In Vivo PET Detection of Lung Micrometastasis in Mice by Targeting Endothelial VCAM-1 Using a Dual-Contrast PET/MRI Probe.

Current clinical diagnostic imaging methods for lung metastases are sensitive only to large tumours (1-2 mm cross-sectional diameter), and early detection can dramatically improve treatment. We have previously demonstrated that an antibody-targeted MRI contrast agent based on microparticles of iron oxide (MPIO; 1 µm diameter) enables the imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Using a mouse model of lung metastasis, upregulation of endothelial VCAM-1 expression was demonstrated in micrometastasis-associated vessels but not in normal lung tissue, and binding of VCAM-MPIO to these vessels was evident histologically. Owing to the lack of proton MRI signals in the lungs, we modified the VCAM-MPIO to include zirconium-89 (89Zr, t1/2 = 78.4 h) in order to allow the in vivo detection of lung metastases by positron emission tomography (PET). Using this new agent (89Zr-DFO-VCAM-MPIO), it was possible to detect the presence of micrometastases within the lung in vivo from ca. 140 µm in diameter. Histological analysis combined with autoradiography confirmed the specific binding of the agent to the VCAM-1 expressing vasculature at the sites of pulmonary micrometastases. By retaining the original VCAM-MPIO as the basis for this new molecular contrast agent, we have created a dual-modality (PET/MRI) agent for the concurrent detection of lung and brain micrometastases.

Targeted opening of the blood-brain barrier using VCAM-1 functionalised microbubbles and "whole brain" ultrasound.

Metastatic tumours in the brain now represent one of the leading causes of death from cancer. Current treatments are largely ineffective owing to the combination of late diagnosis and poor delivery of therapies across the blood-brain barrier (BBB). Conjugating magnetic resonance imaging (MRI) contrast agents with a monoclonal antibody for VCAM-1 (anti-VCAM1) has been shown to enable detection of micrometastases, two to three orders of magnitude smaller in volume than those currently detectable clinically. The aim of this study was to exploit this targeting approach to enable localised and temporary BBB opening at the site of early-stage metastases using functionalised microbubbles and ultrasound. Methods: Microbubbles functionalised with anti-VCAM1 were synthesised and shown to bind to VCAM-1-expressing cells in vitro. Experiments were then conducted in vivo in a unilateral breast cancer brain metastasis mouse model using Gadolinium-DTPA (Gd-DTPA) enhanced MRI to detect BBB opening. Following injection of Gd-DTPA and targeted microbubbles, the whole brain volume was simultaneously exposed to ultrasound (0.5 MHz, 10% duty cycle, 0.7 MPa peak negative pressure, 2 min treatment time). T1-weighted MRI was then performed to identify BBB opening, followed by histological confirmation via immunoglobulin G (IgG) immunohistochemistry. Results: In mice treated with targeted microbubbles and ultrasound, statistically significantly greater extravasation of Gd-DTPA and IgG was observed in the left tumour-bearing hemisphere compared to the right hemisphere 5 min after treatment. No acute adverse effects were observed. There was no investigation of longer term bioeffects owing to the nature of the study. Conclusion: The results demonstrate the feasibility of using targeted microbubbles in combination with low intensity ultrasound to localise opening of the BBB to metastatic sites in the brain. This approach has potential application in the treatment of metastatic tumours whose location cannot be established a priori with conventional imaging methods.

Inflammatory bowel disease induces pathological α-synuclein aggregation in the human gut and brain.

AIMS: According to Braak's hypothesis, it is plausible that Parkinson's disease (PD) originates in the enteric nervous system (ENS) and spreads to the brain through the vagus nerve. In this work, we studied whether inflammatory bowel diseases (IBDs) in humans can progress with the emergence of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. METHODS: We have analysed the gut and the ventral midbrain from subjects previously diagnosed with IBD and form a DSS-based rat model of gut inflammation in terms of α-syn pathology. RESULTS: Our data support the existence of pathogenic α-syn in both the gut and the brain, thus reinforcing the potential role of the ENS as a contributing factor in PD aetiology. Additionally, we have analysed the effect of a DSS-based rat model of gut inflammation to demonstrate (i) the appearance of P-α-syn inclusions in both Auerbach's and Meissner's plexuses (gut), (ii) an increase in α-syn expression in the ventral mesencephalon (brain) and (iii) the degeneration of nigral dopaminergic neurons, which all are considered classical hallmarks in PD. CONCLUSION: These results strongly support the plausibility of Braak's hypothesis and emphasise the significance of peripheral inflammation and the gut-brain axis in initiating α-syn aggregation and transport to the substantia nigra, resulting in neurodegeneration.

