Kimberley S. Samkoe

Imaging and Photodynamic Therapy: Mechanisms, Monitoring, and Optimization

The purpose of this review is to present the current state of the role of imaging in photodynamic therapy (PDT). In order for the reader to fully appreciate the context of the discussions embodied in this article we begin with an overview of the PDT process, starting with a brief historical perspective followed by detailed discussions of specific applications of imaging in PDT. Each section starts with an overview of the specific topic and, where appropriate, ends with summary and future directions. The review closes with the authors’ perspective of the areas of future emphasis and promise. The basic premise of this review is that a combination of imaging and PDT will provide improved research and therapeutic strategies.

Review of Neurosurgical Fluorescence Imaging Methodologies

Fluorescence imaging in neurosurgery has a long historical development, with various biomarkers and biochemical agents being used, and numerous technological approaches. This review focuses on contrast agents, summarizing endogenous fluorescence, exogenously stimulated fluorescence, and exogenous contrast agents, and then on tools used for imaging. It ends with a summary of key clinical trials that lead to consensus studies. The practical utility of protoporphyrin IX (PpIX) as stimulated by administration of δ-aminolevulinic acid has had substantial pilot clinical studies and basic science research completed. Recently, multicenter clinical trials using PpIX fluorescence to guide resection have shown efficacy for improved short-term survival. Exogenous agents are being developed and tested preclinically, and hopefully hold the potential for long-term survival benefit if they provide additional capabilities for resection of microinvasive disease or certain tumor subtypes that do not produce PpIX or help delineate low-grade tumors. The range of technologies used for measurement and imaging varies widely, with most clinical trials being carried out with either point probes or modified surgical microscopes. Currently, optimized probe approaches are showing efficacy in clinical trials, and fully commercialized imaging systems are emerging, which will clearly help to lead adoption into neurosurgical practice.

In Vivo Quantification of Tumor Receptor Binding Potential with Dual-Reporter Molecular Imaging

PurposeReceptor availability represents a key component of current cancer management. However, no approaches have been adopted to do this clinically, and the current standard of care is invasive tissue biopsy. A dual-reporter methodology capable of quantifying available receptor binding potential of tumors in vivo within a clinically relevant time scale is presented.ProceduresTo test the methodology, a fluorescence imaging-based adaptation was validated against ex vivo and in vitro measures of epidermal growth factor receptor (EGFR) binding potential in four tumor lines in mice, each line expected to express a different level of EGFR.ResultsA strong correlation was observed between in vivo and ex vivo measures of binding potential for all tumor lines (r = 0.99, p < 0.01, slope = 1.80 ± 0.48, and intercept = −0.58 ± 0.84) and between in vivo and in vitro for the three lines expressing the least amount of EGFR (r = 0.99, p < 0.01, slope = 0.64 ± 0.32, and intercept = 0.47 ± 0.51).ConclusionsBy providing a fast and robust measure of receptor density in tumors, the presented methodology has powerful implications for improving choices in cancer intervention, evaluation, and monitoring, and can be scaled to the clinic with an imaging modality like SPECT.

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo

The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies.

Microscopic lymph node tumor burden quantified by macroscopic dual-tracer molecular imaging

Lymph node biopsy is employed in many cancer surgeries to identify metastatic disease and to determine cancer stage, yet morbidity and diagnostic delays associated with lymph node biopsy could be avoided if noninvasive imaging of nodal involvement were reliable. Molecular imaging has potential in this regard; however, variable delivery and nonspecific uptake of imaging tracers have made conventional approaches ineffective clinically. Here we present a method of correcting for nonspecific uptake with injection of a second untargeted tracer that allows for quantification of tumor burden in lymph nodes. We confirmed the approach in an athymic mouse model of metastatic human breast cancer by targeting epidermal growth factor receptor, a cell surface receptor overexpressed by many cancers. We observed a significant correlation between in vivo (dual-tracer) and ex vivo measures of tumor burden (r = 0.97, P < 0.01), with an ultimate sensitivity of approximately 200 cells (potentially more sensitive than conventional lymph node biopsy).

IMAGING TUMOR VARIATION IN RESPONSE TO PHOTODYNAMIC THERAPY IN PANCREATIC CANCER XENOGRAFT MODELS

PURPOSE A treatment monitoring study investigated the differential effects of orthotopic pancreatic cancer models in response to interstitial photodynamic therapy (PDT), and the validity of using magnetic resonance imaging as a surrogate measure of response was assessed. METHODS AND MATERIALS Different orthotopic pancreatic cancer xenograft models (AsPC-1 and Panc-1) were used to represent the range of pathophysiology observed in human beings. Identical dose escalation studies (10, 20, and 40J/cm) using interstitial verteporfin PDT were performed, and magnetic resonance imaging with T2-weighted and T1-weighted contrast were used to monitor the total tumor volume and the vascular perfusion volume, respectively. RESULTS There was a significant amount of necrosis in the slower-growing Panc-1 tumor using high light dose, although complete necrosis was not observed. Lower doses were required for the same level of tumor kill in the faster-growing AsPC-1 cell line. CONCLUSIONS The tumor growth rate and vascular pattern of the tumor affect the optimal PDT treatment regimen, with faster-growing tumors being relatively easier to treat. This highlights the fact that therapy in human beings shows a heterogeneous range of outcomes, and suggests a need for careful individualized treatment outcomes assessment in clinical work.

