Background: Tractography has become a standard technique for planning neurosurgical operations in the past decades. This technique relies on diffusion magnetic resonance imaging. The cutoff value for the fractional anisotropy (FA) has an important role in avoiding false-positive and false-negative results. However, there is a wide variation in FA cutoff values. Methods: We analyzed a prospective cohort of 14 patients (six males and eight females, 50.1 ± 4.0 years old) with intracerebral tumors that were mostly gliomas. Magnetic resonance imaging (MRI) was obtained within 7 days before and within 7 days after surgery with T1 and diffusion tensor image (DTI) sequences. We, then, reconstructed the corticospinal tract (CST) in all patients and extracted the FA values within the resulting volume. Results: The mean FA in all CSTs was 0.4406 ± 0.0003 with the fifth percentile at 0.1454. FA values in right-hemispheric CSTs were lower (p < 0.0001). Postoperatively, the FA values were more condensed around their mean (p < 0.0001). The analysis of infiltrated or compressed CSTs revealed a lower fifth percentile (0.1407 ± 0.0109 versus 0.1763 ± 0.0040, p = 0.0036). Conclusion: An FA cutoff value of 0.15 appears to be reasonable for neurosurgical patients and may shorten the tractography workflow. However, infiltrated fiber bundles must trigger vigilance and may require lower cutoffs.

Diffuse Gliome bei Erwachsenen sind aggressive, unheilbare Hirntumore. Humanisierte Mausmodelle helfen, molekulare Mechanismen zu verstehen und therapeutische Ziele zu identifizieren, aber der Vergleich mit Proben von Patienten gestaltet sich schwierig. Ich habe eine computergestützte Plattform namens CAPE entwickelt, um Tumormodelle und Patienten-Expressionsprofile mit Hilfe der nicht-negativen Matrixfaktorisierung zu vergleichen. Die Anwendung von CAPE auf humanisierte Maus-Gliom-Avatar-Modelle (GSA) und diffuse Glioma-Patienten zeigte eine starke Übereinstimmung zwischen den Modellen und dem proneuralen Glioblastom-Subtyp. CAPE hat gezeigt, dass durch die Transplantation der Erwerb neuer Tumorzustände in den Modellen verbessert wurde. Durch die Kombination von reporterbasiertem genetischem Tracing und CAPE zeigte sich, dass eine Untergruppe der in vivo GSA-Populationen mit Patienten zusammenfällt, die astrozytische Merkmale aufweisen. Die Behandlung von GSA-Modellen in vitro mit menschlichem Serum, TNFα oder ionisierender Strahlung führte zu einer Verschiebung in den mesenchymalen Zustand. Einzelzell-Transkriptomik annotierte GSA-Populationen unter verschiedenen Bedingungen und zeigte alle Glioblastomzustände in vivo und bei Aktivierung durch externe Faktoren. Der Vergleich von GSA-Einzelzellpopulationen und Patienten bestätigte diese Identitäten. Die Studie etablierte einen umfassenden Rahmen für die Erprobung und Validierung von Verbesserungen der Tumormodelle, um Patienten besser abzubilden, und erweiterte das Verständnis der Tumorbiologie und Ansprechen auf Therapie. / Adult-type diffuse gliomas are aggressive, incurable adult brain cancers. Humanized mouse models help understand molecular mechanisms and identify therapeutic targets, but comparing them with patient samples is difficult. I developed a computational framework, CAPE, for comparing tumor models and patient expression profiles using non-negative matrix factorization. Applying CAPE to humanized mouse glioma subtype avatar models (GSA) and adult-type diffuse glioma patients revealed a strong resemblance between models and proneural glioblastoma subtype. CAPE showed that transplantation improved new tumor state acquisition in models. Combining genetic tracing reporter phenotypic selection with CAPE showed a subset of in vivo GSA populations clustering with patients having astrocytic-like identities. In vitro treatment of GSA models with human serum, TNFα, or ionizing radiation led to a mesenchymal state shift upon reporter selection. Single-cell transcriptomics annotated GSA populations in different conditions, revealing all glioblastoma states in vivo and upon external factor activation. Comparing GSA single-cell populations and patients confirmed these identities. The study established a comprehensive framework for testing and validating tumor model improvements to resemble patients, advancing tumor biology and treatment response understanding.

