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Cancer continues to be a leading cause of death worldwide despite the increasing research advances into novel treatments. Thermal ablation of tumors is a relatively established method for the destruction of many tumor types, despite inherent shortcomings including incomplete tumor treatment and non-specific treatment. Novel therapies are currently studied including nanoparticle-based therapies to overcome these limitations. One field of research is focused on utilizing non-lethal hyperthermia to enhance carried chemotherapeutic drugs. Additionally a novel field of non-thermal ablations termed Irreversible Electroporation has recently been developed to treat tumors by irreversibly destroying cell membrane function through short electrical pulses. The goal of the present study is to research two novel potential treatments for cancer that do not require thermal destruction of tissue. Firstly, we developed and tested novel ways to load the antineoplastic agent Cisplatin into SWNHs to test the ability to thermally enhance carried drugs with non-lethal, mild hyperthermia. We attached the imaging agent Quantum Dots (QDs) to the particles to understand how hyperthermia affects cellular uptake, minimizing thermal enhancement. Results of this study highlight the need for better biomimetic in vitro models of the tumors to study how hyperthermia affects tissue level transport of nanoparticles. In the second aim we utilized a perfusable 3D collagen in vitro model of the tumor microenvironment, previously developed by our group to study tumor angiogenesis, to study nanoparticle transport. We demonstrated the ability of this model to study key mass transport obstacles nanoparticles face in the tumor including extravasation from a leaky, pro-angiogenic vasculature, diffusion in the extracellular matrix, and cellular uptake in a 3D environment. This model was then utilized in the third aim to study how mild hyperthermia affects transport of SWNHs. Results from this aim were valuable in showing the utility of the 3D in vitro model to controllably test the effects of external stimuli on transport of particles and shows how mild hyperthermia can selectively allow increased permeability of SWNHs in the tumor, increasing selectivity of nanoparticle transport to the targeted tissue. Lastly, we tested the non-thermal ablation, high-frequency irreversible electroporation (H-FIRE) in a 3D tumor platform and in an in vivo swine model to better understand the ability of H-FIRE to produce repeatable destruction of hepatocellular carcinoma, a disease state growing in incidence rate. We then used H-FIRE in an outpatient treatment for infiltrative skin tumors in equines, showcasing the ability to deliver high voltage, short duration pulses in a clinical setting without muscle contractions. Ultimately, the results of this study the engineering studies that must occur to optimize novel treatments utilizing non-lethal hyperthermia, or non-thermal death mechanism to treat cancer. The studies show the usefulness of more complex 3D in vitro models of tumors for early development of novel therapies and the utility of in vivo models to validate studies. / Ph. D. / Cancer continues to be a leading cause of death worldwide despite the increasing research advances into novel targeted treatments. Physical tumor destruction remains a viable therapeutic modality despite new molecular and biological targets. Specifically, thermal ablation of tumors is a relatively established therapy of numerous tumor types, despite inherent shortcomings which include incomplete tumor and margin treatment, contraindications from sensitive nearby structures, and the non-specific nature of thermal energy. Within the field of thermal cancer therapies, there is a new focus on utilizing nanoparticles to deliver non-lethal hyperthermia that enhance carried chemotherapeutic drugs. Additionally, a novel field of non-thermal ablations termed Irreversible Electroporation has recently been developed to treat tumors by irreversibly destroying cell membrane function through externally applied short electrical pulses. The goal of the present study is to research two novel potential treatments for cancer that destroy tumors with energy which do not require complete thermal destruction. We first developed and tested novel methods to load the antineoplastic agent Cisplatin into carbon nanoparticles (SWNHs) to thermally enhance loaded drugs with non-lethal, mild hyperthermia. We attached the imaging agent Quantum Dots (QDs) to the carbon particles in order to understand how hyperthermia affects cellular uptake, minimizing thermal enhancement. Results of this study highlight the need for better biomimetic in vitro models of the tumors to study how hyperthermia affects tissue level transport of nanoparticles. In the second aim we utilized a perfusable 3D collagen in vitro model of the tumor microenvironment, previously developed by our group to study tumor angiogenesis, to study nanoparticle transport. We demonstrated the ability of this model to study key mass transport obstacles nanoparticles face in the tumor including extravasation from a leaky, pro-angiogenic tumor vasculature, diffusion in the dense extracellular matrix, and cellular uptake in a 3D environment. This model was then utilized in the third aim to study how mild hyperthermia affects transport of SWNHs. Results from this aim were valuable in showing the utility of the 3D in vitro model to controllably test the effects of external stimuli on transport of particles and demonstrates the mechanism for how mild hyperthermia can selectively allow increased permeability of SWNHs in the tumor microenvironment by increasing selectivity of nanoparticle transport to the targeted tissue. Ultimately this work highlights the need for early nanoparticle optimization in more biomimetic testing platforms in order to better develop based on the physiological boundaries to therapy delivery. Lastly, we tested the non-thermal ablation, high-frequency irreversible electroporation (H-FIRE) in a 3D tumor platform and in an in vivo swine model to better understand the ability of H-FIRE to produce repeatable destruction of hepatocellular carcinoma, a disease state growing in incidence rate. We then used H-FIRE in an outpatient treatment for infiltrative skin tumors in equines, showcasing the ability to deliver high voltage, short duration pulses in a clinical setting without muscle contractions which could potentially expand the application of non-thermal ablation into indications currently limited by the difficult anesthetic protocol. Ultimately, the results of this study demonstrate the tie between engineering solutions to model the tumor microenvironment with the basic science of therapy discovery and optimization that must occur to optimize novel treatments utilizing non-lethal hyperthermia, or non-thermal death mechanism to treat cancer. The studies show the usefulness of more complex 3D in vitro models of tumors for early development of novel therapies and the utility of in vivo models to validate studies.

