energy depletion in KHS101-treated GBM cells. Consistently, KHS101 induced key mitochondrial unfolded protein response factor DDIT3 in vitro and in vivo, and significantly reduced intracranial GBM xenograft tumor growth upon systemic administration, without discernible side effects. These findings suggest targeting of HSPD1-dependent oncometabolic pathways as an anti-GBM therapy.

Brain tumours are one of the most aggressive forms of cancer with limited treatment options. Current treatment options include surgical resection of the main tumour mass, radiation and chemotherapy treatments. Surgical resection will inadvertently leave ~ 5 % of cancer cells within difficult anatomical locations in the brain, while radiation and chemotherapy treatments will often lead to unwanted systemic toxicities. In an attempt to minimise systemic toxicities, we aim to develop a targeted approach for the delivery of therapeutic agents using haematopoietic stem cells (HSCs) as cellular delivery vehicles. We first evaluated the suitability of HSCs as drug delivery vehicles for brain tumours in a syngeneic mouse model. Murine Sca1+ HSCs isolated from mouse bone marrow (BM) were transduced with the lentiviral vector pFUGW expressing green fluorescent protein (GFP) and transplanted into lethally irradiated C57BL/6J mice. This resulted in successful BM engraftment as confirmed by flow cytometry. Subsequently, brain tumours in these mice were generated by intracranial implantation of primary glioma cell lines (GL261 and CT-2A) to address primary brain tumours or the murine breast carcinoma cell line, EO771, to address breast cancer brain metastases. Our studies show that the progeny of GFP-tagged HSCs strongly infiltrated primary brain tumours and brain metastases, with the main immune cell infiltrating population being CD11b+/F4/80+ macrophages. To address the issue of targeted therapy delivery specifically to brain lesions, we sought to identify gene promoters whose activity is specifically upregulated in the HSC progeny that infiltrates brain tumours. Gene expression analysis identified 3 genes whose expression is specifically upregulated in brain tumour infiltrating macrophages as opposed to macrophages infiltrating other tissues, namely bone marrow and spleen. To validate promoter specificity in our mouse models, we generated promoter:reporter constructs to drive GFP expression downstream of these 3 identified promoters. To this end, C57BL/6J mice were lethally irradiated and reconstituted with genetically modified murine Sca1+ HSCs expressing GFP downstream of the 3 identified promoters. Following successful bone marrow reconstitution, we tested promoter specificity in mouse models of breast cancer brain metastases. Our results show that 2 of the promoters display specific activity in the brain as opposed to other tissues (blood, bone marrow, spleen, lungs, liver, heart), suggesting that HSC progenitors can drive delivery of therapeutics specifically to the brain downstream of these 2 promoters. We now plan to test the delivery of therapeutic agents downstream of these tissue-specific promoters. This study demonstrates a proof-of-concept for using HSCs as cellular delivery vehicles for the targeted delivery of therapeutics to the brain to overcome the blood-brain barrier and has potential for combination therapy approaches in the future.

Central nervous system (CNS) metastases develop in ~15% of metastatic breast cancer patients and are associated with a very poor prognosis due to the lack of effective therapies. Intracranial metastases are mainly located within the brain parenchyma, at the leptomeninges or at the dura. Although simultaneous involvement of multiple intracranial locations is very common in breast cancer, the experimental studies have been focusing mainly on parenchymal brain metastases. To address the knowledge gap in understanding of metastases at other CNS locations, we developed a preclinical mouse model of intracranial breast cancer metastases with simultaneous involvement of brain parenchyma and the dura. This enabled us to subsequently study and compare cancer cell phenotypes and inflammatory tumour microenvironment at the two intracranial locations. Simultaneous colonization of brain parenchyma and the dura was achieved by administration of cancer cells into the internal carotid artery of mice. This resulted in a significantly higher dural as compared to the parenchymal tumour burden across 3 different breast cancer models. The inflammatory tumour microenvironment in dural and parenchymal brain metastases was subsequently analysed by flow cytometry. Although model-specific differences in metastases-infiltrating immune cell populations were detected, microglia/macrophages were the most abundant cell population across all 3 models. In contrast to primary tumours, the understanding of macrophages within metastases is very limited and comparison of macrophage phenotypes between different metastatic sites is lacking. Through analysis of multiple intra- and extra-cellular markers by flow cytometry, we discovered significant differences in the activation state of metastasis-associated macrophages (MAMs) at the two sites. Interestingly, the phenotype of parenchymal MAMs was causally linked to the metastatic site-specific cancer cell features. Gene expression analysis revealed that cancer cells that have metastasized to brain parenchyma evolved different molecular profiles compared with metastatic cells isolated from the dura. Significant differences in several inflammation-related pathways, NF-kB1 activity and cytokine profiles were detected. The most significantly upregulated cytokine in brain parenchyma- versus dura-derived cancer cells was Lymphotoxin β and a gain-of-function approach demonstrated a direct involvement of this factor in the polarization of parenchymal MAMs towards the M2 state. Because macrophages and their activation state have been implicated in tumor growth and modulation of responses to therapies, the understanding of metastatic site-specific MAM differences is important for the design of treatment strategies that can effectively target metastases at distinct anatomical locations.

