Click-assembled, oxygen-sensing nanoconjugates for depth-resolved, near-infrared imaging in a 3D cancer model.

Hypoxia is an important contributing factor to the development of drug-resistant cancer, yet few nonperturbative tools exist for studying oxygenation in tissues. While progress has been made in the development of chemical probes for optical oxygen mapping, penetration of such molecules into poorly perfused or avascular tumor regions remains problematic. A click-assembled oxygen-sensing (CAOS) nanoconjugate is reported and its properties demonstrated in an in vitro 3D spheroid cancer model. The synthesis relies on the sequential click-based ligation of poly(amidoamine)-like subunits for rapid assembly. Near-infrared confocal phosphorescence microscopy was used to demonstrate the ability of the CAOS nanoconjugates to penetrate hundreds of micrometers into spheroids within hours and to show their sensitivity to oxygen changes throughout the nodule. This proof-of-concept study demonstrates a modular approach that is readily extensible to a wide variety of oxygen and cellular sensors for depth-resolved imaging in tissue and tissue models.

In vitro optimization of EtNBS-PDT against hypoxic tumor environments with a tiered, high-content, 3D model optical screening platform.

Hypoxia and acidosis are widely recognized as major contributors to the development of treatment resistant cancer. For patients with disseminated metastatic lesions, such as most women with ovarian cancer (OvCa), the progression to treatment resistant disease is almost always fatal. Numerous therapeutic approaches have been developed to eliminate treatment resistant carcinoma, including novel biologic, chemo, radiation, and photodynamic therapy (PDT) regimens. Recently, PDT using the cationic photosensitizer EtNBS was found to be highly effective against therapeutically unresponsive hypoxic and acidic OvCa cellular populations in vitro. To optimize this treatment regimen, we developed a tiered, high-content, image-based screening approach utilizing a biologically relevant OvCa 3D culture model to investigate a small library of side-chain modified EtNBS derivatives. The uptake, localization, and photocytotoxicity of these compounds on both the cellular and nodular levels were observed to be largely mediated by their respective ethyl side chain chemical alterations. In particular, EtNBS and its hydroxyl-terminated derivative (EtNBS-OH) were found to have similar pharmacological parameters, such as their nodular localization patterns and uptake kinetics. Interestingly, these two molecules were found to induce dramatically different therapeutic outcomes: EtNBS was found to be more effective in killing the hypoxic, nodule core cells with superior selectivity, while EtNBS-OH was observed to trigger widespread structural degradation of nodules. This breakdown of the tumor architecture can improve the therapeutic outcome and is known to synergistically enhance the antitumor effects of front-line chemotherapeutic regimens. These results, which would not have been predicted or observed using traditional monolayer or in vivo animal screening techniques, demonstrate the powerful capabilities of 3D in vitro screening approaches for the selection and optimization of therapeutic agents for the targeted destruction of specific cellular subpopulations.

Label-Free, Longitudinal Visualization of PDT Response In Vitro with Optical Coherence Tomography.

A major challenge in creating and optimizing therapeutics in the fight against cancer is visualizing and understanding the microscale spatiotemporal treatment response dynamics that occur in patients. This is especially true for photodynamic therapy (PDT), where therapeutic optimization relies on understanding the interplay between factors such as photosensitizer localization and uptake, in addition to light dose and delivery rate. In vitro 3D culture systems that recapitulate many of the biological features of human disease are powerful platforms for carrying out detailed studies on PDT response and resistance. Current techniques for visualizing these models, however, often lack accuracy due to the perturbative nature of the sample preparation, with light attenuation complicating the study of intact models. Optical coherence tomography (OCT) is an ideal method for the long-term, non-perturbative study of in vitro models and their response to PDT. Monitoring the response of 3D models to PDT by time-lapse OCT methods promises to provide new perspectives and open the way to cancer treatment methodologies that can be translated towards the clinic.

An Integrin-Targeted, Highly Diffusive Construct for Photodynamic Therapy.

