Browsing by Author "Dewhirst, Mark W"

Radiation therapy (RT) has been a standard-of-care treatment for many localized cancers for decades. Despite being an effective treatment modality for many clinical presentations, the efficacy of RT against cancer can be limited due to local recurrence, metastatic spread, and radiation resistance from tumor hypoxia. These limitations provide opportunity for innovative approaches to enhance the overall efficacy of RT. This thesis explores the potential novel approach to enhancing RT through the paradigm changing approach of adding a phototherapeutic component initiated simultaneously with RT. X-ray Psoralen Activated Cancer Therapy (X-PACT) is one such approach, where diagnostics-energy kilovoltage (kV) x-ray coupled with energy modulators (phosphors) converts kV photon to ultraviolet (UV) light, which in turn activates psoralen. Radiotherapy Enhanced with Cherenkov photo-Activation (RECA) is another approach, where therapeutic megavoltage (MV) x-ray generates UV light via Cherenkov phenomenon. Both approaches could increase local control in RT, increase treatment effectiveness in hypoxic tumors, and amplify anti-cancer systemic response. The overarching hypothesis that drives this dissertation is that X-PACT and RECA can activate psoralen to enhance cytotoxicity in-vitro and tumor growth control in-vivo compared to RT alone. In line with this hypothesis, this work explores the feasibility of both X-PACT and RECA via in-vitro and in-vivo verification as well as optimization of radiation techniques to maximize the therapeutic benefit of the approach.

X-PACT and RECA in-vitro / in-vivo studies indicate radiotherapy enhancement is plausible with psoralens activated by secondary UV light production from radiation, though further investigation is required to establish feasibility of RECA in-vivo. For X-PACT in-vitro, a substantial reduction in cell viability and increase in apoptosis was observed in various murine cancer cells (4T1, KP-B, and CT2A) when treated with a combination of 50µg/mL phosphor, 10µM psoralen (8-MOP), and 1Gy of 80kVp x-ray (viability < 20%), compared to any of these components alone (viability > 70%). This suggests a synergistic interaction between the components congruent with the X-PACT scheme, where x-ray induces phosphor UV emission, which in turn activates psoralen. The X-PACT in-vivo mice study showed improved survival with X-PACT versus saline control with flank 4T1 tumors (30.7 days for X-PACT vs. 21.6 days for saline) for survival criteria of 1000, 1500, and 2000mm3, respectively. For RECA, in-vitro results seem promising, where reductions in viability of 20% and 9.5% were observed for 4T1 and B16 murine cancer cell lines treated with RECA (radiation + trioxsalen, a potent psoralen derivative) versus radiation alone. A substantial increase in MHC I expression was observed for B16 cells treated with RECA versus those treated with radiation alone. A small RECA in-vivo pilot study using 8-MOP was inconclusive. Further in-vivo trials with a greater number mice per arm of are required to establish the RECA feasibility to enhance radiotherapy.

Feasibility of treatment optimization for both X-PACT and RECA were demonstrated with kV and MV beams respectively, by optimization of optical output per radiation dose delivered. It was found that in both X-PACT and RECA scheme, the energy of the photon radiation beam (i.e. tube voltage and LINAC energy settings) affected optical output the most. With kV beams for X-PACT, accurate beam delivery within the target volume to reduce normal tissue damage typically expected of kV beams was demonstrated with a 3D-printing-based preclinical irradiation scheme, which is expected to help X-PACT translation into the clinics. In addition, for X-PACT, novel MV-responding phosphors were characterized under MV radiation beam, suggesting the possibility of MV-radiation-mediated X-PACT. Immediate future studies should investigate the efficacy of the optimized X-PACT and RECA, as well as MV X-PACT in-vitro and in-vivo. Studies beyond these immediate ones should investigate X-PACT and RECA efficacy against hypoxic and metastatic tumor sites, where radiation can traditionally fail.

Tumor hypoxia is an enduring problem for traditional cancer therapies such as radiation and chemotherapy. This obstacle has traditionally been attributed to the widespread presence of chronic, diffusion-limited hypoxia in solid tumors; recent data suggests that tumor hypoxia may also be spatially and temporally variable. In this work we characterize the presence of spatial and temporal fluctuations in hypoxia, as well as use mathematical modeling to predict the impact of fluctuations on the hypoxic cytotoxin, tirapazamine, and examine potential mechanisms of fluctuations in tumor oxygenation. Using phosphorescence lifetime imaging on preclinical tumors, we show that instabilities in tumor oxygenation are a prevalent characteristic of three tumor lines and that previous characterization of tumor hypoxia as being primarily diffusion-limited does not accurately portray the tumor microenvironment. Then, using a one-dimensional theoretical model, we examine the effects of fluctuating hypoxia on metabolized tirapazamine concentration; we find that fluctuating oxygen reduces the concentration of metabolized tirapazamine at distances farther from the source, thereby limiting its ability to reach and kill the most hypoxic cells. Finally, we use a three-dimensional Green's function oxygen transport model to explore the effects of temporal fluctuations in hemoglobin saturation, blood flow, and overall perfusion on tumor tissue oxygenation. Results from the model predict that hemoglobin saturation has a dominant effect on tissue oxygenation. These studies collectively suggest that the pervasive temporal and spatial heterogeneity in tumor oxygenation are highly therapeutically relevant, and future clinical and preclinical studies are needed.

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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:

1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes

2. Establish the effects of Mb-containing polymersomes on tumor physiology

3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors

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.