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease.

At late stage, nearly all breast cancers are heterogeneous and refractory to treatment, like metaplastic breast cancer is at an early stage. These rare carcinomas are highly aggressive and de-differentiated. They are enriched for mesenchymal and stem cell features and essentially fail current therapies. As metaplastic tumors provide a time-compressed picture of breast cancer progression early on, understanding these tumors will yield insight into mechanisms that drive breast cancer into advanced stages and treatment resistance. To investigate a genetic basis for heterogeneity in metaplastic breast cancer, we established a progression model comprising three cell lines. The cell lines were derived from a primary tumor, a local recurrence and a pleural effusion of a 40-year old patient. The primary tumor was a stage III invasive metaplastic, triple negative, inflammatory breast cancer, resected after neoadjuvant chemotherapy (capecitabine and taxotere, then adriamycin and one cycle of bevacizumab). The local recurrence, biopsied seven months post mastectomy, developed after the patient received adjuvant carboplatin and gemcitabine for 3 cycles and then radiation to the chest wall. At this time, the patient had lung metastases and was treated with taxol and bevacizumab yielding a mixed response. Local invasive growth continued and a malignant pleural effusion developed four months later. Analyzing the genetic and molecular characteristics of this progression model in vitro, its tumorigenicity and metastasis in vivo, and interrogating lead findings in a growing collection of metaplastic tumors helps us to dissect the genetic heterogeneity in breast cancer, and potentially to identify the cell types that drive disease progression and treatment resistance. Our gene expression analyses and genomic evaluations identified epithelial to mesenchymal transition (EMT) as a key characteristic in the progression and treatment resistance of this cancer. Major changes in cytoskeletal genes, chemokines and their receptors, amplification of drug transporter proteins, metalloproteinases and matrix proteins seen with increasing motility and invasiveness along with recruitment of host inflammatory responses in the in vivo model, loss of chromosomal regions harboring known and putative tumor suppressors, and deletions of genes encoding proteins for metabolic inactivation of sex hormones in the breast tissue, along with specific loss of clusters of desmosomal genes are guiding our understanding of metaplastic breast cancer progression. The results provide insight into the development, the extremely invasive nature, and treatment resistance of these tumors. Our collaborative network of clinicians, pathologists, translational genomic researchers and bioinformatics specialists will enable us to identify and prioritize genetic events as disease drivers, prognostic biomarkers of disease progression, and determinants of treatment resistance. Our goal is to identify molecular and functional targets for effective therapy and evaluate them in the clinic. Lessons learned from metaplastic breast cancer will improve our understanding of breast cancer progression in general, and could translate into effective treatments for advanced breast cancer where current standard of care is failing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD03-07.