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Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults for which overall prognosis remains poor despite recent treatment advances, thus emphasizing the need for developing effective therapeutic agents. Styryl sulfones belong to a class of non-ATP competitive antineoplastic agents in early stage clinical trials. Detailed investigation of the pharmacokinetics (PKs) and pharmacodynamics (PDs) of novel agents in the preclinical stage plays a pivotal role in drug development that could be applied to guide clinical trials. The main goal of the project was to undertake comprehensive PK and PD evaluation of new agents for brain tumor therapy and in the process establish a PK/PD strategy for the development of such novel agents. The current project was aimed to evaluate the potential of a styryl benzyl sulfone compound, ON01910.Na, as a chemotherapeutic agent for the treatment of GBMs using PK/PD approaches. First, the systemic pharmacokinetics of ON01910 was characterized following single dose intravascular administration of ON01910.Na in healthy mice over a 50-fold dose range of 5 mg/kg - 250 mg/kg. Secondly, an evaluation of the brain and brain tumor disposition of ON01910 was conducted in an orthotopic U87 glioma model in mice using a steady-state dosing regimen, and, in addition, using the same brain tumor model its pharmacodynamic and antiangiogenic activity were determined following multiple dosing. ON01910 exhibited nonlinear pharmacokinetics in the dose range of 50 mg/kg - 250 mg/kg. It showed inadequate brain and brain tumor penetration and insignificant antiangiogenic and antiproliferative activity. The limited brain tumor penetration and activity of ON01910 in the intracerebral glioma model led to the evaluation of ON013105, a prodrug of its more lipophilic anticancer congener, ON013100. A similar PK/PD approach as for ON01910.Na was applied wherein systemic pharmacokinetic properties of ON013105 and its active form, ON013100 in healthy mice, as well as an analysis of their brain and brain tumor distribution following steady-state dosing regimen were determined following administration of prodrug. The active form, ON013100 showed appreciable brain and brain tumor penetration while the prodrug did not. Subsequent pharmacodynamic investigations conducted in vitro identified phosphorylated-ERK (pERK) as a PD marker. To assess time-dependent PK/PD characteristics, mice bearing intracerebral U87 glioma were administered ON013105 at 100 mg/kg intravenously and plasma, brain and brain tumor concentrations of ON013105 and its active form, ON013100 were quantitated as well as tumoral pERK levels. Further, a PK-PD model was developed that characterized plasma, brain and brain tumor concentration-time profiles of ON013105 and ON013100 and tumoral pERK levels. In summary, a PK/PD-driven approach was applied to evaluate and select novel compounds that may have potential in the treatment of brain tumors. The progression of studies yielded one compound, ON013100 that possessed favorable brain tumor distribution and showed PD activity that warrant continued evaluation. / Pharmaceutical Sciences

Malignant gliomas (MG), most notably glioblastoma multiforme (GBM), are among the most aggressive of all malignancies. High-grade variants of this type of brain cancer are generally considered incurable with singular or multimodal therapies. Many patients with GBM die within one year of diagnosis, and the 5-year survival rate in people is approximately 10%. Despite extensive research in diagnostic and therapeutic technologies, very few developments have emerged that significantly improve survival over the last seven decades. Irreversible electroporation (IRE) is a new non-thermal focal tissue ablation technique that uses low-energy electric pulses to destabilize cell membranes, thus achieving tissue death. The procedure is minimally invasive and is performed through small electrodes inserted into the tissue with treatment duration of about one minute. The pulses create an electric field that induces an increase in the resting transmembrane potential (TMP) of the cells in the tissue. The induced increase in the TMP is dependent on the electric pulse parameters. Depending on the magnitude of the induced TMP the electric pulses can have no effect, transiently increase membrane permeability or cause spontaneous death. In this dissertation we hypothesize that irreversible electroporation is capable of ablating normal (gray and white matter) and pathological (MG and/or GBM) brain tissue in a highly focused non-thermal manner that is modulated through pulse parameters and electrode configuration. Through a comprehensive experimental and numerical investigation, we tested and attained results strongly supporting our hypothesis. Specifically, we developed numerical models that were capable of simulating an entire IRE treatment protocol and would take into account pulse parameters (e.g. duration, frequency, repetition rate and strength) in addition to the dynamic changes in tissue electrical conductivity due to electroporation and joule heating, as well as biologically relevant processes such as blood perfusion and metabolic heat. We also provided a method to isolate the IRE effects from undesired thermal damage in models that were validated with real-time temperature measurements during the delivery of the pulses. Finally we outlined a procedure to use 3D volumetric reconstructions of IRE lesions using patient specific MRI scans in conjunction with the models described for establishing field thresholds or performing treatment planning prior to the surgical procedure; thus supplying the readers with the tools and understanding necessary to design appropriate treatment protocols for their specific application. Experimentally we presented the first systematic in vivo study of IRE in normal canine brain and the multimodal treatment of a canine MG patient. We confirmed that the procedure can be applied safely in the brain and was well tolerated clinically. The lesions created with IRE were sub-millimeter in resolution and we achieved 75% tumor volume reduction within 3 days post-IRE in the patient. In addition to the sharp delineation between necrotic and normal brain, the treatments spared the major blood vessels, making it appropriate for treatment of tumors adjacent to, or enveloping critical vascular structures. We believe that irreversible electroporation will play a key role in the treatment of intracranial disorders including malignant brain cancer in which the intent is to focally kill undesired tissue while minimizing damage to surrounding healthy tissue. / Ph. D.