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.

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).

Thus, the conclusions for each aim are as follows:

1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes

NIR imaging techniques were optimized for studying polymersomes, demonstrating long plasma circulation times and accumulation within tumors.

2. Establish the effects of Mb-containing polymersomes on tumor physiology

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.

3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors

Combination therapy of PEMs with RT results in enhanced tumor growth delay in aggressive 4T1 mammary carcinomas compared with RT or PEMs alone.

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.

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.

Alternative mRNA processing can result in the generation of multiple, qualitatively different RNA transcripts from the same gene and is a powerful engine of complexity in higher organisms. Recent deep sequencing studies have indicated that essentially all human genes containing more than a single exon generate multiple RNA transcripts. Functional roles of alternative processing have been established in virtually all areas of biological regulation, particularly in development and cancer. Changes in alternative mRNA processing can now be detected from over a billion dollars' worth of conventional gene expression microarray data archived over the past 20 years using a program we created called SplicerAV. Application of SplicerAV to publicly available microarray data has granted new insights into previously existing studies of oncogene over-expression and clinical cancer prognosis.

Adaptation of SplicerAV to the new Affymetrix Human Exon arrays has resulted in the creation of SplicerEX, the first program that can automatically categorize microarray detected changes in alternative processing into biologically pertinent categories. We use SplicerEX's automatic event categorization to identify changes in global mRNA processing during B cell transformation and show that the conventional U133 platform is able to detect 3' located changes in mRNA processing five times more frequently than the Human Exon array.

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumor. Relying on the EPR effect (Enhanced Permeability and Retention), a host of nanoparticles (from micelles and dendrimers to liposomes and lipidic nanoparticles) have been developed and tested for passive accumulation into tumor interstitium. Unfortunately, most nanoparticles achieve only suboptimal drug delivery to tumors, due to heterogeneity of tumor vessel permeability, limited nanoparticle penetration, and relatively slow drug release. However, recent developments in nanoparticle technology have occurred with the design and testing of a fast drug-releasing liposome triggered by local heat. This temperature-sensitive liposome formulation loaded with doxorubicin (Dox-TSL) has already shown substantial anti-tumor efficacy and is currently in clinical trials.

Previous pre-clinical work to understand the mechanism of efficacy has illustrated increases in overall drug concentration in the tumor, and an anti-vascular effect not observed with heat alone. These initial studies have also suggested that these liposomes may be the most efficacious when they are injected into a pre-heated tumor, with the hypothesis that in this treatment scheme the liposomes may be releasing inside the tumor vasculature. However, whether intravascular release is indeed occurring, and the subsequent implications this paradigm change in drug delivery could have are still unanswered questions.

The experiments presented herein aimed to investigate two effects: the existence and influence of intravascular drug release on drug delivery and distribution within the tumor, and the effect of drug delivery on subsequent anti-vascular effects. To investigate drug delivery, two mouse models were used. Dorsal window chambers implanted with FaDu human squamous carcinomas were used with real-time intravital confocal microscopy to evaluate time-resolved delivery of doxorubicin and liposome extravasation over the first 20 minutes of treatment. As a complimentary mouse model, flank FaDu tumors were also treated with Dox-TSL or treatment controls (doxorubicin with and without heat and Doxil with heat), and subsequently sectioned and histologicaly imaged to evaluate drug delivery and penetration depth, as well as impact on hypoxia and perfusion parameters. To investigate vascular effects, a GFP-eNos transgenic mouse model was used, also with window chamber confocal microscopy, to evaluate morphological changes occurring in the tumor vasculature following treatment.

The results presented herein demonstrate that contrary to the traditional liposome paradigm of extravasation and subsequent drug release, thermally sensitive liposomes release drug inside the tumor vasculature, and that the released free drug diffuses into the tumor interstitium. Real-time confocal imaging of doxorubicin delivery to murine tumor window chambers illustrates that intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by free drug diffusion. Histological analysis further confirms this finding, illustrating that drug delivered with Dox-TSL intravascular release can result in drug penetration levels up to 80 µm from vessels, in comparison with 40 µm achievable with free drug with heat. Further, Dox-TSL delivers drug to a higher percentage of a tumor's hypoxic area than possible with free drug with or without heat. Endothelial cells display marked morphological changes apparent immediately following treatment, with significant vascular destruction at 6 hours. However, heat had a similar influence on vascular morphology, underscoring the complexity of the anti-vascular effect, particularly in the more sensitive vasculature of a mouse model compared with reported human vascular heat tolerances. This work establishes intravascular release as a new paradigm in drug delivery to solid tumors, resulting in improved drug bioavailability, penetration depth, and enhanced delivery of drug to hypoxic regions of tumors.

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Background: PD-L1 (Programmed Death Ligand 1) is an immune checkpoint molecule that is commonly expressed on the surface of cancer cells. When it interacts with its receptor – the PD-1 molecule, which is commonly expressed on the surface of immune cells, such as T-cells, it will then deliver a negative signal which in turn inactivates the T-cell function, depresses the T-cell expansion, and dampens the overall tumor response. Anti-PD-L1 antibody blocks the direct interaction between the PD-L1 and PD-1 molecules, thus inhibits the PD-L1 signaling pathways, enabling the immune system, and hence the anti-tumor immunity, to eliminate the cancer cells. We refer to these mechanisms as the Immune Checkpoint Blockade (ICB).