Galectin-3, a rising star in modulating microglia activation under conditions of neurodegeneration.

The advent of high-throughput single-cell transcriptomic analysis of microglia has revealed different phenotypes that are inherently associated with disease conditions. A common feature of some of these activated phenotypes is the upregulation of galectin-3. Representative examples of these phenotypes include disease-associated microglia (DAM) and white-associated microglia (WAM), whose role(s) in neuroprotection/neurotoxicity is a matter of high interest in the microglia community. In this review, we summarise the main findings that demonstrate the ability of galectin-3 to interact with key pattern recognition receptors, including, among others, TLR4 and TREM2 and the importance of galectin-3 in the regulation of microglia activation. Finally, we discuss increasing evidence supporting the involvement of this lectin in the main neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain injury, and stroke.

Arginine deprivation alters microglial polarity and synergizes with radiation to eradicate non-arginine-auxotrophic glioblastoma tumors.

New approaches for the management of glioblastoma (GBM) are an urgent and unmet clinical need. Here, we illustrate that the efficacy of radiotherapy for GBM is strikingly potentiated by concomitant therapy with the arginine-depleting agent ADI-PEG20 in a non-arginine-auxotrophic cellular background (argininosuccinate synthetase 1 positive). Moreover, this combination led to durable and complete radiological and pathological response, with extended disease-free survival in an orthotopic immune-competent model of GBM, with no significant toxicity. ADI-PEG20 not only enhanced the cellular sensitivity of argininosuccinate synthetase 1-positive GBM to ionizing radiation by elevated production of nitric oxide (˙NO) and hence generation of cytotoxic peroxynitrites, but also promoted glioma-associated macrophage/microglial infiltration into tumors and turned their classical antiinflammatory (protumor) phenotype into a proinflammatory (antitumor) phenotype. Our results provide an effective, well-tolerated, and simple strategy to improve GBM treatment that merits consideration for early evaluation in clinical trials.

A novel molecular magnetic resonance imaging agent targeting activated leukocyte cell adhesion molecule as demonstrated in mouse brain metastasis models.

Molecular magnetic resonance imaging (MRI) allows visualization of biological processes at the molecular level. Upregulation of endothelial ALCAM (activated leukocyte cell adhesion molecule) is a key element for leukocyte recruitment in neurological disease. The aim of this study, therefore, was to develop a novel molecular MRI contrast agent, by conjugating anti-ALCAM antibodies to microparticles of iron oxide (MPIO), for detection of endothelial ALCAM expression in vivo. Binding specificity of ALCAM-MPIO was demonstrated in vitro under static and flow conditions. Subsequently, in a proof-of-concept study, mouse models of brain metastasis were induced by intracardial injection of brain-tropic human breast carcinoma, lung adenocarcinoma or melanoma cells to upregulate endothelial ALCAM. At selected time-points, mice were injected intravenously with ALCAM-MPIO, and ALCAM-MPIO induced hypointensities were observed on T2*-weighted images in all three models. Post-gadolinium MRI confirmed an intact blood-brain barrier, indicating endoluminal binding. Correlation between endothelial ALCAM expression and ALCAM-MPIO binding was confirmed histologically. Statistical analysis indicated high sensitivity (80-90%) and specificity (79-83%) for detection of endothelial ALCAM in vivo with ALCAM-MPIO. Given reports of endothelial ALCAM upregulation in numerous neurological diseases, this advance in our ability to image ALCAM in vivo may yield substantial improvements for both diagnosis and targeted therapy.

VCAM-1 targeted alpha-particle therapy for early brain metastases.