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer with an average survival time of 15 months. GBM is considered incurable with even the most aggressive multimodal therapies and is characterized by near universal recurrence. Irreversible electroporation (IRE) is a cellular ablation method currently being investigated as a therapy for a variety of cancers. Application of IRE involves insertion of electrodes into tissue to deliver pulsed electric fields (PEFs), which destabilize the cell membrane past the point of recovery, thereby inducing cell death. While this treatment modality has numerous advantages, the lack of selectivity for malignant cells limits its application in the brain where damage to healthy tissue is especially deleterious. In this dissertation we hypothesize that a form of IRE therapy, high-frequency IRE (H-FIRE), may be able to act as a selective targeted therapy for GBM due to its ability to create an electric field inside a cell to interact with altered inner organelles. Through a comprehensive investigation involving experimental testing combined with numerical modeling, we have attained results in strong support of this hypothesis. Using tissue engineered hydrogels as our platform for therapy testing, we demonstrate selective ablation of GBM cells. We develop mathematical models that predict the majority of the electric field produced by H-FIRE pulses reach the inside of the cell. We demonstrate that the increased nuclear to cytoplasm ratio (NCR) of malignant GBM cells compared to healthy brain—evidenced in vivo and in in vitro tissue mimics—is correlated with greater ablation volumes and thus lower electric field thresholds for cell death when treated with H-FIRE. We enhance the selectivity achieved with H-FIRE using a molecularly targeted drug that induces an increase in NCR. We tune the treatment pulse parameters to increase selective malignant cell killing. Finally, we demonstrate the ability of H-FIRE to ablate therapy-resistant GBM cells which are a focus of many next-generation GBM therapies. We believe the evidence presented in this dissertation represents the beginning stages in the development of H-FIRE as a selective therapy to be used for treatment of human brain cancer. / Ph. D. / Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer with an average survival time of 15 months. GBM is considered incurable with even the most aggressive multimodal therapies and is characterized by near universal recurrence. Irreversible electroporation (IRE) is a therapy currently being developed for the treatment of a variety of cancers. Application of IRE involves the delivery of energy directly into the tumor tissue in the form of pulsed electric fields (PEFs). These PEFs destabilize the cell membrane past the point of recovery, thereby inducing cell death. Though this treatment modality has numerous advantages, the lack of selectivity for malignant cells limits its application in the brain where damage to healthy tissue is especially deleterious. In this dissertation we hypothesize that a form of IRE therapy, high-frequency IRE (H-FIRE), may be able to act as a selective targeted therapy for GBM due to its ability to create electric fields inside cells. Because cancer is characterized by alterations in inner organelles compared to healthy cells, electric fields inside the cell may be able to target these alterations resulting in selective malignant cell killing. Through a comprehensive investigation involving experimental testing combined with numerical modeling, we have attained results in strong support of this hypothesis. We have successfully demonstrated selective ablation of malignant GBM cells. We have shown that the increased nuclear to cytoplasm ratio (NCR) of malignant GBM cells compared to healthy brain—evidenced in vivo and in in vitro tissue mimics—is correlated with greater ablation volumes and thus lower electric field thresholds for cell death when treated with H-FIRE. We have enhanced the selectivity v achieved with H-FIRE using a molecularly targeted drug that induces an increase in NCR. We have tuned the treatment parameters to increase selective malignant cell killing. Finally, we have demonstrated the ability of H-FIRE to ablate therapy-resistant GBM cells which are a focus of many next-generation GBM therapies. We believe the evidence presented in this dissertation represents the beginning stages in the development of H-FIRE as a selective therapy to be used for treatment of human brain cancer.

