Intracellular Paired-Agent Imaging (iPAI) quantifies intracellular drug targets using fluorescently-labeled small molecule imaging agents. iPAI has the potential to predict therapeutic response for individual patients but requires a patient derived xenograft (PDX) model that accurately mimics in vivo tumor heterogeneity yet is easily accessible for intravenous drug administration and rapid image collection. The chicken embryo chorioallantoic membrane (CAM) provides an intermediate, cost-effective model that provides vascularized, in vivo tumors in a matter of days (typically ~72 h) from implantation. Here, we investigate the implantation of thin (< 1 mm) cross-sectional slices (1-2 cm) of freshly excised tumor tissues from mouse xenografts. Multispectral iPAI will be performed to quantify the distribution and heterogeneity of drug targets within the tumor cross-section.
Targeting the aberrant epidermal growth factor receptor (EGFR) signaling pathway is an attractive choice for many cancers (e.g., non-small cell lung carcinoma (NSCLC) and head and neck squamous cell carcinoma (HNSCC)). Despite the development of promising therapeutics, incomplete target engagement and acquired resistance (e.g., mutagenesis and intracellular signaling pathway rewiring) ensure that curative options still elude patients. To address limitations posed by standard drug evaluation assays (e.g., western blot, bulk plasma monitoring, immunohistochemistry), we have developed a novel dynamic, fluorescence-based platform termed intracellular paired agent imaging (iPAI). iPAI quantifies intracellular protein target engagement using two matched small-molecule, cell membrane-permeable agents: one targeted to the protein of interest and one untargeted, which accounts for non-specific therapeutic uptake. Currently, our iPAI panel includes successfully characterized tyrosine kinase inhibitors targeting the kinase binding domain of numerous proteins in the EGFR pathway, including erlotinib (EGFR). Here, we present a pharmacokinetic uptake study using our novel iPAI erlotinib reagents: a targeted erlotinib probed conjugated to silicon tetramethylrhodamine (Erl- SiTMR-T) and an untargeted reagent conjugated to tetramethylrhodaime (Erl-TMR-UT). An initial uptake study in a cell derived xenograft (CDX) model of NSCLC was performed by administering the Erl iPAI reagents systemically via tail vein injection, where drug uptake was quantified in the tumor over time. Excitingly, evidence of heterogeneous uptake was observed in the iPAI injected cohort, displaying distinct drug-uptake within a single tumor. Characterization of additional iPAI agents targeting downstream effectors (e.g., AKT, PI3K, MEK and ERK) is ongoing and will allow us to visualize complex drug-target interactions and quantify their downstream signaling partners during treatment regimens for NSCLC and other cancers. Together, we anticipate these iPAI probes will improve understanding of current limitations in personalized cancer therapy.
Small molecule kinase inhibitors (SMKIs) drugs have the potential to offer exquisite specificity in controlling aberrant intracellular signaling pathways in cancer and other disease states. However, while nearly 50 SMKIs have been FDA-approved, patient responses have been variable, and sensitive populations not easy to identify. For instance, in non-small-cell lung cancer, only 30% of patients respond to the epidermal growth factor receptor (EGFR) targeted SMKI, erlotinib, yet the level of erlotinib uptake is a poor indicator of treatment efficacy. The development of fluorescently-labeled SMKIs that maintain their viability as drugs has facilitated the use of paired-agent molecular imaging protocols that are able to discriminate, in vivo, between imaging agent uptake and binding. Here we present a mathematical framework of SMKI transport and binding, in vivo, and derive a kinetic model for extracting SMKI binding potential (BP) from kinetic fluorescent-SMKI imaging data-proposed as a more effective indicator of potential therapeutic response than SMKI uptake alone. The accuracy and precision of the SMKI BP kinetic model was demonstrated in simulation studies and in an in ovo xenograft experiment. In simulation, the SMKI BP estimates were within 20 5% of expected values over a large range of physiologically relevant blood flow, vascular permeability and cell permeability; and over a range of SMKI affinity, cell membrane permeability, and blood plasma pharmacokinetics. The in ovo experiment bolstered the simulation findings, demonstrating a statistically significant spatial correlation (r > 0.9, p < 0.01) between EGFR concentration measured by a validated extracellular approach and the SMKI BP approach.
Quantification of protein concentrations is often a static and tissue destructive technique. Paired-agent imaging (PAI) using matched targeted and untargeted agents has been established as a dynamic method for quantifying the extracellular domain of epidermal growth factor receptor (EGFR) in vivo in a variety of tumor lines. Here we extend the PAI model to simultaneously quantify the extracellular and intracellular regions of EGFR using novel cell membrane permeable fluorescent small molecules, TRITC-erlotinib (targeted) and BODIPY-N-erlotinib (non-binding control isoform) synthesized in house. An EGFR overexpressing squamous cell carcinoma cell xenograft tumor, A431, was implanted on the chorioallantoic membrane (CAM) of the embryonated chicken egg. In total six fluorescent molecules were administered and monitored over 1 h using multi-spectral imaging. EGFR concentrations were determined using both extracellular and intracellular PAI methods. The fluorescent molecules used for extracellular PAI were ABY-029, an anti- EGFR Affibody molecule conjugated to IRDye 800CW, and a Control Imaging Agent Affibody molecule conjugated to IRDye 680RD. The intracellular PAI (iPAI) fluorescent molecules were cell membrane penetrating TRITC-erlotinib, BODIPY-N-erlotinb, and BODIPY TR carboxylate, as well as cell membrane impermeant control agent, Alexa Fluor 647 carboxylate. Results from simultaneous imaging of both the extracellular and intracellular binding domains of EGFR indicate that concentrations of intracellular EGFR are higher than extracellular. This is anticipated as EGFR exists in two distinct populations in cells, cell membrane bound and internalized, activated protein. iPAI is a promising new method for quantifying intracellular proteins in a rapid tumor model on the chicken CAM.
Surgical nerve damage due to difficulty with identification remains a major risk for postsurgical complications and decreased quality of life. Fluorescence-guided surgery offers a means to specifically highlight tissues of interest such as nerves and a number of fluorescence-guided surgical systems are in clinical trial or are approved for clinical use. However, no clinically approved nerve-specific fluorophores exist. In addition, many preclinical nerve-specific fluorophores tend to accumulate in adipose tissue due to the molecular composition similarities between the two tissues, making it challenging to generate a specific nerve signal. To alleviate this difficulty, we have synthesized a library of oxazine fluorophores based on the Nile Red scaffold, with the goal of strong adipose specificity without nerve uptake to facilitate ratiometric imaging. The library was screened for tissue specificity ex vivo and in vivo, enabling quantification of adipose-, nerve- and muscle-specific uptake as well as selection of the best candidate for adipose selectivity without nerve signal. We showed our selected Nile Red fluorophore improved nerve contrast using ratiometric imaging, especially nerve-to-adipose contrast as compared to the parent Nile Red compound or nerve-specific imaging alone. This adipose-specific Nile Red derivative could be used in future fluorescence-guided surgery applications where adipose- or nerve-specific contrast is required.
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