Brain metastases occur in 20 to 40% of patients with metastatic breast cancer. The process is complex and depends on successful cancer cell evasion from the primary tumor, distribution and survival within the blood stream and cerebral microvasculature, penetration of the blood brain barrier and proliferation within the brain microenvironment. The initial steps of brain colonization are difficult to study in vivo. Therefore, in vitro assays have been developed to mimic this process. Most commonly, in vitro studies of brain colonization focus on tumor cell adhesion to brain endothelial cells and transendothelial migration. We previously investigated breast cancer brain colonization from the blood stream in vivo and defined the time and process of brain entry for five different cancer cell lines in a mouse model. We now investigated if in vitro approaches can reliably emulate the initial steps that determine successful brain colonization in vivo. To this end, we optimized an in vitro model of the vascular blood brain barrier and compared the brain invasion properties of the in vivo characterized cell models with their ability to interact with and penetrate the blood brain barrier model in vitro. Our results show that the in vitro findings correlate only poorly with the vivo results. The limitations of the in vitro approaches are discussed in light of the in vivo processes. We conclude that investigation of mechanisms supporting the earliest steps of breast cancer brain metastasis from the blood stream will depend on in vivo analyses.

SummaryGlioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.

The incidence of brain metastasis is rising and poses a severe clinical problem, as we lack effective therapies and knowledge of mechanisms that control metastatic growth in the brain. Here we demonstrate a crucial role for high-affinity tumor cell integrin α v β 3 in brain metastatic growth and recruitment of blood vessels. Although α v β 3 is frequently up-regulated in primary brain tumors and metastatic lesions of brain homing cancers, we show that it is the α v β 3 activation state that is critical for brain lesion growth. Activated, but not non-activated, tumor cell α v β 3 supports efficient brain metastatic growth through continuous up-regulation of vascular endothelial growth factor (VEGF) protein under normoxic conditions. In metastatic brain lesions carrying activated α v β 3 , VEGF expression is controlled at the post-transcriptional level and involves phosphorylation and inhibition of translational respressor 4E-binding protein (4E-BP1). In contrast, tumor cells with non-activated α v β 3 depend on hypoxia for VEGF induction, resulting in reduced angiogenesis, tumor cell apoptosis, and inefficient intracranial growth. Importantly, the microenvironment critically influences the effects that activated tumor cell α v β 3 exerts on tumor cell growth. Although it strongly promoted intracranial growth, the activation state of the receptor did not influence tumor growth in the mammary fat pad as a primary site. Thus, we identified a mechanism by which metastatic cells thrive in the brain microenvironment and use the high-affinity form of an adhesion receptor to grow and secure host support for proliferation. Targeting this molecular mechanism could prove valuable for the inhibition of brain metastasis.

AF6 and its rat homologue afadin are multidomain proteins localized at cell junctions and involved in intercellular adhesion. AF6 interacts via its PDZ domain with nectin-1 at epithelial adherens junctions. Nectin-1 serves as a mediator of cell-to-cell spread for Herpes simplex virus 1 (HSV-1). We analyzed the role of AF6 protein in the viral spread and nectin-1 clustering at cell–cell contacts by knockdown of AF6 in epithelial cells. AF6 knockdown reduced efficiency of HSV-1 spreading, however, the clustering of nectin-1 at cell–cell contacts was not affected. Thus, AF6 protein is important for spreading of HSV-1 in epithelial cells, independently of nectin clustering, possibly by stabilization of the E-cadherin-dependent cell adhesion.

African trypanosomes are extracellular blood parasites that cause sleeping sickness in humans and Nagana in cattle. The therapeutics used to control and treat these diseases are very ineffective and thus, the development of new drugs is urgently needed. We have previously suggested to use trypanosome-specific RNA aptamers as tools for the development of novel trypanocidal compounds. Here, we report the selection of a 2'-NH(2)-modified RNA aptamer that binds to live trypanosomes with an affinity of 70 +/- 15 nM. The aptamer adopts a stable G-quartet structure and has a half-life in human serum of > 30 h. RNA binding is restricted to the flagellar attachment zone, located between the cell body and the flagellum of the parasite. We demonstrate that antigen-tagged preparations of the aptamer can bind to live trypanosomes and that they can be used to re-direct immunoglobulins to the parasite surface.