High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation therapeutic that applies bipolar, high-frequency pulsed electric fields to tumors, triggering the formation of irreversible membrane pores and to induce tumor cell death. H-FIRE has demonstrated pre-clinical and clinical utility as a therapeutic for brain tumors, including gliomas. H-FIRE has been shown to induce precise, uniform ablation within the tumor tissue, as well as local changes to the tumor microenvironment and systemic changes to the immune landscape. Namely, disruption of the peritumoral blood-brain barrier (BBB) following H-FIRE ablation of brain tumors, and infiltration and activation of the innate immune system are clinically observed following H-FIRE tumor ablation. Such effects persist long after death of the treated tumor, and therefore an understanding of the mechanisms underlying these local and systemic changes are critical for the development of H-FIRE. Using in vitro models of glioma and lung carcinoma-derived brain metastases, we investigate the interactions between cancer cells that have been ablated with H-FIRE and the brain tumor microenvironments. Specifically, we demonstrate that H-FIRE-treated cancer cells can recover treatment-induced damage and proliferative capacity after treatment with specific electric field doses, while higher doses inhibit such recovery. This suggests that after H-FIRE ablation of brain tumors, tumor cells can still secrete factors to trigger alterations in their local and systemic environments. We then specifically investigate the role of tumor-derived extracellular vesicles (TDEVs) in mediating these changes, namely pBBB disruption and changes in innate immunity. We find that, following H-FIRE ablation of brain cancer cells, treated cells immediately release TDEVs that disrupt the blood-brain barrier (BBB) endothelium in vitro, and are uniquely internalized by cerebral endothelial cells in vitro, despite reduced release of TDEVs after H-FIRE. We further demonstrate that H-FIRE significantly alters the proteomic payloads of TDEVs. When TDEVs released by sham- and H-FIRE-treated glioma cells are delivered to healthy rats, only TDEVs released by H-FIRE-ablated cells are retained in the brain, suggesting changes to TDEV organotropism after H-FIRE ablation of glioma. Further, once retained in the brain, these post-H-FIRE TDEVs cluster near cerebral endothelial cells, similarly to in vitro. Although the TDEVs released by H-FIRE ablated glioma cells do not disrupt the BBB in vivo, Iba1+ cells were increased in the brains of rats that received TDEVs released by H-FIRE-ablated glioma cells. Together, these data suggest that H-FIRE immediately alters the secretion and proteome of TDEVs, facilitating changes in TDEV organotropism and cellular tropism and immune cell recruitment to the tumor microenvironment. Together, this research indicates mechanisms by which tumor cells continue to modulate their local and systemic environments via the action of TDEVs, which is critical information for the continued development of H-FIRE and its optimization with adjuvant therapeutics for the treatment of malignant brain tumors. / Doctor of Philosophy / All cells secrete extracellular vesicles, which are packets of information that function as communication highways between cells. In cancer, tumor-derived extracellular vesicles (TDEVs) reprogram local and distant cells to support tumor growth. However, they have also been shown to change local and systemic functions, such as blood vessel function and immune response, after tumors are treated with therapeutics. Therefore, a full understanding of the role of TDEVs in how tumors communicate with the body after cancer treatment is necessary when developing new anti-cancer therapeutics. Here, in developing high-frequency irreversible electroporation (H-FIRE), a novel anti-tumor therapeutic for the treatment of malignant brain tumors, we explore how TDEVs released by brain cancer cells treated with H-FIRE interact with various cell types and structures in the body, and how these interactions may affect the response to treatment. Using a glioma model of primary brain cancer, and a lung carcinoma model of brain metastases, we first explore how tumor cells may be able to recover from damage after treatment with H-FIRE. We discover that brain cancer cells treated with specific doses of H-FIRE recover cell damage and continue to proliferate, but cells treated with higher doses of H-FIRE cannot recover these functions. The fact that tumor cells may be able to recover after H-FIRE suggests that cancer cells may still secrete factors, such as TDEVs, that interact with cells in the microenvironment after tumor treatment. We investigated the role of TDEVs released by brain cancer cells treated with H-FIRE to determine whether they cause changes in surrounding cells and structures in the brain cancer microenvironment. We determined that brain cancer cells treated with H-FIRE release TDEVs that carry proteins different from those carried by TDEVs routinely released by untreated cells. We further found that these TDEVs disrupt the blood-brain barrier (BBB) endothelium in vitro, and are uniquely internalized by cells of the endothelium. When these TDEVs were administered to the brains of healthy rats, they were retained in the brain, clustered near the endothelium, and recruited immune cells from circulation into the brain. Conversely, TDEVs that were routinely released from the brain cancer cells, in the absence of H-FIRE treatment, exhibited none of these functions. Taken together, these results show that H-FIRE changes TDEVs in numerous ways: after H-FIRE, the TDEVs may gravitate toward particular organs and cell types, and recruit immune cells. All of these changes can impact the overall therapeutic response after H-FIRE, and may also be specifically optimized and targeted with additional therapeutics to make H-FIRE more effective for brain cancer.

