Tumor endothelial marker imaging in melanomas using dual-tracer fluorescence molecular imaging

Abstract

Purpose: Cancer-specific endothelial markers available for intravascular binding are promising targets for new molecular therapies. In this study, a molecular imaging approach of quantifying endothelial marker concentrations (EMCI) is developed and tested in highly light-absorbing melanomas. The approach involves injection of targeted imaging tracer in conjunction with an untargeted tracer, which is used to account for nonspecific uptake and tissue optical property effects on measured targeted tracer concentrations.

Procedures: Theoretical simulations and a mouse melanoma model experiment were used to test out the EMCI approach. The tracers used in the melanoma experiments were fluorescently labeled anti-Plvap/PV1 antibody (plasmalemma vesicle associated protein Plvap/PV1 is a transmembrane protein marker exposed on the luminal surface of endothelial cells in tumor vasculature) and a fluorescent isotype control antibody, the uptakes of which were measured on a planar fluorescence imaging system.

Results: The EMCI model was found to be robust to experimental noise under reversible and irreversible binding conditions and was capable of predicting expected overexpression of PV1 in melanomas compared to healthy skin despite a 5-time higher measured fluorescence in healthy skin compared to melanoma: attributable to substantial light attenuation from melanin in the tumors.

Conclusions: This study demonstrates the potential of EMCI to quantify endothelial marker concentrations in vivo, an accomplishment that is currently unavailable through any other methods, either in vivo or ex vivo.

Fig. 1

Compartment models for an endothelial marker targeted tracer (a), and an untargeted tracer (b). The rate constants k₁–k₅ represent (1) extravasation of the tracer from the blood (C_p) to the extravascular space (C_e,1 for the targeted tracer and C_e,2 for the untargeted tracer), (2) efflux of the tracer from the extravascular space to the blood, (3) binding to endothelial markers (C_b), (4) dissociation from endothelial marker into blood, and (5) dissociation from endothelial marker into extravascular space. Below each compartment model diagram are the differential equations that express the rate of change of tracer concentration in each compartment. The colored boxes highlight the relationship between tracer concentrations in each compartment and the signal measured in a given region of interest (ROI_T for the targeted in red and ROI_U for the untargeted tracer in green) as a function of time, t. The parameters η_T and η_U represent all factors associated with the relation of measured signal to tracer concentration for the targeted and untargeted tracers, respectively, such as detection efficiency, quantum efficiency of the tracers (for fluorescence imaging), tissue absorption of signal, and uneven excitation of tracer (for fluorescence).

Fig. 2

Simulated targeted tracer curves (blue, red, and black) and an untargeted tracer curve (green), prior to noise addition, are presented in a. The blue curves correspond to k₃ = 0.05 min⁻¹, the red to k₃ = 0.3 min⁻¹, and the black to k₃ = 0.5 min⁻¹. The solid targeted tracer curves correspond to k₄ = 0 min⁻¹ (irreversible binding) and the dotted lines to k₄ = 0.1 min⁻¹ (reversible binding). Results from fitting the endothelial marker concentration imaging (EMCI) algorithm to the simulated solid curves (irreversible binding) in a to estimate k₃ over a range of k₃ inputs are presented in b. The blue data represents the results for irreversible binding data simulated with a K₁ = 0.007 min⁻¹ while the red data corresponds to the same data simulated with a K₁ = 0.001 min⁻¹. The dashed line represents the line of identity between estimated and true k₃.

Fig. 3

Fitting parameter results from applying the endothelial marker concentration imaging (EMCI) algorithm and the single time point EMCI (EMCI_stp) to simulated targeted and untargeted tracer curves for reversible binding (k₄ = 0.1 min⁻¹) are presented in the top two rows and the bottom row, respectively. Each column represents results for different input values of k3: first column k₃ = 0.05 min⁻¹; second column k₃ = 0.3 min⁻¹; third column k₃ = 0.5 min⁻¹. All results are presented in a histogram format where the results from repeated random noise additions to simulated curves were tallied to depict the accuracy and precision of the approaches for estimated endothelial marker concentration-related results. The EMCI fit parameters of k₃ (blue data) and k₄ (red data) are presented in the top row with the simulated input values (“true” values) denoted by the dashed vertical lines. The results of k₃/k₄ for EMCI are presented in the second row of histograms (dark red data), a ratio that is often referred to as the “binding potential” (BP) for its proportionality to available target concentration. Similarly, the results of k₃/k₄ for EMCI_stp are presented in the third row of histograms (green data).

Fig. 4

A gray-scale white-light image of a shaved melanoma mouse positioned in the small animal fluorescence imaging system is presented in a. The yellow arrow highlights the location of the melanoma. Fluorescent images of the untargeted (green-scale) and targeted (red-scale) tracer uptakes at 100-min post-injection are presented in b and c, respectively. The images are equally scaled. Typical uptake curves of the targeted (red curve) and untargeted (green curve) tracer are presented in a region of healthy skin (d) and in the tumor (e). Note the scale of the tumor curves is approximately an order of magnitude lower than those in the healthy skin.

Fig. 5

Imaging results of melanoma mice administered 5-, 20-, and 50-μg of tracer are presented in rows 1, 2, and 3, respectively. The first column presents a gray-scale white-light image of each melanoma and surrounding healthy tissue (yellow arrow locates the melanoma). The second and third columns of images present the fluorescence uptake of the untargeted and targeted tracers, respectively, at 100-min post tracer injection (note that the 5-μg images are scaled to 0.02 rather than 0.2 in the 20- and 50-μg images). The fourth column of images presents the k₃ results from pixel-by-pixel analysis using the endothelial marker concentration imaging algorithm. The fifth column presents the same data overlaid on the white-light image.

Fig. 6

A boxplot presenting the ratio of PV1-targeted fluorescence (orange data) and untargeted fluorescence (purple data) uptake at 100-min post tracer injection in the healthy skin vs. the melanoma tissue is presented in a. This fluorescence uptake is plotted in correlation with the injected concentration of tracer for the healthy skin tissue (red data) and the melanoma tissue (blue data) in b. Solid lines represent linear regressions of the data. Boxplots of the endothelial marker concentration imaging algorithm estimates of the binding parameter k₃ are presented in c for melanoma (blue data) and healthy skin (red data). Correlations between the k₃ estimates in each tissue and the injected tracer concentration are presented in d with the same color code as in c.