African trypanosomes cause sleeping sickness in humans and Nagana in cattle. The parasites multiply in the blood and escape the immune response of the infected host by antigenic variation. Antigenic variation is characterized by a periodic change of the parasite protein surface, which consists of a variant glycoprotein known as variant surface glycoprotein (VSG). Using a SELEX (systematic evolution of ligands by exponential enrichment) approach, we report the selection of small, serum-stable RNAs, so-called aptamers, that bind to VSGs with subnanomolar affinity. The RNAs are able to recognize different VSG variants and bind to the surface of live trypanosomes. Aptamers tethered to an antigenic side group are capable of directing antibodies to the surface of the parasite in vitro. In this manner, the RNAs might provide a new strategy for a therapeutic intervention to fight sleeping sickness.

During angiogenesis, Rho-GTPases influence endothelial cell migration and cell–cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell–cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis., Blood vessel development depends upon endothelial cell migration and adhesion, which are regulated by Rho-GTPases. Here the authors identify Rho-GTPase guanine nucleotide exchange factors that specifically control lateral filopodial contacts and are required for lumen formation during angiogenesis.

The shift from a normal to high glycolytic state (Warburg effect) is a central hallmark of almost every cancer and a strong driving force for cancer cell survival and disease progression. Importantly, studies - including our own - have shown that normalization of metabolic state in cancer cells can halt or reverse malignancy. To date, the intrinsic and extrinsic factors that drive metabolic shifts in cancer cells are not entirely clear. A better understanding of how the metabolic state of cancer cells is regulated will give us important tools to inhibit disease progression. We have established a novel model of breast cancer progression consisting of three cancer cell lines established from the primary tumor (TES1) and two sites of metastasis (TES2b and TESPE) of the same patient. We compared gene expression profiles between primary and recurrent tumor cells to identify factors that regulate disease progression and dissemination. Key differences between cells derived from primary and recurrent disease were an activation of genes involved in glycolysis and genes involved in activation and maintenance of the epithelial to mesenchymal transition (EMT) program, a powerful driver of cancer metastasis. Our analyses on a functional relationship between EMT and activation of glycolysis uncovered a direct link between the malignancy-driving EMT mechanism in breast cancer progression and metabolic reprogramming of the tumor cells. Specifically, we identified Twist1, a pro-metastatic master regulator of EMT as a direct modulator of a key glycolytic enzyme known to modulate the rate of glycolysis, 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3). We found that Twist1 binds directly to the promoter region of PFKFB3 and induces expression of the enzyme. Additionally, inhibition of Twist1 expression in TES2b (TES2b-shTwist1) cells decreased glycolytic flux, while Twist1 overexpression in HMLE immortalized human breast epithelial cells (HMLE-Twist1) increased glycolytic activity and simultaneously decreased cellular respiration. Metabolomics analyses of HMLE vs. HMLE-Twist1 cells using Gas Chromatography Mass Spectroscopy (GCMS) indicated that Twist1 expression is sufficient to induce drastic changes in the overall metabolic state of the cells, including glutamine metabolism and availability of TCA cycle intermediates and amino acids. Together, our findings are significant as they underscore the importance and potency of EMT activation in cancer and uncover a novel mechanism by which EMT activation through Twist1 drives metabolic alterations that fuel cancer progression and metastasis. Results from this study will ultimately aid in the discovery and development of new paradigms to fight cancer mortality rates around the world. Citation Format: Esmeralda Casas, Antonio F. Santidrian, Mihaela Lorger, Ali Torkamani, Boris Ratnikov, Sheena M. Saayman, Julie B. Steele, Smith W. Jeffrey, Brunhilde Felding. Twist1 modifies the metabolic state of breast cancer cells through transcriptional activation of a key regulator of glycolysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 57.

