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A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.

Background Re-excision rates following breast conserving surgery (BCS) remain as high as ~ 35%, with positive margins detected during follow-up histopathology. Additional breast cancer resection surgery is not only taxing on the patient and health care system, but also delays adjuvant therapies, increasing morbidity and reducing the likelihood of a positive outcome. The ability to precisely resect and visualize tumor margins in real time within the surgical theater would greatly benefit patients, surgeons and the health care system. Current tumor margin assessment technologies utilized during BCS involve relatively lengthy and labor-intensive protocols, which impede the surgical work flow. Methods In previous work, we have developed and validated a fluorescence imaging method termed dual probe difference specimen imaging (DDSI) to accurately detect benign and malignant tissue with direct correlation to the targeted biomarker expression levels intraoperatively. The DDSI method is currently on par with touch prep cytology in execution time (~ 15-min). In this study, the main goal was to shorten the DDSI protocol by decreasing tissue blocking and washing times to optimize the DDSI protocol to Results We evaluated the utility of the shortened DDSI staining methodology using xenografts grown from cell lines with varied epidermal growth factor receptor (EGFR) expression levels, comparing accuracy through receiver operator characteristic (ROC) curve analyses across varied tissue blocking and washing times. An optimized 8-min DDSI methodology was developed for future clinical translation. Conclusions Successful completion of this work resulted in substantial shortening of the DDSI methodology for use in the operating room, that provided robust, highly receptor specific, sensitive diagnostic capabilities between benign and malignant tissues.

The current five-year survival rate of less than 5% for pancreatic ductal adenocarcinoma (PDAC) is compounded by late diagnosis, a lack of PDAC-specific intraoperative guidance to ensure complete resection, and the ineffectiveness of current therapies. Previously, utilizing compound 1, a fluorophore with inherent PDAC selectivity, PDAC was visualized both in vivo and ex vivo in a murine model. In the current study, human PDAC tissue is targeted. Compound 1 selectively stains ducts of the adenocarcinoma versus the surrounding stroma, enabling the imaging of PDAC in frozen tissue sections with high contrast. To enhance the potential of 1 for intraoperative applications, the ex vivo staining protocol was optimized for rapid margin assessment, with a final staining time of ~15 min. To measure diagnostic performance, the area under a receiver operating characteristic (ROC) curve was measured for the identification of ductal adenocarcinoma vs. stroma. The bright fluorescence contrast enabled quantitative determination of PDAC (or precancerous PanIN lesions) versus healthy pancreas tissue in human tissue array samples.

Significance: Surgery is often paramount in the management of many solid organ malignancies because optimal resection is a major factor in disease-specific survival. Cancer surgery has multiple challenges including localizing small lesions, ensuring negative surgical margins around a tumor, adequately staging patients by discriminating positive lymph nodes, and identifying potential synchronous cancers. Intraoperative molecular imaging (IMI) is an emerging potential tool proposed to address these issues. IMI is the process of injecting patients with fluorescenttargeted contrast agents that highlight cancer cells prior to surgery. Over the last 5 to 7 years, enormous progress has been achieved in tracer development, near-infrared camera approvals, and clinical trials. Therefore, a second biennial conference was organized at the University of Pennsylvania to gather surgical oncologists, scientists, and experts to discuss new investigative findings in the field. Our review summarizes the discussions from the conference and highlights findings in various clinical and scientific trials.Aim: Recent advances in IMI were presented, and the importance of each clinical trial for surgical oncology was critically assessed. A major focus was to elaborate on the clinical endpoints that were being utilized in IMI trials to advance the respective surgical subspecialties.Approach: Principal investigators presenting at the Perelman School of Medicine Abramson Cancer Center's second clinical trials update on IMI were selected to discuss their clinical trials and endpoints.Results: Multiple phase III, II, and I trials were discussed during the conference. Since the approval of 5-ALA for commercial use in neurosurgical malignancies, multiple tracers and devices have been developed to address common challenges faced by cancer surgeons across numerous specialties. Discussants also presented tracers that are being developed for delineation of normal anatomic structures that can serve as an adjunct during surgical procedures.Conclusions: IMI is increasingly being recognized as an improvement to standard oncologic surgical resections and will likely advance the art of cancer surgery in the coming years. The endpoints in each individual surgical subspecialty are varied depending on how IMI helps each specialty solve their clinical challenges. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License.

Fluorescence-guided surgery (FGS) to aid in the precise visualization of vital nerve structures in real-time intraoperatively could greatly improve patient outcomes. We took a medicinal chemistry approach that facilitated the design of our first-in-class NIR nerve-binding small molecule fluorophore libraries with excitation and emission profiles compatible with the “700-” and “800-” nm fluorescence imaging channels in the clinical grade FGS systems. Molecular engineering of the lead candidates allowed for the development of water-soluble nerve-specific contrast agents with improved safety profile that has great potential for clinical translation in the near future.

