Glioblastoma (GBM) is the most common and aggressive primary brain tumor and is comprised of transcriptionally heterogeneous cells and a complex microenvironment. Despite decades of research effort, few treatments significantly benefit clinical outcomes. This may be, in part, due to the lack of tools to directly measure functional responses of these heterogeneous cell types under therapy. This thesis aims to advance the understanding of cell type-specific therapeutic response by the development and application of integrated single cell imaging and RNA sequencing technology. Chapter 1 provides an overview of GBM and its heterogeneity, how investigation of cell type-specific phenotypes would benefit the development of GBM treatments, and current sequencing and imaging technologies to examine cell phenotypes with single-cell resolution. Chapter 2 presents a new microfluidic technology for joint single cell imaging and RNA sequencing that can link imaging-based phenotypes and transcriptional identity of the same individual cells with high throughput, molecular capture efficiency, linking accuracy, and user-friendliness. Chapters 3 and 4 present applications of this technology in investigating cell type-specificities of GBM treatments. Chapter 3 focuses on the specificities of 5-aminolevulinic acid (5-ALA), an FDA approved fluorogenic agent, used in fluorescence guided surgery and reveals 5-ALA labeling is not specific to transformed glioma cells, which encourages further studies to systematically compare its performance with potential alternatives. Chapter 4 focuses on the specificities of drug responses by presenting a functional drug screening approach that directly links cell states measured by apoptosis indicators with transcriptional states, which greatly enhances the interpretability of single cell-resolved drug perturbation assays.

In the twenty-first century, advances in synthetic biology and molecular tools to implement programmable behavior into microbes have fueled significant efforts to develop microbial-based therapeutics. Bacteria and viruses have been explored independently for their ability to replicate and induce cytotoxic effects in cancer cells selectively. This dissertation aims to co-opt the anti-tumor capabilities of gram-negative Salmonella enterica subspecies enterica serotype Typhimurium (referred to as Salmonella typhimurium moving forward) and picornaviruses (small RNA viruses with positive sense genomes) to develop a potent, single bacterial-viral consortium- based system to treat solid tumors.I first describe our efforts to co-opt S. typhimurium’s natural internalization into hosts and intracellular space-sensing to deliver self-amplifying picornaviral RNA. Protein effectors that promote intracellular survival of S. typhimurium within the Salmonella-Containing-Vacuole (SCV) are transcribed by Salmonella Pathogenicity Island-2 (SPI-2) promoters, which turn on after sensing the intracellular pH, ion concentrations, and oxidative stressors. These effectors are then translocated into the host’s cytoplasm by a needle apparatus that connects the SCV and cytoplasm, which is also transcribed by SPI-2 promoters. By using the SPI-2 promoter PsseA to drive the expression of fluorescent reporters and membrane-disrupting proteins (eukaryotic and prokaryotic), efficient escape of Salmonella-produced proteins into tumor-host cells was established. RNA delivery into host cells was also made possible by a secondary SPI-2 promoter, PsseJ, which transcribes RNA polymerase T7 (T7), which then transcribes a T7-promoter-driven Poliovirus replicon or full-length Senecavirus A (SVA). Inoculation of this engineered S. typhimurium strain on a panel of cancer cell lines identified the system’s ability to deliver viral replicons and full-length viruses in a small cell cancer cell line, H446. In a murine model, S. typhimurium delivery of SVA was then shown to clear xenografted H446 tumors. Motivated by the possibility of delivering other picornaviral species with similar anti-tumor properties, but documented healthy tissue cytotoxicity, S. typhimurium was further engineered to control SVA viral spread. By driving Tobacco Etch Virus (TEV) protease expression via a second SseA promoter, and replacing a natural cleavage site on SVA with the TEV-cleavage domain, we demonstrate TEV-dependent SVA spread in H446 cells. I conclude with efforts on engineering TEV-dependent-SVA transgene expression to confer greater antitumor properties. Interferon-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been reported to attenuate H446 growth in vitro. Expression of interferon-gamma off SVA would produce a direct selective pressure against viral replication and virion production. However, a fusion of human GM-CSF to Nano-Luciferase protein on the TEV-dependent SVA genome maintained luminescent signals, GM-CSF activity, and TEV-dependent spread, providing a framework to survey anti-tumor properties of SVA-transgenes. Furthermore, I address our development of syngeneic models for Salmonella-mediated delivery of SVA, an important step towards clinical applications of the system as immunocompetent models more closely correlate to immunocompetent patient populations. SVA’s efficient entry and replication in neuroendocrine-derived tissue identified murine neuroblastoma N1E-115 cells as a suitable cell line for SVA cytotoxicity studies. However, the ability of these cells to support bacterial-viral superinfections is unknown. Here, we show that Salmonella-mediated launch of SVA leads to viral spread that can attenuate heterologous hind flank tumor growth and improve their survival along with mice engrafted with orthotopic intracranial brain tumors.