Central nervous system metastases develop in ~15% of metastatic breast cancer patients and are associated with a very poor prognosis due to the lack of effective therapies. Intracranial metastases are mainly located within the brain parenchyma, in the skull, at the leptomeninges or at the dura. Although simultaneous involvement of multiple intracranial locations is very common in breast cancer, the experimental studies have been focusing mainly on parenchymal brain metastases. To address the knowledge gap in understanding of metastases at other CNS locations, we developed preclinical mouse models of intracranial breast cancer metastases with simultaneous involvement of brain parenchyma and the dura. This enabled us to subsequently study and compare cancer cell phenotype and inflammatory tumour microenvironment at the two intracranial locations. Simultaneous colonization of brain parenchyma and the dura was achieved by administration of cancer cells into the internal carotid artery of mice. This resulted in a significantly higher dural as compared to the parenchymal tumour burden across 3 different breast cancer models. The inflammatory tumour microenvironment in dural and parenchymal brain metastases was subsequently analysed by flow cytometry. Although model-specific differences in metastases-infiltrating immune cell populations were detected, microglia/macrophages were the most abundant cell population across all 3 models. Further analysis of this cell population revealed that microglia were the predominant population within parenchymal metastases, while they were almost absent from the dural metastases. Macrophages were present at both locations and were significantly more abundant at the dura. In comparison to parenchymal macrophages, dural macrophages expressed significantly higher levels of MHCII and CD11c, which was suggestive of a higher antigen presenting capacity. We next investigated site-specific cancer cell phenotypes. To this end, dura- and brain parenchyma-tropic cancer cell variants were generated through 3 rounds of in vivo selection and investigated by gene expression profiling. Among others, this revealed significant differences in inflammation-related pathways, expression of inflammatory cytokines and differences in activity of transcription factors NFKB1 and TCF4. This was in line with distinct expression of antigen presenting cell markers in dural versus parenchymal macrophages. Further investigation into polarization of macrophages revealed site-specific polarization patterns, with dural macrophages being skewed towards M1 phenotype as compared to the parenchymal macrophages. In the above study we established pre-clinical models of intracranial breast cancer metastases with simultaneous involvement of brain parenchyma and the dura. Our study demonstrated that cancer cells at those two clinically relevant intracranial locations acquire different site-specific phenotypes, which was reflected in distinct polarization phenotypes of macrophages. Due to distinct phenotypes of cancer cells as well as macrophages, parenchymal brain metastases and dural metastases may respond differently to certain therapies (e.g. immune therapies). Therefore, these site-specific differences may need to be considered in the treatment of “multi-site” central nervous system metastases. Citation Format: Nora Rippaus, Jennifer Williams, David Taggart, Tereza Andreou, Krzysztof Wronski, Mihaela Lorger. Dural and parenchymal brain metastases in breast cancer are characterized by distinct inflammatory tumour microenvironments. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B37.

Integrins have been identified as major contributors to the progression of brain cancer. This includes the development and spreading of primary brain tumors and, more recently, also metastatic disease originating from solid tumors outside the brain. In these processes, integrin functions affect not only the tumor cells themselves but also the reactions of host cells that respond to the presence of developing lesions within the brain microenvironment. Integrin functions can promote cell survival, proliferation, invasion and regulatory adjustments of signaling cascades ­triggered by other receptor types, including those for growth factors. Supported by the promise of ongoing clinical trials, specific targeting of certain integrins and their functions could thus prove successful for new therapeutic approaches against brain cancer and metastatic disease in the central nervous system.

Brain metastases are difficult to treat and mostly develop late during progressive metastatic disease. Patients at risk would benefit from the development of prevention and improved treatments. This requires knowledge of the initial events that lead to brain metastasis. The present study reveals cellular events during the initiation of brain metastasis by breast cancer cells and documents the earliest host responses to incoming cancer cells after carotid artery injection in immunodeficient and immunocompetent mouse models. Our findings capture and characterize heterogeneous astrocytic and microglial reactions to the arrest and extravasation of cancer cells in the brain, showing immediate and drastic changes in the brain microenvironment on arrival of individual cancer cells. We identified reactive astrocytes as the most active host cell population that immediately localizes to individual invading tumor cells and continuously associates with growing metastatic lesions. Up-regulation of matrix metalloproteinase-9 associated with astrocyte activation in the immediate vicinity of extravasating cancer cells might support their progression. Early involvement of different host cell types indicates environmental clues that might codetermine whether a single cancer cell progresses to macrometastasis or remains dormant. Thus, information on the initial interplay between brain homing tumor cells and reactive host cells may help develop strategies for prevention and treatment of symptomatic breast cancer brain metastases.