Intracellular paired-agent imaging (iPAI) utilizes fluorescent-labeled small molecule therapeutics to measure drug target engagement. Our new water-soluble, cell-permeant fluorophores termed Sulfo-Rh and Sulfo-SiRh show substantially improved optical stability against solvent polarity changes over tetramethylrhodamine (TMR) and silicon-substituted TMR, respectively, while retaining the desirable photophysical properties of the base compounds, including brightness, to facilitate stable biological imaging. Targeted and untargeted versions of an EGFR tyrosine kinase inhibitor labeled with these fluorophores, demonstrated similarity to the parent drug in competitive binding and cytotoxicity assays. Utilizing iPAI to visualize drug target engagement enables quantitative small molecule imaging in living systems.

This Conference Plenary, “Near-infrared nerve-specific probes to guide surgery,” was recorded for the Photonics West 2021 Digital Forum

Nerve damage plagues surgical outcomes, significantly affecting post-surgical quality of life. Intraoperative nerve detection is difficult since neuroanatomy is varilable between patients, and nerves are typically protected deep within the tissue. Fluorescence-guided surgery (FGS) offers a potential means for enhanced intraoperative nerve identification and preservation. We have developed the first near infrared (NIR) nerve-specific fluorophores for use during FGS. Lead optimization has yielded water soluble derivatives with excellent safety and pharmacology parameters. Work is underway to plan and execute preclinical toxicity testing to enable first-in-human clincial trials.

Personalized molecularly targeted therapy has largely not lived up to its promise of providing curative therapies due to a lack of durable therapeutic response driven by a lack of drug target engagement (i.e. sublethal drug delivery to the tumor) and cell signaling reprogramming as a mechanism of acquired resistance. To simultaneously measure both of these factors, we have developed and optimized a fluorescence imaging platform, Therapeutic Response Imaging through Proteomic and Optical Drug Distribution (TRIPODD), resulting in the only methodology capable of simultaneous quantification of single-cell DTE and protein expression with preserved spatial context within a tumor.

Improved therapeutic strategies require unraveling the complex interactions between drug target availability and therapeutic response. Herein, we propose a novel fluorescence imaging platform capable of measuring both with single cell resolution.

Personalized medicine has largely failed to produce curative therapies in advanced cancer patients. Evaluation of in situ drug target availability (DTA) concomitant with local protein expression is critical to an accurate assessment of therapeutic efficacy, but tools capable of both are currently lacking. We developed and optimized a fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution), resulting in the only methodology capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. Using TRIPODD, we demonstrate the feasibility of combining two complementary fluorescence imaging techniques, intracellular paired agent imaging (iPAI) and cyclic immunofluorescence (cyCIF), conducted with oligonucleotide-conjugated antibodies (Ab-oligos) on tissue samples. We successfully performed sequential imaging on a single tissue section of iPAI to capture single-cell DTA and local protein expression heterogeneity using Ab-oligo cyCIF. Fluorescence imaging data acquisition was followed by spatial registration resulting in high dimensional data correlating DTA to protein expression at the single-cell level where uptake of a targeted probe alone was not well correlated to protein expression. Herein, we demonstrated the utility of TRIPODD as a powerful imaging platform capable of interpreting tumor heterogeneity for a mechanistic understanding of therapeutic response and resistance through quantification of drug target availability and proteomic response with preserved spatial context at single-cell resolution.

Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and have vastly affected how cancer is diagnosed and treated. However, the translation of complex genomic analyses to pathological diagnosis remains challenging using conventional immunofluorescence (IF) staining, which is typically limited to 2-5 antigens. Numerous strategies to increase distinct antigen detection on a single sample have been investigated, but all have deleterious effects on the tissue limiting the maximum number of biomarkers that can be imaged on a single sample and none can be seamlessly integrated into routine clinical workflows. To facilitate ready integration into clinical histopathology, we have developed a novel cyclic IF (cycIF) technology based on antibody conjugated oligonucleotides (Ab-oligos). In situ hybridization of complementary oligonucleotides (oligos) facilitates biomarker labeling for imaging on any conventional fluorescent microscope. We have validated a variety of oligo configurations and their respective signal removal strategies capable of diminishing fluorescent signal to levels of autofluorescence before subsequent staining cycles. Robust signal removal is performed without the employment of harsh conditions or reagents, maintaining tissue integrity and antigenicity for higher dimensionality immunostaining of a single sample. Our platform Ab-oligo cycIF technology uses conventional fluorophores and microscopes, allowing for dissemination to a broad audience and congruent integration into clinical histopathology workflows.

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.

In the past several decades, a number of novel fluorescence image-guided surgery (FGS) contrast agents have been under development, with many in clinical translation and undergoing clinical trials. In this review, we have identified and summarized the contrast agents currently undergoing clinical translation. In total, 39 novel FGS contrast agents are being studied in 85 clinical trials. Four FGS contrast agents are currently being studied in phase III clinical trials and are poised to reach FDA approval within the next two to three years. Among all novel FGS contrast agents, a wide variety of probe types, targeting mechanisms, and fluorescence properties exists. Clinically available FGS imaging systems have been developed for FDA approved FGS contrast agents, and thus further clinical development is required to yield FGS imaging systems tuned for the variety of contrast agents in the clinical pipeline. Additionally, study of current FGS contrast agents for additional disease types and development of anatomy specific contrast agents is required to provide surgeons FGS tools for all surgical specialties and associated comorbidities. The work reviewed here represents a significant effort from many groups and further development of this promising technology will have an enormous impact on surgical outcomes across all specialties.

