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Radiation and Heat Improve the Delivery and Efficacy of Nanotherapeutics by Modulating Intra-Tumoral Fluid Dynamics Shawn Stapleton, Michael Dunne, Michael Milosevic, Charles W. Tran, Matthew J. Gold, Ali Vedadi, Trevor McKee, Pamela S. Ohashi, Christine Allen, and David Anthony Jaffray ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b06301 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018
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Radiation and Heat Improve the Delivery and Efficacy of Nanotherapeutics by Modulating IntraTumoral Fluid Dynamics Author Names: Shawn Stapleton1,2,*†, Michael Dunne3†, Michael Milosevic2,4Charles W. Tran5, Matthew J. Gold6, Ali Vedadi2, Trevor Mckee2, Pamela S. Ohashi5,6, Christine Allen3, David A. Jaffray1,2,4,7 AUTHOR ADDRESS: 1
Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada Radiation Medicine Program, Princess Margaret Cancer Centre, Toronto, ON, Canada 3 Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada 4 Department of Radiation Oncology, University of Toronto, Toronto, ON, Canada 5 Department of Immunology, University of Toronto, Toronto, ON, Canada 6 The Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, Toronto, ON, Canada 7 Techna Institute, University Health Network, Toronto, ON, Canada 2
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Corresponding author Contributed Equally
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Abstract
Nanomedicine drug delivery systems are capable of transporting significant payloads to solid tumors. However, only a modest increase in anti-tumor efficacy relative to the standard of care has been observed. In this study, we demonstrate that a single dose of radiation or mild hyperthermia can substantially improve tumor uptake and distribution of nanotherapeutics, resulting in improved treatment efficacy. The delivery of nanomedicine was driven by a reduction in interstitial fluid pressure (IFP), and small perturbation of steady-state fluid flow. The transient effects on fluid dynamics in tumors with high IFP was also shown to dominate over immune cell endocytic capacity, another mechanism suspected of improving drug delivery. Furthermore, we demonstrate the specificity of this mechanism by showing that delivery of nanotherapeutics to low IFP tumors with high leukocyte infiltration do not benefit from pretreatment with radiation or heat. These results demonstrate that focusing on small perturbations to steady-state fluid dynamics, rather than large sustained effects or uncertain immune cell recruitment strategies, can impart a vulnerability to tumors with high IFP and enhance nanotherapeutic drug delivery and treatment efficacy.
Keywords: nanomedicine, liposomes, radiation, hyperthermia, interstitial fluid pressure, cancer, image-guided drug delivery
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Nanomedicine treatment of cancer exploits several advantageous properties of nanometer-scale drug delivery systems, including: improved drug solubility, protection from degradation of drug, extension of the pharmacokinetics (PK), selective and enhanced delivery of drugs to the tumor, potential to overcome resistance mechanisms, and reduced toxicity.1 However, the improved precision of drug delivery provided by nanomedicine does not always translate into improved treatment response and outcome.2 This is exemplified by a Phase III trial wherein Caelyx™, a prototypical long-circulating liposome carrier of doxorubicin, was found to provide comparable efficacy to doxorubicin, as a first-line therapy for metastatic breast cancer.3 Several studies have demonstrated abundant heterogeneity in nanomedicine deposition both within and between tumors in the preclinical and clinical setting.4-7 Therapeutic strategies that improve the tumor accumulation and intra-tumoral distribution of nanomedicine have resulted in increased tumor response.8-12 This is predicated on the notion that increased drug exposure (i.e. higher concentration and/or longer duration of drug/target engagement) leads to improved treatment response.13 While these studies have demonstrated the effect of modulating nanotherapeutic drug delivery, little is currently known about the mechanisms involved, or the optimal strategy for their combination.
