A computer-controlled system, based on the design of S. Brown et al. at the Ontario Cancer Institute, Canada, has been developed for uniform heating of mouse tumors. A steady hyperthermic temperature was maintained in the tumor by combining waterbath heating with RF-heating. The RF field was provided by a transmitter which was connected to two steel capacitive plates via a matching circuit. 50% isotonic saline kept at a temperature 2°C below that in the tumor was circulated in the waterbath. The saline prevented overheating of the skin and provided coupling between the RF field and the tumor. A computer program has been written to measure continually the temperature in the tumor with implanted fine thermocouples. The program also controlled the average RF power delivered to the tumor by switching the transmitter on and off at appropriate intervals. The system has been tested on tumor xenografts growing in the thigh of nude mice. A steady temperature of 42° or 44°c has been maintained in the tumor for up to an hour. The system could be used to study the effect of hyperthermia on the uptake of radiolabelled tumor-associated antibodies and the treatment of tumors by such antibodies. / Thesis / Master of Science (MS)

DOCTOR OF PHILOSOPHY (2019) McMaster University, Hamilton, Ontario (Medical Sciences) TITLE: Serotonergic antagonists affect the activity of breast tumor initiating cells in human and mouse models of breast cancer. AUTHOR: William D. Gwynne, BSc SUPERVISOR: Dr. John A. Hassell NUMBER OF PAGES: XXI; 255 / Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death amongst women worldwide. The relatively unchanging breast cancer-associated mortality rate is in part due to the existence of rare tumor cells (breast tumor initiating cells; BTIC) that possess stem-like properties permitting them to survive therapy and initiate disease recurrence. Hence, identifying agents capable of eradicating these cells would be a favourable therapeutic strategy to improve the durability of breast cancer remissions. To achieve the latter objective our lab screened over 35,000 small molecules for their capacity to inhibit the viability of BTIC-enriched mouse tumor cells. Unexpectedly, several antagonists of the serotonin (5-hydroxytryptamine; 5-HT) transporter and select receptors were among the hit compounds identified in the screen. This thesis aims to establish a connection between serotonergic activity and BTIC. We accomplished the latter by assessing whether components of the 5-HT signaling system are expressed in mouse and human breast tumor cells and whether inhibition of their activity affects BTIC frequency using multiple orthogonal assays. Our data suggest that breast tumor cells of both mouse and human origin express the components necessary for 5-HT synthesis, activity and metabolism and that inhibition of these proteins with selective antagonists reduces the capacity of these cells to form tumorspheres. We demonstrate that highly selective antagonists of SERT and HTR5A target BTIC as established ex vivo cell transplantation assays. We also discovered that these agents synergize with chemotherapy in vivo to affect the growth of mouse breast tumor allografts and human breast tumor xenografts. To validate the molecular targets of these agents, we attempted to phenocopy their effects in functional assays by knocking out their respective genes using CRISPR-Cas9 technology. Collectively, this thesis contributes to an understanding of how 5-HT signaling affects BTIC and identifies serotonergic antagonists as novel anticancer agents. / Dissertation / Doctor of Philosophy (PhD) / Despite improvements in screening technologies and the development of targeted therapies breast cancer remains the second leading cause of cancer-related death among Canadian women. Whereas the current standard of care is effective at treating the majority of patients diagnosed with breast cancer, there remains a substantial proportion of patients that experience relapse after undergoing therapy. Recurrence is due in part to the existence of rare, stem-like tumor cells, termed breast tumor-initiating cells (BTIC) that are insensitive to existing anticancer agents. Hence, identifying drugs capable of targeting these cells is a desirable goal. To pursue the latter, our lab screened approximately 35,000 compounds for their capacity to affect the growth of BTIC-enriched tumor cell populations. Among the hit compounds were antagonists of the serotonin transporter and serotonin receptors, including FDA-approved psychiatric medications. Here, we explore a connection between serotonin-related proteins and BTIC activity with the aim of identifying novel therapeutic agents.