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 drag 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 drag 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.

Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and identify druggable therapeutic targets. Tyrosine kinase inhibitors (TKI) are a class of drugs seeking to inhibit signaling pathways critical to sustaining proliferative signaling, resisting cell death, and the other hallmarks of cancer. While tumors may initially respond to TKI therapy, disease progression is near universal due to mechanisms of acquired resistance largely involving cellular signaling pathway reprogramming. With the ultimate goal of improved TKI therapeutic efficacy our group has developed intracellular paired agent imaging (iPAI) to quantify drug target interactions and oligonucleotide conjugated antibody (Ab-oligo) cyclic immunofluorescence (cycIF) imaging to characterize perturbed signaling pathways in response to therapy. iPAI uses spectrally distinct, fluorescently labeled targeted and untargeted drug derivatives, correcting for non-specific drug distribution and facilitating quantitative assessment of the drug binding before and after therapy. Ab-oligo cycIF exploits in situ hybridization of complementary oligonucleotides for biomarker labeling while oligonucleotide modifications facilitate signal removal for sequential rounds of fluorescent tagging and imaging. Ab-oligo CycIF is capable of generating extreme multi-parametric images for quantifying total and phosphorylated protein expression to quantify protein activation, expression, and spatial distribution. Together iPAI and Ab-oligo cycIF can be applied to interrogate drug uptake and target binding as well as changes to heterogenous cell populations within tumors that drive variable therapeutic responses in patients.

Therapeutic resistance plagues cancer outcomes, challenging treatment particularly in aggressive disease. A unique method to decipher drug interactions with their targets and inform therapy is to employ fluorescence-based screening tools; however, to implement productive screening assays, adequate model systems must be developed. Patient-derived pancreatic cancer models (e.g., cell culture, patient-derived xenograft mouse models, and organoids) have been traditionally utilized to predict personalized therapeutic response. However, cost, long read out times and the inability to fully recapitulate the tumor microenvironment have rendered most models incompatible with clinical decision making for pancreatic ductal adenocarcinoma (PDAC) patients. Tumor explant cultures, where patient tissue can be kept viable for up to weeks, have garnered interest as a platform for delivering personalized therapeutic prediction on a clinically relevant timeline. To fully explore this ex vivo platform, a series of studies were completed to quantitatively compare in vivo models with tumor explants, examining gemcitabine therapeutic efficacy, small molecule uptake and drug-target engagement using a novel fluorescently-labeled gemcitabine conjugate. This initial work shows promise for patient-specific therapeutic selection, where tumor explant drug distribution and response recapitulated the in vivo behavior and could provide a valuable platform for understanding mechanisms of therapeutic response and resistance.

Nerve-binding fluorophores with near-infrared (NIR; 650 to 900 nm) emission could reduce iatrogenic nerve injury rates by providing surgeons precise, real-time visualization of the peripheral nervous system. Unfortunately, current systemically administered nerve contrast agents predominantly emit at visible wavelengths and show nonspecific uptake in surrounding tissues such as adipose, muscle, and facia, thus limiting detection to surgically exposed surface-level nerves. Here, a focused NIR fluorophore library was synthesized and screened through multi-tiered optical and pharmacological assays to identify nerve-binding fluorophore candidates for clinical translation. NIR nerve probes enabled micrometer-scale nerve visualization at the greatest reported tissue depths (~2 to 3 mm), a feat unachievable with previous visibly emissive contrast agents. Laparoscopic fluorescent surgical navigation delineated deep lumbar and iliac nerves in swine, most of which were invisible in conventional white-light endoscopy. Critically, NIR oxazines generated contrast against all key surgical tissue classes (muscle, adipose, vasculature, and fascia) with nerve signal-to-background ratios ranging from ~2 (2- to 3-mm depth) to 25 (exposed nerve). Clinical translation of NIR nerve-specific agents will substantially reduce comorbidities associated with surgical nerve damage.

Nerves are extremely difficult to identify and are often accidently damaged during surgery, leaving patients with lasting pain and numbness. Herein, a novel near-infrared (NIR) nerve-specific fluorophore, LGW01–08, was utilized for enhanced nerve identification using fluorescence guided surgery (FGS), formulated using clinical translatable strategies. Formulated LGW01–08 was examined for toxicology, pharmacokinetics (PK), and pharmacodynamics (PD) parameters in preparation for future clinical translation. Optimal LGW01–08 imaging doses were identified in each formulation resulting in a 10x difference between the toxicity to imaging dose window. Laparoscopic swine surgery completed using the da Vinci surgical robot (Intuitive Surgical) demonstrated the efficacy of formulated LGW01–08 for enhanced nerve identification. NIR fluorescence imaging enabled clear identification of nerves buried beneath ~3 mm of tissue that were unidentifiable by white light imaging. These studies provide a strong basis for future clinical translation of NIR nerve-specific fluorophores for utility during FGS to improve patient outcomes.