BACKGROUND: Brain metastases (BM) develop frequently in patients with breast cancer. Despite the use of external beam radiotherapy (EBRT), the average overall survival is short (6 months from diagnosis). The therapeutic challenge is to deliver molecularly targeted therapy at an early stage when relatively few metastatic tumor cells have invaded the brain. Vascular cell adhesion molecule 1 (VCAM-1), overexpressed by nearby endothelial cells during the early stages of BM development, is a promising target. The aim of this study was to investigate the therapeutic value of targeted alpha-particle radiotherapy, combining lead-212 (212Pb) with an anti-VCAM-1 antibody (212Pb-αVCAM-1). METHODS: Human breast carcinoma cells that metastasize to the brain, MDA-231-Br-GFP, were injected into the left cardiac ventricle of nude mice. Twenty-one days after injection, 212Pb-αVCAM-1 uptake in early BM was determined in a biodistribution study and systemic/brain toxicity was evaluated. Therapeutic efficacy was assessed using MR imaging and histology. Overall survival after 212Pb-αVCAM-1 treatment was compared with that observed after standard EBRT. RESULTS: 212Pb-αVCAM-1 was taken up into early BM with a tumor/healthy brain dose deposition ratio of 6 (5.52e108 and 0.92e108) disintegrations per gram of BM and healthy tissue, respectively. MRI analyses showed a statistically significant reduction in metastatic burden after 212Pb-αVCAM-1 treatment compared with EBRT (P < 0.001), translating to an increase in overall survival of 29% at 40 days post prescription (P < 0.01). No major toxicity was observed. CONCLUSIONS: The present investigation demonstrates that 212Pb-αVCAM-1 specifically accumulates at sites of early BM causing tumor growth inhibition.

Reformulating Pro-Oxidant Microglia in Neurodegeneration.

In neurodegenerative diseases, microglia-mediated neuroinflammation and oxidative stress are central events. Recent genome-wide transcriptomic analyses of microglial cells under different disease conditions have uncovered a new subpopulation named disease-associated microglia (DAM). These studies have challenged the classical view of the microglia polarization state's proinflammatory M1 (classical activation) and immunosuppressive M2 (alternative activation). Molecular signatures of DAM and proinflammatory microglia (highly pro-oxidant) have shown clear differences, yet a partial overlapping gene profile is evident between both phenotypes. The switch activation of homeostatic microglia into reactive microglia relies on the selective activation of key surface receptors involved in the maintenance of brain homeostasis (a.k.a. pattern recognition receptors, PRRs). Two relevant PRRs are toll-like receptors (TLRs) and triggering receptors expressed on myeloid cells-2 (TREM2), whose selective activation is believed to generate either a proinflammatory or a DAM phenotype, respectively. However, the recent identification of endogenous disease-related ligands, which bind to and activate both TLRs and TREM2, anticipates the existence of rather complex microglia responses. Examples of potential endogenous dual ligands include amyloid ß, galectin-3, and apolipoprotein E. These pleiotropic ligands induce a microglia polarization that is more complicated than initially expected, suggesting the possibility that different microglia subtypes may coexist. This review highlights the main microglia polarization states under disease conditions and their leading role orchestrating oxidative stress.

VCAM-1-targeted MRI Enables Detection of Brain Micrometastases from Different Primary Tumors.

PURPOSE: A major issue for the effective treatment of brain metastasis is the late stage of diagnosis with existing clinical tools. The aim of this study was to evaluate the potential of vascular cell adhesion molecule 1 (VCAM-1)-targeted MRI for early detection of brain micrometastases in mouse models across multiple primary tumor types.Experimental Design: Xenograft models of brain micrometastasis for human breast carcinoma (MDA231Br-GFP), lung adenocarcinoma (SEBTA-001), and melanoma (H1_DL2) were established via intracardiac injection in mice. Animals (n = 5-6/group) were injected intravenously with VCAM-1-targeted microparticles of iron oxide (VCAM-MPIO) and, subsequently, underwent T 2*-weighted MRI. Control groups of naïve mice injected with VCAM-MPIO and tumor-bearing mice injected with nontargeting IgG-MPIO were included. RESULTS: All models showed disseminated micrometastases in the brain, together with endothelial VCAM-1 upregulation across the time course. T 2*-weighted MRI of all tumor-bearing mice injected with VCAM-MPIO showed significantly more signal hypointensities (P < 0.001; two-sided) than control cohorts, despite a lack of blood-brain barrier (BBB) impairment. Specific MPIO binding to VCAM-1-positive tumor-associated vessels was confirmed histologically. VCAM-1 expression was demonstrated in human brain metastasis samples, across all three primary tumor types. CONCLUSIONS: VCAM-1-targeted MRI enables the detection of brain micrometastases from the three primary tumor types known to cause the majority of clinical cases. These findings represent an important step forward in the development of a broadly applicable and clinically relevant imaging technique for early diagnosis of brain metastasis, with significant implications for improved patient survival.

