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The editorial highlights articles in a JBO special section, as well as emerging trends in small-animal molecular imaging.The editorial highlights articles in a JBO special section, as well as emerging trends in small-animal molecular imaging.

Non-destructive fluorophore diffusion across cell membranes to provide an unbiased fluorescence intensity readout is critical for quantitative imaging applications in live cells and tissues. Commercially available small-molecule fluorophores have been engineered for biological compatibility, imparting high water solubility by modifying rhodamine and cyanine dye scaffolds with multiple sulfonate groups. The resulting net negative charge, however, often renders these fluorophores cell-membrane-impermeant. Here we report the design and development of our biologically compatible, water-soluble and cell-membrane-permeable fluorophores, termed OregonFluor (ORFluor). By adapting previously established ratiometric imaging methodology using bio-affinity agents, it is now possible to use small-molecule ORFluor-labelled therapeutic inhibitors to quantitatively visualize their intracellular distribution and protein target-specific binding, providing a chemical toolkit for quantifying drug target availability in live cells and tissues.Water-soluble, cell-permeable, inert fluorescent tags called OregonFluors have been developed to withstand environmental changes while resistant towards non-specific binding with subcellular structures. These tags enable quantitative imaging of drug target availability in cells and tissues, providing a route for the future assessment of personalized therapies.

Paired-agent fluorescent molecular imaging approaches involve co-administration of a control (untargeted) imaging agent with a molecularly targeted agent to account for non-specific effects and quantify binding potential (BP)-a parameter proportional to the concentration of the targeted biomolecule. Accurate BP estimation often requires correction for differences in targeted and control agent plasma input functions (PIFs). We provide a simulation-based evaluation of whether dual-channel pulse dye densitometry (PDD) can be used to measure the PIFs of co-administered targeted and control imaging agents, to enable accurate BP estimation. Monte-Carlo simulations of light propagation were carried out using the anatomy and optical properties of a finger, as well as experimentally measured PIFs of co-administered anti-epidermal growth factor receptor fluorescent affibody, ABY-029, and IRDye 680LT, a control imaging agent from past mouse experiments. The accuracy of PIF shape estimation from PDD and PIF difference correction was evaluated by assessing BP estimation accuracy in a simulated \"tumor\" tissue. \"Tumor\" BP measurements using deconvolution correction with noise-free PIFs versus PDD-measured PIFs were compared. The relative error in PDD PIF deconvolution BP estimation was . No statistical difference was found between the estimated BP via deconvolution correction with true PIFs and the estimated BP via the reconstructed PIFs using the proposed PAF-PDD methodology. These results highlight the potential for developing a PDD instrument that can directly measure targeted and control agent PIFs and be used to correct for any PIF differences between agents for more quantitative estimates of BP in paired-agent imaging studies.

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.

Binding kinetics play an important role in cancer diagnosis and therapeutics. However, current methods of quantifying binding kinetics fail to consider the three-dimensional environment that drugs and imaging agents experience in biological tissue. In response, a methodology to assay agent binding and dissociation in 3-D tissue culture was developed using paired-agent molecular imaging principles. To test the methodology, the uptakes of ABY-029 (an IRDye 800CW-labeled epidermal growth factor receptor (EGFR)-targeted antibody mimetic) and IRDye-700DX carboxylate in 3-D spheroids were measured in four different human cancer cell lines throughout staining and rinsing. A compartment model (optimized for the application) was then fit to the kinetic curves of both imaging agents to estimate binding and dissociation rate constants of the EGFR-targeted ABY-029 agent. A statistically significant correlation was observed between apparent association rate constant ( k 3 ) and the receptor concentration experimentally and in simulations ( r  = 0.99, p  < 0.05). A statistically significant difference was found between effective k 3 (apparent rate constant of ABY-029 binding to EGFR) values for cell lines with varying levels of EGFR expression ( p  < 0.05), with no significant difference found between cell lines and controls for other fit parameters. Additionally, a similar binding affinity profile compared to a gold standard method was determined by this model. This low-cost methodology to quantify imaging agent or drug binding affinity in clinically relevant 3-D tumor spheroid models can be used to guide timing of imaging in molecular guided surgery and could have implications in drug development.