Molecular probes that selectively highlight pancreatic cancer (PC) tissue have the potential to improve pancreatic ductal adenocarcinoma (PDAC) margin assessment through the selective highlighting of individual PC cells. Herein, we report a simple and unique family of systematically modified red and near-infrared fluorescent probes that exhibit a field-effect-derived redshift. Two of thirteen probes distributed to the normal mouse pancreas following systemic administration. One selectively accumulated in genetically modified mouse models of PDAC. The probe exhibited intracellular accumulation and enabled visualization of four levels of the structure, including the whole organ, resected tissue, individual cells, and subcellular organelles. In contrast to the small-molecule probes reported previously, it possesses an inherent affinity toward PDAC cells and thus does not require conjugation to any targeting agent. The fluorescent probe can thus promote new strategies not only for precision image-guided surgery, but also for PC detection, monitoring of therapeutic outcomes, and basic research.

Identification of tumor margins in the operating room in real time is a critical challenge for surgical procedures that serve as cancer cure. Breast conserving surgery (BCS) is particularly affected by this problem, with current re-excision rates above 25%. Due to a lack of clinically available methodologies for detection of involved or close tumor margins, much effort is focused on developing intraoperative margin assessment modalities that can aid in addressing this unmet clinical need. BCS provides a unique opportunity to design contrast-based technologies that are able to assess tumor margins independent from the patient, providing a rapid pathway from bench to bedside at a much lower cost. Since resected tissue is removed from the patient's blood supply, non-specific contrast agent uptake becomes a challenge due to the lack of clearance. Therefore, a dual probe, ratiometric fluorescence imaging approach was taken in an effort to reduce non-specific signal, and provide a modality that could demonstrate rapid, robust margin assessment on resected patient samples. Termed, dual-stain difference specimen imaging (DDSI), DDSI includes the use of spectrally unique, and fluorescently labeled target-specific, as well as non-specific biomarkers. In the present study, we have applied epidermal growth factor receptor (EGFR) targeted DDSI to tumor xenografts with variable EGFR expression levels using a previously optimized staining protocol, allowing for a quantitative assessment of the predictive power of the technique under biologically relevant conditions. Due to the presence of necrosis in the model tumors, ring analysis was employed to characterize diagnostic accuracy as measured by receiver operator characteristic (ROC) curve analysis. Our findings demonstrate the robust nature of the DDSI technique even in the presence of variable biomarker expression and spatial patterns. These results support the continued development of this technology as a robust diagnostic tool for tumor margin assessment in resected specimens during BCS.

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.

Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)-based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches.

Complete removal of malignant tissue during primary breast cancer resection is a critical prognostic indicator of local recurrence and overall patient survival, making intraoperative tumor margin assessment essential. Positive margin status following breast-conserving surgery (BCS) is a common difficulty reported in 20-60% of patients, with re-excision rates >55%. Re-excision increases the risk of morbidity and delays the use of adjuvant therapy, thus significant efforts are underway to develop successful intraoperative margin assessment strategies to eliminate repeat surgery. One novel strategy uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI) where a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent probe are topically applied to the fresh resected specimen. While conceptually simple, resected specimen staining is dominated by non-specific uptake of fluorescent probes in normal tissue, requiring the use of a dual probe strategy for accuracy. DDSI permits fluorescence images from both the targeted and untargeted probes to be used to calculate a normalized difference image, facilitating quantitative identification of targeted probe tumor distribution in the specimen. 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 validation. Current work is focused on development of targeted and untargeted small molecule affinity tags, facilitating access to intracellular breast cancer biomarkers and quantitative assessment of DDSI signal in the context of varied biomarker expression level.

Nerve damage plagues surgical outcomes, significantly affecting post-surgical quality of life. Surprisingly, no method exists to enhance direct nerve visualization in the operating room, and nerve detection is completed through a combination of palpation and visualization when possible. Fluorescence image-guided surgery offers a potential means of enhanced nerve identification and preservation, however a clinically approved nerve-specific contrast agent does not yet exist. Several classes of nerve-specific fluorophores have recently been demonstrated including the distyrylbenzenes (DSB), select oxazines (oxazine 4 perchlorate), and certain cyanines (3,3’-diethylthiatricarbocyanine iodine), which could provide intraoperative guidance. The nerve-sparing radical prostatectomy is a surgical procedure that could benefit from fluorescence image-guided nerve identification. Although the nerve-sparing surgical technique was developed over 30 years ago, nerve damage following radical prostatectomy is reported in some form in up to 60% of patients one to two years post-surgery. To facilitate clinical translation of fluorescence image guided surgery to the nerve sparing prostatectomy, a direct administration methodology was developed that allows selective nerve highlighting with a significantly lower fluorophore dose than systemic administration, where large animal studies have confirmed the technique’s translatability. Tissue penetration will be critical for clinical feasibility of the direct administration methodology and novel formulation strategies have been explored to enhance tissue penetration for identifying buried nerves. In addition, several biomolecular targets of Oxazine 4, a promising candidate for nerve-specific fluorophore development into a near-infrared (NIR) agent, have been identified, providing insight into the mechanism of nerve-specificity. Fluorophore development has made progress towards the goal of creating a NIR nerve-specific fluorophore and determining the structure-activity relationship responsible for nerve binding.