The failure of clinically approved nanotherapeutics to increase antitumor efficacy relative to the standard of care is related, at least in part, to microenvironment effects on nanotherapeutic uptake and distribution.14-17 Variability in nanotherapeutic delivery and uptake are primarily driven by the complex interplay between transport barriers imposed by the tumor microenvironment and the presence of endocytic immune cells, such as tumor associated
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macrophages (TAMs).7, 18-19 The selective accumulation of nanomedicines in solid tumors has classically been described by the enhanced permeability and retention (EPR) effect.20 The EPR effect attributes preferential accumulation of nanotherapeutics to high vascular permeability and lack of functional lymphatic vessels in tumors. However, we now understand nanotherapeutic transport to be a substantially more complex process that also involves the interplay between tumor microenvironment factors that ultimately manifest in heterogeneously perfused tissue and elevated interstitial fluid pressure (IFP), whereby vascular permeability is a driving factor that can have both a positive and negative impact on nanotherapeutic delivery.1 Although less well understood and with mixed findings, some studies suggest that tumor stromal cells and TAMs play a role in mediating the transport of nanotherapeutics due to their pro-angiogenic phenotype, and drug release following uptake by endocytosis.14,
21-23
Vascular density, perfusion, and
elevated IFP have all been shown to strongly influence the limited bulk and intra-tumoral delivery of nanotherapeutics to tumors.7 Furthermore, nanotherapeutics tend to localize in close proximity to tumor blood vessels, which is consistent with an IFP-related limitation in convective transport through the interstitium and should be more pronounced in the tumor center than at the border.4
In order to overcome the barriers to nanotherapeutic delivery, we explored the use of radiation and heat to modulate drug delivery kinetics in two preclinical tumor model systems. A more thorough understanding of how heat, radiation and nanotherapeutics interact with the tumor microenvironment will drive the evolution of optimal/integrated treatment approaches, and biomarkers of response to facilitate precision nanotherapeutic approaches for individual patients. Herein we specifically investigate the effects of radiation and heat on intra-tumoral fluid
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dynamics, and the recruitment of tumor-associated myeloid cells. To explore and establish specificity of these effects, we used a xenogeneic tumor model with high IFP and limited leukocyte infiltration, and a syngeneic tumor model with low IFP and extensive leukocyte infiltration. We demonstrate that heat and radiation can shift high IFP tumors towards a vulnerable state that allows for improved intra-tumoral delivery of nanotherapeutics. The improved nanotherapeutic delivery of cytotoxic agents enhanced treatment response. We further demonstrate through measurements and mathematical modeling a different mechanism whereby the enhanced intra-tumoral delivery of nanotherapeutics can be driven by small spatio-temporal fluctuations in fluid dynamics caused by radiation and heat. We show that the myeloid cell population is efficient at internalizing the nanotherapeutic in both xenogeneic and syngeneic tumor models; however, radiation and heat did not lead to substantial myeloid cell recruitment and myeloid cells did not contribute significantly to nanotherapeutic accumulation and efficacy. As a consequence of our study we also demonstrate the power of imaging in guiding the delivery of radiation in combination with nanotherapeutics, and quantitatively assessing the intra-tumoral distribution of nanotherapeutics. The later may play an important role in predicting response to treatment. Ultimately, this work demonstrates the immediate translational impact of combining radiation or heat with nanoparticles in patients wherein a nanomedicine has been ineffective due to poor intra-tumoral drug delivery in high IFP tumors.
Results Radiation and Heat Improves the Bulk and Intra-Tumoral Accumulation of NPs in High IFP Tumors
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We first established the effects of radiation and heat on the intra-tumoral transport of nanotherapeutics in a tumor model with high IFP, namely the MDA-MB-231 metastatic breast adenocarcinoma model. The spatio-temporal kinetics of an imageable liposome formulation, previously developed in our lab,6, 24 was tracked using computed tomography (CT) imaging (Fig. 1A). A cohort of mice were pre-treated with 15 Gy radiation 24 h prior to the administration of CT-liposomes. A small animal image-guided radiation system (described below) was used to delivery radiation locally with CT-image guidance ensuring conformal irradiation of the tumor volume.25 The treatment protocols for radiation and heat were chosen based on previous preclinical reports26-28 as well as our own measurements that have shown that radiation and heat result in a substantial and enduring decrease in IFP (Fig. S1). Additionally, similar radiation and heat protocols have been reported to increase the bulk accumulation of nanoparticles in several pre-clinical tumor models.9 Control mice were not subjected to any pre-treatment.