In the current study we tested the hypothesis that tumorigenic transformation of T cells may result from aberrant regulation of autocrine growth factor production. We describe for the first time characterization of several normal T cell lines that underwent spontaneous transformation resulting from constitutive expression of interleukin-2 (IL-2) and interleukin 2 receptor (IL-2R) genes. Such cell lines could induce tumors <i>in vivo</i> and furthermore, the growth of such transformed cells both <i>ex vivo</i> and <i>in vivo</i> could be inhibited by monoclonal antibodies (mAbs) or antisense oligonucleotides specific for IL-2 or IL-2R. Interestingly, an <i>in vivo</i> originated T cell lymphoma, LSA, was also found to be dependent on constitutive production of IL-2 and Interleul(in-4 (IL-4). Together, these data demonstrated that dysregulation in the production or responsiveness to autocrine T cell growth factors, plays an important role in T cell transformation and tumorigenicity. Due to their ability to produce T cell growth factors, such tumor cells were found to be highly immunogenic. Thus it was surprising that some T cell lymphomas could still grow in an immunocompetent host and kill the host. To this effect, we investigated additional mechanisms used by such tumor cell lines to grow in an immunocompetent host. It was noted that the tumor cells used mechanisms such as failure to express MHC and production of immunosuppressive cytokines, such as, transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), to evade the action of the immune system. In addition, the T cell lymphomas constitutively expressed F as-ligand and triggered apoptosis of host T cells that expressed Fas. Together, the current study suggests that cytokines produced by the tumor cells play an important role in tumorigenic transformation as vvell as maintenance of tumor growth in an immunocompetent host. / Ph. D.

Tumor necrosis factor-a (TNFa) is primarily a macrophagederived cytokine. TNFa, in vitro, kills or inhibits growth of approximately one third of surveyed transformed cell lines dincluding the L929 and WEHI 164 murine fibrosarcoma cell lines. Very little is known about the mechanisms of TNFa action. However, recently, it has been theorized that TNFa has no activity of its own and that the receptor for TNFa on the cell surface, when properly triggered, activates the cellular mechanisms which may result in the cell's death. The objective of this study was to produce an antiidiotypic antibody to TNFa to be used as a tool to study the mechanisms of TNFa action. A hybridoma that secretes an antiidiotypic antibody to TNFa (Ab2J1) has been produced and isolated following standard procedures. This antibody was found to be of isotype IgG2a as determined by an indirect ELISA test. The Ab2J1 exhibited TNFa target cell-killing capabilities in vitro. The TNFa-resistant cell lines, SP2jO and NS-1 were resistant to Ab2J1 and TNFa sensitive cells, L929 and WEHI 164, were sensitive to Ab2J1. The cell killing activity of both TNFa and Ab2f3 could be neutralized by a monoclonal anti-TNFa antibody. Both TN Fa and Ab2f3 acted in parallel having an effect on the killing of Brucella abortus strain RB51 by peritoneal macrophages, whereas neither TNFa nor Ab2f3 had an effect on the killing of strain 2308 by macrophages. These results, again indicate that TNFa and Ab2f3 have parallel dbactericidal effects and that Ab2f3 is capable of mimicking TNFa activity. The Ab2J1 was further characterized by gel electrophoresis and Western blot and was found to have two subunits of 25 and 50 kDa molecular weights similar to IgG. This anti-idiotypic antibody to TNFa may help in understanding the mechanisms of the cytotoxic activity of TNFa. / Master of Science

Breast cancer is the most common cancer among women. Statistics released by the American Cancer Society (1999) show that every 1 in 8 women in the United States is likely to get breast cancer during her lifetime. Thermography, also known as thermal or infrared imaging, is a procedure to determine if an abnormality is present in the breast tissue temperature distribution, which may indicate the presence of an embedded tumor. In the year 1982, the United States Food and Drug Administration (FDA) approved thermography as an adjunct method of detecting breast cancer, which could be combined with other established techniques like mammography. Although thermography is currently used to indicate the presence of an abnormality, there are no standard protocols to interpret the abnormal thermal images and determine the size and location of an embedded tumor. This research explores the relationship between the physical characteristics of an embedded tumor and the resulting temperature distributions on the skin surface. Experiments were conducted using a resistance heater that was embedded in agar in order to simulate the heat produced by a tumor in the biological tissue. The resulting temperature distribution on the surface was imaged using an infrared camera. In order to estimate the location and heat generation rate of the source from these temperature distributions, a genetic algorithm was used as the estimation method. The genetic algorithm utilizes a finite difference scheme for the direct solution of Pennes bioheat equation. It was determined that a genetic algorithm based approach is well suited for the estimation problem since both the depth and the heat generation rate of the heat source were accurately predicted. Thermography can prove to be a valuable tool in locating tumors if combined with such an algorithm. / Master of Science