Pancreatic ductal adenocarcinoma (PDAC) has the poorest five-year survival rate of any cancer. Surgical resection is the only curative treatment but, is only possible in 15-20% of cases. Systemic therapies that demonstrate some efficacy are limited to chemotherapeutics, such as Gemcitabine, Abraxane (nab-Paclitaxel) and FOLFIRINOX, a multidrug chemotherapy regimen. Numerous studies have tried to apply multi-agent therapies to advance standard of care for metastatic PDAC. However, consistent improvement in survival remains poor. Of broader interest, there is an urgent need for improved treatment selection and early assessment of therapeutic responses as mean survival time following diagnosis is less than one year. Paired agent imaging (PAI) is a spatially-resolved, quantitative imaging methodology, which enables quantification of bound and unbound drugs on a cell by cell basis, using a targeted and control (untargeted) imaging agent. PAI has the potential to provide a spatially resolved map of drug engagement and treatment efficacy for PDAC. Herein, we have developed fluorescently labeled, paired targeted and control Gemcitabine, which have been characterized for their similarity to the parent Gemcitabine using competitive binding, liquid chromatography mass spectroscopy based uptake and pharmacokinetic studies, and cytotoxicity assays. Using our validated targeted and control agents, we have generated maps of targeted and untargeted Gemcitabine in murine models of PDAC, demonstrating that therapeutic efficacy is correlated to the bound drug fractions. We anticipate that PAI using fluorescently labeled targeted and control drug derivatives will be useful for future studies to prediction therapy combinations for personalized PDAC therapy.

Tools for rapid therapeutic selection for pancreatic ductal adenocarcinoma (PDAC) patients are vital to improved survival as PDAC is the third leading cause of cancer death, with an average life expectancy of 5-7 months post diagnosis. Treatment options are limited to surgery and chemotherapy, where Gemcitabine has been the cornerstone of therapy since 1997. However, Gemcitabine resistance prevents PDAC cure and mechanisms of resistance are not fully understood. Yet, with average survival times still

Nerve damage during surgery is a common morbidity experienced by patients that leaves them with chronic pain and/or loss of function. Currently, no clinically approved imaging technique exists to enhance nerve visualization in the operating room. Fluorescence image-guided surgery has gained in popularity and clinical acceptance over the past decade with a handful of imaging systems approved for clinical use. However, contrast agent development to complement these fluorescence-imaging systems has lagged behind with all currently approved fluorescent agents providing untargeted blood pool information. Nerve-specific fluorophores are known, however translations of these agents to the clinic has been complicated by their lipophilic nature, which necessitates specialized formulation strategies for successful systemic administration. To date the known nerve-specific fluorophores have only been demonstrated preclinically due to the necessity of a dimethyl sulfoxide containing formulation for solubilization. In the current study, a polymeric micellar (PM) formulation strategy was developed for a representative nerve-specific fluorophore from the distyrylbenzene family, BMB. The PM formulation strategy was able to solubilize BMB and demonstrated improved nerve-specific accumulation and fluorescence intensity when the same fluorophore dose was administered to mice utilizing the previous formulation strategy. The success of the PM formulation strategy will be important for moving toward clinical translation of these novel nerve-specific probes as it is nontoxic and biodegradable and has the potential to decrease the necessary dose for imaging while also improving the safety profile.

Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels. Images of the same molecule in the two channels are then combined to determine both the location and the identity of the molecule. The single-objective configuration of our approach allows for flexible sample geometry and the use of a live-cell incubation chamber required for live-cell SMT. Despite a lower photon yield, we achieve excellent spatial (20–40 nm) and spectral (10–15 nm) resolutions comparable to those obtained with dual-objective, spectrally resolved Stochastic Optical Reconstruction Microscopy. Furthermore, motions of the fluorescent molecules did not cause loss of spectral resolution owing to the dual-channel spectral calibration. We demonstrate SMT in three (and potentially more) colors using spectrally proximal fluorophores and single-laser excitation, and show that trajectories of each species can be reliably extracted with minimal cross talk.

