Receptor-targeted Fluorescent Probes Research Articles

Fluorescent tamoxifen derivatives as biophotonic probes for the study of human breast cancer and estrogen-receptor directed photosensitizers

Currently, breast cancer is the most common type of cancer worldwide, being the so-called estrogen receptor (ER) positive the subtype with highest prevalence. For decades, the most successful strategy to prevent recurrence of ER + breast cancers is tamoxifen coadjuvant therapy. However, the involvement of the different estrogen receptors (ERα, ERβ and GPER) and the interaction in their signaling pathways, the evidence for side-effects in chronic tamoxifen treatments, as well as the appearance of tamoxifen-resistant breast cancers, have encouraged the need for novel receptor-targeted fluorescent probes. In line with this, multifunctional antiestrogen conjugates, exhibiting fluorescent properties while retaining the ability to antagonize estrogen actions, have been synthesized and proven to be particularly useful in the study of the molecular biology of ER + breast cancers. These novel fluorescent tamoxifen derivatives (FTDs) exhibit pharmacological features of pure antiestrogens, with similar or even greater affinity for ERα than tamoxifen, inhibit ER-dependent gene transcription and cell proliferation, and are devoid of uterotrophic effects. In the present study we have aim at providing a detailed view of their biophotonic potential, including their spectroscopic properties, their usefulness for fluorescent labelling of cellular compartments and intracellular targets, their application in the identification of non-ER antiestrogen binding sites thought fluorescence competition assays, and finally, in their ability to function as efficient ER-targeted photosensitizers.

First Clinical Results of Fluorescence Lifetime-enhanced Tumor Imaging Using Receptor-targeted Fluorescent Probes.

Fluorescence molecular imaging, using cancer-targeted near infrared (NIR) fluorescent probes, offers the promise of accurate tumor delineation during surgeries and the detection of cancer specific molecular expression in vivo. However, nonspecific probe accumulation in normal tissue results in poor tumor fluorescence contrast, precluding widespread clinical adoption of novel imaging agents. Here we present the first clinical evidence that fluorescence lifetime (FLT) imaging can provide tumor specificity at the cellular level in patients systemically injected with panitumumab-IRDye800CW, an EGFR-targeted NIR fluorescent probe. We performed wide-field and microscopic FLT imaging of resection specimens from patients injected with panitumumab-IRDye800CW under an FDA directed clinical trial. We show that the FLT within EGFR-overexpressing cancer cells is significantly longer than the FLT of normal tissue, providing high sensitivity (>98%) and specificity (>98%) for tumor versus normal tissue classification, despite the presence of significant nonspecific probe accumulation. We further show microscopic evidence that the mean tissue FLT is spatially correlated (r > 0.85) with tumor-specific EGFR expression in tissue and is consistent across multiple patients. These tumor cell-specific FLT changes can be detected through thick biological tissue, allowing highly specific tumor detection and noninvasive monitoring of tumor EFGR expression in vivo. Our data indicate that FLT imaging is a promising approach for enhancing tumor contrast using an antibody-targeted NIR probe with a proven safety profile in humans, suggesting a strong potential for clinical applications in image guided surgery, cancer diagnostics, and staging.

Design and Application of Receptor-Targeted Fluorescent Probes Based on Small Molecular Fluorescent Dyes.

In recent years, a variety of receptor-targeted fluorescent probes have been developed and widely used to realize the visualization of certain receptors, which facilitates the early diagnosis and treatment of diseases. In this Review, we focus on the recent achievements in design, chemical structure, imaging characterization, and potential applications of receptor-targeted fluorescent probes from the past 10 years. The development and application of receptor-targeted fluorescent probes will expand our knowledge of the distribution and function of disease-related receptors, shed light on the drug discovery for clinical diseases where receptors are implicated, and feed into the diagnosis and treatment of a plethora of diseases, including tumors.

Peptide Receptor-Targeted Fluorescent Probe: Visualization and Discrimination between Chronic and Acute Ulcerative Colitis.