The Multifarious Role of Microglia in Brain Metastasis.

The immune landscape in brain metastasis is a very heterogeneous framework. Amongst a broad plethora of cells within the tumor microenvironment, the presence of activated microglia has been perfectly described. The innate role of microglial cells is to detect and eliminate any insults that may disturb the regular behavior of the brain. As part of its defensive role, it releases pro- and anti-inflammatory cytokines that aim to modulate the inflammatory scenario at the metastatic foci. However, the long term effects that these cells may exert on the metastatic progression is not clear. One of the biggest challenges in the field is to distinguish between brain resident microglial cells and infiltrated bone-marrow derived macrophages. Part of this issue is the fact that both cell types share similar phenotypes. Current studies are based on the modulation of the immune response against cancer cells (immunotherapy). However, most of current clinical trials and newly developed drugs focus on the adaptive immune response (e.g., immune blockade check-points). Additionally, the unique structure of the central nervous system with the presence of the blood-brain barrier have hindered a significant advance in novel therapies against brain metastasis. In this manuscript, we describe current advances in characterization of tumor-associated microglia and macrophages, the importance of microglia during the anti-cancerous response, and the future direction for the development of new strategies against this complex disease.

Optimization of molecularly targeted MRI in the brain: empirical comparison of sequences and particles.

BACKGROUND: Molecular MRI is an evolving field of research with strong translational potential. Selection of the appropriate MRI sequence, field strength and contrast agent depend largely on the application. The primary aims of the current study were to: 1) assess the sensitivity of different MRI sequences for detection of iron oxide particles in mouse brain; 2) determine the effect of magnetic field strength on detection of iron oxide particles in vivo; and 3) compare the sensitivity of targeted microparticles of iron oxide (MPIO) or ultra-small superparamagnetic iron oxide (USPIO) for detection of vascular cell adhesion molecule-1 (VCAM-1) in vivo. METHODS: Mice were injected intrastriatally with interleukin 1ß to induce VCAM-1 expression on the cerebral vasculature. Subsequently, animals were injected intravenously with either VCAM-MPIO or VCAM-USPIO and imaged 1 or 13 hours post-injection, respectively. MRI was performed at 4.7, 7.0, or 9.4 T, using three different T2*-weighted sequences: single gradient echo 3D (GE3D), multi-gradient echo 3D (MGE3D) and balanced steady-state free precession 3D (bSSFP3D). RESULTS: MGE3D yielded the highest signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) for the detection of iron oxide particles. All sequences showed a significant increase in SNR and CNR from 4.7 to 7.0 T, but no further improvement at 9.4 T. However, whilst targeted MPIO enabled sensitive detection of VCAM-1 expression on the cerebral vasculature, the long half-life (16.5 h vs 1.2 min) and lower relaxivity per particle (1.29×10-14 vs 1.18×10-9 Hz L/particle) of USPIO vs. MPIO rendered them impractical for molecular MRI. CONCLUSION: These findings demonstrate clear advantages of MPIO compared to USPIO for molecularly-targeted MRI, and indicate that the MGE3D sequence is optimal for MPIO detection. Moreover, higher field strengths (7.0/9.4 T) showed enhanced sensitivity over lower field strengths (4.7 T). With the development of biodegradable MPIO, these agents hold promise for clinical translation.

Dosimetric evaluation of radionuclides for VCAM-1-targeted radionuclide therapy of early brain metastases.