Pancreatic ductal adenocarcinomas (PDAC) are notoriously difficult to treat and in general, molecular targeted therapies have failed even when the targeted protein is overexpressed in the tumor tissue. Genetic mutations in extracellular receptors and downstream signaling proteins (i.e., RAS signaling pathway) and convoluted intracellular cross-talk between cell signaling pathways are likely reasons that these promising therapies fail. Monitoring the complex relationship between intracellular protein signaling is difficult and to-date, standard techniques that are used (Western blot, flow cytometry, immunohistochemistry, etc.) are invasive, static and do not accurately represent in vivo structure-function relationships. Here, we describe the development of an in ovo avatar using patient derived tumors grown on the chicken chorioallantoic membrane (CAM) and the novel fluorescence-based Quantitative Protein Expression Tracking (QUIET) methodology to bridge the gap between oncology, genomics and patient outcomes. Previously developed paired-agent imaging, was extended to a three-compartment model system in QUIET, which utilizes three types of imaging agents: novel fluorophore conjugated cell permeable targeted and untargeted small molecule paired-agents, in addition to a tumor perfusion agent that is not cell membrane permeable. We have demonstrated the ability to quantify the intracellular binding domain of a trans-membrane protein in vitro using cell permeable fluorescent agents (erlotinib-TRITC and control isotype-BODIPY FL). In addition, we have demonstrated imaging protocols to simultaneously image up to 6 spectrally distinct organic fluorophores in in ovo avatars using the Nuance EX (Perkin Elmer) and established proof-of-principle intracellular and extracellular protein concentrations of epidermal growth factor receptor using QUIET and traditional paired-agent imaging.

Nerve damage remains a major morbidity following nerve sparing radical prostatectomy, significantly affecting quality of life post-surgery. Nerve-specific fluorescence guided surgery offers a potential solution by enhancing nerve visualization intraoperatively. However, the prostate is highly innervated and only the cavernous nerve structures require preservation to maintain continence and potency. Systemic administration of a nerve-specific fluorophore would lower nerve signal to background ratio (SBR) in vital nerve structures, making them difficult to distinguish from all nervous tissue in the pelvic region. A direct administration methodology to enable selective nerve highlighting for enhanced nerve SBR in a specific nerve structure has been developed herein. The direct administration methodology demonstrated equivalent nerve-specific contrast to systemic administration at optimal exposure times. However, the direct administration methodology provided a brighter fluorescent nerve signal, facilitating nerve-specific fluorescence imaging at video rate, which was not possible following systemic administration. Additionally, the direct administration methodology required a significantly lower fluorophore dose than systemic administration, that when scaled to a human dose falls within the microdosing range. Furthermore, a dual fluorophore tissue staining method was developed that alleviates fluorescence background signal from adipose tissue accumulation using a spectrally distinct adipose tissue specific fluorophore. These results validate the use of the direct administration methodology for specific nerve visualization with fluorescence image-guided surgery, which would improve vital nerve structure identification and visualization during nerve sparing radical prostatectomy.

Millions of archived formalin-fixed, paraffin-embedded (FFPE) specimens contain valuable molecular insight into healthy and diseased states persevered in their native ultrastructure. To diagnose and treat diseases in tissue on the nanoscopic scale, pathology traditionally employs electron microscopy (EM), but this platform has significant limitations including cost and painstaking sample preparation. The invention of single molecule localization microscopy (SMLM) optically overcame the diffraction limit of light to resolve fluorescently labeled molecules on the nanoscale, leading to many exciting biological discoveries. However, applications of SMLM in preserved tissues has been limited. Through adaptation of the immunofluorescence workflow on FFPE sections milled at histological thickness, cellular architecture can now be visualized on the nanoscale using SMLM including individual mitochondria, undulations in the nuclear lamina, and the HER2 receptor on membrane protrusions in human breast cancer specimens. Using astigmatism imaging, these structures can also be resolved in three dimensions to a depth of ~800 nm. These results demonstrate the utility of SMLM in efficiently uncovering ultrastructural information of archived clinical samples, which may offer molecular insights into the physiopathology of tissues to assist in disease diagnosis and treatment using conventional sample preparation methods.

Background The aim of this study was to demonstrate feasibility of migration and in situ chemotherapy delivery to regional lymph nodes (LN) in a large animal model using an expansile polymer nanoparticle (eNP) delivery system. Study Design Dual-labeled 50-nm and 100-nm eNP were prepared by encapsulating an IR-813 near-infrared (NIR) fluorescent dye within coumarin-conjugated expansile polymer nanoparticles (NIR-C-eNP). NIR imaging and fluorescent microscopy were used to identify intralymphatic migration of NIR-nanoparticles to draining inguinal or mesenteric LN after injection in swine hind legs or intestine. Nanoparticle-mediated intranodal delivery of chemotherapy was subsequently assessed with Oregon Green paclitaxel-loaded NIR-eNP (NIR-OGpax-eNP). Results NIR imaging demonstrated direct lymphatic migration of 50-nm, but not 100-nm, NIR-C-eNP and NIR-OGpax-eNP to the draining regional LNs after intradermal injection in the hind leg or subserosal injection in intestine. Fluorescent microscopy demonstrated that IR-813 used for NIR real-time trafficking colocalized with both the coumarin-labeled polymer and paclitaxel chemotherapy and was identified within the subcapsular spaces of the draining LNs. These studies verify nodal migration of both nanoparticle and encapsulated payload, and confirm the feasibility of focusing chemotherapy delivery directly to regional nodes. Conclusions Regionally-targeted intranodal chemotherapy can be delivered to draining LNs for both skin and solid organs using 50-nm paclitaxel-loaded eNP.