The inflammatory activity of ulcerative colitis plays an important role in the medical treatment. However, accurate and real-time monitoring of the colitis activity with noninvasive bioimaging method is still challenging, especially in distinguishing between chronic and acute colitis. As a good receptor, the oligopeptide transporter (PepT1) is overexpressed in the colonic epithelial cells of chronic ulcerative colitis, which can deliver tripeptide KPV (Lys-Pro-Val, the C-terminal sequence of α-MSH) into cytosol in the intestine. Herein, we report a PepT1 peptide receptor-targeted fluorescent probe, dicyanomethylene-4H-pyran (DCM)-KPV, with the strategy of conjugating the KPV into the DCM chromophore. The diagnostic fluorescent probe bestows a specific receptor-targeted interaction with PepT1 through the KPV moiety, possessing several beneficial characteristics, such as efficient long emission, low photobleaching, negligible cytotoxicity, and high cytocompatibility in living cells. We build the overexpressed PepT1 on the cytomembrane of ulcerative colitis model Caco-2 cell as the efficient receptor to accumulate the targeted tripeptide KPV in the cytoplasm and nucleus. With the co-localization of DCM-KPV and the DNA-specific fluorophore, DAPI, the specifically long emission from chromophore DCM and efficient receptor-targeted peptide KPV, the fluorescent probe of DCM-KPV makes a breakthrough to the direct noninvasive observation of the accumulation in colon inflammation regions via intestinal mucosa, even successfully distinguishing the chronic, acute ulcerative colitis and normal groups. Compared with the traditional unenhanced magnetic resonance imaging and hematoxylin and eosin (H&E) staining, we make full use of exploiting the specific target-receptor interaction between the tripeptide unit, KPV, and the oligopeptide transporter, PepT1, for sensing selectivity. The desirable diagnostic ability of DCM-KPV can guarantee the real-time tracking and visualization of the role of intracellular KPV on ulcerative colitis, which provides an alternative to replace the time-consuming and tissue sampling-invasive H&E staining diagnosis.

Optimizing fresh specimen staining for rapid identification of tumor biomarkers during surgery.

Rationale: Positive margin status due to incomplete removal of tumor tissue during breast conserving surgery (BCS) is a prevalent diagnosis usually requiring a second surgical procedure. These follow-up procedures increase the risk of morbidity and delay the use of adjuvant therapy; thus, significant efforts are underway to develop new intraoperative strategies for margin assessment to eliminate re-excision procedures. One strategy under development uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI). DDSI uses a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent companion imaging agent topically applied to fresh resected specimens, where the fluorescence from each probe is imaged and a normalized difference image is computed to identify tumor-target distribution in the specimen margins. While previous reports suggested this approach is a promising new tool for surgical guidance, advancing the approach into the clinic requires methodical protocol optimization and further validation.Methods: In the present study, we used breast cancer xenografts and receiver operator characteristic (ROC) curve analysis to evaluate a wide range of staining and imaging parameters, and completed a prospective validation study on multiple tumor phenotypes with different target expression. Imaging fluorophore-probe pair, concentration, and incubation times were systematically optimized using n=6 tissue specimen replicates per staining condition. Resulting tumor vs. normal adipose tissue diagnostic performance were reported and staining patterns were validated via receptor specific immunohistochemistry colocalization. Optimal staining conditions were tested in receptor positive and receptor negative cohorts to confirm specificity.Results: The optimal staining conditions were found to be a one minute stain in a 200 nM probe solution (area under the curve (AUC) = 0.97), where the choice of fluorescent label combination did not significantly affect the diagnostic performance. Using an optimal threshold value determined from ROC curve analysis on a training data set, a prospective study on xenografts resulted in an AUC=0.95 for receptor positive tumors and an AUC = 0.50 for receptor negative (control) tumors, confirming the diagnostic performance of this novel imaging technique.Conclusions: DDSI provides a robust, molecularly specific imaging methodology for identifying tumor tissue over benign mammary adipose tissue. Using a dual probe imaging strategy, nonspecific accumulation of targeted probe was corrected for and tumor vs. normal tissue diagnostic potential was improved, circumventing difficulties with ex vivo tissue specimen staining and allowing for rapid clinical translation of this promising technology for tumor margin detection during BCS procedures.

Estrogen receptor-targeted fluorescent probes and bioapplications

Owing to their high sensitivity and applicability, fluorescent probes have been considered as an inevitable research tool in biology, chemistry, biomedical science, and materials science. Herein, we present the recent progress on fluorescent probes for the estrogen receptor. In particular, their classification, optical properties, biological activity, such as binding assays, ER detection/imaging and estrogen signaling studies were reviewed. The development of these fluorescent probes will provide new clues and improved therapeutics that target the estrogen receptor.