Methods and Materials: In the current study, we report the effects of combination treatment of radiation and ICB on tumor metastasis. A single radiation dose of 10 Gy was used to irradiate the dorsal fat pad region of mice, into which the E0771-luc breast cancer cells were injected. For the ICB treatment, anti-PD-L1 antibody was used. We established a spontaneous metastatic model using E0771-luc breast cancer cells. As the tumor grew, primary tumor growth was monitored using calipers. We then examined the metastatic lung lesions by using In-Vivo Imaging System (IVIS), the signals from IVIS were then quantified in terms of total photon flux (photons/second). An India-Ink Assay was also employed to further verify the lung mass formations. In addition, variation of primary tumors’ hemoglobin saturation levels and total hemoglobin levels were recorded before and after treatments using zenascope, for the sake of assessing the vascular oxygenation and vascularity information, respectively.

Principal Findings: Data from calipers’ measurement showed that the RT-alone group did not show any effects on tumor growth. Tha anti-PD-L1-alone group showed a slightly delayed tumor growth. The combination treatment showed that 3 out of 5 mice showed tumor growth delay, while the tumor regrew after 20 days. Furthermore, survival curves indicated that there is no significant difference among all groups, indicating that radiation treatment or anti-PD-L1 antibody or the combination of both treatments did not affect the time at which the mice reach their endpoints (tumor volume ≥ 1500mm3). In addition, data from the IVIS indicated that the total photon flux emitted from primary tumors varied dramatically among experimental subjects within the same group. There were also extremely low or no luciferase signals from the lung. Statistically, two-way ANOVA for IVIS data showed that there are no significant differences between the RT-alone or anti-PD-L1-alone or RT + anti-PD-L1 group and the control group, for both primary tumors and lung tissue, suggesting that all kinds of treatments used in the current study neither helped eliminate the primary tumor cells nor reduced the burden of metastatic cancer cells in lungs, compared to the control group. Interestingly, results from the India-ink Assay showed that grossly visible lung nodules were not observed in all lungs of the mice, suggesting that the primary tumors in the dorsal fat pad region did not result in grossly visible lung metastases in any groups. Furthermore, analysis for the Zenascope data showed that there was a gradual increase in Hb-Sat(%) in mice for the control group, while a gradual decrease in Hb-Sat(%) for the anti-PD-L1-alone group. The RT-alone group did not show a clear response of change in Hb-Sat. For the combination treatment group, 3 out of 4 mice demonstrated a relatively flat response of change in Hb-Sat(%). Lastly, the total Hb levels in the control group, the anti-PD-L1-alone group and the combination treatment group remained relatively stable over the treatment time. For the RT-alone group, 3 out of 5 mice showed almost no changes, while the other two demonstrated a huge increase in total Hb levels on day 0, and day 2, respectively, but the levels went back to almost the pre-treatment values after day 3.

Conclusions: The combination treatment of 10 Gy of radiation and anti-PD-L1 antibodyimmunotherapy did not show significant effects on E0771 primary tumor growth when using an orthotopic tumor model. The time required for the tumor volume to exceed an endpoint of 1500mm3 was not significantly affected by all of the treatment methods used in the current study. The results from the IVIS and the India-ink Assay suggest that E0771 might not a good model for lung metastasis. However, the treatment response and the E0771 model were affected by a number of technical problems that render the evaluation inconclusive. Solutions to some of these technical problems have been provided, enabling future reseachers to replicate and improve on this study and futher determine the treatment response and the usefulness of the E0771 model. Regarding the zenascope measurement, the changes in Hb-Sat(%) may be correlated with the blood vessel growth within the primary tumors, while the changes in total Hb were almost negligible. Nevertheless, several limitions when performing the zenascope measurement have been listed, including the pigmentation and fur of the skin of mice, the motion of the mice and/or the operator’s hands, as well as the uncertainties in placing the optical probe onto the tumor. Further research is needed to uncover the promise of this combined therapy and to further verify the correlation among the changes in Hb-Sat(%), the changes in total Hb levels, and the tumor physiological characteristics.

Mild hyperthermia (39°C-43°C) has numerous therapeutic benefits as an adjuvant therapy in the treatment of a variety of tumor types. Hyperthermia increases tumor blood flow and vascular permeability, promoting drug delivery and tumor oxygenation. Hyperthermia enhances the uptake and efficacy of numerous chemotherapeutic agents, including cisplatin, resulting in increased cytotoxicity. In addition to these biological responses, hyperthermia can be used as a drug-release trigger for temperature-sensitive nanoparticles, resulting in an improved and more targeted drug delivery system. Cisplatin was chosen because 1) it shows broad spectrum activity against a wide range of heatable cancers (i.e., those in sites such as the pancreas, colon and rectum, cervix and bladder, and 2) the same hyperthermic temperatures that enable temperature-sensitive lipsome-drug release also enhance cisplatin-induced cytotoxicity.

The role of hyperthermia in enhancing cisplatin delivery and cytotoxicity was investigated at both the cellular and tissue levels. While hyperthermia treatment is applicable to a variety of tumor types, the focus of this work was on bladder cancer. The synergistic effects of hyperthermia and cisplatin were investigated, along with the role of copper transport protein 1 (Ctr1) in this process. In addition, cisplatin was encapsulated within temperature-sensitive liposomes, which were used in combination with hyperthermia for targeted drug delivery. These studies demonstrated that the combination of cisplatin and hyperthermia improved drug delivery, and potentially anti-tumor efficacy, and that targeted delivery was enhanced through incorporation of temperature-sensitive liposomes. As many current methods for administering bladder hyperthermia have drawbacks, such as invasiveness and regional heating, the final aim of this study was to develop and test a less-invasive and more focused preclinical bladder heating device in a rat model.