Intraoperative margin assessment is imperative to cancer cure but is a continued challenge to successful surgery. Breast conserving surgery is a relevant example, where a cosmetically improved outcome is gained over mastectomy, but re-excision is required in >25% of cases due to positive or closely involved margins. Clinical translation of margin assessment modalities that must directly contact the patient or required administered contrast agents are time consuming and costly to move from bench to bedside. Tumor resections provide a unique surgical opportunity to deploy margin assessment technologies including contrast agents on the resected tissues, substantially shortening the path to the clinic. However, staining of resected tissues is plagued by nonspecific uptake. A ratiometric imaging approach where matched targeted and untargeted probes are used for staining has demonstrated substantially improved biomarker quantification over staining with conventional targeted contrast agents alone. Our group has developed an antibody-based ratiometric imaging technology using fluorescently labeled, spectrally distinct targeted and untargeted antibody probes termed dual-stain difference specimen imaging (DDSI). Herein, the targeted biomarker expression level and pattern are evaluated for their effects on DDSI diagnostic potential. Epidermal growth factor receptor expression level was correlated to DDSI diagnostic potential, which was found to be robust to spatial pattern expression variation. These results highlight the utility of DDSI for accurate margin assessment of freshly resected tumor specimens.

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.

SMMART is a precision medicine research program focused on understanding the evolution of actionable biology and mechanisms of resistance in human tumors during therapy. This is accomplished through in depth functional, ‘omic and multiscale image analysis of longitudinal samples acquired during treatment. Here we present a case report detailing the insights that can be gained from the comparative analysis of pre- and post-treatment biopsy specimens in a late-stage metastatic breast cancer patient. To understand the molecular evolution of cancer, we interrogated genomics with targeted and whole exome sequencing, transcriptomics with RNA and gene-fusion sequencing, and proteomics with reverse phase protein arrays. To understand cellular organization and architectural changes, we employed multi-scale imaging tools, including scanning electron microscopy (SEM), cyclic immunofluorescence, immune cell profiling with cyclic immunohistochemistry, and traditional pathological assessment. During the course of treatment, we monitored patient response to therapy with clinical imaging, circulating tumor DNA sequencing and cancer protein assessment. Individual assays revealed key aspects of how this individual's cancer evolved under therapeutic pressure. For example, mutational profiling revealed the patterns of clonal evolution and the acquisition of new genetic driver events. 2D and 3D SEM showed changes in ECM organization, macropinocytosis, mitochondrion size, number and density and number and organization of filopodia-like protrusions. We used a 30-color cyclic immunofluorescence analysis to identify differences in cancer cell proliferation and differentiation state, as well as the composition and organization of infiltrating immune cells. In addition, integrative analyses of multiple data types provided insight into the evolution of actionable biology within this patient's disease. This included changes in the suitability of the patient for immune checkpoint inhibitors as well as specific tyrosine kinase inhibitors. The comprehensive molecular and architectural characterization of an individual patient's cancer at multiple time points provides biologically novel and clinically relevant insight into the ways in which cancers become resistant to treatment. Citation Format: Brett Johnson, Jamie Keck, Max Morris, Kiara Siex, Annette Kolodzie, Swapnil Parmar, Jessica Riesterer, Koei Chin, Summer Gibbs, Laura Heiser, Paul Spellman, Kyle Ellrott, Ozgun Babur, Emek Demir, Adam Margolin, Jeremy Goecks, Lisa Coussens, Raymond Bergan, Joe Gray. SMMART: Serial measurements of molecular and architectural responses to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3296.

Rationale and Objectives Noninvasive molecular imaging of glioma tumor receptor activity was assessed with diagnostic in vivo fluorescence monitoring during targeted therapy. The study goals were to assess the range of use for treatment monitoring and stratification of tumor types using epidermal growth factor (EGF) receptor (EGFR) status with administration of fluorescently labeled EGF and determine its utility for tumor detection compared to magnetic resonance imaging (MRI). Materials and Methods EGFR+ and EGFR− glioma tumor lines (human glioma [U251-GFP] and rat gliosarcoma [9L-GFP], respectively) were used to assess these goals, having a 20-fold difference between their EGF uptakes. Results Treatment with cetuximab in the EGFR+ tumor-bearing animals led to decreased EGF tumor uptake, whereas for the EGFR− tumors, no change in fluorescence signal followed treatment. This diagnostic difference in EGFR expression could be used to stratify the tumor-bearing animals into groups of potential responders and nonresponders, and receiver-operating characteristic curve analysis revealed an area under the curve (AUC) of 0.92 in separating these tumors. The nonlocalized growth pattern of U251-GFP tumors resulted in detection difficulty on standard MRI, but high EGFR expression made them detectable by fluorescence imaging (AUC = 1.0). The EGFR+ U251-GFP tumor–bearing animals could be noninvasively stratified into treated and untreated groups on the basis of fluorescence intensity difference ( P = .035, AUC = 0.90). Conclusions EGFR expression was tracked in vivo with fluorescence and determined to be of use for the stratification of EGFR+ and EGFR− tumors, the detection of EGFR+ tumors, and monitoring of molecular therapy.