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Fig. 1. Radiation and heat improve the bulk accumulation and intra-tumoral distribution of nanoparticles in tumors with high IFP. (A) Outline of the experimental protocol employed for pre-treatment of all tumors with radiation or heat, followed by injection of nanoparticles. (B) Representative CT-images showing improved accumulation and intra-tumoral distribution of nanotherapeutic (regions of signal enhancement within the red contour outlining the tumor) in the MDA-MB-231 tumor model. Pre-treatment with radiation and heat improved the bulk tumor accumulation (C), and the intra-tumoral distribution (D) relative to control MDA-MB-231 7 ACS Paragon Plus Environment
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tumors. Error bars represent standard error of the mean (SEM) of five mice. Scale bar represents 2mm.
Radiation and heat significantly enhanced bulk accumulation and intra-tumoral distribution of the CT-liposomes in tumors exhibiting high IFP, with heat having the largest effect (Fig. 1B, C and D). A 2-fold increase in peak tumor accumulation was observed after heating, reaching 14.6±0.8% of the injected dose (ID)/g by 48 to 72 h. The latter demonstrated rapid accumulation kinetics compared to both pre-treatment with radiation and control tumors. Radiation pre-treated tumors had a 1.3 fold increase in bulk accumulation of liposomes, peaking at 12.4±1.1 % ID/g by 72 h compared to control mice which peaked at 9.5±0.8 %ID/g at the same time point (Fig. 1C). The increased bulk accumulation was not dependent on variations in systemic PK between treatment groups and the concentration of CT-liposomes in the vasculature was subtracted from the bulk accumulation curves (Fig. S2). Radiation and heat also had profound effects on the intra-tumoral distribution of liposomes. Visually, we observed that CT-liposomes were able to accumulate at greater concentrations in the central regions of pre-treated tumors, whereas control tumors had predominantly peripheral accumulation (Fig. 1B). We observed a 1.2 to 2.0-fold and a 1.3 to 2.2-fold increase in the enhanced volume fraction following radiation and heat, respectively (Fig. 1D). These results indicate that pre-treatment with radiation and heat improves the intra-tumoral accumulation of liposomes in the high IFP MDA-MB-231 tumor model, providing for the ability of nanotherapeutics to reach previously inaccessible regions.
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Fig. 2. Increasing nanoparticle delivery improves response due to improved drug distribution. (A) Radiation or heat followed by Doxil increases the delay in tumor growth
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compared to individual treatments in MDA-MB-231 tumors. Volume is given as fold increase 3
from initial value (112.2 ± 20.5 mm ). Error bars represent the standard deviation. (B) The improved response is driven by improved intra-tumoral nanoparticle exposure in the tumor. (C) More nanoparticles are able to access the center of the tumor and increase exposure when radiation and heat are used as a pre-treatment. Radial analysis further demonstrates that nanoparticles have increased ability to penetrate the tumor center following radiation and heat. (D) The liposome imaging agent is an accurate surrogate of nanotherapeutic delivery (Doxil), localizing in the same cells as Doxil. Doxorubicin is predominantly in the cytoplasm and to a lesser extent within the nucleus.
Radiation and Heat Improve the Efficacy of Nanotherapeutics in High IFP Tumors We next investigated if the improved intra-tumoral accumulation of nanotherapeutics following radiation and heat resulted in improved response in high IFP tumors. We performed an efficacy study with liposomal doxorubicin (Doxil®) in a separate cohort of MDA-MB-231 tumor-bearing mice. The mice were administered Doxil (6 mg/kg) using the same radiation and heat pre-treatment protocols as previously described (Fig. 1A). Four sets of controls were used: saline alone, Doxil alone, 15 Gy radiation alone, and heat alone. Pre-treating tumors with radiation or heat followed by Doxil resulted in a significant tumor growth delay compared to untreated, Doxil alone, radiation alone, and heat alone treatment groups (Fig. 2A). Mice treated with either radiation or heat and Doxil survived significantly longer (i.e. median survival of 91 days and 63 days, respectively; Fig. S3A; p