Brain metastases develop frequently in patients with breast cancer, and present a pressing therapeutic challenge. Expression of vascular cell adhesion molecule 1 (VCAM-1) is upregulated on brain endothelial cells during the early stages of metastasis and provides a target for the detection and treatment of early brain metastases. The aim of this study was to use a model of early brain metastasis to evaluate the efficacy of α-emitting radionuclides, 149Tb, 211At, 212Pb, 213Bi and 225Ac; ß-emitting radionuclides, 90Y, 161Tb and 177Lu; and Auger electron (AE)-emitters 67Ga, 89Zr, 111In and 124I, for targeted radionuclide therapy (TRT). METHODS: Histologic sections and two photon microscopy of mouse brain parenchyma were used to inform a cylindrical vessel geometry using the Geant4 general purpose Monte Carlo (MC) toolkit with the Geant4-DNA low energy physics models. Energy deposition was evaluated as a radial function and the resulting phase spaces were superimposed on a DNA model to estimate double-strand break (DSB) yields for representative ß- and α-emitters, 177Lu and 212Pb. Relative biological effectiveness (RBE) values were determined by only evaluating DNA damage due to physical interactions. RESULTS: 177Lu produced 2.69 ± 0.08 DSB per GbpGy, without significant variation from the lumen of the vessel to a radius of 100 µm. The DSB yield of 212Pb included two local maxima produced by the 6.1 MeV and 8.8 MeV α-emissions from decay products, 212Bi and 212Po, with yields of 7.64 ± 0.12 and 9.15 ± 0.24 per GbpGy, respectively. Given its higher DSB yield 212Pb may be more effective for short range targeting of early micrometastatic lesions than 177Lu. CONCLUSION: MC simulation of a model of early brain metastases provides invaluable insight into the potential efficacy of α-, ß- and AE-emitting radionuclides for TRT. 212Pb, which has the attributes of a theranostic radionuclide since it can be used for SPECT imaging, showed a favorable dose profile and RBE.

Anti-inflammatory Microglia/Macrophages As a Potential Therapeutic Target in Brain Metastasis.

Brain metastasis is a common complication of cancer patients and is associated with poor survival. Histological data from patients with brain metastases suggest that microglia are the major immune population activated around the metastatic foci. Microglia and macrophages have the ability to polarize to different phenotypes and to exert both tumorigenic and cytotoxic effects. However, the role of microglia/macrophages during the early stages of metastatic growth in the brain has not yet been determined. The aim of this study was to profile microglial/macrophage activation in a mouse model of breast cancer brain metastasis during the early stages of tumor growth, and to assess the role of the anti-inflammatory microglial/macrophage population, specifically, during this phase. Following intracerebral injection of 5 × 103 4T1-GFP mammary carcinoma cells into female BALB/c mice, robust microglial/macrophage activation around the 4T1 metastatic foci was evident throughout the time-course studied (28 days) and correlated positively with tumor volume (R2 = 0.67). Populations of classically (proinflammatory) and alternatively (anti-inflammatory) activated microglia/macrophages were identified immunohistochemically by expression of either induced nitric oxide synthase/cyclooxygenase 2 or mannose receptor 1/arginase 1, respectively. Temporally, levels of both pro- and anti-inflammatory cells were broadly stable across the time-course. Subsequently, selective depletion of the anti-inflammatory microglia/macrophage population by intracerebral injection of mannosylated clodronate liposomes significantly reduced metastatic tumor burden (p < 0.01). Moreover, increased levels of apoptosis were associated with tumors in clodronate liposome treated animals compared to controls (p < 0.05). These findings suggest that microglia/macrophages are important effectors of the inflammatory response in the early stages of brain metastasis, and that targeting the anti-inflammatory microglial/macrophage population may offer an effective new therapeutic avenue for patients with brain metastases.

Disruption of tumour-host communication by downregulation of LFA-1 reduces COX-2 and e-NOS expression and inhibits brain metastasis growth.

Over 20% of cancer patients will suffer metastatic spread to the brain, and prognosis remains poor. Communication between tumour cells and host tissue is essential during metastasis, yet little is known of the processes underlying such interactions in the brain.Here we test the hypothesis that cross-talk between tumour cells and host brain cells, through tumour cell leukocyte function associated protein-1 (LFA-1), is critical in metastasis development. Temporal expression of LFA-1 and its major ligand intercellular adhesion molecule-1 (ICAM-1) was determined in two different mouse models of brain metastasis. Marked upregulation of both proteins was found, co-localising with astrocytes, microglia and tumour cells themselves. Silencing of LFA-1 expression in MDA231Br-GFP cells prior to intracerebral injection resulted in > 70% reduction in tumour burden compared to control MDA231Br-GFP cells (p < 0.005, n = 5). Subsequent qRT-PCR analysis of brain tissue revealed significant reductions in COX-2, VEGF and eNOS from host brain tissue, but not tumour cells, in mice injected with LFA-1 knockdown cells (p < 0.0001, n = 5). Finally, expression of both LFA-1 and ICAM-1 was demonstrated in human brain metastasis samples.The results of this study suggest LFA-1 as a new target in brain metastasis therapy and highlight the potential synergy with current anti-COX-2 and anti-NOS therapies.