Making up just over 3% of all new cancer cases in the United States, pancreatic cancer is not inherently a common malignant disease. Yet, it continuously is shown to be one of the most lethal and common causes of cancer death. Early detection is critical among all cancer types. However, oncologists and researchers have struggled to find effective strategies or tests to detect cancer of the pancreas early on in development. Thus, the cancer is often found late stage and requires significant chemotherapy intervention. These multi-drug treatment cocktails have shown benefit, but only in added months and not years to a patient’s life. Significant adverse effects often limit the full effective doses of treatment. In order to limit these adverse effects, as well as increase the effectiveness of treatment, we have designed, optimized, and tested unique drug carriers known as polymersomes. Using characteristics of the localized environment surrounding pancreatic tumors and the cells found therein, we created targeted therapies that are responsive and relatively selective toward cancerous cells. Herein, are found two distinct polymersomes. The first, is a low oxygen reactive drug carrier with an additional small peptide molecule that is able to penetrate dense tumor tissue and has shown decreased tumor growth of as much as 260% as compared to control samples in an animal model of pancreatic cancer. The chemical make-up of this polymersome allows for extended circulation time and a high accumulation at the tumor site. A second design, uses an intracellular enzyme to destabilize the polymersomes’ structure, which in turn, releases a selected chemotherapy drug near its intended site of action. This strategy, has shown a 10 fold increase in potency of the chemotherapy drug, as compared to when the drug is given alone and showed decreased toxicity to non-cancerous cells. It is certain that thoughtful drug delivery strategies and not just drug molecule design will be instrumental in the paradigm shift of pancreatic cancer from likely death to survival. / NIH grant 1 R01GM 114080; Grand Challenge Initiative; Office of the Dean, College of Health Professions

As immunotherapy grows in popularity as a cancer treatment option, we need to further understand how immune cells interact with the tumor microenvironment and influence tumor progression. The goal of this thesis was to characterize the different immune, cellular, and structural components within the breast tumor tissues of two orthotopic (MCaP0008 and M3C) and one spontaneous (MMTV-PyVT) murine models of immunogenic breast cancer. Identification of the tumor components in question, including CD3+ lymphocytes, CD11b+ myeloid cells, CD31+ endothelial cells, αSMA+ cancer associated fibroblasts, Ki67+ cells, cleaved caspase-3+ cells, collagen-1, and hyaluronan, were done by immunohistochemistry (IHC)-immunofluorescence (IF) staining of frozen tumor tissues with appropriate antibodies and imaging with multispectral confocal microscopy. Quantification and further data analysis were performed using a custom MATLAB program designed by Dr. Mei Rosa Ng. Gaining understanding of these stromal compositions will allow for better utilization of these breast cancer mouse models in future experiments. / 2019-10-31

Doctor of Philosophy / Over 1.9 million new cases of cancer will pop up just this year alone. The prevalence of cancer, however, has not been met with the same magnitude of effective treatments, resulting in over 600,000 deaths in the United States. Before current treatments can be improved and new treatments can be developed, it is critical that we increase our understanding of what drives cancer to be so aggressive and maintain a fighting chance within the body despite our complex immune systems. The severity of cancer is not just a product of the cancer cell itself, but rather the components that make up the cell that define and drive metastatic behaviors and drug resistance. In order to improve diagnoses, prognoses, and treatment planning, the intracellular drivers of the disease must be better understood. Cells, electrical circuits in nature, reflect unique electrical properties dictated by their biophysical composition. These electrical properties can be revealed and exploited to characterize and treat contributors to disease progression. Using electric fields applied in several modalities, this work explores the electrical entities of malignant cell types towards improving in vitro treatment planning and developing a treatment modality cognizant of subcellular drivers. This dissertation details the use of dielectrophoresis and electroporation to detect and treat intracellular changes associated with poor prognosis.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第25167号 / 医博第5053号 / 新制||医||1071(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 椛島 健治, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

