In situ single-cell therapeutic response imaging facilitated by the TRIPODD fluorescence imaging platform.

Purpose: Small molecule drugs such as tyrosine kinase inhibitors (TKIs) targeting tumoral molecular dependencies have become standard of care for numerous cancer types. Notably, epidermal growth factor receptor (EGFR) TKIs (e.g., erlotinib, afatinib, osimertinib) are the current first-line treatment for non-small cell lung cancer (NSCLC) due to their improved therapeutic outcomes for EGFR mutated and overexpressing disease over traditional platinum-based chemotherapy. However, many NSCLC tumors develop resistance to EGFR TKI therapy causing disease progression. Currently, the relationship between in situ drug target availability (DTA), local protein expression and therapeutic response cannot be accurately assessed using existing analytical tools despite being crucial to understanding the mechanism of therapeutic efficacy. Procedure: We have previously reported development of our fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution) that is capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. TRIPODD combines two complementary fluorescence imaging techniques: intracellular paired agent imaging (iPAI) to measure DTA and cyclic immunofluorescence (cyCIF), which utilizes oligonucleotide conjugated antibodies (Ab-oligos) for spatial proteomic expression profiling on tissue samples. Herein, TRIPODD was modified and optimized to provide a downstream analysis of therapeutic response through single-cell DTA and proteomic response imaging. Results: We successfully performed sequential imaging of iPAI and cyCIF resulting in high dimensional imaging and biomarker assessment to quantify single-cell DTA and local protein expression on erlotinib treated NSCLC models. Pharmacodynamic and pharmacokinetic studies of the erlotinib iPAI probes revealed that administration of 2.5 mg/kg each of the targeted and untargeted probe 4 h prior to tumor collection enabled calculation of DTA values with high Pearson correlation to EGFR, the erlotinib molecular target, expression in the tumors. Analysis of single-cell biomarker expression revealed that a single erlotinib dose was insufficient to enact a measurable decrease in the EGFR signaling cascade protein expression, where only the DTA metric detected the presence of bound erlotinib. Conclusion: We demonstrated the capability of TRIPODD to evaluate therapeutic response imaging to erlotinib treatment as it relates to signaling inhibition, DTA, proliferation, and apoptosis with preserved spatial context.

Nerve Visualization using Phenoxazine-Based Near-Infrared Fluorophores to Guide Prostatectomy.

Fluorescence-guided surgery (FGS) is poised to revolutionize surgical medicine through near-infrared (NIR) fluorophores for tissue- and disease-specific contrast. Clinical open and laparoscopic FGS vision systems operate nearly exclusively at NIR wavelengths. However, tissue-specific NIR contrast agents compatible with clinically available imaging systems are lacking, leaving nerve tissue identification during prostatectomy a persistent challenge. Here, it is shown that combining drug-like molecular design concepts and fluorophore chemistry enabled the production of a library of NIR phenoxazine-based fluorophores for intraoperative nerve-specific imaging. The lead candidate readily delineated prostatic nerves in the canine and iliac plexus in the swine using the clinical da Vinci Surgical System that has been popularized for minimally invasive prostatectomy procedures. These results demonstrate the feasibility of molecular engineering of NIR nerve-binding fluorophores for ready integration into the existing surgical workflow, paving the path for clinical translation to reduce morbidity from nerve injury for prostate cancer patients.

Nerve-Sparing Gynecologic Surgery Enabled by A Near-Infrared Nerve-Specific Fluorophore Using Existing Clinical Fluorescence Imaging Systems.

Patients undergoing gynecological procedures suffer from lasting side effects due to intraoperative nerve damage. Small, delicate nerves with complex and nonuniform branching patterns in the female pelvic neuroanatomy make nerve-sparing efforts during standard gynecological procedures such as hysterectomy, cystectomy, and colorectal cancer resection difficult, and thus many patients are left with incontinence and sexual dysfunction. Herein, a near-infrared (NIR) fluorescent nerve-specific contrast agent, LGW08-35, that is spectrally compatible with clinical fluorescence guided surgery (FGS) systems is formulated and characterized for rapid implementation for nerve-sparing gynecologic surgeries. The toxicology, pharmacokinetics (PK), and pharmacodynamics (PD) of micelle formulated LGW08-35 are examined, enabling the determination of the optimal imaging doses and time points, blood and tissue uptake parameters, and maximum tolerated dose (MTD). Application of the formulated fluorophore to imaging of female rat and swine pelvic neuroanatomy validates the continued clinical translation and use for real-time identification of important nerves such as the femoral, sciatic, lumbar, iliac, and hypogastric nerves. Further development of LGW08-35 for clinical use will unlock a valuable tool for surgeons in direct visualization of important nerves and contribute to the ongoing characterization of the female pelvic neuroanatomy to eliminate the debilitating side effects of nerve damage during gynecological procedures.

OregonFluor enables quantitative intracellular paired agent imaging to assess drug target availability in live cells and tissues.

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.

TRIPODD: a Novel Fluorescence Imaging Platform for In Situ Quantification of Drug Distribution and Therapeutic Response.

PURPOSE: 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. PROCEDURE: 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. RESULTS: 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. CONCLUSION: 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.

Near-infrared nerve-binding fluorophores for buried nerve tissue imaging.

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.

In Vivo Nerve-Specificity of Rhodamines and Si-rhodamines.

Accidental nerve damage or transection of vital nerve structures remains an unfortunate reality that is often associated with surgery. Despite the existence of nerve-sparing techniques, the success of such procedures is not only complicated by anatomical variance across patients but is also highly dependent on a surgeon's first-hand experience that is acquired over numerous procedures through trial and error, often with highly variable success rates. Fluorescent small molecules, such as rhodamines and fluoresceins have proven incredibly useful for biological imaging in the life sciences, and they appeared to have potential in illuminating vital nerve structures during surgical procedures. In order to make use of the current clinically relevant imaging systems and to provide surgeons with fluorescent contrast largely free from the interference of hemoglobin and water, it was first necessary to spectrally tune known fluorescent scaffolds towards near infrared (NIR) wavelengths. To determine whether the well-documented Si-substitution strategy could be applied towards developing a NIR fluorophore that retained nerve-specific properties of candidate molecules, an in vivo comparison was made between two compounds previously shown to highlight nervous structures - TMR and Rhodamine B - and their Si-substituted derivatives.

Investigation of Oxazine and Rhodamine Derivatives as Peripheral Nerve Tissue Targeting Contrast Agent for In Vivo Fluorescence Imaging.

Accidental nerve transection or injury is a significant morbidity associated with many surgical interventions, resulting in persistent postsurgical numbness, chronic pain, and/or paralysis. Nerve-sparing can be a difficult task due to patient-to-patient variability and the difficulty of nerve visualization in the operating room. Fluorescence image-guided surgery to aid in the precise visualization of vital nerve structures in real time during surgery could greatly improve patient outcomes. To date, all nerve-specific contrast agents emit in the visible range. Developing a near-infrared (NIR) nerve-specific fluorophore is poised to be a challenging task, as a NIR fluorophore must have enough "double-bonds" to reach the NIR imaging window, contradicting the requirement that a nerve-specific agent must have a relatively low molecular weight to cross the blood-nerve-barrier (BNB). Herein we report our efforts to investigate the molecular characteristics for the nerve-specific oxazine fluorophores, as well as their structurally analogous rhodamine fluorophores. Specifically, optical properties, physicochemical properties and their in vivo nerve specificity were evaluated herein.