Manuel Sarmiento Soto

Molecular MRI enables early and sensitive detection of brain metastases

Metastasis to the brain is a leading cause of cancer mortality. The current diagnostic method of gadolinium-enhanced MRI is sensitive only to larger tumors, when therapeutic options are limited. Earlier detection of brain metastases is critical for improved treatment. We have developed a targeted MRI contrast agent based on microparticles of iron oxide that enables imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Our objectives here were to determine whether VCAM-1 is up-regulated on vessels associated with brain metastases, and if so, whether VCAM-1–targeted MRI enables early detection of these tumors. Early up-regulation of cerebrovascular VCAM-1 expression was evident on tumor-associated vessels in two separate murine models of brain metastasis. Metastases were detectable in vivo using VCAM-1–targeted MRI 5 d after induction (<1,000 cells). At clinical imaging resolutions, this finding is likely to translate to detection at tumor volumes two to three orders of magnitude smaller (0.3–3 × 105 cells) than those volumes detectable clinically (107–108 cells). VCAM-1 expression detected by MRI increased significantly (P < 0.0001) with tumor progression, and tumors showed no gadolinium enhancement. Importantly, expression of VCAM-1 was shown in human brain tissue containing both established metastases and micrometastases. Translation of this approach to the clinic could increase therapeutic options and change clinical management in a substantial number of cancer patients.

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 (105 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 β4 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/mm2 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.

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

Summary 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, β-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 β-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.

Glial Activation in the Early Stages of Brain Metastasis: TSPO as a Diagnostic Biomarker

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 123I-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 123I-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 123I-DPA-713 in the intracerebrally induced model of brain metastasis, which was significantly greater than that observed in controls (P < 0.05). 123I-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.

Structural and functional effects of metastases in rat brain determined by multimodal MRI

Metastasis to the brain results in significant impairment of brain function and poor patient survival. Currently, magnetic resonance imaging (MRI) is under‐utilised in monitoring brain metastases and their effects on brain function. Here, we sought to establish a model of focal brain metastasis in the rat that enables serial multimodal structural and functional MRI studies, and to assess the sensitivity of these approaches to metastatic growth. Female Berlin–Druckrey‐IX rats were injected intracerebrally with metastatic ENU1564 cells in the ventroposterior medial nucleus (VPM) of the thalamus, a relay node of the whisker‐to‐barrel cortex pathway. Animals underwent multimodal structural and vascular MRI, as well as functional MRI of the cortical blood oxygenation level dependent (BOLD) responses to whisker pad stimulation. T2, diffusion, magnetisation transfer and perfusion weighted MRI enabled differentiation between a central area of more advanced metastatic growth and penumbral regions of co‐optive perivascular micrometastatic growth, with magnetisation transfer MRI being the most sensitive to micrometastatic growth. Areas of cortical BOLD activation in response to whisker pad stimulation were significantly reduced in the hemisphere containing metastases in the VPM. The reduction in BOLD response correlated with metastatic burden in the thalamus, and was sensitive to the presence of smaller metastases than currently detectable clinically. Our findings suggest that multimodal MRI provides greater sensitivity to tumour heterogeneity and micrometastatic growth than single modality contrast‐enhanced MRI. Understanding the relationships between these MRI parameters and the underlying pathology may greatly enhance the utility of MRI in diagnosis, staging and monitoring of brain metastasis.

Magnetic resonance imaging reveals therapeutic effects of interferon-beta on cytokine-induced reactivation of rat model of multiple sclerosis

Interferon-β (IFN-β) drugs are considered to derive their beneficial effects on multiple sclerosis (MS) progression via their antiinflammatory properties, but the precise mechanism of action remains unclear. Here, we sought to discover how IFN-β impacts on inflammation-associated aggravation of MS-like lesions in rat. Animals with dormant focal experimental allergic encephalomyelitis (EAE) lesions were challenged intravenously with a replication-deficient adenovirus vector carrying interleukin (IL)-1β cDNA (AdIL-1β). Aggravation of inflammation and demyelination within the focal EAE lesion was observed after AdIL-1β injection with associated changes in tissue structure detected by diffusion and magnetization transfer imaging. Postgadolinium-DTPA T1-weighted images revealed contrast enhancement in the ipsilateral meninges, indicating breakdown of the blood–cerebrospinal fluid barrier, and increased left/right regional cerebral blood volume ratio was also observed after AdIL-1β injection. To determine the role of IFN-β on reactivation of the EAE lesion, rats were treated with therapeutic doses of IFN-β and focal EAE lesions showed significantly reduced reactivation in response to systemic AdIL-1β injection. In conclusion, these findings indicate a central role for peripheral IL-1β expression in the mechanism of MS lesion reactivation and that the therapeutic effects of IFN-β may, at least in part, reflect suppression of the effects of peripheral inflammation on MS lesion pathogenesis.

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.

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; β-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 β- 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-, β- 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-VCAM-1 targeted MRI allows earlier detection of brain metastases in a xenograft melanoma mouse model

BACKGROUND Malignant melanoma frequently metastasises to the brain, with between 10% to 40% of patients estimated to develop intracranial metastases following diagnosis. Although treatments are available for managing brain metastases, their frequently late stage of detection limits the therapeutic potential. Anti-VCAM-1 antibody conjugated microparticles of iron oxide (VCAM-MPIO) have previously been shown to enable detection of early intracranial disease with a breast cancer model.

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 1β 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.