- David Větvička, M.Sc . Many researchers have, in the past, focused on pathophysiological features of tumor tissue, various tumor-nonmalignant cell interactions, and secretion of active molecules within the tumor mass. All these aspects of tumor structure are known as tumor microenvironment. The composition of particular tumor ecosystem is highly variable, with differences between various tumor types, even between patients with the same diagnosis, and in separate areas of the same tumor. Moreover, further changes in tumor microenvironment often occur during the progression of the disease. Studies of tumor microenvironment have revealed both novel targets for therapy and new prognostic markers. New therapy modalities are being developed to target these discovered features, including drugs functioning to boost anti-malignancy immunity, to block pro-metastatic potential, or to utilize the unique features of this pathological environment established by the tumor. These are obviously of great interest and harbor high potential for better management of malignant diseases. The focus of this thesis is to study the interactions of polymeric drug delivery systems within the tumor microenvironment and to utilize various features of this specific niche for drug delivery research. We have followed three...

O microambiente tumoral é composto por células, como fibroblastos, células do sistema imune, células endoteliais e pericitos, envoltas por uma matriz extracelular, além de possuir fatores solúveis que participam da comunicação celular. Nas últimas décadas, têm-se entendido cada vez melhor seu papel na iniciação e progressão dos tumores. É de fundamental importância, portanto, entender a biologia dos seus componentes e como podem agir em favor do desenvolvimento tumoral. Diversos trabalhos demonstram que há uma associação entre a presença dos pericitos nos vasos tumorais com a agressividade e prognóstico de alguns tipos de câncer. Uma vez ativadas, além do papel estrutural, essas células modulam as atividades das células endoteliais durante a formação de novos vasos, além de adquirirem propriedades como proliferação e migração. Neste contexto, os pericitos passam a secretar fatores importantes na comunicação célula-a-célula e liberam enzimas moduladoras na matriz extracelular. A lisil oxidase (LOX) é uma das principais enzimas que atuam sobre a matriz extracelular. Já está bem descrito que, quando superexpressa em células tumorais, a LOX pode alterar a migração e invasão dessas células, promovendo a geração de metástases. Entretanto, pouco se sabe a respeito da atuação dessa enzima sobre os demais componentes celulares do estroma tumoral, como os pericitos. Sendo assim, o presente trabalho teve como objetivo principal verificar se enzima LOX é relevante para a ativação de propriedades dos pericitos que possam contribuir para suas funções pró-tumorigênicas, como migração, proliferação e formação de vasos. Os resultados foram gerados avaliando essas atividades dos pericitos após pré-tratamento de 24 horas com β-aminopropionitrile (βAPN), um inibidor irreversível da LOX. Foram utilizadas duas linhagens de pericitos derivados de tecido normal (adiposo e muscular) e duas linhagens de pericitos provenientes de tecido tumores do sistema nervoso central (neuroblastoma e ependimoma). Este composto foi capaz de diminuir a capacidade de migração das células de todas as linhagens testadas e, de maneira geral, tornou o processo de formação de estruturas tubulares in vitro menos eficiente. Entretanto, não foram observadas alterações na proliferação celular. Os dados indicam, portanto, que a enzima LOX pode ser importante para a ativação dos pericitos e, possivelmente, influenciem no seu comportamento no microambiente tumoral / The tumor microenvironment is composed of non-cancer cells, such as fibroblasts, immune cells, endothelial cells and pericytes, surrounded by an extracellular matrix, in addition to soluble factors involved in cellular crosstalk. In the last decades, it has been better understood its role in the initiation and progression of tumors. It is critical, therefore, to understand the biology of its components and how they can act in favor of tumor development. Several studies show an association between the presence of pericytes in tumor vessels with aggressiveness and prognosis of some cancers. Once activated, these cells modulate the activities of endothelial cells during the new vessels formation, and acquire properties as proliferation and migration. In this context, pericytes triggers the secretion of important factors in cell-to-cell communication and release modulating enzymes of extracellular matrix. The lysyl oxidase (LOX) is one of the main enzymes that act on the extracelular matrix. It is well described that when overexpressed in tumor cells, LOX can alter the migration and invasion of these cells, promoting