Recent Advances in Receptor-Targeted Fluorescent Probes for In Vivo Cancer Imaging

Receptor-targeted optical imaging of cancer is emerging as an attractive strategy for early cancer diagnosis and surgical guidance. The success of such strategy depends largely upon the development of receptor-targeted fluorescent probes with high specificity and binding affinity to the target receptors. Recently, a host of such probes have been reported to target cancer-specific receptors, such as somatostatin receptors (SSTRs), integrin receptors, cholecystokinin-2 (CCK(2)) receptor, gastrin-releasing peptide (GRP) receptor, endothelin A (ET(A)) receptor, translocator protein (TSPO) receptor, epidermal growth factor (EGF) receptor, human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF) receptor, folate receptor (FR), transferrin receptor (TFR), low-density lipoprotein (LDL) receptors, type I insulin-like growth factor receptor (IGF1R), vasoactive intestinal peptide (VIP) receptors, urokinase plasminogen activator (uPA) and estrogen receptor (ER). This review will describe the recent advances in synthetic targeting optical imaging probes and demonstrate their in vivo imaging potentials. Moreover, current status of near infrared (NIR) fluorescent dyes, targeting moieties and coupling reactions, as well as strategies for designing targeted probes, will also be discussed.

Enhancing Surgical Vision by Using Real-Time Imaging of αvβ3-Integrin Targeted Near-Infrared Fluorescent Agent

This study was designed to improve the surgical procedure and outcome of cancer surgery by means of real-time molecular imaging feedback of tumor spread and margin delineation using targeted near-infrared fluorescent probes with specificity to tumor biomarkers. Surgical excision of cancer often is confronted with difficulties in the identification of cancer spread and the accurate delineation of tumor margins. Currently, the assessment of tumor borders is afforded by postoperative pathology or, less reliably, intraoperative frozen sectioning. Fluorescence imaging is a natural modality for intraoperative use by directly relating to the surgeon's vision and offers highly attractive characteristics, such as high-resolution, sensitivity, and portability. Via the use of targeted probes it also becomes highly tumor-specific and can lead to significant improvements in surgical procedures and outcome. Mice bearing xenograft human tumors were injected with αvβ3-integrin receptor-targeted fluorescent probe and in vivo visualized by using a novel, real-time, multispectral fluorescence imaging system. Confirmatory ex vivo imaging, bioluminescence imaging, and histopathology were used to validate the in vivo findings. Fluorescence images were all in good correspondence with the confirming bioluminescence images in respect to signal colocalization. Fluorescence imaging detected all tumors and successfully guided total tumor excision by effectively detecting small tumor residuals, which occasionally were missed by the surgeon. Tumor tissue exhibited target-to-background ratio of ~4.0, which was significantly higher compared with white-light images representing the visual contrast. Histopathology confirmed the capability of the method to identify tumor negative margins with high specificity and better prediction rate compared with visual inspection. Real-time multispectral fluorescence imaging using tumor specific molecular probes is a promising modality for tumor excision by offering real time feedback to the surgeon in the operating room.

Synthesis, characterization, and biological properties of cyanine-labeled somatostatin analogues as receptor-targeted fluorescent probes.

We present the synthesis and characterization of the somatostatin receptor-specific peptide H(2)N-(D-Phe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, which is labeled with a carboxylated indodicarbo- and an indotricarbocyanine dye at the N-terminal amino group. The preparation was performed by automated solid-phase synthesis, with subsequent attachment of the cyanine dye and cleavage of the entire conjugate from the resin. The compounds display high molar absorbance and fluorescence quantum yields typical for cyanine dyes and are thus suitable receptor-targeted contrast agents for molecular optical imaging. The ability of these agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, in which the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. In contrast, the corresponding linearized derivative of the sequence H(2)N-(D-Phe)-Met-Phe-(D-Trp)-Lys-Thr-Met-Thr-OH produced only minimal cell fluorescence, hence confirming the specificity of the cyclic somatostatin analogue. Intracellular localization could be visualized by near-infrared (NIR) fluorescence microscopy. In conclusion, receptor-specific peptides are promising tools for designing site-directed optical contrast agents for use in molecular optical imaging.