Targeted antineoplastic agents show great promise in the treatment of cancer, having the ability to impart cytotoxicity only to specific tumor types. However, these therapies do not experience uniform uptake throughout tumors, leading to sub-lethal cell killing that can impart treatment resistance, and cause problematic off-target effects. Here we demonstrate a photodynamic therapy construct that integrates both a cyclic RGD moiety for integrin-targeting, as well as a 5 kDa PEG chain that passivates the construct and enables its rapid diffusion throughout tumors. PEGylation of the photosensitizer construct was found to prevent photosensitizer aggregation, boost the generation of cytotoxic reactive radical species, and enable the rapid uptake of the construct into cells throughout large (>500 µm diameter) 3D tumor spheroids. Replacing the cyclic RGD with the generic RAD peptide led to the loss of cellular uptake in 3D culture, demonstrating the specificity of the construct. Photodynamic therapy with the construct was successful in inducing cytotoxicity, which could be competitively blocked by a tenfold concentration of free cyclic RGD. This construct is a first-of-its kind theranostic that may serve as a new approach in our growing therapeutic toolbox.

Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT.

Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.

PLGA nanoparticle encapsulation reduces toxicity while retaining the therapeutic efficacy of EtNBS-PDT in vitro.

Photodynamic therapy regimens, which use light-activated molecules known as photosensitizers, are highly selective against many malignancies and can bypass certain challenging therapeutic resistance mechanisms. Photosensitizers such as the small cationic molecule EtNBS (5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride) have proven potent against cancer cells that reside within acidic and hypoxic tumour microenvironments. At higher doses, however, these photosensitizers induce "dark toxicity" through light-independent mechanisms. In this study, we evaluated the use of nanoparticle encapsulation to overcome this limitation. Interestingly, encapsulation of the compound within poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA-EtNBS) was found to significantly reduce EtNBS dark toxicity while completely retaining the molecule's cytotoxicity in both normoxic and hypoxic conditions. This dual effect can be attributed to the mechanism of release: EtNBS remains encapsulated until external light irradiation, which stimulates an oxygen-independent, radical-mediated process that degrades the PLGA nanoparticles and releases the molecule. As these PLGA-encapsulated EtNBS nanoparticles are capable of penetrating deeply into the hypoxic and acidic cores of 3D spheroid cultures, they may enable the safe and efficacious treatment of otherwise unresponsive tumour regions.

Killing hypoxic cell populations in a 3D tumor model with EtNBS-PDT.

An outstanding problem in cancer therapy is the battle against treatment-resistant disease. This is especially true for ovarian cancer, where the majority of patients eventually succumb to treatment-resistant metastatic carcinomatosis. Limited perfusion and diffusion, acidosis, and hypoxia play major roles in the development of resistance to the majority of front-line therapeutic regimens. To overcome these limitations and eliminate otherwise spared cancer cells, we utilized the cationic photosensitizer EtNBS to treat hypoxic regions deep inside in vitro 3D models of metastatic ovarian cancer. Unlike standard regimens that fail to penetrate beyond ∼150 µm, EtNBS was found to not only penetrate throughout the entirety of large (>200 µm) avascular nodules, but also concentrate into the nodules' acidic and hypoxic cores. Photodynamic therapy with EtNBS was observed to be highly effective against these hypoxic regions even at low therapeutic doses, and was capable of destroying both normoxic and hypoxic regions at higher treatment levels. Imaging studies utilizing multiphoton and confocal microscopies, as well as time-lapse optical coherence tomography (TL-OCT), revealed an inside-out pattern of cell death, with apoptosis being the primary mechanism of cell killing. Critically, EtNBS-based photodynamic therapy was found to be effective against the model tumor nodules even under severe hypoxia. The inherent ability of EtNBS photodynamic therapy to impart cytotoxicity across a wide range of tumoral oxygenation levels indicates its potential to eliminate treatment-resistant cell populations.