Oliver J. Klein

Click-Assembled, Oxygen-Sensing Nanoconjugates for Depth-Resolved, Near-Infrared Imaging in a 3 D 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.

Longitudinal, quantitative monitoring of therapeutic response in 3D in vitro tumor models with OCT for high-content therapeutic screening

In vitro three-dimensional models of cancer have the ability to recapitulate many features of tumors found in vivo, including cell-cell and cell-matrix interactions, microenvironments that become hypoxic and acidic, and other barriers to effective therapy. These model tumors can be large, highly complex, heterogeneous, and undergo time-dependent growth and treatment response processes that are difficult to track and quantify using standard imaging tools. Optical coherence tomography is an optical ranging technique that is ideally suited for visualizing, monitoring, and quantifying the growth and treatment response dynamics occurring in these informative model systems. By optimizing both optical coherence tomography and 3D culture systems, it is possible to continuously and non-perturbatively monitor advanced in vitro models without the use of labels over the course of hours and days. In this chapter, we describe approaches and methods for creating and carrying out quantitative therapeutic screens with in vitro 3D cultures using optical coherence tomography to gain insights into therapeutic mechanisms and build more effective treatment regimens.

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.

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.

3D-resolved targeting of photodynamic therapy using temporal focusing.

A method for selectively inducing apoptosis in tumor nodules is presented, with close-to-cellular level resolution, using 3D-resolved widefield temporal focusing illumination. Treatment times on the order of seconds were achieved using Verteporfin as the photosensitizer, with doses of 30 μg ml-1 and below. Results were achieved on both 2D and 3D cell cultures, demonstrating that treatment was possible through approximately one hundred microns of dense tumor nodules.

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.

Abstract B44: Mapping, targeting, and eliminating therapeutically unresponsive ovarian cancer with high content imaging and photodynamic therapy

Ovarian cancer is the 5th most common cancer among women, in which epithelial ovarian cancer (EOC) is the most common type, accounting for 90% of cases. Currently, the standard treatment for ovarian cancer is surgical debulking followed by chemotherapy. Although initially EOC patients are highly responsive to standard platinum-derived chemotherapy, the majority of cancer patients will eventually relapse and experience chemoresistance. In spite of numerous efforts to improve EOC treatment, the five-year overall survival rate is still disappointing at 30%. It is therefore necessary to examine the fundamental causes of treatment resistance and use this information to design new therapeutic strategies. Recently, the concept of tumor-initiating cells and their supporting tumor microenvironment has been proposed as potential targets for cancer treatment due to their observed chemoresistance and ability to repopulate tumors. These cells are proposed to reside deep within tumors, potentially in oxygen-deprived environments. We recently observed that a light-based treatment modality known as photodynamic therapy (PDT) may be uniquely suited to targeting and eliminating this cellular population. PDT applies a light activated drug (photosensitizer) to selectively target and kill cancer cells through generation of reactive oxygen species (ROS), making it an attractive alternative cancer treatment modality with the potential to overcome adaptive therapeutic resistance. Characteristics of PDT, including the direct destruction of cellular organelles, selective tumor accumulation, and photochemistry-driven tumor killing mechanism give PDT advantages over traditional chemotherapy and radiotherapy. We have found that a lysosome targeted photosensitizer known as EtNBS specifically localizes and accumulates into the acidic and hypoxic tumor microenvironment in 3D cultures to successfully kill otherwise unresponsive cellular populations. EtNBS-mediated PDT is thus a potential modality in treating tumor initiating cells. In addition, the quantitative and label-free optical coherence tomography (OCT) imaging modality has recently been demonstrated to provide real time information regarding tumor tissue structure and therapeutic dynamics, making it a potential tool to study tumors and their associated change during the course of tumor development and treatment. Here, we utilized a 3D in vitro metastatic ovarian cancer culture model, with an oxygen sensor and OCT imaging as a combinational platform to study tumor initiating cells and the effects of their associated tumor microenvironment on therapeutic response under standard and EtNBS-PDT treatment. By mapping tumor response with this integrated platform, this work represents a mechanism- based targeted approach that will hopefully enable the identification of cellular targets to overcome disease relapse and adaptive chemoresistance. Citation Format: Hsin-I Hung, Oliver J. Klein, Yookyung Jung, Kashmira S. Kulkarni, Bo R. Rueda, Rosemary Foster, Conor L. Evans. Mapping, targeting, and eliminating therapeutically unresponsive ovarian cancer with high content imaging and photodynamic therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B44.

EtNBS and EtNBS-COOH PDT efficacy in ovarian cancer cells

The effective treatment of metastatic cancer continues to be a challenge due to the highly invasive nature of metastatic lesions and their propensity to develop therapeutic resistance. Optimal therapeutic regimens for peritoneal metastases should have both rapid uptake and penetrate throughout cancerous lesions. Using in vitro models of ovarian cancer, we have found that the cores of tumor nodules are both hypoxic and acidic, rendering most chemotherapeutic agents ineffective, and considerably reducing the therapeutic efficacy of the majority of photodynamic therapy (PDT) and radiation therapy regimens. PDT using EtNBS, a cationic photosensitizer, is a promising approach for treating these hypoxic and acidic nodule cores as it rapidly accumulates into the hypoxic cores of tumor nodules, and is effective even in completely anoxic environments. To improve the uptake rate of EtNBS into cells, we utilized a carboxylic acid terminated derivative of EtNBS that is zwitterionic at biological pH levels. In this study, we investigated the effect of this structural change on PDT efficacy in ovarian cancer cells.