Hyperthermia sensitizes cells to the cytotoxic effects of the commonly used chemotherapeutic agent cisplatin by increasing drug accumulation and subsequent platinum-DNA adduct formation. However, the molecular mechanisms underlying this enhancement remain unclear. Understanding the fundamental mechanisms involved in the synergistic interaction is necessary to increase the therapeutic benefits of this combination in the clinic. The synergism between the anti-cancer benefits of cisplatin and the drug delivery benefits of hyperthermia may offer a novel and more effective treatment for many cancer patients. We hypothesized that hyperthermia increases cisplatin accumulation and efficacy in part by modulating the function of Ctr1, a major regulator of cellular cisplatin uptake. To test this hypothesis, we examined the significance of Ctr1 during combined hyperthermia and cisplatin therapies and assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerization in enhancing cisplatin cytotoxicity. We observed increased Ctr1 multimerization following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C), suggesting that this may be a mechanism for increased cisplatin uptake in heat-treated cells. The impact of increased Ctr1 multimerization was evaluated by measuring platinum accumulation in wild-type (WT) and Ctr1-/- cells. WT cells contained greater levels of platinum compared to Ctr1-/- cells. A further increase in platinum was observed following hyperthermia treatment, but only in the WT cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT cells with a dose-modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells. Our data suggest that heat increases Ctr1 activity by increasing multimerization, resulting in enhanced drug accumulation. Although we recognize that the effect of heat on cells is multi-factorial, our results support the hypothesis that Ctr1 is, in part, involved in the synergistic interaction observed with cisplatin and hyperthermia treatment.

In addition to assessing cisplatin delivery at the cellular level, we evaluated cisplatin delivery at the tissue level, using novel cisplatin-loaded temperature-sensitive liposomes. We hypothesized that delivering cisplatin encapsulated in liposomes under hyperthermic conditions would improve the pharmacokinetic profiles of cisplatin, increase drug delivery to the tumor, decrease normal tissue toxicity, and enhance the anti-tumor activity of cisplatin. We successfully prepared temperature-sensitive liposomes loaded with cisplatin and demonstrated that heat (42°C) sensitizes cisplatin-resistant cells to the cytotoxic effects of cisplatin in vitro.

Decreased toxicity was observed in animals treated with the cisplatin liposome (± heat) compared to the free drug treatments. A pharmacokinetic study of cisplatin-loaded temperature-sensitive liposomes and free drug was performed in tumor-bearing mice under normothermic and hyperthermic conditions. Cisplatin half-life in plasma was increased following liposome treatment compared to free cisplatin, and cisplatin delivery to the tumors was greatest in mice that received liposomal cisplatin under hyperthermia. These initial in vivo data demonstrate the potential effectiveness of this cisplatin-loaded liposome formulation in the treatment of certain types of cancer. To assess the anti-cancer efficacy of the liposome treatment, a tumor growth delay study was conducted and demonstrated equivalent efficacy for the cisplatin-loaded temperature-sensitive liposome compared to free drug.

In addition to the liposome work, we developed and evaluated a novel heating device for the bladder. Despite the evidence that hyperthermia is an effective adjuvant treatment strategy, current clinical heating devices are inadequate, warranting the development of a new and improved system. We induced hyperthermia using ferromagnetic nanoparticles and an alternating magnetic field device developed by Actium Biosystems. Initial preclinical studies in a rat model demonstrated preferential bladder heating. However, our preliminary studies show severe toxicity with the direct instillation of the nanoparticles in the bladder, and further studies are needed to potentially modify the nanoparticle coating, the catheterization procedure, as well as to develop a different animal model.

Pathophysiologic lactate accumulation is characteristic of solid tumors and has been associated with metastases and poor overall survival in cancer patients. In recent years, there has been a resurgence of interest in tumor lactate metabolism. In the past, our group has shown that lactate can be used as a fuel in some cancer cell lines; however, survival responses to exogenous lactate alone are not well-described. We hypothesized that lactate utilization and cellular responses to exogenous lactate were varied and dynamic, dependent upon factors such as lactate concentration, duration of lactate exposure, and of expression of the lactate transporter, monocarboxylate transporter 1 (MCT1). We hypothesized that pharmacological inhibition of MCT1 with a small molecule, competitive MCT1 inhibitor, α-cyano-4-hydroxycinnamic acid (CHC), could elicit cancer cell death in high lactate conditions typical of that seen in breast cancer.

My work focused on defining: 1. Lactate levels in locally advanced breast cancer (LABC); 2. Lactate uptake and catabolism in a variety of cancer cell lines; 3. The effect of exogenous lactate on cancer cell survival; 4. Whether the lactate-transporters, MCT1 and MCT4 can be used as markers of cycling hypoxia.

Lactate levels in LABC biopsies were assessed ex vivo by bioluminescence. NMR techniques were employed extensively to determine metabolites generated from 13C-labeled lactate. Cell viability in response to extracellular lactate ( ± glucose and ± CHC) was measured with Annexin V / 7-AAD staining to assess acute survival responses and clonogenic assays to evaluate long-term colony forming ability after lactate treatment. MCT1 and MCT4 protein expression was evaluated in cancer cell lines with Western blots after exposure to chronic or cycling hypoxia. Immunofluorescence was employed to assess MCT1 and MCT4 expression in head and neck cancer biopsies, and the expression patterns of the transporters were correlated to areas of hypoxia, as indicated by hypoxia marker EF5.

