Whole-body fluorescence cryotomography identifies a fast-acting, high-contrast, durable contrast agent for fluorescence-guided surgery.

Imaging of tumor-specific fluorescent contrast agents to guide tumor removal has been shown to improve outcomes and is now standard practice for some neurosurgical procedures. However, many agents require administration hours before surgery, a practical challenge, and may exhibit inconsistent concordance with contrast-enhanced MRI (CE-MRI), the current standard for diagnosing and guiding glioma removal. A fluorescent agent that accurately marks tumor shortly after administration and is otherwise similar to CE-MRI would help overcome these shortcomings.

Comparing fluorescent contrast agents for fluorescence guided surgery using 3-D cryo-imaging.

Fluorescence cryo-imaging is a high-resolution optical imaging technique that produces 3-D whole-body biodistributions of fluorescent molecules within an animal specimen. To accomplish this, animal specimens are administered a fluorescent molecule or reporter and are frozen to be autonomously sectioned and imaged at a temperature of -20°C or below. Thus, to apply this technique effectively, administered fluorescent molecules should be relatively invariant to low temperature conditions for cryo-imaging and ideally the fluorescence intensity should be stable and consistent in both physiological and cryo-imaging conditions.

Dynamic oxygen assessment techniques enable determination of anesthesia's impact on tissue.

Tissue oxygenation is well understood to impact radiosensitivity, with reports demonstrating a significant effect of breathing condition and anesthesia type on tissue oxygenation levels and radiobiological response. However, the temporal kinetics of intracellular and extracellular oxygenation have never been quantified, on the timescale that may affect radiotherapy studies. C57BL/6 mice were anesthetized using isoflurane at various percentages or ketamine/xylazine (ket/xyl: 100/10 mg/kg) (N = 48).

Comparing spatial distributions of ALA-PpIX and indocyanine green in a whole pig brain glioma model using 3D fluorescence cryotomography.

Significance: ALA-PpIX and second-window indocyanine green (ICG) have been studied widely for guiding the resection of high-grade gliomas. These agents have different mechanisms of action and uptake characteristics, which can affect their performance as surgical guidance agents. Elucidating these differences in animal models that approach the size and anatomy of the human brain would help guide the use of these agents.

Two-color diffuse flow cytometer.

Significance: Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal.

Evaluating Receptor-Specific Fresh Specimen Staining for Tumor Margin Detection in Clinical Breast Specimens.

Purpose: Reliable and rapid identification of tumor in the margins of breast specimens during breast-conserving surgery to reduce repeat surgery rates is an active area of investigation. Dual-stain difference imaging (DDSI) is one of many approaches under evaluation for this application. This technique aims to topically apply fluorescent stain pairs (one targeted to a receptor-of-interest and the other a spectrally distinct isotype), image both stains, and compute a normalized difference image between the two channels.

A new candidate agent for fluorescence guided neurosurgery produces high, persistent tumor contrast shortly after administration.

Neurosurgical fluorescence guidance relies on contrast agents to identify tumor regions to aid in increasing the extent of resection. Existing contrast agents for this indication each have their own limitation: unpredictable contrast from tumor heterogeneity, significant extravasation into the background brain and long incubation times. An ideal contrast agent should have high and rapid contrast that persists well into the surgical procedure.

Monitoring cancer cell surface receptor expression during anti-angiogenesis therapy in vivo.

Concurrent administration of cancer therapeutics with tumor vasculature targeting treatment has been shown to improve overall survival in multiple human cancer types, as such combinations aim to destroy different compartments of tumors. Anti-angiogenesis therapeutics designed to inhibit tumor induced vessel sprouting have also been shown to re-model the tumor vasculature through a transient vessel normalization effect, which leads to improved perfusion of oxygen and drug in tumor. However, the effects that this normalized vasculature has on the availability of cancer receptor, such as EGFR, is unknown.

Examining the Feasibility of Quantifying Receptor Availability Using Cross-Modality Paired-Agent Imaging.

Purpose: The ability to noninvasively quantify receptor availability (RA) in solid tumors is an aspirational goal of molecular imaging, often challenged by the influence of non-specific accumulation of the contrast agent. Paired-agent imaging (PAI) techniques aim to compensate for this effect by imaging the kinetics of a targeted agent and an untargeted isotype, often simultaneously, and comparing the kinetics of the two agents to estimate RA. This is usually accomplished using two spectrally distinct fluorescent agents, limiting the technique to superficial tissues and/or preclinical applications.

