A pan-cancer dye for solid-tumour screening, resection and wound monitoring via short-wave and near-infrared fluorescence imaging.

The efficacy of fluorescence-guided surgery in facilitating the real-time delineation of tumours depends on the optical contrast of tumour tissue over healthy tissue. Here we show that CJ215-a commercially available, renally cleared carbocyanine dye sensitive to apoptosis, and with an absorption and emission spectra suitable for near-infrared fluorescence imaging (wavelengths of 650-900 nm) and shortwave infrared (SWIR) fluorescence imaging (900-1,700 nm)-can facilitate fluorescence-guided tumour screening, tumour resection and the assessment of wound healing. In tumour models of either murine or human-derived breast, prostate and colon cancers and of fibrosarcoma, and in a model of intraperitoneal carcinomatosis, imaging of CJ215 with ambient light allowed for the delineation of nearly all tumours within 24 h after intravenous injection of the dye, which was minimally taken up by healthy organs. At later timepoints, CJ215 provided tumour-to-muscle contrast ratios up to 100 and tumour-to-liver contrast ratios up to 18. SWIR fluorescence imaging with the dye also allowed for quantifiable non-contact wound monitoring through commercial bandages. CJ215 may be compatible with existing and emerging clinical solutions.

A pan-cancer agent for screening, resection and wound monitoring via NIR and SWIR imaging.

Fluorescence guided surgery (FGS) facilitates real time tumor delineation and is being rapidly established clinically. FGS efficacy is tied to the utilized dye and provided tumor contrast over healthy tissue. Apoptosis, a cancer hallmark, is a desirable target for tumor delineation. Here, we preclinically in vitro and in vivo, validate an apoptosis sensitive commercial carbocyanine dye (CJ215), with absorption and emission spectra suitable for near infrared (NIR, 650-900nm) and shortwave infrared (SWIR, 900-1700nm) fluorescence imaging (NIRFI, SWIRFI). High contrast SWIRFI for solid tumor delineation is demonstrated in multiple murine and human models including breast, prostate, colon, fibrosarcoma and intraperitoneal colorectal metastasis. Organ necropsy and imaging highlighted renal clearance of CJ215. SWIRFI and CJ215 delineated all tumors under ambient lighting with a tumor-to-muscle ratio up to 100 and tumor-to-liver ratio up to 18, from 24 to 168 h post intravenous injection with minimal uptake in healthy organs. Additionally, SWIRFI and CJ215 achieved non-contact quantifiable wound monitoring through commercial bandages. CJ215 provides tumor screening, guided resection, and wound healing assessment compatible with existing and emerging clinical solutions.

Membrane-derived particles shed by PSMA-positive cells function as pro-angiogenic stimuli in tumors.

Cell membrane-derived particles (Mp) are rounded membrane-enclosed particles that are shed from tumor cells. Mp are formed from tumor membranes and are capable of tumor targeting and immunotherapeutic agents because they share membrane homology with parental cells; thus, they are under consideration as a drug delivery vehicle. Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein with enzymatic functionality, is highly expressed in Mp and extracellular vesicles (EV) from prostate cancer (PCa) with poor clinical prognosis. Although PSMA expression was previously shown in EV and Mp isolated from cell lines and from the blood of patients with high-grade PCa, no pathophysiological effects have been linked to PCa-derived Mp. Here, we compared Mp from PSMA-expressing (PSMA-Mp) and PSMA-non-expressing (WT-Mp) cells side by side in vitro and in vivo. PSMA-Mp can transfer PSMA and new phenotypic characteristics to the tumor microenvironment. The consequence of PSMA transfer to cells and increased secretion of vascular endothelial growth factor-A (VEGF-A), pro-angiogenic and pro-lymphangiogenic mediators, with increased 4E binding protein 1 (4EBP-1) phosphorylation.

Ambient Light Resistant Shortwave Infrared Fluorescence Imaging for Preclinical Tumor Delineation via the pH Low-Insertion Peptide Conjugated to Indocyanine Green.

