This PDF file contains the front matter associated with SPIE Proceedings Volume 11624, including the Title Page, Copyright information, and Table of Contents.

Welcome and Introduction to SPIE Conference 11624

This is a brief introduction to the Visualizing and Quantify Drugs in Tissue Conference. It will briefly review the conference and its schedule and invite participation during the day.

Being able to visualize and quantify where an active ingredient and/or excipient resides within a multiphasic formulation (e.g., a semisolid emulsion), how the drug diffuses and is released from the dosage form, and the delivery and disposition of the drug at the intended site of action can be a powerful tool the development of locally acting drug products such as a dermatological cream. The presentation will focus on how techniques for the evaluation of cutaneous pharmacokinetics have evolved over the years, and how noninvasive imaging-based techniques are being utilized to mechanistically understand drug availability and drug disposition following topical application.

Intrathecal (IT) administration is a drug delivery method with great potential for the treatment of a broad range of neurodegenerative disorders. Imaging is a useful tool for assessing the biodistribution of intrathecally administered molecules, especially for longer-lived radionuclides. However, longer-lived radionuclides are often associated with higher radiation absorbed dose, a quantitative measure computed from serial imaging data. We have developed a custom dosimetry model to support intrathecal administration. In this work, emphasis is placed on recent model refinements to improve absorbed dose estimates to the spinal cord and its implication for dosimetry calculation in translational imaging trials.

Confocal Raman spectroscopy (CRS) enables the real-time profiling of substances penetrating to the skin without sample pre-treatment or labelling. Until now, CRS had been used as a semi-quantitative method, which posed challenges for evaluating topical formulations and assessing bioequivalence. We present a novel approach of CRS for quantitative analysis of skin delivery. The quantitative CRS in vivo has been correlated with the well-established in vitro Franz-diffusion experiments, indicating the potential of CRS for determining skin delivery. We anticipate CRS providing a rapid and non-invasive method that will be an attractive alternative to the clinical studies currently used in bioequivalence testing.

Fluorescence imaging of cancers using continuous wave (CW) detection of receptor targeted probes offer poor sensitivity and specificity due to background autofluorescence and non-specific probe accumulation. Here we show that fluorescence lifetime (FLT) imaging can significantly improve tumor contrast using epidermal growth factor receptor (EGFR) and programed death ligand 1 (PD-L1) targeted probes in a preclinical model of human breast cancer. Our results suggest that these probes have significantly longer FLTs in tumors than in normal tissue and the FLT enhancement is receptor dependent. We also show potential for simultaneous quantification of EGFR and PD-L1 using in vivo FLT multiplexing.

Nanoscale materials are routinely developed, characterized, and evaluated as diagnostic and therapeutic agents for disease treatment in humans. However, the size and composition of many such agents result in poor clearance profiles within biological tissues, thereby posing profound challenges to translational clinical applications. Herein, we present a hyperspectral imaging technique capable of quantifying plasmonic nanoparticle biodistribution with single particle limit of detection and microanatomical detail. Using this method, we find that, although intravenous administration of plasmonic nanoparticles remains largely infeasible from a biodistribution perspective, alternate routes relevant to treatment of oral and gastrointestinal diseases are within translational reach.

Diffuse optics are deep-tissue non-invasive monitoring techniques that quantify total hemoglobin concentration, blood oxygen saturation and blood flow. Although many demonstrated that diffuse optics are sensitive to changes induced by neoadjuvant chemotherapy in human with breast cancer, the effects of chemotherapeutic drug type, dose, duration, and timing on longitudinal hemodynamic responses are still not well-established. To investigate these effects, we performed longitudinal monitoring of hemodynamic parameters on syngeneic rodent models of breast cancer treated with various chemotherapeutic drugs often used in the clinic. Various doses and combination were explored based on the clinically equivalent dose and median lethal dose.

Structural complexity and heterogeneity may play critical roles in pathophysiology and therapeutic effect, at length scales ranging from subcellular (nm) to whole organ (cm). Here, we report on efforts to utilize a multidimensional spectral FRET imaging approach for visualization of the effects of pharmacologic treatments on tissues. We have developed a transgenic rat line expressing a cAMP FRET reporter for visualization of second messenger signaling in tissues in response to treatment and pathological conditions. When utilized with vascular and lung preparations, transgenic FRET tissues displayed the ability to elicit agonist-induced cAMP responses in single cells.

Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary RNA sequences. Triantennary N-acetyl galactosamine (GN)-conjugated ASOs show greatly improved potency via Asialoglycoprotein receptor (ASGR)-mediated uptake in hepatocytes. Here, we compare the uptake kinetics and subsequent distribution of untargeted ASOs to that of GN-ASOs in mouse macrophages and hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon excited fluorescence imaging. While the CARS modality captured the changing lipid distributions and overall morphology of the cell, two-photon fluorescence imaging measured the uptake and the subsequent distribution of the fluorescently labeled (Alexa-488) ASOs inside the cells.

Overexpression of Human EGF Receptor 2 (HER2) in cancer is a marker of aggressive metastatic disease and poor prognosis. Anti-HER2 humanized monoclonal antibody trastuzumab (TZM) has been successfully used in the clinic over the last decades. However, a large fraction of eligible patients display resistance to this therapy. This calls for a deeper investigation of HER2 interaction with other members of HER tyrosine kinase receptors and modulation of their endocytic trafficking upon TZM treatment. Forster resonance energy transfer Fluorescence lifetime microscopy (FLIM- FRET) offers a robust approach to monitor HER2 homo and heterodimerization via the reduction of donor fluorophore lifetime. The objective of this study was to assess the dynamics of HER receptor homo and heterodimerization behavior via FLIMFRET by using near-infrared (NIR) FRET pair labeled anti-HER2 and anti-EGFR therapeutic antibodies in HER2- overexpressing breast cancer cells. In addition, we tested our new deep learning platform DL4FLIM adapted for automated analysis of all datasets. Herein, we report a first attempt to quantify NIR FRET pair labeled cetuximab (CTM, as a donor) and TZM (as an acceptor) binding to their receptors EGFR and HER2 respectively in AU565 cells. As a control, we also performed and human isotype IgG FLIM -FRET and found it completely non-specific. Our data demonstrate both the occurrence of FRET between NIR-labeled probes CTM and TZM as well as between CTM-CTM bound to their respective receptors. This proof-of -principal study demonstrated feasibility of monitoring HER2 hetero receptor FRET FLIM to better understand mechanism of TZM resistance.

Recent advances in tissue engineering and microfabrication have led to development of novel Complex In Vitro models (CIVMs) that more closely mimic pathophysiological functions of human tissues and organs. CIVMs can provide deeper insights into the mechanisms of human disease and pharmacological properties of new drug candidates during early stages of development. In this study, a multimodal optical imaging platform was used for characterizing the structural and functional features of a liver-on-a-chip model (CN Bio Innovations, UK).

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.

Machine learning and deep learning are ubiquitous across a wide variety of scientific disciplines, including medical imaging. An overview of multiple application areas along the imaging chain where deep learning methods are utilized in discovery and clinical quantitative imaging trials is presented. Example application areas along the imaging chain include quality control, preprocessing, segmentation, and scoring. Within each area, one or more specific applications is demonstrated, such as automated structural brain MRI quality control assessment in a core lab environment, super-resolution MRI preprocessing for neurodegenerative disease quantification in translational clinical trials, and multimodal PET/CT tumor segmentation in prostate cancer trials. The quantitative output of these algorithms is described, including their impact on decision making and relationship to traditional read-based methods. Development and deployment of these techniques for use in quantitative imaging trials presents unique challenges. The interplay between technical and scientific domain knowledge required for algorithm development is highlighted. The infrastructure surrounding algorithm deployment is critical, given regulatory, method robustness, computational, and performance considerations. The sensitivity of a given technique to these considerations and thus complexity of deployment is task- and phase-dependent. Context is provided for the infrastructure surrounding these methods, including common strategies for data flow, storage, access, and dissemination as well as application-specific considerations for individual use cases.

Many topically applied drugs are naturally fluorescent, but quantifying their uptake into skin via fluorescence emission is complicated by both weak fluorescence and often-overwhelming skin autofluorescence. Fluorescence lifetime imaging microscopy (FLIM) has been found capable of identifying and separating the fluorescence of multiple drugs from skin autofluorescence via their different spectral and lifetime properties. This investigation was focused on combining epi-fluorescence microscopy with deep learning for the quantification of naturally fluorescent topically applied drugs. This combination of deep learning and fluorescence imaging may allow for straightforward relative quantification of fluorescent drugs in tissue using only simple, readily available imaging tools.

Fighting cancer involves more and more combination of modalities and drugs to maximize the long-term tumor resistance and cure. The rationale for combination therapy is to use treatment modalities or drug combinations that work by different mechanisms, decreasing the likelihood that resistant cancer cells will develop. The combination of light induced therapy like photodynamic therapy (PDT) and chemotherapy has the potential to overcome the limitations traditionally associated with light-based therapies and simultaneously limit the well-known adverse effects of chemotherapy by controlling local release and dose. Modulight ML7710i medical laser systems have not only been shown to unleash the cytotoxic potential of different photochemotherapeutic compounds but also to effectively monitor the drug release process providing clinicians real-time information on treatment progress and preliminary projections on treatment outcome. In vitro and in vivo experiments suggest that Modulight ML7710i lasers are capable of inducing drug release from liposomes with different mechanisms depending on the nano-construct and laser wavelength. Near infrared wavelengths such as 808 nm are capable of disturbing liposomal bilayer upon light energy conversion to heat by dyes like indocyanine green.¹ Red wavelengths such as 665 nm in turn can induce photodynamic effect also causing drug release from hydrophobic core of the liposome.² Modulight ML7710i medical lasers are being validated for both use-cases and also for use with other dyes. The only limitation in using treatment monitoring capability is that the chemotherapeutic must have fluorescent potential. Modulight medical lasers can host multiple wavelengths within one system so that the drug release and the excitation can happen with different wavelengths if required.

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.