Glioblastoma (GBM) is the most aggressive and malignant glioma. It accounts for 48.6% of all primary, malignant gliomas with a median survival of 15 months. Infiltration into the surrounding parenchyma is a hallmark of GBM. Radiotherapy is used to address the invasion; however, recent studies have implicated that radiation contributes to increased invasiveness of glioma. Although the effect of radiation on cells has been studied extensively, its effect on the transport of fluid is not well characterized. Transport in the brain which has significant roles in physiology, GBM pathophysiology, and GBM treatment. Thus, understanding the effect of radiation on transport within the lesion and surrounding interstitium will be beneficial in characterizing the effects of radiotherapy in GBM patients. This dissertation seeks to explore the relationship between radiation, transport, and movement of glioma cells and includes the following: 1) Characterizing in vitro motility metrics of glioma stem cell lines in and relating them to in vivo invasion. 2) Studying the effect of radiation on motility, flow-mediated invasion, extracellular matrix components, and transport within the lesion and interstitium. 3) Assessing transport in clinical images and relating transport parameters to progression of GBM. 4) Developing a novel pipeline for applying vector field topology to the study of interstitial fluid flow in glioma. Surprisingly, we found that motility metrics in vitro have a negative correlation trend with in vivo invasion. Next, we found that radiation causes a transient increase in advective flow, and a more sustained decrease in diffusivity in a murine glioma model. Tenascin C was found to correlate significantly with invasion and diffusivity, indicating that it might be a link between radiation, transport, and invasion. Furthermore, interstitial fluid flow was calculated and assessed in clinical images. This showed that interstitial fluid flow velocity magnitude in the tumor correlates with overall survival in GBM patients. Lastly, vector field topology was introduced as a novel method of studying transport that provides more detailed information to identify potential drivers of transport within a flow field. Altogether, this work presents novel insight into the effects of radiation on invasion and transport in GBM. Hopefully, this work can provide a foundation to build upon in efforts of improving treatment planning and clinical outcomes for GBM patients. / Doctor of Philosophy / Glioblastoma (GBM) is the most aggressive glioma. It accounts for 48.6% of all primary, malignant gliomas with a median survival of 15 months. The movement of cancer cells into the surrounding tissue is a defining factor of GBM. Radiotherapy is used after surgery to treat the remaining cancer cells in tissue surrounding the tumor; however, recent studies have implicated that radiation contributes to increased movement of glioma into surrounding tissue. Although the effect of radiation on cells has been studied extensively, its effect on transport of fluid is not well characterized. Interstitial fluid flow in the brain has significant roles in healthy bodily functions, GBM disease state, and GBM treatment. Thus, understanding the effect of radiation on transport within the tumor and surrounding tissue is beneficial in better characterizing the effects of radiotherapy. This dissertation seeks to explore the relationship between radiation, transport, and movement of glioma cells and includes the following: 1) Characterizing in vitro motility metrics of glioma cells in and relating them to in vivo movement into healthy tissue. 2) Studying the effect of radiation on motility, flow-mediated infiltration into healthy tissue, tissue matrix components, and fluid flow within the tumor and surrounding tissue. 3) Assessing transport in clinical images and relating transport parameters to progression of GBM. 4) Developing a novel pipeline for applying vector field topology to the study of interstitial fluid flow in glioma. Surprisingly, we found that motility metrics in vitro have a negative correlation trend with in vivo invasion. Next, we found that radiation causes a transient increase in flow velocity magnitude, and a more sustained decrease in diffusivity in a murine glioma model. Tenascin C, a component of the tissue matrix, was found to correlate significantly with invasion and diffusivity. This indicates that Tenascin C might be a link between radiation, transport, and invasion. Furthermore, interstitial fluid flow was calculated and assessed in clinical images which showed that interstitial fluid flow velocity magnitude in the tumor correlates with survival. Lastly, vector field topology was introduced as a novel method of studying fluid flow in glioma that provides more detailed information regarding the flow field. Altogether, this work presents novel insight into the effects of radiation on fluid flow and cellular movement in GBM. Hopefully, this work can provide a foundation to build upon in efforts of improving treatment planning and clinical outcomes for GBM patients.