Shortwave infrared (900-1,700 nm) fluorescence imaging (SWIRFI) has shown significant advantages over visible (400-650 nm) and near-infrared (700-900 nm) fluorescence imaging (reduced autofluorescence, improved contrast, tissue resolution, and depth sensitivity). However, there is a major lag in the clinical translation of preclinical SWIRFI systems and targeted SWIRFI probes. Methods: We preclinically show that the pH low-insertion peptide conjugated to indocyanine green (pHLIP ICG), currently in clinical trials, is an excellent candidate for cancer-targeted SWIRFI. Results: pHLIP ICG SWIRFI achieved picomolar sensitivity (0.4 nM) with binary and unambiguous tumor screening and resection up to 96 h after injection in an orthotopic breast cancer mouse model. SWIRFI tumor screening and resection had ambient light resistance (possible without gating or filtering) with outstanding signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) values at exposures from 10 to 0.1 ms. These SNR and CNR values were also found for the extended emission of pHLIP ICG in vivo (>1,100 nm, 300 ms). Conclusion: SWIRFI sensitivity and ambient light resistance enabled continued tracer clearance tracking with unparalleled SNR and CNR values at video rates for tumor delineation (achieving a tumor-to-muscle ratio above 20). In total, we provide a direct precedent for the democratic translation of an ambient light resistant SWIRFI and pHLIP ICG ecosystem, which can instantly improve tumor resection.

Vascularized tumor on a microfluidic chip to study mechanisms promoting tumor neovascularization and vascular targeted therapies.

The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of metastatic renal cell carcinoma spheroids and endothelial cells in a 3D environment. Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprout towards the tumor and mimic a vascular network. Bevacizumab, an angiogenic inhibitor, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessel's functionality, drug delivery, and molecular expression of PSMA. Finally, observations in the vascularized tumor on chip permitted direct and conclusive quantification of this therapy in weeks as opposed to months in a comparable animal model. Teaser: Vascularized tumor on microfluidic chip provides opportunity to study targeted therapies and improves preclinical drug discovery.

Prospective testing of clinical Cerenkov luminescence imaging against standard-of-care nuclear imaging for tumour location.

In oncology, the feasibility of Cerenkov luminescence imaging (CLI) has been assessed by imaging superficial lymph nodes in a few patients undergoing diagnostic 18F-fluoro-2-deoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT). However, the weak luminescence signal requires the removal of ambient light. Here we report the development of a clinical CLI fiberscope with a lightproof enclosure, and the clinical testing of the setup using five different radiotracers. In an observational prospective trial (ClinicalTrials.gov identifier NCT03484884 ) involving 96 patients with existing or suspected tumours, scheduled for routine clinical FDG PET or 131I therapy, the level of agreement of CLI with standard-of-care imaging (PET or planar single-photon emission CT) for tumour location was 'acceptable' or higher (≥3 in the 1-5 Likert scale) for 90% of the patients. CLI correlated with the concentration of radioactive activity, and captured therapeutically relevant information from patients undergoing targeted radiotherapy or receiving the alpha emitter 223Ra, which cannot be feasibly imaged clinically. CLI could supplement radiological scans, especially when scanner capacity is limited.

Identification of alternative protein targets of glutamate-ureido-lysine associated with PSMA tracer uptake in prostate cancer cells.

Prostate-specific membrane antigen (PSMA) is highly overexpressed in most prostate cancers and is clinically visualized using PSMA-specific probes incorporating glutamate-ureido-lysine (GUL). PSMA is effectively absent from certain high-mortality, treatment-resistant subsets of prostate cancers, such as neuroendocrine prostate cancer (NEPC); however, GUL-based PSMA tracers are still reported to have the potential to identify NEPC metastatic tumors. These probes may bind unknown proteins associated with PSMA-suppressed cancers. We have identified the up-regulation of PSMA-like aminopeptidase NAALADaseL and the metabotropic glutamate receptors (mGluRs) in PSMA-suppressed prostate cancers and find that their expression levels inversely correlate with PSMA expression and are associated with GUL-based radiotracer uptake. Furthermore, we identify that NAALADaseL and mGluR expression correlates with a unique cell cycle signature. This provides an opportunity for the future study of the biology of NEPC and potential therapeutic directions. Computationally predicting that GUL-based probes bind well to these targets, we designed and synthesized a fluorescent PSMA tracer to investigate these proteins in vitro, where it shows excellent affinity for PSMA, NAALADaseL, and specific mGluRs associated with poor prognosis.

Cytomegalovirus infection of glioblastoma cells leads to NF-κB dependent upregulation of the c-MET oncogenic tyrosine kinase.