Lactate concentrations in LABC biopsied ranged from 0 - 12.3 µmol/g of tissue. The LABC dataset was too small to derive statistical power to test if lactate accumulation in LABC biopsies was associated with poor patient outcome or other clinical parameters of known prognostic significance. All cell lines tested (normal and cancer) showed uptake and metabolism of labeled lactate, with dominant generation of alanine and glutamate; however, relative rates and the diversity of metabolites generated was different among cell lines. MCF7 cells showed greater overall lactate uptake (mean = 18mM) over five days than MDA-MB-231 cells (mean = 5.5mM). CHC treatment effectively prevented lactate uptake in cancer cells when lactate concentrations were ≤20mM.

Cell survival was dependent upon lactate concentration and glucose availability. Acute responses to exogenous lactate did not reflect the long-term consequences of lactate exposure. Acutely, HMEC and R3230Ac cells were tolerant of all lactate concentrations tested (0-40mM) regardless of presence or absence of glucose. MCF7 and MDA-MB-231 cells were tolerant of lactate within the concentration ranges seen in biopsies. Cytotoxicity was seen after 24 hr incubation with 40mM lactate (-glucose), but this concentration is three times higher than any measurement made in human biopsies of LABC. Similarly, HMEC and MCF7 cells showed significantly decreased colony formation in response to 40mM exogenous lactate (+ glucose) while R3230Ac and MDA-MB-231 cells showed no impairment in colony-forming abilities with any lactate concentration (+ glucose). 5mM CHC significantly increased cell death responses independent of lactate treatment, indicating off-target effects at high concentrations.

MCT1 was found to be expressed in a majority of the cell lines tested, except for MDA-MB-231 cells. Cancer cells exposed to exogenous lactate showed upregulation of MCT1 but not MCT4. Chronic hypoxia resulted in an increase in protein expression of MCT4 but a decrease in MCT1 expression in cancer cell lines. The time course of regulation of protein levels of each transporter suggested the possibility of expression of both transporters during cycling hypoxia. When cancer cells were exposed to cycling hypoxia, both transporters showed upregulation. In head and neck tumor biopsies, MCT1 expression was significantly positively correlated to aerobic tumor regions and inversely correlated to hypoxic tumor regions.

Cancer cell responses to exogenous lactate were not uniform. Some cell lines demonstrated a lactate-tolerant and/or a lactate-consuming phenotype while other cell lines demonstrated lactate-intolerant and/or non-lactate-consuming phenotype. My work indicates that exogenous lactate was well-tolerated at clinically relevant concentrations , especially in the presence of glucose. Evidence of glutamate metabolism from lactate indicated that exogenous lactate partially progresses through the TCA cycle, suggesting that lactate may be utilized for fuel. The cell death elicited from 5mM CHC treatment was not dependent upon presence of lactate, indicating that manipulation of lactate metabolism may not be the best option for targeting cancer metabolism. When attempting to manipulate lactate metabolism in tumors, microenvironmental factors, such as hypoxia and glucose, must be taken into account in order to ensure a predictable and favorable outcome. Together, these results illustrate the importance of characterizing tumor metabolism before therapeutic intervention.

Use of chemotherapeutics in treatment of solid tumors suffers from insufficient and heterogeneous drug delivery, systemic toxicity and lack of knowledge of delivered drug concentration. The overall objectives of this work were: 1) to address these shortcomings through development and characterization of a treatment system capable of real-time spatiotemporal control of drug distribution and 2) to investigate the role of MR-image-able tissue transport parameters in predicting drug distribution following hyperthermia-triggered drug release from nanoparticles. Towards these objectives, a combination of potentially synergetic technologies was used: 1) image-able low temperature-sensitive liposomes (iLTSLs) for drug delivery, 2) quantitative drug delivery and transport parameter imaging with magnetic resonance imaging (MRI), and 3) control over drug release with magnetic resonance-guided high intensity focused ultrasound (MR-HIFU). The overall hypothesis of this work is that the drug distribution in the targeted zone spatially correlates with the image-able transport-related parameters as well as contrast enhancement due to release of contrast agent during treatment.

We began by developing and characterizing iLTSLs, which were designed using a lipid formulation similar to one that is in clinical trials in the US (ThermoDox®) and a gadolinium-based MR contrast agent that is in widespread clinical use (Prohance®) and least likelihood of toxicity due to nephrogenic systemic fibrosis (NSF). The resulting liposome was found to stably encapsulate both an anthracycline chemotherapeutic, doxorubicin, and the MR contrast agent. Release rates were similar for these two species in physiologic buffer as well as in human plasma. The next step towards control and imaging of release with this drug delivery system (DDS) was development of algorithms that allowed for large-volume mild hyperthermia with MR-HIFU that would be required to move this combination of technologies into the clinic.

Optimal drug delivery with iLTSL requires a sustained period of heating of the entire target to the range of temperatures that are optimal for liposomal release and maintenance of perfusion (40 - 45 C). The MR-HIFU technology was developed and used mainly for rapid thermal ablation or mechanical disruption of tissue in small ellipsoid volumes. Variability and size of common clinical lesions called for modifications that would enable stable conformal heating of large tumor volumes to the sub-ablative temperature range of mild hyperthermia (40 - 45 C). Therefore, we set out to develop an algorithm that would allow rapid attainment and maintenance of mild hyperthermia in larger volumes of variable shape that were typically encountered in the clinic. We approached this goal through a series of successive steps that addressed different aspects of mild hyperthermia treatment: 1) controlled heating to mild hyperthermia, 2) conformity of heating and 3) ability to heat large volumes.