Mouse Models of Brain Metastasis for Unravelling Tumour Progression.

Secondary tumours in the brain account for 40 % of triple negative breast cancer patients, and the percentage may be higher at the time of autopsy. The use of in vivo models allow us to recapitulate the molecular mechanisms potentially used by circulating breast tumour cells to proliferate within the brain.Metastasis is a multistep process that depends on the success of several stages including cell evasion from the primary tumour, distribution and survival within the blood stream and cerebral microvasculature, penetration of the blood-brain barrier and proliferation within the brain microenvironment. Cellular adhesion molecules are key proteins involved in all of the steps in the metastatic process. Our group has developed two different in vivo models to encompass both seeding and colonisation stages of the metastatic process: (1) haematogenous dissemination of tumour cells by direct injection into the left ventricle of the heart, and (2) direct implantation of the tumour cells into the mouse brain.This chapter describes, in detail, the practical implementation of the intracerebral model, which can be used to analyse tumour proliferation within a specific area of the central nervous system and tumour-host cell interactions. We also describe the use of immunohistochemistry techniques to identify, at the molecular scale, tumour-host cell interactions, which may open new windows for brain metastasis therapy.

Alternate RASSF1 Transcripts Control SRC Activity, E-Cadherin Contacts, and YAP-Mediated Invasion.

Tumor progression to invasive carcinoma is associated with activation of SRC family kinase (SRC, YES, FYN) activity and loss of cellular cohesion. The hippo pathway-regulated cofactor YAP1 supports the tumorigenicity of RAS mutations but requires both inactivation of hippo signaling and YES-mediated phosphorylation of YAP1 for oncogenic activity. Exactly how SRC kinases are activated and hippo signaling is lost in sporadic human malignancies remains unknown. Here, we provide evidence that hippo-mediated inhibition of YAP1 is lost upon promoter methylation of the RAS effector and hippo kinase scaffold RASSF1A. We find that RASSF1A promoter methylation reduces YAP phospho-S127, which derepresses YAP1, and actively supports YAP1 activation by switching RASSF1 transcription to the independently transcribed RASSF1C isoform that promotes Tyr kinase activity. Using affinity proteomics, proximity ligation, and real-time molecular visualization, we find that RASSF1C targets SRC/YES to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, ß-catenin, and YAP1. RASSF1A restricts SRC activity, preventing motility, invasion, and tumorigenesis in vitro and in vivo, with epigenetic inactivation correlating with increased inhibitory pY527-SRC in breast tumors. These data imply that distinct RASSF1 isoforms have opposing functions, which provide a biomarker for YAP1 activation and explain correlations of RASSF1 methylation with advanced invasive disease in humans. The ablation of epithelial integrity together with subsequent YAP1 nuclear localization allows transcriptional activation of ß-catenin/TBX-YAP/TEAD target genes, including Myc, and an invasive phenotype. These findings define gene transcript switching as a tumor suppressor mechanism under epigenetic control.

ASPP2 controls epithelial plasticity and inhibits metastasis through ß-catenin-dependent regulation of ZEB1.

Epithelial to mesenchymal transition (EMT), and the reverse mesenchymal to epithelial transition (MET), are known examples of epithelial plasticity that are important in kidney development and cancer metastasis. Here we identify ASPP2, a haploinsufficient tumour suppressor, p53 activator and PAR3 binding partner, as a molecular switch of MET and EMT. ASPP2 contributes to MET in mouse kidney in vivo. Mechanistically, ASPP2 induces MET through its PAR3-binding amino-terminus, independently of p53 binding. ASPP2 prevents ß-catenin from transactivating ZEB1, directly by forming an ASPP2-ß-catenin-E-cadherin ternary complex and indirectly by inhibiting ß-catenin's N-terminal phosphorylation to stabilize the ß-catenin-E-cadherin complex. ASPP2 limits the pro-invasive property of oncogenic RAS and inhibits tumour metastasis in vivo. Reduced ASPP2 expression results in EMT, and is associated with poor survival in hepatocellular carcinoma and breast cancer patients. Hence, ASPP2 is a key regulator of epithelial plasticity that connects cell polarity to the suppression of WNT signalling, EMT and tumour metastasis.