Tichauer et al . describe a dual-tracer approach to quantify cancer cell receptor concentrations, in this case epidermal growth factor receptor, in lymph nodes, that can also correct for nonspecific uptake. 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).

Pancreatic cancer tumors are known to be avascular, but their neovascular capillaries are still chaotic leaky vessels. Capillary permeability could have significant value for therapy assessment, and its quantification might be possible with macroscopic imaging of indocyanine green (ICG) kinetics in tissue. The capacity of using standard fluorescence surgical systems for ICG kinetic imaging as a probe for capillary leakage was evaluated using a clinical surgical fluorescence imaging system, as interpreted through vascular permeability modeling. Xenograft pancreatic adenocarcinoma models were imaged in mice during bolus injection of ICG to capture the kinetics of uptake. Image analysis included ratiometric data, normalization, and match to theoretical modeling. Kinetic data were converted into the extraction fraction of the capillary leakage. Pancreatic tumors were usually less fluorescent than the surrounding healthy tissues, but still the rate of tumor perfusion could be assessed to quantify capillary extraction. Model simulations showed that flow kinetics stabilized after about 1 min beyond the initial bolus injection and that the relative extraction fraction model estimates matched the experimental data of normalized uptake within the tissue. The kinetics in the time period of 1 to 2 min post-injection provided optimal differential data between AsPC1 and BxPC3 tumors, although high individual variation exists between tumors. ICG kinetic imaging during the initial leakage phase was diagnostic for quantitative vascular permeability within pancreatic tumors. Methods for autogain correction and normalized model-based interpretation allowed for quantification of extraction fraction and difference identification between tumor types in early timepoints.

Lymph node biopsy is a primary means of staging breast cancer, yet standard pathological techniques are time-consuming and typically sample less than 1% of the total node volume. A low-cost fluorescence optical projection tomography (OPT) protocol is demonstrated for rapid imaging of whole lymph nodes in three dimensions. The relatively low scattering properties of lymph node tissue can be leveraged to significantly improve spatial resolution of lymph node OPT by employing angular restriction of photon detection. It is demonstrated through porcine lymph node metastases models that simple filtered-backprojection reconstruction is sufficient to detect and localize 200-μm-diameter metastases (the smallest clinically significant) in 1-cm-diameter lymph nodes.

Significance: Fluorescence guidance in cancer surgery (FGS) using molecular-targeted contrast agents is accelerating, yet the influence of individual patients’ physiology on the optimal time to perform surgery post-agent-injection is not fully understood. Aim: Develop a mathematical framework and analytical expressions to estimate patient-specific time-to-maximum contrast after imaging agent administration for single- and paired-agent (coadministration of targeted and control agents) protocols. Approach: The framework was validated in mouse subcutaneous xenograft studies for three classes of imaging agents: peptide, antibody mimetic, and antibody. Analytical expressions estimating time-to-maximum-tumor-discrimination potential were evaluated over a range of parameters using the validated framework for human cancer parameters. Results: Correlations were observed between simulations and matched experiments and metrics of tumor discrimination potential (p  <  0.05). Based on human cancer physiology, times-to-maximum contrast for peptide and antibody mimetic agents were <200  min, >15  h for antibodies, on average. The analytical estimates of time-to-maximum tumor discrimination performance exhibited errors of <10  %   on average, whereas patient-to-patient variance is expected to be greater than 100%. Conclusion: We demonstrated that analytical estimates of time-to-maximum contrast in FGS carried out patient-to-patient can outperform the population average time-to-maximum contrast used currently in clinical trials. Such estimates can be made with preoperative DCE-MRI (or similar) and knowledge of the targeted agent’s binding affinity.