To achieve controlled heating to mild hyperthermia we implemented a simple binary mild hyperthermia feedback mechanism that adequately maintained mild hyperthermia for extended periods of time in small ellipsoidal volumes. We then developed a conformal small-volume mild hyperthermia algorithm that could provide spatial control over heating in an environment with spatially heterogeneous perfusion. This algorithm used electronic steering of the HIFU focus to heat each MR image voxel with different power, depending on temperature measured within that voxel. Finally, to heat large volumes conformally, we developed an algorithm that combined mechanical displacement of the MR-HIFU transducer (to cover large areas) with electronic deflection of the HIFU beam (to heat sub-volumes conformally). This advancement allowed us to quickly attain mild hyperthermia (<8.1 min to steady state) in larger volumes (cross-sectional area = 8.4 cm, ~12 times larger than previous methods).

Following their characterization, we examined iLTSL pharmacokinetics and combined MR-HIFU large volume mild hyperthermia with iLTSL to deliver doxorubicin to large Vx2 carcinomas in the hindlimb muscle of rabbits. To determine MR image-able correlates to the intratumoral drug distribution, we assessed the spatial pattern of drug distribution with fluorescence microscopy and examined spatial correlations of this pattern to several parameters measured with MRI, including the spatial distributions of temperature, contrast enhancement following injection of iLTSL, dynamic contrast-enhanced MRI (DCE-MRI) parameters, and maps of apparent diffusion coefficient (ADC). Dynamic contrast-enhanced MRI parameters have been used extensively in literature to approximate a mixture of parameters critical to drug delivery, such as perfusion (F), permeability-vascular surface area product (PS) and vascular volume and ADC has been previously correlated with cellular density in tumors. Possible utility of such spatial correlations was examined for future use in treatment planning, intraprocedural feedback control and post-treatment evaluation.

Highly perfused peripheral regions of Vx2 tumors in rabbit hindlimb displayed high Ktrans and ve, indicative of high perfusion. Maps of ADC obtained with low b-values also showed high ADC in the periphery of these tumors, indicating high perfusion there. ADC maps that were weighted more towards diffusion (using higher b-values) showed that diffusion was largest in the tumor core, indicating destruction of the cellular membranes and greater mobility of water. Microscopic examination of excised tumors was spatially registered to the MRI datasets and showed that most of the tumor core is necrotic, though some highly vascularized and viable tissue was present in strands or segments that traversed the necrotic regions. Those segments also showed bright doxorubicin fluorescence following treatment with MR-HIFU and iLTSL. The two control groups - free drug and iLTSL without mild hyperthermia - showed minimal to no doxorubicin fluorescence in the tumor.

Susceptibility effects due to use of contrast agent caused large errors (up to 15 °C) in MR thermometry measurements. To address this phenomenon, experiments were designed to arrive at steady state heating (target temperature = 41 °C), and employ an algorithm to learn the spatiotemporal distribution of power that was needed to maintain steady state heating. This heating pattern was then played back several times to verify maintenance of steady state, and if satisfactory, image-able liposomes were injected. Since temperature feedback was replaced by the learned steady-state heating, injection of image-able liposomes likely did not alter the heating performance. Following injection, changes in T1 and magnetic susceptibility were most pronounced in regions that previously showed greatest enhancement during DCE-MRI and displayed larger values of ADC with perfusion-weighted, low b-value scans. Maps of T1 were obtained in real time using a variable flip angle sequence during heating, and were corrected for inhomogeneity of the B1 field and calibrated against a more accurate, T1 mapping technique.

After treatment with MR-HIFU and iLTSL, the drug was preferentially distributed in the viable tissue, in and around the tumor. Doxorubicin fluorescence was greatest in zones that were heated, though the drug distribution did not display a clear boundary between heated and unheated tissue. While iLTSL provided intraprocedural feedback via enhancement of T1-weighted image intensity, susceptibility-related effects of iLTSL on MR thermometry complicate their prospects of clinical use, where precise temperature feedback is required for control of therapy and MR thermometry techniques that are in widespread use would be affected. Spatial correlations between drug delivery with iLTSL and MR-imageable parameters may serve as a predictive tool to identify areas that will not receive adequate drug. Such a-priori knowledge of correlates to the approximate tumor drug distribution has the potential to inform treatment planning by revealing the extent to which drug dose could be painted with a combination of LTSL and MR-HIFU. These studies point to an adjustment of course in further development of drug dose painting this combination of technologies, towards informing treatment planning, and not only painting the dose, but predicting it. These results also point to the need to develop rational combinations other treatments, such as ablation and radiation, to treat regions that will not receive sufficient drug.