MRI-coupled Fluorescence Tomography Quantifies EGFR Activity in Brain Tumors

RATIONALE AND OBJECTIVES This report demonstrates the diagnostic potential of magnetic resonance imaging (MRI)-coupled fluorescence molecular tomography (FMT) to determine epidermal growth factor receptor (EGFR) status in brain cancer. MATERIALS AND METHODS Two orthotopic glioma xenograft models were used in this study: one represented high EGFR expression and the other low expression. Nude mice were inoculated with cells from either one of the tumor lines or were used in a sham surgery control group. Animals were imaged using a unique MRI-FMT scanner 48 hours after intravenous injection of a near-infrared fluorophore bound to epidermal growth factor (EGF) ligand. Coronal images of fluorescence activity of the injected dye in the mouse brain were recovered using the MRI images as anatomical templates. RESULTS In vivo images of fluorescence activity showed significant differences between animal populations, an observation confirmed by receiver operating characteristic analysis that revealed 100% sensitivity and specificity between animal groups implanted with EGFR((+)) and EGFR((-)) tumor lines. Similar performance was observed between EGFR((+)) and sham surgery control animals. CONCLUSIONS This preclinical study suggests that MRI-FMT with fluorescent EGF provides excellent discrimination between tumors based on EGFR status. Reliable quantification of receptor status using minimally invasive techniques would be an important innovation for investigating new and existing cancer treatments that target these cellular mechanisms in research animals, and may be applied to identify receptor amplification in human brain cancer patients. This study represents the first systematic multianimal validation of receptor-specific imaging using MRI-guided fluorescence tomography.

Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

Object Fluorescence imaging has the potential to significantly improve neurosurgical resection of oncologic lesions through improved differentiation between normal and cancerous tissue at the tumor margins. In order to successfully mark glioma tissue a fluorescent tracer must have the ability to penetrate through the blood brain barrier (BBB) and provide delineation in the tumor periphery where heterogeneously intact BBB may exist. In this study it was hypothesized that, due to its smaller size, fluorescently labeled anti-EGFR Affibody protein (∼7 kDa) would provide a more clear delineation of the tumor margin than would fluorescently labeled cetuximab, a full antibody (∼150 kDa) to the epidermal growth factor receptor (EGFR). Methods Cetuximab and anti-EGFR targeted Affibody were conjugated to two different fluorescent dyes (both emitting in the near-infrared) and injected intravenously into 6 athymic mice which were inoculated orthotopically with green fluorescent protein (GFP) expressing human U251 glioma cells. Each mouse was sacrificed at 1-h post injection, at which time brains were removed, snap frozen, sectioned and quantitatively analyzed for fluorescence distribution. Results Ex vivo analysis showed on average, nearly equal concentrations of cetuximab and Affibody within the tumor (on average Affibody made up 49±6% of injected protein), however, the cetuximab was more confined to the center of the tumor with Affibody showing significantly higher concentrations at the tumor periphery (on average Affibody made up 72±15% of injected protein in the outer 50 um of the tumor). Further ex vivo analysis of detection studies showed that the Affibody provided superior discrimination for differentiation of tumor from surrounding normal brain. Conclusions The present study indicates that fluorescently labeled anti-EGFR Affibody can provide significantly better delineation of tumor margins than a fluorescently labeled anti-EGFR antibody and shows considerable potential for guiding margin detection during neurosurgery.

Nanoparticle uptake in tumors is mediated by the interplay of vascular and collagen density with interstitial pressure

UNLABELLED Nanoparticle delivery into solid tumors is affected by vessel density, interstitial fluid pressure (IFP) and collagen, as shown in this article by contrasting the in vivo macroscopic quantitative uptake of 40 nm fluorescent beads in three tumor types.The fluorescence uptake was quantified on individual animals by normalization with the transmitted light and then normalized to normal tissue uptake in each mouse. Mean data for uptake in individual tumor lines then showed expected trends with the largest uptake in the most vascularized tumor line. Tumor lines with increased collagen were also consistent with highest interstitial fluid pressure and correlated with lowest uptake of nanoparticles. The data is consistent with a delivery model indicating that while vascular permeability is maximized by neovascular growth, it is inhibited by collagen content and the resulting interstitial pressure. Imaging of these parameters in vivo can lead to better individual noninvasive methods to assess drug penetration in situ. FROM THE CLINICAL EDITOR In this manuscript the dependence of nanoparticle delivery is addressed from the standpoint of vascular factors (the more vascularized, the better delivery) and as a function of collagen density and interstitial pressure (the higher these are, the worse the delivery).

Quantitative imaging of scattering changes associated with epithelial proliferation, necrosis, and fibrosis in tumors using microsampling reflectance spectroscopy

Highly localized reflectance measurements can be used to directly quantify scatter changes in tissues. We present a microsampling approach that is used to raster scan tumors to extract parameters believed to be related to the tissue ultrastructure. A confocal reflectance imager was developed to examine scatter changes across pathologically distinct regions within tumor tissues. Tissue sections from two murine tumors, AsPC-1 pancreas tumor and the Mat-LyLu Dunning prostate tumor, were imaged. After imaging, histopathology-guided region-of-interest studies of the images allowed analysis of the variations in scattering resulting from differences in tissue ultra-structure. On average, the median scatter power of tumor cells with high proliferation index (HPI) was about 26% less compared to tumor cells with low proliferation index (LPI). Necrosis exhibited the lowest scatter power signature across all the tissue types considered, with about 55% lower median scatter power than LPI tumor cells. Additionally, the level and maturity of the tumors fibroplastic response was found to influence the scatter signal. This approach to scatter visualization of tissue ultrastructure in situ could provide a unique tool for guiding surgical resection, but this kind of interpretation into what the signal means relative to the pathology is required before proceeding to clinical studies.