Atypische teratoide Rhabdoidtumore sind trotz Ausschöpfen der multimodalen Therapieoptionen weiterhin mit einer schlechten Prognose belastet. Gründe hierfür liegen in den oftmals unzureichenden Resektionsmöglichkeiten, dem jungen Erkrankungsalter der PatientInnen und der Resistenz der Tumorzellen gegenüber Chemotherapeutika (Frühwald et al. 2020; Egiz et al. 2022; Richards et al. 2019). Gerade deshalb versucht man durch die aktuelle Forschung zu kindlichen Hirntumoren mit Immuntherapie ein besseres Outcome zu erreichen. Wichtige Grundlagen hierzu sind durch diese Arbeit dargestellt worden. Erstmals wurde gezeigt, dass Tumorzellen der AT/RT sowohl HLA-Klasse I und -Klasse II Antigene präsentieren. Es wurde außerdem die Expression von PD-L1 nachgewiesen. Des Weiteren konnte die Anwesenheit von Immunzellen durch den Nachweis CD 3+ Zellen bewiesen werden. Insgesamt zeigte sich eine große Heterogenität innerhalb des einzelnen und unter den verschiedenen Tumoren. Es zeigte sich eine negative Korrelation zwischen der Expression von MHC I und CD 3+ Zellen, welche insgesamt für einen Tumor Escape Mechanismus sprechen könnte, wie er bereits bei Glioblastomen nachgewiesen wurde (Bagley et al. 2018; Marcu et al. 2021). Es sollte eine Ausweitung der hier begonnen Forschung mit Einbeziehung der personenbezogenen Daten und Vergrößerung der untersuchten Fallzahl erfolgen. / Even with using the multimodal therapy options available, atypical teratoid rhabdoid tumors still have a poor prognosis. This is caused by limited chirurgical resection, the young age of the patient cohort and resistence to chemotherapeutics (Frühwald et al.2020; Egiz et al. 2022; Richards et al. 2019). Hence, current research is trying to acchieve a better outcome through immunotherapy. Important fundamental research is done by this work. For the first time, it has been shown that tumorcells of AT/RT express HLA-class I and -class II antigens. Furthermore PD-L1 is expressed by these tumorcells. The presence of immuncells is proved by the evidence of CD 3+ cells. In total, a great heterogeneity presented in one tumor itself and in between the diffrent tumors examined. This work pointed out a negativ correlation between the expression of MHC I and CD 3+ cells, which could show a tumor escape mechanism, like it is already proven for glioblastoma multiforme (Bagley et al. 2018; Marcu et al. 2021). Beside that here is need for further studys with a bigger case number and link to personal and clinical data.

Mathematical modeling of cancer is of significant interest due to its potential to aid in our understanding of the disease, including investigation into which factors are most important in the progression of cancer. With this knowledge and model different paths of treatment can be examined; (e.g. simulation of different treatment techniques followed by the more costly venture of testing on animal models). Significant work has been done in the field of cancer modeling with models ranging from the more broad systems, avascular tumor models, to smaller systems, models of angiogenic pathways. A preliminary model of a vascularized tumor has been developed; the model is based on fundamental principles of mechanics and will serve as the framework for a more detailed model in the future. The current model is a system of nonlinear partial differential equations (PDEs) separated into two basic sub-models, avascular and angiogenesis. The avascular sub-model is primarily based of Fickian diffusion of nutrients into the tumor. While the angiogenesis sub-model is based on the diffusion and chemotaxis of active sprout tips into the tumor. These two portions of the models allow the effects of microvessels on nutrient concentration within the tumor, as well as the effect of the tumor in driving angiogenesis, to be examined. The results of the model have been compared to experimental measurements of tumor growth over time in animal models, and have been found to be in good agreement with a correlation coefficient of (r2=0.98). / Master of Science

Osteosarcoma (OS) is the most common primary bone tumor with an annual incidence rate of 3-4 individuals per million particularly affecting children and young adults. The 5-year survival rate stands at 60-80% with the current standard of care for human OS patients who do not have metastatic disease at presentation, but this drops to 20% for patients with metastatic disease which frequently occurs in the lungs. OS is much more common in canines, with metastasis being the major contributor to mortality, the same as in humans. Metastatic OS warrants novel treatment strategies to improve prognosis and survival. High-frequency irreversible electroporation (H-FIRE) is a promising, non-thermal, minimally invasive technique that induces cell death by applying pulsed electric fields in targeted regions, potentially triggering an anti-tumor immune response that could also target and prevent metastases. Such a dual functionality of H-FIRE is uniquely suited to treat pulmonary metastatic OS. The goal of this thesis was to study the ablative and immunomodulatory effects of H-FIRE on OS in-vitro with the overall hypothesis that H-FIRE completely ablates OS cells, induces the release of damage-associated molecular patterns (DAMPs), and promotes pro-inflammatory immune activating signatures in macrophages and T cells. Using an in-vitro model, my master's thesis focused on 1) Determining the electric field strength that completely ablates OS cells 2) Evaluating the immunomodulatory effects of H-FIRE by co-culturing H-FIRE treated OS cells with macrophages and T cells separately. Our study has utilized murine, canine, and human OS and immune cells, thus demonstrating a unique cross-species approach, 3) Evaluating DAMPs (ATP, calreticulin, and HMGB1) post-H-FIRE ablation of human OS cells. Overall, our study showed that H-FIRE successfully ablated OS cells in-vitro, induced the release of DAMPs from treated cells, and promoted activation signatures in immune cells. This thesis provides foundational data for future investigations developing H-FIRE as an immunomodulatory strategy for treating metastatic OS. / Master of Science / Osteosarcoma (OS) is the most common primary bone tumor that majorly affects young adults and children with an incidence rate of 3-4 individuals per million per year. When metastasis occurs (i.e. OS spreads from its site of origin to other organs in the body), most frequently to the lungs, patients experience poor chances of recovery and survival. Currently, the treatment protocol followed for patients with metastatic OS largely includes complete surgical removal and chemotherapy both of which can be very grueling for patients. No significant improvement in the overall 5-year survival rate with current mainstay treatment has led to the urgent need of novel treatment modalities for treating patients with pulmonary metastatic OS. High-Frequency Irreversible Electroporation (H-FIRE) is a novel non-thermal tumor ablation strategy that utilizes electrical pulses to create pores on the cell membrane, thus leading to irreversible damage and cell death. These dying tumor cells release certain molecules and proteins that send danger signals to activate the body's own immune system against the tumor. H-FIRE with its dual function of destroying the targeted tumor region via electroporation and distant metastases via activating immune system is uniquely suited to treat pulmonary metastatic OS. This thesis is the first to investigate H-FIRE ablation and immunomodulation for OS. We hypothesized that H-FIRE can completely destructs OS cells, promotes the release of danger signals, and causes immune activation. Using an in-vitro model, this thesis focused on 1) Determining the electric field strength needed for complete OS cell destruction by H-FIRE 2) Evaluating the immune activation potential of H-FIRE by exposing these H-FIRE treated cells to immune cells like macrophages and T cells separately. We utilized human, mouse, and dog-derived OS cells to increase the biological and clinical relevance of our study. 3) Evaluating certain proteins that act as danger signals post-H-FIRE treatment of human OS cells. Overall, our results indicated that H-FIRE can successfully destruct OS cells in-vitro, promotes the release of danger signals, and induces immune activation. This thesis contributes to providing crucial preliminary data in the development of H-FIRE as a novel ablation and immunomodulation treatment strategy for pulmonary metastatic OS.

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

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Atypical teratoid rhabdoid tumor (ATRT) is a highly malignant pediatric central nervous system tumor. The prognosis is often poor, with a 2‐year survival rate estimated at 15%. This dismal prognosis highlights the need to develop new treatment modalities for this devastating pediatric tumor. Recently, a tumor suppressing signaling pathway known as Hippo has emerged as a possible cancer treatment target. The Hippo signaling pathway is involved in organ growth and maintenance, and is dysregulated in many diverse cancers. We used quantitative real‐time PCR to evaluate the mRNA expression profile of Hippo pathway genes. We then used determined the protein expression of various Hippo components using Western blots. The results of this study suggest that Hippo plays a definite role in atypical teratoid rhabdoid tumor.