Advanced metastatic disease is difficult to manage and specific therapeutic targets are rare. We showed earlier that metastatic breast cancer cells use the activated conformer of adhesion receptor integrin alphavbeta3 for dissemination. We now investigated if targeting this form of the receptor can impact advanced metastatic disease, and we analyzed the mechanisms involved. Treatment of advanced multi-organ metastasis in SCID mice with patient-derived scFv antibodies specific for activated integrin alphavbeta3 caused stagnation and regression of metastatic growth. The antibodies specifically localized to tumor lesions in vivo and inhibited alphavbeta3 ligand binding at nanomolar levels in vitro. At the cellular level, the scFs associated rapidly with high affinity alphavbeta3 and dissociated extremely slowly. Thus, the scFvs occupy the receptor on metastatic tumor cells for prolonged periods of time, allowing for inhibition of established cell interaction with natural alphavbeta3 ligands. Potential apoptosis inducing effects of the antibodies through interaction with caspase-3 were studied as potential additional mechanism of treatment response. However, in contrast to a previous concept, neither the RGD-containing ligand mimetic scFvs nor RGD peptides bound or activated caspase-3 at the cellular or molecular level. This indicates that the treatment effects seen in the animal model are primarily due to antibody interference with alphavbeta3 ligation. Inhibition of advanced metastatic disease by treatment with cancer patient derived single chain antibodies against the activated conformer of integrin alphavbeta3 identifies this form of the receptor as a suitable target for therapy.

c-Src tyrosine kinase controls proliferation, cell adhesion, and cell migration and is highly regulated. A novel regulatory mechanism to control c-Src function that has recently been identified involves the C-terminal amino acid sequence Gly-Glu-Asn-Leu (GENL) of c-Src as ligand for PDZ domains. Herein, we determined the biological relevance of this c-Src regulation in human breast epithelial cells. The intact GENL sequence maintained c-Src in an inactive state in starved cells and restricted c-Src functions that might lead to metastatic transformation under normal growth conditions. c-Src with a C-terminal Leu/Ala mutation in GENL (Src-A) promoted the activation and translocation of cortactin and focal adhesion kinase and increased the motility and persistence of cell migration on the basement membrane. Src-A promoted increased extracellular proteolytic activity, and in acinar cultures, it led to the escape of cells through the basement membrane into the surrounding matrix. We ascribe the regulatory function of C-terminal Leu to the role of GENL in modulating c-Src activity downstream of cell matrix adhesion. We propose that the C terminus of c-Src via its GENL sequence presents a mechanism that restricts c-Src in epithelia and prevents progression toward an invasive phenotype.

High-grade brain tumours are characterized by inter- and intratumour heterogeneity and remain incurable despite current multi-modality treatment. The cellular aggressiveness within brain tumours includes tumour cell invasion of healthy brain tissue, resistance to conventional therapy, and greatly enhanced growth potential. This has been attributed to undifferentiated/stem cell-like features and clonal expansion under therapeutic pressure. The evolving notion of cancer cell plasticity implies that tumour cells may alter their phenotype both randomly and in response to external cues. In this context, we aim to elucidate the molecular mechanisms regulating glioma cell fate. We investigate selective small molecule-induced phenotypic changes such as ‘forced’ differentiation, surface-molecule expression, neurite outgrowth, autophagy, and apoptosis using patient-derived glioma models. Notably, these models display variability in cellular stemness/differentiation characteristics as well as glioma subtype-related classifiers. We therefore further analyse these small molecule-induced phenotypes at the single cell level using gene expression analysis. In addition to chemical/genetic-inspired approaches in vitro, we evaluate the tumour growth and invasion potential of glioma cell phenotypes in vivo. Together, these approaches have revealed molecular vulnerabilities (for example the TACC3-ARNT and HIF axis) that are required for aggressive glioma cell behaviour and may be pharmacologically exploitable for combination therapy.