Hyperspectral imaging and spectral unmixing for improving whole-body fluorescence cryo-imaging.

Whole-animal fluorescence cryo-imaging is an established technique that enables visualization of the biodistribution of labeled drugs, contrast agents, functional reporters and cells in detail. However, many tissues produce endogenous autofluorescence, which can confound interpretation of the cryo-imaging volumes. We describe a multi-channel, hyperspectral cryo-imaging system that acquires densely-sampled spectra at each pixel in the 3-dimensional stack.

Noninvasive quantification of target availability during therapy using paired-agent fluorescence tomography.

Immuno-oncological treatment strategies that target abnormal receptor profiles of tumors are an increasingly important feature of cancer therapy. Yet, assessing receptor availability (RA) and drug-target engagement, important determinants of therapeutic efficacy, is challenging with current imaging strategies, largely due to the complex nonspecific uptake behavior of imaging agents in tumors. Herein, we evaluate whether a quantitative noninvasive imaging approach designed to compensate for nonspecific uptake, MRI-coupled paired-agent fluorescence tomography (MRI-PAFT), is capable of rapidly assessing the availability of epidermal growth factor receptor (EGFR) in response to one dose of anti-EGFR antibody therapy in orthotopic brain tumor models.

Heterogeneity of circulating tumor cell dissemination and lung metastases in a subcutaneous Lewis lung carcinoma model.

Subcutaneous () tumor models are widely used in pre-clinical cancer metastasis research. Despite this, the dynamics and natural progression of circulating tumor cells (CTCs) and CTC clusters (CTCCs) in peripheral blood are poorly understood in these models. In this work, we used a new technique called 'diffuse flow cytometry' (DiFC) to study CTC and CTCC dissemination in an Lewis lung carcinoma (LLC) model in mice.

A paired-agent fluorescent molecular imaging strategy for quantifying antibody drug target engagement in window chamber xenograft models.

A paired-agent fluorescent molecular imaging strategy is presented as a method to measure drug target engagement in whole tumor imaging. The protocol involves dynamic imaging of a pair of targeted and control imaging agents prior to and following antibody therapy. Simulations demonstrated that antibody "drug target engagement" can be estimated within a 15%-error over a wide range of tumor physiology (blood flow, vascular permeability, target density) and antibody characteristics (affinity, binding rates).

Noninvasive imaging of dual-agent uptake in glioma and normal tissue using MRI-coupled fluorescence tomography.

As the role of immuno-oncological therapeutics expands, the capacity to noninvasively quantify molecular targets and drug-target engagement is increasingly critical to drug development efforts and treatment monitoring. Previously, we showed that MRI-coupled dual-agent fluorescence tomography (FMT) is capable of estimating the concentration of epidermal growth factor receptor (EGFR) in orthotopic glioma models noninvasively. This approach uses the dynamic information of two fluorescent agents (a targeted agent and untargeted isotype) to estimate tumor receptor concentration in vivo.

Effect of staining temperature on topical dual stain imaging of tissue specimens for tumor identification.

In the pursuit of reducing re-excision rates in breast conserving surgery, a dual probe specimen staining technique has emerged as a promising approach to identify positive margins during surgery. This approach generally involves staining the tissue with a fluorescent dye targeted to a biomarker of interest, such as a cell surface receptor, and an untargeted counterpart, imaging both dyes and using the two images together to compensate for instrumentation inhomogeneities and non-specific uptake. A growing body of literature suggests that this approach can effectively discriminate tumor and normal tissue in gross fresh specimens in reasonable timeframes.

On the use of fluorescein-based contrast agents as analogs to MRI-gadolinium agents for imaging brain tumors.

Magnetic resonance imaging (MRI) of gadolinium (Gd)-based contrast agents plays a central role in managing the treatment of intracranial tumors. These images are involved in diagnosis, surgical planning, surgical navigation, and postoperative assessment of extent of resection. Replicating the information from Gd-MRI in the visual surgical field using fluorescent agents that behave similar to gadolinium in vivo would represent a major advance for surgical intervention of these tumors, and could provide robust compensation information to update pre-operative MRI images during surgery.

Estimating drug delivery using hybrid system for simultaneous dynamic MRI and fluorescence tomography.

Optical tomography is often coupled with high resolution imaging modality like MRI to provide functional information associated with specific anatomical structure noninvasively. MRI-coupled paired agent fluorescence molecular tomography (MRI-PAFT) is a hybrid imaging modality capable of noninvasively quantifying drug-target engagement in vivo utilizing a targeted and an untargeted fluorescence agent. This study compares the uptake kinetics of MRI contrast agent and fluorescence agents in tumor and normal tissue, and demonstrates the potential of utilizing MRI contrast agent kinetic and targeted fluorescence agent kinetics to quantify targeted tumor receptor concentration in glioma tumor model.