Selective therapeutic targeting of tumors requires identification of differences between the homeostatic requirements of cancer and host cells. One such difference is the manner in which cancer cells acquire energy. Cancer cells often grow in an environment of local hypoxia; under these conditions tumor cells depend on glycolysis for energy, but are unable to perform oxidative phosphorylation. Many tumor cells, despite normoxic conditions, continue to perform glycolysis without oxidative phosphorylation. The net result of glycolysis without oxidative phosphorylation is twofold: the need to consume a greater amount of glucose than a non-cancerous host cell, and the burden of increased intracellular lactic acid. The proteins responsible for the transport of lactic acid in and out of cells are known as the monocarboxylate transporters (MCTs). Monocarboxylate Transporter 1 (MCT1) and Monocarboxylate Transporter 4 (MCT4) are the MCTs that play a major role in the transport of lactic acid. Tumor cells depend on MCT1 and MCT4 activity to excrete excess intracellular lactic acid to maintain neutral intracellular pH and homeostasis. Using human neuroblastoma and prostate cancer cell lines this work demonstrates that tumor cells can be selectively targeted tumor under conditions of hypoxia or acidosis in vitro with the drug lonidamine, with a small molecule inhibitor selective for MCT1, or with RNA interference of MCT1. Inhibition of MCT1 activity in neuroblastoma cells under acidic extracellular conditions results in intracellular acidification and cell death. MCT1 mRNA is expressed in human neuroblastoma and positively correlated with clinical risk profile. Inhibition of MCT1 activity in hypoxic prostate cancer cells results in a reduction of lactate excretion, decreased intracellular pH, inhibition of ATP production, and subsequent cell death. MCT1 expression in sections of human prostate tumors has been demonstrated to validate MCT1 as a target in prostate cancer.

Through the Pasteur and Warburg effects, tumors have an increased demand for glucose. Some cancers store glycogen, but the reasons for this are largely unknown. It is hypothesized that tumor glycogen is used to promote tumor survival during transient hypoxia or low glucose, and that the mechanisms by which glycogen is stored is a potential therapeutic target in cancer. Tumors from human cell lines (WiDr, PC3, FaDu) have been grown in nude mice, sectioned and stained to measure glycogen storage. Using consecutive frozen sections, levels of hypoxia, glucose, lactate, ATP, and CD31, an endothelial cell marker, have been determined. These sections have been employed to elucidate the "architecture" of tumor metabolism in terms of vessel distance. Additionally, PAS-stained EF5 labeled human tumor samples were used to obtain calibrated hypoxia measurements to correlate with PAS. These studies demonstrate a correlation between hypoxia and the formation of glycogen deposits in human tumors and nude mouse xenografts. In cell culture, formation of glycogen deposits after exposure to hypoxia has been demonstrated, in addition to expression of glycogen synthase in human cancer cell lines.

The development of novel selective cancer chemotherapeutics will require the identification of differences between cancerous cells and normal host cells to exploit as targets. Several differences in metabolism, including the need to excrete excess lactic acid and store glycogen under hypoxic conditions, are such targets. Novel therapeutics exploiting these targets should be effective against cancer cells and minimally toxic to host cells.

The outcomes for both (i) radiation therapy and (ii) preclinical small animal radio- biology studies are dependent on the delivery of a known quantity of radiation to a specific and intentional location. Adverse effects can result from these procedures if the dose to the target is too high or low, and can also result from an incorrect spatial distribution in which nearby normal healthy tissue can be undesirably damaged by poor radiation delivery techniques. Thus, in mice and humans alike, the spatial dose distributions from radiation sources should be well characterized in terms of the absolute dose quantity, and with pin-point accuracy. When dealing with the steep spatial dose gradients consequential to either (i) high dose rate (HDR) brachytherapy or (ii) within the small organs and tissue inhomogeneities of mice, obtaining accurate and highly precise dose results can be very challenging, considering commercially available radiation detection tools, such as ion chambers, are often too large for in-vivo use.

In this dissertation two tools are developed and applied for both clinical and preclinical radiation measurement. The first tool is a novel radiation detector for acquiring physical measurements, fabricated from an inorganic nano-crystalline scintillator that has been fixed on an optical fiber terminus. This dosimeter allows for the measurement of point doses to sub-millimeter resolution, and has the ability to be placed in-vivo in humans and small animals. Real-time data is displayed to the user to provide instant quality assurance and dose-rate information. The second tool utilizes an open source Monte Carlo particle transport code, and was applied for small animal dosimetry studies to calculate organ doses and recommend new techniques of dose prescription in mice, as well as to characterize dose to the murine bone marrow compartment with micron-scale resolution.

Hardware design changes were implemented to reduce the overall fiber diameter to <0.9 mm for the nano-crystalline scintillator based fiber optic detector (NanoFOD) system. Lower limits of device sensitivity were found to be approximately 0.05 cGy/s. Herein, this detector was demonstrated to perform quality assurance of clinical 192Ir HDR brachytherapy procedures, providing comparable dose measurements as thermo-luminescent dosimeters and accuracy within 20% of the treatment planning software (TPS) for 27 treatments conducted, with an inter-quartile range ratio to the TPS dose value of (1.02-0.94=0.08). After removing contaminant signals (Cerenkov and diode background), calibration of the detector enabled accurate dose measurements for vaginal applicator brachytherapy procedures. For 192Ir use, energy response changed by a factor of 2.25 over the SDD values of 3 to 9 cm; however a cap made of 0.2 mm thickness silver reduced energy dependence to a factor of 1.25 over the same SDD range, but had the consequence of reducing overall sensitivity by 33%.