AF6 is a human multi-domain protein involved in signaling and organization of cell junctions during embryogenesis. Its homologue in rat is called afadin. Three different AF6 transcripts are known, but only isoform 1 (AF6i1) has been characterized as protein. We focused on the AF6 isoform 3 (AF6i3), which differs from the AF6i1 by an additional C-terminal F-actin-binding site. Knockdown of AF6i3 in epithelial cells, which express only this isoform, resulted in impaired E-cadherin-dependent intercellular adhesion due to concomitantly reduced association of E-cadherin with F-actin and p120-catenin. Impaired intercellular adhesion also accelerated wound closure due to increased directionality of cell migration and delayed de novo formation of cell junctions. In contrast to AF6i3, the AF6i1 displayed a reduced association with the actin cytoskeleton and did not stabilize intercellular adhesion. Therefore, we propose that the AF6i3 protein stabilizes E-cadherin-dependent adhesion during dynamic processes, such as wound closure and formation of cell junctions, by linking the E-cadherin-catenin complex to the actin cytoskeleton via its F-actin-binding site.

The logic of using nucleic acids as pharmaceutical reagents is in part based on their capacity to interact with high affinity and specificity with other biological components. Considerable progress has been made over the past 10 years in the development of nucleic acid-based drug molecules using a variety of different technologies. One approach is a combinatorial technology that involves an iterative Darwinian-type in vitro evolution process, which has been termed SELEX for 'systematic evolution of ligands by exponential enrichment'. The procedure is a highly efficient method of identifying rare ligands from combinatorial nucleic acid libraries of very high complexity. It allows the selection of nucleic acid molecules with desired functions and it has been instrumental in the identification of a number of synthetic DNA and RNA molecules, so-called aptamers that recognise ligands of different chemical origin. The method is fast, it does not require special equipment and the selected aptamers typically bind their target with high affinity and high specificity. Here we summarise the recent examples of the SELEX technique within the context of identifying high-affinity ligands against parasite target molecules.

CXCL14 (BRAK) is a cytokine implicated in inflammatory responses, but its contribution to cancer is largely unknown. Recent studies in clinical breast cancer indicate an association between CXCL14 expression and shortened time to metastatic progression, as well as the presence of CXCL14 in a subset of breast epithelial cells with a stem-like phenotype. Using global gene expression profiling, we identified CXCL14 as the most prominently upregulated cytokine in a novel progression model of triple-negative inflammatory and metaplastic breast cancer that we generated. The model comprises tumor cells established from a locally aggressive primary tumor obtained from a patient at the time of initial surgery (TES-1), and tumor cells from the same site at the time of local failure in the chest wall after chemo and radiation therapy (TES-2b). In contrast to TES-1, TES-2b cells are tumorigenic in immuno deficient mice and can be followed and quantified by non-invasive bioluminescence imaging after injecting the F-luc tagged cancer cells into the 4th mammary fat pad. Illumina microarray analysis revealed a 66-fold increase in CXCL14 expression in TES-2b cells over TES-1 cells, ranking it as one of the top 25 genes most differentially expressed among over 25,000 identified gene transcripts. We validated CXCL14 overexpression in TES-2b cells by real time PCR (TaqMan) (64 fold), and found a significant increase in CXCL14 protein secretion in cultured TES-2b cells (3.8 fold) by immuno-capture ELISA. To address a functional role in CXCL14 in the progression of these aggressive breast cancer cells, we stably reduced CXCL14 expression in TES-2b cells by lentiviral transduction with small interfering RNA and confirmed an 80% reduction in gene expression. CXCL14 protein production was concomitantly reduced 2.7 fold. Importantly, xenograft studies revealed a critical role of CXCL14 in the in vivo growth behavior of these breast cancer cells, as the tumor growth rate in the mammary fat pad of CXCL14 knock-down cells was significantly reduced compared to scrambled control vector treated cells. After injecting 5×105 F-luc tagged tumor cells, 4/6 mice had tumors measuring 10 mm in diameter after 11 weeks (requiring sacrifice of the animals) compared with 1/6 in the CXCL14 knock-down group. These initial results indicate a critical role of tumor cell derived CXCL14 in the in vivo survival and proliferation of these breast cancer cells. Our findings provide a basis for further analyses of CXCL14 in breast cancer progression in vivo and for detailed studies of the mechanisms through which CXCL14 may promote disease progression of highly aggressive triple-negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5251. doi:10.1158/1538-7445.AM2011-5251