Invisible NIR fluorescent light can provide high sensitivity, high-resolution, and real-time image-guidance during oncologic surgery, but imaging systems that are presently available do not display this invisible light in the context of surgical anatomy. The FLARE imaging system overcomes this major obstacle.Color video was acquired simultaneously, and in real-time, along with two independent channels of NIR fluorescence. Grayscale NIR fluorescence images were converted to visible "pseudo-colors" and overlaid onto the color video image. Yorkshire pigs weighing 35 kg (n = 5) were used for final preclinical validation of the imaging system. A six-patient pilot study was conducted in women undergoing sentinel lymph node (SLN) mapping for breast cancer. Subjects received (99m)Tc-sulfur colloid lymphoscintigraphy. In addition, 12.5 microg of indocyanine green (ICG) diluted in human serum albumin (HSA) was used as an NIR fluorescent lymphatic tracer.The FLARE system permitted facile positioning in the operating room. NIR light did not change the look of the surgical field. Simultaneous pan-lymphatic and SLN mapping was demonstrated in swine using clinically available NIR fluorophores and the dual NIR capabilities of the system. In the pilot clinical trial, a total of nine SLNs were identified by (99m)Tc- lymphoscintigraphy and nine SLNs were identified by NIR fluorescence, although results differed in two patients. No adverse events were encountered.We describe the successful clinical translation of a new NIR fluorescence imaging system for image-guided oncologic surgery.

The diffuse spread of glioma tumors leads to the inability to image and properly treat this disease. The optical spectral signature of targeted fluorescent probes provides molecular signals from the diffuse morphologies of glioma tumors, which can be a more effective diagnostic probe than standard morphology-based magnetic resonance imaging (MRI) sequences. Three orthotopic xenograft glioma models were used to examine the potential for transmitted optical fluorescence signal detection in vivo, using endogenously produced protoporphyrin IX (PpIX) and exogenously administered fluorescently labeled epidermal growth factor (EGF). Accurate quantification of the fluorescent signals required spectral filtering and signal normalization, and when optimized, it was possible to improve detection of sparse diffuse glioma tumor morphologies. The signal of endogenously produced PpIX provided similar sensitivity and specificity to MRI, while detection with fluorescently labeled EGF provided maximal specificity for tumors with high EGF receptor activity. Optical transmitted fluorescent signal may add significant benefit for clinical cases of diffuse infiltrative growth pattern glioma tumors given sufficient optimization of the signal acquisition for each patient.

The Dunning R3327-MLL is a well established transplanted tumour line, and as such it makes a desirable model for evaluative studies of therapy. In the current study, the interstitial growth characteristics as well as the response of this tumour to a single fraction of high dose rate radiation is investigated.The in vitro response to radiation of the Dunning R3327-MLL was studied via a colony forming assay using a Cs-137 irradiator. In vitro radiosensitivity was determined on tumours implanted intramuscularly in the left gastrocnemius muscle of the rat and irradiated using an Ir-192 afterloader.The results demonstrate a faster growth rate when compared to the reported subcutaneous growth rates. The Dunning R3327-MLL's radiosensitivity is comparable to that of late response tissues. The dose required to achieve a specific radiobiological response (the alpha:beta ratio) of the in vitro cell line is 2.4 Gy, whereas the ratio for the intramuscularly growing tumour was 0.99 Gy.These findings signify the intramuscularly implanted Dunning R3327-MLL tumour model as a desirable model for the study of single fraction high dose rate radiation treatments.

Hematoxylin and eosin (H&E) staining of tissue has been the mainstay of pathology for more than a century. However, the learning curve for H&E tissue interpretation is long while intra- and interobserver variability remains high. Computer-assisted image analysis of H&E sections holds promise for increased throughput and decreased variability, but has yet to demonstrate significant improvement in diagnostic accuracy. Addition of biomarkers to H&E staining can improve diagnostic accuracy, however co-registration of immunohistochemical staining with H&E is problematic as immunostaining is completed on slides that are at best 4 μm apart. Simultaneous H&E and immunostaining would alleviate co-registration problems; however current opaque pigments used for immunostaining obscure H&E. In this study, we demonstrate that diagnostic information provided by two or more independent wavelengths of near-infrared (NIR) fluorescence leave the H&E stain unchanged while enabling computer-assisted diagnosis and assessment of human disease. Using prostate cancer as a model system, we introduce NIR digital pathology and demonstrate its utility along the spectrum from prostate biopsy to whole mount analysis of H&E-stained tissue.

Advances in molecular biology and new developments in imaging, engineering, and novel contrast agents position molecular imaging to play a major role in disease management. This current issue mainly covers ultrasound techniques and radionuclide imaging techniques, and both preclinical research on animal models and clinical studies on cancer patients are presented herein. Molecular imaging has demonstrated its potential in characterizing disease models in small animal preclinical studies. In the clinical setting, many patients have the potential to benefit from these new and quantitative imaging techniques providing improved disease characterization, therapeutic monitoring, and objective prognostic criteria. To realize the full potential of molecular imaging, chemists, molecular biologist, and biomedical engineers will need to work closely with clinicians to translate molecular imaging techniques into clinical applications, ultimately providing improved disease diagnosis, treatment monitoring, and noninvasive prognostic imaging for patients.