AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma

A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our study provides a functional landscape of gliomagenesis suppressors in vivo

OCT Angiography (OCTA) in Retinal Diagnostics

Optical coherence tomography angiography (OCTA) is an imaging modality which can be applied in ophthalmology to provide detailed visualization of the perfusion of vascular networks in the eye. Compared to previous state of the art dye-based imaging, such as fluorescein angiography, OCTA is non-invasive, time-efficient, and it allows for the examination of retinal vasculature in 3D. These advantages of the technique combined with the good usability in commercial devices led to a quick adoption of the new modality in the clinical routine. However, the interpretation of OCTA data is not without problems: Commonly observed image artifacts and the quite involved algorithmic details of OCTA signal construction can make the clinical assessment of OCTA exams challenging. In this article we describe the technical background of OCTA and discuss the data acquisition process, common image visualization techniques, as well as limitations and sources of artifacts of the modality. Examples of clinical cases underline the increasing importance of the OCTA technology in ophthalmology and its relation to dye-based angiography

Dendrimer display of tumor-homing peptides

In vivo selection of phage libraries that display random peptide sequences on their surface has yielded a number of peptides that specifically home to tumor tissue. In this study, two different peptides are introduced to synthetic dendritic scaffolds via oxime chemistry and the resulting compounds are analyzed for tumor homing. Modification of the dendritic wedge with a short, linear peptide that homes to clotted plasma proteins showed that a specific receptor in tumor tissue is recognized, but that the extravasation is likely affected by the size of the construct. In contrast, a positively charged cyclic peptide with cell penetrating properties was capable of directing the entire dendritic architecture toward a specific receptor in tumor lymphatics. These observations are in agreement with results previously reported for micelles and nanoparticles and emphasize the influence of peptide properties and overall size on the biodistribution of multivalent macromolecule

High-resolution optoacoustic imaging of tissue responses to vascular-targeted therapies.

The monitoring of vascular-targeted therapies using magnetic resonance imaging, computed tomography or ultrasound is limited by their insufficient spatial resolution. Here, by taking advantage of the intrinsic optical properties of haemoglobin, we show that raster-scanning optoacoustic mesoscopy (RSOM) provides high-resolution images of the tumour vasculature and of the surrounding tissue, and that the detection of a wide range of ultrasound bandwidths enables the distinction of vessels of differing size, providing detailed insights into the vascular responses to vascular-targeted therapy. Using RSOM to examine the responses to vascular-targeted photodynamic therapy in mice with subcutaneous xenografts, we observed a substantial and immediate occlusion of the tumour vessels followed by haemorrhage within the tissue and the eventual collapse of the entire vasculature. Using dual-wavelength RSOM, which distinguishes oxyhaemoglobin from deoxyhaemoglobin, we observed an increase in oxygenation of the entire tumour volume immediately after the application of the therapy, and a second wave of oxygen reperfusion approximately 24 h thereafter. We also show that RSOM enables the quantification of differences in neoangiogenesis that predict treatment efficacy

Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice

The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1+ cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1–coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[18F]fluorobenzoic acid ([18F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [18F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [18F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions

Targeted nanoparticle enhanced proapoptotic peptide as potential therapy for glioblastoma

Antiangiogenic therapy can produce transient tumor regression in glioblastoma (GBM), but no prolongation in patient survival has been achieved. We have constructed a nanosystem targeted to tumor vasculature that incorporates three elements: (i) a tumor-homing peptide that specifically delivers its payload to the mitochondria of tumor endothelial cells and tumor cells, (ii) conjugation of this homing peptide with a proapoptotic peptide that acts on mitochondria, and (iii) multivalent presentation on iron oxide nanoparticles, which enhances the proapoptotic activity. The iron oxide component of the nanoparticles enabled imaging of GBM tumors in mice. Systemic treatment of GBM-bearing mice with the nanoparticles eradicated most tumors in one GBM mouse model and significantly delayed tumor development in another. Coinjecting the nanoparticles with a tumor-penetrating peptide further enhanced the therapeutic effect. Both models used have proven completely resistant to other therapies, suggesting clinical potential of our nanosystem