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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.

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.

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.

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.

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.

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.

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.