<p>There is a critical need to target tumor hypoxia as patients with hypoxic tumors have worse prognosis due to aggressive phenotypes and resistance to radiotherapy and chemotherapy. The overall goal of this work is to improve response to conventional cancer therapies by targeting tumor hypoxia. This has been carried out and evaluated through the use of polymersome-encapsulated myoglobin (PEMs) with the hypothesis that O2-releasing PEMs will increase tumor oxygenation, and thereby improve response to radiotherapy. Mb was chosen as an O2 carrying protein to deliver to tumors because it has a strong association to O2, providing a mechanism to deliver O2 only within the hypoxic regions of the tumor. Mb was loaded within nanoscale polymeric vesicles that were expected to accumulate within solid tumors due to the enhanced permeability and retention (EPR) effect. This hypothesis has been tested through the following aims:</p><p>1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes</p><p>2. Establish the effects of Mb-containing polymersomes on tumor physiology</p><p>3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors</p><p>These aims have been evaluated through numerous in vivo studies. First, polymersomes of various polymer formulations and diameters ranging from 110-550 nm were prepared with a near-infrared (NIR) -emissive fluorophore. Using live animal fluorescence imaging, I was able to study the biodistribution of the polymersomes following i.v. administration, demonstrating significant polymersome accumulation in orthotopic 4T1 mammary carcinomas. In addition, a novel method for measuring pharmacokinetics was developed, using serial small volume blood draws from individual mice. The plasma fluorescence in microcapillary tubes was used to quantify polymersome concentrations, demonstrating long circulation half-lives that varied from 6-23 h. Toxicity of various polymersome formulations were also studied in vitro and in vivo, revealing negligible toxicities.</p><p>For the second aim, PEMs were administered i.v. in tumor-bearing mice. Unexpectedly, we observed a dramatic gross tumor effect within hours of treatment in both orthotopic 4T1 tumors and flank Renca renal cell carcinomas. Histological analysis revealed endothelial cell apoptosis as early as 1 h following treatment, with scattered tumor cell death throughout the tumor by 4 h. Hematoxylin and eosin staining showed significant necrosis 24 h following PEM treatment. Vascular effects and polymersome distribution were studied in 4T1 window chamber tumors. Following i.v. treatment with PEMs, intravital microscopy was used to image polymersome fluorescence, brightfield transmission was imaged for vessel morphology and blood flow, and a tunable filter was used for determining hemoglobin (Hb) oxygen saturation. Tumor hemorrhaging was observed within hours of PEM treatment, which was not seen with empty polymersomes. This was consistent with the gross tumor effects observed initially. Hb saturation decreased in both the PEM and empty polymersome groups, but not in saline-treated mice. While we expected to observe an increase in tumor oxygenation by using Mb as an oxygen carrier, we actually observed hemorrhage, decreased oxygenation, and central tumor necrosis. In vitro studies using human endothelial cells demonstrated dramatic changes in cell morphology and increased permeability due to Mb and PEM treatments, which appear to be enhanced in an oxidative environment. These in vitro and in vivo observations are similar to what is seen with tumor vascular disrupting agents.</p><p>For the third aim, I combined radiotherapy (RT) and PEM treatment with a new hypothesis. I originally expected the PEMs to increase tumor oxygenation, thus making the tumor more susceptible to RT. However, considering the results from the second aim, this hypothesis was modified: the PEMs would result in necrosis of the tumor core, while RT would target the more oxygenated rim of the tumor, thus leading to improved tumor growth delay compared with PEM or RT alone. This hypothesis was tested in both orthotopic, syngeneic 4T1 tumors as well as flank FaDu xenografts. 4T1 tumor cells were surgically implanted within the dorsal mammary fat pad of mice and grown until ~200 mm3. A CT microirradiator with a square collimator was used in order to locate and specifically irradiate the tumor. Within 1 h following RT, the PEMs were administered i.v.. Mice receiving PEMs with no RT showed a significant decrease in tumor growth compared with saline-treated mice (p = 0.0001 for time to 3x original tumor volume). In addition, the combination of RT plus PEMs reduced tumor growth compared with RT alone (p = 0.0144 for time to 3x original tumor volume). However, this effect was not seen with FaDu tumors. This may have been due to excessive radiation dose or other compounding factors: the timing between RT and PEM treatment was not optimized, and the number of mice per group was small (3-4). </p><p>Thus, the conclusions for each aim are as follows:</p><p>1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes</p><p>NIR imaging techniques were optimized for studying polymersomes, demonstrating long plasma circulation times and accumulation within tumors.</p><p>2. Establish the effects of Mb-containing polymersomes on tumor physiology</p><p>While the hypothesis was that PEMs would accumulate within hypoxic tumors and subsequently increase O2 tension, we observed a rapid decrease in tumor oxygenation followed by a dramatic hemorrhagic effect of Mb polymersomes, which appear to be due to both endothelial cell apoptosis and morphological changes, resulting in central tumor necrosis.</p><p>3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors</p><p>Combination therapy of PEMs with RT results in enhanced tumor growth delay in aggressive 4T1 mammary carcinomas compared with RT or PEMs alone.</p><p>These studies have led to a proposed mechanism for the PEM anti-tumor effect in combination with RT. Prior to PEM administration, RT is administered, resulting in tumor cell kill of the well-oxygenated tumor periphery. Mb polymersomes are then injected i.v. and begin to accumulate within tumors due to the EPR effect. As shown in Aim 1, this accumulation occurs over a short time scale. Within 30 min of PEM treatment, the Mb is believed to act on tumor vessels, resulting in morphological changes and apoptosis of endothelial cells. These effects are expected to increase permeability of the vessels and expose the basement membrane, which leads to clotting and decreased blood flow. Both decreased perfusion and increased permeability are believed to have a catastrophic effect on interior tumor vessels. Hemorrhage results as the endothelial cells die, resulting in tumor core necrosis. Therefore, the result is tumor cell kill at the periphery due to RT and central tumor necrosis due to PEM treatment.</p><p>PEMs have potential in cancer therapy as a new class of VDAs. While the mechanism requires further investigation, this work has demonstrated that PEM treatment results in tumor vessel destruction and central necrosis. PEMs accumulate within tumors, thus minimizing the systemic toxicity of treatment commonly seen with VDAs. By combining PEMs with a therapy that kills the better perfused tumor periphery, PEMs show promise in improving tumor response. Future mechanistic studies will be needed in order to maximize vessel damage and optimize combination dosing schedules to improve outcome.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/9843 |
| Date | January 2015 |
| Creators | Hofmann, Christina Lehmkuhl |
| Contributors | Dewhirst, Mark W |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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