Cytomegalovirus (CMV) is widespread in humans and has been implicated in glioblastoma (GBM) and other tumors. However, the role of CMV in GBM remains poorly understood and the mechanisms involved are not well-defined. The goal of this study was to identify candidate pathways relevant to GBM that may be modulated by CMV. Analysis of RNAseq data after CMV infection of patient-derived GBM cells showed significant upregulation of GBM-associated transcripts including the MET oncogene, which is known to play a role in a subset of GBM patients. These findings were validated in vitro in both mouse and human GBM cells. Using immunostaining and RT-PCR in vivo, we confirmed c-MET upregulation in a mouse model of CMV-driven GBM progression and in human GBM. siRNA knockdown showed that MET upregulation was dependent on CMV-induced upregulation of NF-κB signaling. Finally, proneural GBM xenografts overexpressing c-MET grew much faster in vivo than controls, suggesting a mechanism by which CMV infection of tumor cells could induce a more aggressive mesenchymal phenotype. These studies implicate the CMV-induced upregulation of c-MET as a potential mechanism involved in the effects of CMV on GBM growth.

A review of recent and emerging approaches for the clinical application of Cerenkov luminescence imaging.

Cerenkov luminescence (CL) is a blue-weighted emission of light produced by a vast array of clinically approved radioisotopes and LINAC accelerators. When ß particles (emitted during the decay of radioisotopes) are present in a medium such as water or tissue, they are able to travel faster than the speed of light in that medium and in doing so polarize the molecules around them. Once the particle has left the local area, the polarized molecules relax and return to their baseline state releasing the additional energy as light (luminescence). This blue glow has commonly been used to determine the output of nuclear power plant cores and, in recent years, has found traction in the preclinical and clinical imaging field. This brief review will discuss the technology which has enabled the emergence of the biomedical Cerenkov imaging field, recent pre-clinical studies with potential clinical translation of Cerenkov luminescence imaging (CLI) and the current clinical implementations of the method. Finally, an outlook is given as to the direction in which the field is heading.

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.

Cytomegalovirus promotes murine glioblastoma growth via pericyte recruitment and angiogenesis.

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM); however, a mechanistic connection in vivo has not been established. The purpose of this study is to characterize the effects of murine CMV (MCMV) on GBM growth in murine models. Syngeneic GBM models were established in mice perinatally infected with MCMV. We found that tumor growth was markedly enhanced in MCMV+ mice, with a significant reduction in overall survival compared with that of controls (P < 0.001). We observed increased angiogenesis and tumor blood flow in MCMV+ mice. MCMV reactivation was observed in intratumoral perivascular pericytes and tumor cells in mouse and human GBM specimens, and pericyte coverage of tumor vasculature was strikingly augmented in MCMV+ mice. We identified PDGF-D as a CMV-induced factor essential for pericyte recruitment, angiogenesis, and tumor growth. The antiviral drug cidofovir improved survival in MCMV+ mice, inhibiting MCMV reactivation, PDGF-D expression, pericyte recruitment, and tumor angiogenesis. These data show that MCMV potentiates GBM growth in vivo by increased pericyte recruitment and angiogenesis due to alterations in the secretome of CMV-infected cells. Our model provides evidence for a role of CMV in GBM growth and supports the application of antiviral approaches for GBM therapy.

Altered splicing leads to reduced activation of CPEB3 in high-grade gliomas.

Cytoplasmic polyadenylation element binding proteins (CPEBs) are auxiliary translational factors that associate with consensus sequences present in 3'UTRs of mRNAs, thereby activating or repressing their translation. Knowing that CPEBs are players in cell cycle regulation and cellular senescence prompted us to investigate their contribution to the molecular pathology of gliomas-most frequent of intracranial tumors found in humans. To this end, we performed methylation analyses in the promoter regions of CPEB1-4 and identified the CPEB1 gene to be hypermethylated in tumor samples. Decreased expression of CPEB1 protein in gliomas correlated with the rising grade of tumor malignancy. Abundant expression of CPEBs2-4 was observed in several glioma specimens. Interestingly, expression of CPEB3 positively correlated with tumor progression and malignancy but negatively correlated with protein phosphorylation in the alternatively spliced region. Our data suggest that loss of CPEB3 activity in high-grade gliomas is caused by expression of alternatively spliced variants lacking the B-region that overlaps with the kinase recognition site. We conclude that deregulation of CPEB proteins may be a frequent phenomenon in gliomas and occurs on the level of transcription involving epigenetic mechanism as well as on the level of mRNA splicing, which generates isoforms with compromised biological properties.