the generation of metastases. However, little is known about the role of this enzyme over other cellular components of the tumor stroma, such as pericytes. Therefore, the aim of this study was to verify whether LOX enzyme is relevant to the activation of properties of the pericytes that could contribute to its pro-tumorigenic functions such as migration, proliferation and vessel formation. All the results were generated by evaluation of the activities of these pericytes after 24 hours pretreatment with β-aminopropionitrile (βAPN), an irreversible inhibitor of LOX. This study used two cell lines of pericytes derived from normal tissue (fat and muscle) and two isolated from tissue of the central nervous system. The βAPN was able to reduce the migration of cells of all tested cell lines and, in general, alter the tubular formation in vitro. However, changes in cell proliferation weren′t observed. The data showed, that the LOX family may be important for the activation of pericytes and possibly influence on their behavior in the tumor microenvironment

The recently resurrected cancer stem cell (CSC) theory sheds new light on understanding tumor biology. Most solid tumors have now been shown to contain CSCs, i.e., stem cell-like cancer cells. These cells, although generally rare, appear to be highly tumorigenic and may be the cells that drive tumor formation, maintain tumor homeostasis, and mediate tumor metastasis. In order to test whether any given human tumor cell population has CSC properties, the relatively enriched single tumor cells have to be put into a foreign microenvironment in a recipient animal to test their tumorigenic potential. Furthermore, various in vitro assays can be performed to demonstrate that the presumed CSCs have certain biological properties normally associated with the stem cells (SCs). Herein, I first present a comprehensive review of the experimental methodologies that our lab has been using in assaying putative prostate cancer (PCa) SCs in culture, xenograft tumors, and primary tumor samples. Clonal morphology is one of the critical properties of cultured cancer cells that has been largely ignored. Interestingly, long term-cultured human epithelial cancer cells form holoclones, meroclones, and paraclones, and tumor cell holoclones have been hypothesized to harbor stem-like cells. Using PC3 human prostate carcinoma cells as a model, we provide direct experimental evidence that tumor cell holoclones contain stem-like cells that can initiate serially transplantable tumors. Importantly, holoclones derived from either cultured PC3 cells or holoclone-initiated tumors can be serially passaged and regenerate all three types of clones. In contrast, meroclones and paraclones cannot be continuously propagated and fail to initiate tumor development. Phenotypic characterizations reveal high levels of CD44, [alpha]2[beta]1 integrin, and [beta]-catenin expression in holoclones, whereas meroclones and paraclones show markedly reduced expression of these markers. These observations have important implications in understanding morphologic heterogeneities and tumorigenic hierarchies in human epithelial cancer cells. PCa metastasis represents the worst outcome, and, if unchecked, will eventually kill the patient. Although many PCa cell-intrinsic molecules and end-organ factors have been implicated in the metastatic dissemination of PCa cells, the role of primary tumor microenvironment and the nature of the metastatic PCa cells remain poorly defined. By establishing a reliable and quantifiable experimental PCa metastasis model in NOD/SCID mice, we show that PCa cells implanted orthotopically (i.e., in the prostate) metastasize much more extensively and widely than those implanted ectopically (i.e., subcutaneously or s.c). Microarray-based gene expression profiling reveals that the orthotopically implanted human PCa cells prominently overexpress not only several classes of molecules involved in proteolysis/invasion/angiogenesis and inflammation, but also numerous developmental and SC regulating genes. These latter observations suggest that the orthotopic microenvironment (i.e. mouse prostate) appears to be promoting the manifestation of CSC phenotypes and these CSCs might be involved in enhanced metastasis in the orthotopic microenvironment and later distant organ metastasis. In support, shRNA-mediated knockdown in many metastatic and CSC genes greatly inhibits PCa cell metastasis. Importantly, PCa cells that express high levels of osteopontin (OPN) or CD24, when prospectively purified out and used in spontaneous metastasis assays, demonstrate high metastatic capacities characteristic of metastatic CSCs. In sharp contrast, PCa cells negative for OPN and CD24 expression show little metastatic property. Finally, we provide multiple pieces of additional evidence that metastatic/metastasizing PCa cells possess CSC properties. / text