For preclinical measurements, dose accuracy of the NanoFOD was within 1.3% of MOSFET measured dose values in a cylindrical mouse phantom at 225 kV for x-ray irradiation at angles of 0, 90, 180, and 270˝. The NanoFOD exhibited small changes in angular sensitivity, with a coefficient of variation (COV) of 3.6% at 120 kV and 1% at 225 kV. When the NanoFOD was placed alongside a MOSFET in the liver of a sacrificed mouse and treatment was delivered at 225 kV with 0.3 mm Cu filter, the dose difference was only 1.09% with use of the 4x4 cm collimator, and -0.03% with no collimation. Additionally, the NanoFOD utilized a scintillator of 11 µm thickness to measure small x-ray fields for microbeam radiation therapy (MRT) applications, and achieved 2.7% dose accuracy of the microbeam peak in comparison to radiochromic film. Modest differences between the full-width at half maximum measured lateral dimension of the MRT system were observed between the NanoFOD (420 µm) and radiochromic film (320 µm), but these differences have been explained mostly as an artifact due to the geometry used and volumetric effects in the scintillator material. Characterization of the energy dependence for the yttrium-oxide based scintillator material was performed in the range of 40-320 kV (2 mm Al filtration), and the maximum device sensitivity was achieved at 100 kV. Tissue maximum ratio data measurements were carried out on a small animal x-ray irradiator system at 320 kV and demonstrated an average difference of 0.9% as compared to a MOSFET dosimeter in the range of 2.5 to 33 cm depth in tissue equivalent plastic blocks. Irradiation of the NanoFOD fiber and scintillator material on a 137Cs gamma irradiator to 1600 Gy did not produce any measurable change in light output, suggesting that the NanoFOD system may be re-used without the need for replacement or recalibration over its lifetime.

For small animal irradiator systems, researchers can deliver a given dose to a target organ by controlling exposure time. Currently, researchers calculate this exposure time by dividing the total dose that they wish to deliver by a single provided dose rate value. This method is independent of the target organ. Studies conducted here used Monte Carlo particle transport codes to justify a new method of dose prescription in mice, that considers organ specific doses. Monte Carlo simulations were performed in the Geant4 Application for Tomographic Emission (GATE) toolkit using a MOBY mouse whole-body phantom. The non-homogeneous phantom was comprised of 256x256x800 voxels of size 0.145x0.145x0.145 mm3. Differences of up to 20-30% in dose to soft-tissue target organs was demonstrated, and methods for alleviating these errors were suggested during whole body radiation of mice by utilizing organ specific and x-ray tube filter specific dose rates for all irradiations.

Monte Carlo analysis was used on 1 µm resolution CT images of a mouse femur and a mouse vertebra to calculate the dose gradients within the bone marrow (BM) compartment of mice based on different radiation beam qualities relevant to x-ray and isotope type irradiators. Results and findings indicated that soft x-ray beams (160 kV at 0.62 mm Cu HVL and 320 kV at 1 mm Cu HVL) lead to substantially higher dose to BM within close proximity to mineral bone (within about 60 µm) as compared to hard x-ray beams (320 kV at 4 mm Cu HVL) and isotope based gamma irradiators (137Cs). The average dose increases to the BM in the vertebra for these four aforementioned radiation beam qualities were found to be 31%, 17%, 8%, and 1%, respectively. Both in-vitro and in-vivo experimental studies confirmed these simulation results, demonstrating that the 320 kV, 1 mm Cu HVL beam caused statistically significant increased killing to the BM cells at 6 Gy dose levels in comparison to both the 320 kV, 4 mm Cu HVL and the 662 keV, 137Cs beams.

Purpose

Methods and materials

Results

Conclusions

The vascular contribution to tumor radiation response is controversial, but may have profound clinical implications. This is especially true of a new class of radiation therapies which employ spatial fractionation techniques--high radiation doses delivered in a spatially modulated pattern across the tumor. Window chamber tumor models may prove useful in investigating vascular parameters due to their facilitation of non-invasive, serial measurements of living tumors. However, presently there do not exist automated and accurate algorithms capable of quantitatively analyzing window chamber data.

Here we attempt to address these two problems through (1) the generation of novel optical data processing techniques for the quantification of vascular structural and functional parameters, and (2) the application of these methods to the study of vascular radiation effects in window chamber models.

Results presented here demonstrate the versatility and functionality of the data processing methods that we have developed. In the first part of Aim 1, we have developed a vessel segmentation algorithm specifically designed for processing tumor vessels, which present a challenge to existing algorithms due to their highly branching, tortuous structure. This provides us with useful information on vascular structural parameters. In the second part of Aim 1, we demonstrate a complementary vascular functional analysis algorithm, which generates quantitative maps of speed and direction. We prove the versatility of this method by applying it to a number of different studies, including hemodynamic analysis in the dorsal window chamber, the pulmonary window, and after neural electro-stimulation. Both the structural and functional techniques are shown capable of generating accurate and unbiased vascular structural and functional information. Furthermore, that automated nature of these algorithms allow for the rapid and efficient processing of large data sets. These techniques are validated against existing techniques.

The application of these methods to the study of vascular radiation effects produced invaluable quantitative data which suggest startling tumor adaptations to radiation injury. Window chamber grown tumors were treated with either widefield, microbeam, or mock irradiation. After microbeam treatment, we observed a profound angiogenic effect within the radiation field, and no signs of vascular disruption. Upregulation of HIF-1, primarily in the tumor rim, suggested that this response may have been due to bystander mechanisms initiated by oxidative stress. This HIF-1 response may have also initiated an epithelial-mesenchymal transition in the cells of the tumor rim, as post-treatment observation revealed evidence of tumor cell mobilization and migration away from the primary tumor to form secondary satellite clusters. These data indicate the possibility of significant detrimental effects after microbeam treatment facilitated through a HIF-1 response.