Glial activation in the early stages of brain metastasis: TSPO as a diagnostic biomarker.

UNLABELLED: Metastatic spread of cancer cells to the brain is associated with high mortality, primarily because current diagnostic tools identify only well-advanced metastases. Brain metastases have been shown to induce a robust glial response, including both astrocyte and microglial activation. On the basis of these findings, we hypothesized that this stromal response may provide a sensitive biomarker of tumor burden, in particular through the use of SPECT/PET imaging agents targeting the translocator protein (TSPO) that is upregulated on activated glia. Our goals, therefore, were first to determine the spatial and temporal profile of glial activation during early metastasis growth in vivo and second to assess the potential of the radiolabeled TSPO ligand (123)I-DPA-713 for early detection of brain metastases. METHODS: Metastatic mouse mammary carcinoma 4T1-green fluorescent protein cells were injected either intracerebrally or intracardially into female BALB/c mice to induce brain metastases. Astrocyte and microglial activation was assessed immunohistochemically over a 28-d period, together with immunofluorescence detection of TSPO upregulation. Subsequently, SPECT imaging and autoradiography were used to determine in vivo binding of (123)I-DPA-713 at metastatic sites. RESULTS: Dynamic astrocyte and microglial activation was evident throughout the early stages of tumor growth, with the extent of astrocyte activation correlating significantly with tumor size (P < 0.0001). Microglial activation appeared to increase more rapidly than astrocyte activation at the earlier time points, but by later time points the extent of activation was comparable between the glial cell types. Upregulation of TSPO expression was found on both glial populations. Both autoradiographic and in vivo SPECT data showed strong positive binding of (123)I-DPA-713 in the intracerebrally induced model of brain metastasis, which was significantly greater than that observed in controls (P < 0.05). (123)I-DPA-713 binding was also evident autoradiographically in the intracardially induced model of brain metastasis but with lower sensitivity because of smaller tumor size (∼ 100-µm diameter vs. ∼ 600-µm diameter in the intracerebral model). CONCLUSION: These data suggest that the glial response to brain metastasis may provide a sensitive biomarker of tumor burden, with a tumor detection threshold lying between 100 and 600 µm in diameter. This approach could enable substantially earlier detection of brain metastases than the current clinical approach of gadolinium-enhanced MR imaging.

Functional role of endothelial adhesion molecules in the early stages of brain metastasis.

BACKGROUND: Cellular adhesion molecules (CAMs), which are normally associated with leukocyte trafficking, have also been shown to play an essential role in tumor metastasis to non-CNS sites. However, the role played by CAMs in brain metastasis is largely unexplored. It is known that leukocyte recruitment to the brain is very atypical and that mechanisms of disease in peripheral tissues cannot be extrapolated to the brain. Here, we have established the spatiotemporal expression of 12 key CAMs in the initial phases of tumor seeding in 2 different models of brain metastasis. METHODS: BALB/c or SCID mice were injected intracardially (10(5) cells/100 µL phosphate-buffered saline with either 4T1-GFP or MDA231BR-GFP cells, respectively (n = 4-6/group), and expression of the CAMs was determined by immunohistochemistry and immunofluorescence colocalisation. RESULTS: Endothelial expression of E-selectin, VCAM-1, ALCAM, ICAM-1, VLA-4, and ß4 integrin was markedly increased early in tumor seeding. At the same time, the natural ligands to these adhesion molecules were highly expressed on the metastatic tumor cells both in vitro and in vivo. Two of these ligands showed particularly high tumor cell expression (ALCAM and VLA-4), and consequently their functional role in tumor seeding was determined. Antibody neutralization of either ALCAM or VLA-4 significantly reduced tumor seeding within the brain (>60% decrease in tumor number/mm(2) brain; P < .05-0.01). CONCLUSIONS: These findings suggest that ALCAM/ALCAM and VLA-4/VCAM-1 interactions play an important functional role in the early stages of metastasis seeding in the brain. Moreover, this work identifies a specific subset of ligand-receptor interactions that may yield new therapeutic and diagnostic targets for brain metastasis.