Developing a novel hyperspectral imaging cryomacrotome for whole body fluorescence imaging.

The ability to directly measure whole-body fluorescence can enable tracking of labeled cells, metastatic spread, and drug bio-distribution. We describe the development of a new hyperspectral imaging whole body cryo-macrotome designed to acquire 3-D fluorescence volumes in large specimens (whole animals) at high resolution. The use of hyperspectral acquisition provides full spectra at every voxel, enabling spectral decoupling of multiple fluorohpores and autofluorescence.

Estimating paired-agent uptake in altered tumor vasculature using MRI-coupled fluorescence tomography.

Angiogenesis inhibiting cancer therapy has become a standard treatment for many cancer types. The ability to examine the effects of these drugs in tumors noninvasively could help assess efficacy early in the treatment course or identify optimal times to introduce other combinatorial treatments. Herein, we examine whether a paired agent MRI-coupled fluorescence tomography approach can be used to monitor the effects of anti-angiogenesis therapy.

Characterizing short-wave infrared fluorescence of conventional near-infrared fluorophores.

The observed behavior of short-wave infrared (SWIR) light in tissue, characterized by relatively low scatter and subdiffuse photon transport, has generated considerable interest for the potential of SWIR imaging to produce high-resolution, subsurface images of fluorescence activity in vivo. These properties have important implications for fluorescence-guided surgery and preclinical biomedical research. Until recently, translational efforts have been impeded by the conventional understanding that fluorescence molecular imaging in the SWIR regime requires custom molecular probes that do not yet have proven safety profiles in humans.

Real-time in vivo Cherenkoscopy imaging during external beam radiation therapy.

Cherenkov radiation is induced when charged particles travel through dielectric media (such as biological tissue) faster than the speed of light through that medium. Detection of this radiation or excited luminescence during megavoltage external beam radiotherapy (EBRT) can allow emergence of a new approach to superficial dose estimation, functional imaging, and quality assurance for radiation therapy dosimetry. In this letter, the first in vivo Cherenkov images of a real-time Cherenkoscopy during EBRT are presented.

Targeting of systemically-delivered magnetic nanoparticle hyperthermia using a noninvasive, static, external magnetic field.

One of the greatest challenges of nanoparticle cancer therapy is the delivery of adequate numbers of nanoparticles to the tumor site. Iron oxide nanoparticles (IONPs) have many favorable qualities, including their nontoxic composition, the wide range of diameters in which they can be produced, the cell-specific cytotoxic heating that results from their absorption of energy from a nontoxic, external alternating magnetic field (AMF), and the wide variety of functional coatings that can be applied. Although IONPs can be delivered via an intra-tumoral injection to some tumors, the resulting tumor IONP distribution is generally inadequate; additionally, local tumor injections do not allow for the treatment of systemic or multifocal disease.

Iron oxide nanoparticle enhancement of radiation cytotoxicity.

Iron oxide nanoparticles (IONPs) have been investigated as a promising means for inducing tumor cell-specific hyperthermia. Although the ability to generate and use nanoparticles that are biocompatible, tumor specific, and have the ability to produce adequate cytotoxic heat is very promising, significant preclinical and clinical development will be required for clinical efficacy. At this time it appears using IONP-induced hyperthermia as an adjunct to conventional cancer therapeutics, rather than as an independent treatment, will provide the initial IONP clinical treatment.

Imaging and modification of the tumor vascular barrier for improvement in magnetic nanoparticle uptake and hyperthermia treatment efficacy.

The predicted success of nanoparticle based cancer therapy is due in part to the presence of the inherent leakiness of the tumor vascular barrier, the so called enhanced permeability and retention (EPR) effect. Although the EPR effect is present in varying degrees in many tumors, it has not resulted in the consistent level of nanoparticle-tumor uptake enhancement that was initially predicted. Magnetic/iron oxide nanoparticles (mNPs) have many positive qualities, including their inert/nontoxic nature, the ability to be produced in various sizes, the ability to be activated by a deeply penetrating and nontoxic magnetic field resulting in cell-specific cytotoxic heating, and the ability to be successfully coated with a wide variety of functional coatings.

Humanization of yeast to produce complex terminally sialylated glycoproteins.

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation.