Bryan Q. Spring

Imaging and Photodynamic Therapy: Mechanisms, Monitoring, and Optimization

The purpose of this review is to present the current state of the role of imaging in photodynamic therapy (PDT). In order for the reader to fully appreciate the context of the discussions embodied in this article we begin with an overview of the PDT process, starting with a brief historical perspective followed by detailed discussions of specific applications of imaging in PDT. Each section starts with an overview of the specific topic and, where appropriate, ends with summary and future directions. The review closes with the authors’ perspective of the areas of future emphasis and promise. The basic premise of this review is that a combination of imaging and PDT will provide improved research and therapeutic strategies.

In vivo high-resolution fluorescence microendoscopy for ovarian cancer detection and treatment monitoring

Background:In patients with advanced ovarian cancer (OvCa), microscopic residual tumour nodules that remain after surgical debulking frequently escape detection by current treatment assessment methods and lead to disease recurrence. The aim of this study was to evaluate the use of high-resolution fibre-optic fluorescence imaging of the clinically approved photodynamic therapy (PDT) agent benzoporphyin-derivative monoacid ring A (BPD-MA) for detection of microscopic OvCa and for monitoring treatment response.Methods:Our fluorescence microendoscope consists of a flexible imaging fibre coupled to a custom epi-fluorescence system optimised for imaging BPD-MA, which, after a single administration, serves as both an imaging agent and a light-activated therapeutic agent. After characterisation in an in vitro OvCa 3D model, we used the flexible imaging fibre to minimally invasively image the peritoneal cavity of a disseminated OvCa murine model using BPD-MA administered intraperitoneally (i.p.). To evaluate longitudinal changes in response to treatment, we compared sets of images obtained before and after PDT with those from untreated mice imaged at the same time points.Results:By comparison with histopathology, we report an 86% sensitivity for tumour detection in vivo using the microendoscope. Using a custom routine to batch process-image data in the monitoring study, treated mice exhibited an average decrease of 58.8% in tumour volumes compared with an increase of 59.3% in untreated controls (P<0.05).Conclusions:Our findings indicate the potential of this approach as a reporter of treatment outcome that could aid in the rational design of strategies to mitigate recurrent OvCa.

A photoactivable multi-inhibitor nanoliposome for tumour control and simultaneous inhibition of treatment escape pathways

Nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour-selective drug release. However, few are effective because cancer cells develop ways to resist and evade treatment. Here, we introduce a photoactivatable multi-inhibitor nanoliposome (PMIL) that imparts light-induced cytotoxicity in synchrony with photo-initiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways. The PMIL consists of a nanoliposome doped with a photoactivatable chromophore (benzoporphyrin derivative, BPD) in the lipid bilayer, and a nanoparticle containing cabozantinib (XL184)—a multikinase inhibitor—encapsulated inside. Near infrared tumour irradiation, following intravenous PMIL administration, triggers photodynamic damage of tumour cells and microvessels, and simultaneously initiates release of XL184 inside the tumour. A single PMIL treatment achieves prolonged tumour reduction in two mouse models and suppresses metastatic escape in an orthotopic pancreatic tumour model. The PMIL offers new prospects for cancer therapy by enabling spatiotemporal control of drug release whilst reducing systemic drug exposure and associated toxicities.

Engineering Redox-Sensitive Linkers for Genetically Encoded FRET-Based Biosensors

The ability to sense intracellular or intraorganellar reduction/oxidation conditions would provide a powerful tool for studying normal cell proliferation, differentiation, and apoptosis. Genetically encoded biosensors enable monitoring of the intracellular redox environment. We report the development of chimeric polypeptides useful as redox-sensitive linkers in conjunction with Förster resonance energy transfer (FRET). α-helical linkers differing in length were combined with motifs that are sensitive to the redox state of the environment. The first category of linkers included a redox motif found in the thioredoxin family of oxidoreductases. This motif was flanked by two α-helices of equal length. The second and third categories of redox linkers were composed of α-helices with embedded adjacent and dispersed vicinal cysteine residues, respectively. The linkers containing redox switches were placed between a FRET pair of enhanced cyan and yellow fluorescent proteins and these constructs were tested subsequently for their efficacy. A robust method of FRET analysis, the (ratio) A method, was used. This method uses two fluorescence spectra performed directly on the FRET construct without physical separation of the fluorophores. The cyan/yellow construct carrying one of the designed redox linkers, RL5, exhibited a 92% increase in FRET efficiency from its reduced to oxidized states. Responsiveness of the cyan-RL5-yellow construct to changes in the intracellular redox environment was confirmed in mammalian cells by flow cytometry.

Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates

Significance Residual micrometastases following standard therapies limit our ability to cure many cancers. This article demonstrates a new therapy and visualization platform that targets residual cancer micrometastases with enhanced sensitivity and selectivity based on “tumor-targeted activation.” This targeted activation feature not only provides a potent therapeutic arm that is effective against chemoresistant disease while minimizing side effects due to nonspecific toxicities but also enables micrometastasis imaging in common sites of disease recurrence to screen patients harboring residual tumor deposits. This approach offers promise for treating and monitoring drug-resistant micrometastases presently “invisible” to clinicians. Drug-resistant micrometastases that escape standard therapies often go undetected until the emergence of lethal recurrent disease. Here, we show that it is possible to treat microscopic tumors selectively using an activatable immunoconjugate. The immunoconjugate is composed of self-quenching, near-infrared chromophores loaded onto a cancer cell-targeting antibody. Chromophore phototoxicity and fluorescence are activated by lysosomal proteolysis, and light, after cancer cell internalization, enabling tumor-confined photocytotoxicity and resolution of individual micrometastases. This unique approach not only introduces a therapeutic strategy to help destroy residual drug-resistant cells but also provides a sensitive imaging method to monitor micrometastatic disease in common sites of recurrence. Using fluorescence microendoscopy to monitor immunoconjugate activation and micrometastatic disease, we demonstrate these concepts of “tumor-targeted, activatable photoimmunotherapy” in a mouse model of peritoneal carcinomatosis. By introducing targeted activation to enhance tumor selectively in complex anatomical sites, this study offers prospects for catching early recurrent micrometastases and for treating occult disease.

Image analysis for denoising full-field frequency-domain fluorescence lifetime images

Video‐rate fluorescence lifetime‐resolved imaging microscopy (FLIM) is a quantitative imaging technique for measuring dynamic processes in biological specimens. FLIM offers valuable information in addition to simple fluorescence intensity imaging; for instance, the fluorescence lifetime is sensitive to the microenvironment of the fluorophore allowing reliable differentiation between concentration differences and dynamic quenching. Homodyne FLIM is a full‐field frequency‐domain technique for imaging fluorescence lifetimes at every pixel of a fluorescence image simultaneously. If a single modulation frequency is used, video‐rate image acquisition is possible. Homodyne FLIM uses a gain‐modulated image intensified charge‐coupled device (ICCD) detector, which unfortunately is a major contribution to the noise of the measurement. Here we introduce image analysis for denoising homodyne FLIM data. The denoising routine is fast, improves the extraction of the fluorescence lifetime value(s) and increases the sensitivity and fluorescence lifetime resolving power of the FLIM instrument. The spatial resolution (especially the high spatial frequencies not related to noise) of the FLIM image is preserved, because the denoising routine does not blur or smooth the image. By eliminating the random noise known to be specific to photon noise and from the intensifier amplification, the fidelity of the spatial resolution is improved. The polar plot projection, a rapid FLIM analysis method, is used to demonstrate the effectiveness of the denoising routine with exemplary data from both physical and complex biological samples. We also suggest broader impacts of the image analysis for other fluorescence microscopy techniques (e.g. super‐resolution imaging).

Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments

We report the optimization of a novel redox-sensitive probe with enhanced dynamic range and an exceptionally well-positioned oxidative midpoint redox potential. The present work characterizes factors that contribute to the improved Förster resonance energy transfer (FRET) performance of this green fluorescent protein (GFP)-based redox sensor. The α-helical linker, which separates the FRET donor and acceptor, has been extended in the new probe and leads to a decreased FRET efficiency in the linkers reduced, ‘FRET-off’ state. Unexpectedly, the FRET efficiency is increased in the new linkers oxidized, ‘FRET-on’ state compared with the parent probe, in spite of the longer linker sequence. The combination of a lowered baseline ‘FRET-off’ and an increased ‘FRET-on’ signal significantly improves the dynamic range of the probe for a more robust discrimination of its reduced and oxidized linker states. Mutagenesis of the cysteine residues within the α-helix linker reveals the importance of the fourth, C-terminal cysteine and the relative insignificance of the second cysteine in forming the disulfide bridge to clamp the linker into the high-FRET, oxidized state. To further optimize the performance of the redox probe, various cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) FRET pairs, placed at opposite ends of the improved redox linker (RL7), were quantitatively compared and exchanged. We found that the CyPet/YPet and ECFP/YPet FRET pairs when attached to RL7 do not function well as sensitive redox probes due to a strong tendency to form heterodimers, which disrupt the α-helix. However, monomeric versions of CyPet and YPet (mCyPet and mYPet) eliminate dimerization and restore redox sensitivity of the probe. The best performing probe, ECFP-RL7-EYFP, exhibits an approximately six-fold increase in FRET efficiency in vitro when passing from the oxidized to the reduced state. We determined the midpoint redox potential of the probe to be −143 ± 6 mV, which is ideal for measuring glutathione (GSH/GSSG) redox potentials in oxidative compartments of mammalian cells (e.g. the endoplasmic reticulum).

Optical Imaging, Photodynamic Therapy and Optically Triggered Combination Treatments.

AbstractOptical imaging is becoming increasingly promising for real-time image-guided resections, and combined with photodynamic therapy (PDT), a photochemistry-based treatment modality, optical approaches can be intrinsically “theranostic.” Challenges in PDT include precise light delivery, dosimetry, and photosensitizer tumor localization to establish tumor selectivity, and like all other modalities, incomplete treatment and subsequent activation of molecular escape pathways are often attributable to tumor heterogeneity. Key advances in molecular imaging, target-activatable photosensitizers, and optically active nanoparticles that provide both cytotoxicity and a drug release mechanism have opened exciting avenues to meet these challenges. The focus of the review is optical imaging in the context of PDT, but the general principles presented are applicable to many of the conventional approaches to cancer management. We highlight the role of optical imaging in providing structural, functional, and molecular information regarding photodynamic mechanisms of action, thereby advancing PDT and PDT-based combination therapies of cancer. These advances represent a PDT renaissance with increasing applications of clinical PDT as a frontline cancer therapy working in concert with fluorescence-guided surgery, chemotherapy, and radiation.

Simultaneous delivery of cytotoxic and biologic therapeutics using nanophotoactivatable liposomes enhances treatment efficacy in a mouse model of pancreatic cancer

A lack of intracellular delivery systems has limited the use of biologics such as monoclonal antibodies (mAb) that abrogate molecular signaling pathways activated to promote escape from cancer treatment. We hypothesized that intracellular co-delivery of the photocytotoxic chromophore benzoporphyrin derivative monoacid A (BPD) and the anti-VEGF mAb bevacizumab in a nanophotoactivatable liposome (nanoPAL) might enhance the efficacy of photodynamic therapy (PDT) combined with suppression of VEGF-mediated signaling pathways. As a proof-of-concept we found that nanoPAL-PDT induced enhanced extra- and intracellular bevacizumab delivery and enhanced acute cytotoxicity in vitro. In an in vivo subcutaneous mouse model of pancreatic ductal adenocarcinoma, nanoPAL-PDT achieved significantly enhanced tumor reduction. We attribute this to the optimal incorporation of insoluble BPD into the lipid bilayer, enhancing photocytotoxicity, and the simultaneous spatiotemporal delivery of bevacizumab, ensuring efficient neutralization of the rapid but transient burst of VEGF following PDT. From the Clinical Editor: Most patients with pancreatic ductal adenocarcinoma (PDAC) by the time present the disease it is very advanced, which unavoidably translates to poor survival. For these patients, use of traditional chemotherapy often becomes ineffective due to tumor resistance to drugs. Photodynamic therapy (PDT) can be an effective modality against chemo-resistant cancers. In this article, the authors investigated the co-delivery of a photocytotoxic agent and anti-VEGF mAb using liposomes. This combination was shown to results in enhanced tumor killing. This method should be applicable to other combination of treatments.

What is behind all those lifetimes anyway, and where do we go from here?

Fluorescence lifetime-resolved imaging microscopy (FLIM) has made tremendous strides in the last two decades. Exciting applications are being presented weekly, an extensive diversity of instrumentation and commercial devices have appeared and have improved dramatically, sophisticated algorithms for analysis and interpretation are now available, and FLIM is being coupled to other imaging modalities, such as spectral dispersion and anisotropy. In other words, FLIM has matured considerably, and is approaching a point where it can be routinely applied by researchers who are not involved with the instrumentation and analysis side of things. The number of interested users in FLIM has almost certainly surpassed that of the audience that previously employed single channel fluorescence lifetime measurements (in a cuvette). The reason is, of course, the imaging capability of FLIM and its exciting possibilities for biological applications, especially FRET. In this lecture I will attempt to give an overview of where we have come from together with a personal judgment on where we stand, propose possible areas of growth that will be of importance for the future of FLIM, and discuss some specific challenges that remain, especially considering the unique nature of the FLIM measurement and the complex biological and medical systems that require innovative and novel solutions from the FLIM community.