Brain metastases occur mostly in patients with melanoma or primary tumors in the lung, breast or prostate. As therapies against primary tumors and extracranial metastases are improving and patients live longer, the incidence of brain metastases is increasing and brain lesions are seen in 20 to 40 % of patients with advanced metastatic disease. Unfortunately, no therapies are available to treat brain metastasis efficiently.Existing in vivo models of brain metastasis focus mainly on established macrometastases, while very little is known about the initial events of tumor cell brain colonization. In the present study, we used non-invasive bioluminescence imaging and detailed immunohistochemistry to capture the early events of tumor cell interaction with the cerebral microvasculature and invasion of the brain tissue. We used MDA-MB-435 metastatic cancer cells as a model because of their aggressive metastatic phenotype, and injected the cells into the internal carotid artery of immune deficient mice. Our results show that single cancer cells extravasate into the brain parenchyma 3 to 7 days after they arrest within brain capillaries and disrupt the vessel wall, bursting the microvasculature. Cancer cell arrest and extravasation occur exclusively in small vessels that are surrounded by pericytes and lack smooth-muscle cells. Early brain colonization consistently induces local activation of astrocytes, involving heterogeneous astrocyte populations. Reactive astrocytes express high levels of MMP-9 that may assist tumor cell invasion and provide first pro-angiogenic clues. Activated astrocytes consistently persist in the vicinity of growing metastases, and potentially contribute to metastatic proliferation. Occasionally, early host responses also include microglia with stellate or a reactive amoeboid morphology. Importantly, in addition to yielding actively proliferating macrometastases, tumor cell extravasation also leads to widespread single tumor cells dispersed throughout the brain tissue. The majority of these cells remains viable but stays quiescent and does not proliferate. We speculate that the diversity in early local responses of the brain microenvironment to initial cancer cell invasion contributes to the fate of extravasated tumor cells. An early involvement of diverse host cell types might provide distinct environmental clues and co-determine whether a single cell progresses to macrometastasis or remains dormant. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4162.

BACKGROUND Brain metastases (BrM) are an unmet clinical need with poor prognosis. 60% of melanoma patients develop BrM. BrM are strongly understudied due to frequent exclusion from clinical trials, and hence treatment options commonly lag behind. Antibodies targeting the immune-inhibitory receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have demonstrated efficacy against melanoma BrM. Despite this, therapeutic responses are highly variable, and it is unknown why therapy fails in a high proportion of patients. Improved therapeutic strategies require a thorough understanding of potentially exploitable mechanisms of therapeutic efficacy. Our data previously implicated different immune cells, foremost CD8+ T cells, but also NK cells, in the intracranial efficacy and enhanced survival benefit of immune checkpoint blockade (ICB). Our aim here is to investigate the role of NK cells in mediating the response to ICB in melanoma BrM. MATERIAL AND METHODS To study the role of NK cells in the response to ICB in melanoma BrM, a tumour transplantation model of B16 melanoma with simultaneous extracranial (i.e., flank) and brain tumours in C57BL/6 mice was utilised. NK cells were depleted through administration of anti-asialo-GM1 NK cell-depleting antibodies. Confirmation of NK cell depletion and quantification of intratumoral immune cell populations was performed using flow cytometry. Intratumoral gene expression of key chemokines and immune mediator genes was assessed using RT-qPCR and mRNA-seq. RESULTS Highly variable response to ICB with respect to intratumoral accumulation of CD8+ T cells allowed separation of mice into responders and non-responders and revealed genes and pathways associated with response to ICB. NK cell depletion reversed the ICB-mediated increase in the accumulation of CD8+ T cells and significantly reduced the expression of genes associated with response in intracranial and extracranial tumours. The ICB-mediated significant increase in gene expression of various chemokines (i.e., Cxcl9/10) and immune mediators (i.e., Ifng, Prf1 and Gzmb) was significantly abrogated upon NK cell depletion. CONCLUSION NK cells play a critical role in the underlying mechanisms of ICB efficacy through their modulation of the tumour microenvironment and enhancement of CD8+ T cell accumulation in intracranial tumours. Targeting of NK cells may allow potentiation of ICB therapy in the brain, as well as at extracranial sites.