One of the most challenging questions in the study of cancer is the origin and the nature of the cells that initiate cancer. Accumulated studies have provided many molecular origins of cancers but we still do not know what kind of cells in the tissues transform to cancer cells. Therefore, identifying the cellular origin of these cells is critical for the development of better prognosis, diagnosis and treatment of cancer. A stem cell origin of cancer has been postulated over 150 years. Recent cancer stem cell studies have opened a new window on aspects of the cellular origin of cancer. In this communication, we will address two possible cellular origins of cancer in epithelial tumor development using mouse skin cancer model: tissue specific stem cells, and cells from other organs. To demonstrate contribution of the tissue specific stem cells in tumor development, we monitored the contribution of keratin-15 positive hair follicle bulge stem cells to skin tumor development in the multistage skin carcinogenesis model with Krtl- 15CrePRl;R26R transgenic mice. We found that labeled progeny of the keratin-15 positive bulge stem cells migrate into papillomas and these cells contribute to almost all papilloma samples by 20 weeks of promotion. Additionally, in contrast to the transient contribution of bulge-derived cells in skin wound healing, consistent percentage of the bulge-derived cells stay in the papillomas over 20 weeks. Furthermore, papillomas have heterogeneous expression of the codon 61 signature Ha-ras mutation, with approximately 30 percent of bulge-derived regions expressing the mutation. To determine the contribution of exogenous sources in skin tumor development, we examined bone marrow-derived cells (BMDCs) in the skin tumors from the allogeneic gender-mismatched bone marrow transplantation recipient mice after chemical skin carcinogenesis. We observed that genetically marked (EGFP) BMDCs were detected in the epithelial part of skin wounds and also skin tumors, and we found greater degree of BMDC contribution in chronic ulcer-related skin lesions. Lastly, an in-vitro assay demonstrated plasticity of BMDCs by inducing keratin-14 expressing cells from mesenchymal stem cells. These results demonstrated that hair follicle bulge stem cells and also BMDCs are able to contribute to skin tumor development.

Purpose: Control of micrometastatic ovarian cancer in the peritoneal cavity remains a major objective in post-surgical treatment. The purpose of this project was to investigate the efficacy and toxicity of targeted alpha therapy (TAT) for ovarian cancer in vitro and in vivo in animal models and to select the optimal targeting vector for an ovarian cancer clinical trial. Animal models of ovarian, breast and prostate cancer were developed and for further TAT; a phase I melanoma clinical trial was supported, paving the way for an ovarian cancer clinical trial. Methods: The expression of the turnor-associated antigens (Her2, MUC1, uPAfuPAR) on cancer cell line, animal model xenografts and human ovarian cancer tissue was tested by immunostaining. MTS and TUNEL assays were used to evaluate cell killing of alpha conjugates in monolayer and spheroids. Toxicity and maximum tolerance doses for different vectors were tested and determined in vivo. Pharmacokinetics was studied for different time points and different parameters. The antiproliferative effect of 213Bi-C595 and 213Bi-PAI2 was tested at 9 days post-peritoneal cell inoculation of the ovarian cancer cell line OVCAR3. The treatment efficacy of 213Bi-Herceptin was tested at a 2 days post-subcutaneous breast cancer cell BT474 inoculation. Mice were injected (i.p) with various concentrations of alpha conjugates (AC). Changes in cancer progression were assessed by girth size and tumor size. Results: uPA/uPAR and MUCI are expressed on ovarian cancer cell lines and more than 45% ovarian cancer tissue, while HER2 was only positive in one cell line and was positive in less than 15% of ovarian cancer tissues. The ACs can target and kill cancer cells in vitro in a dose dependent fashion. TUNEL positive cells were found after incubation with the different ACs. PAI2 and C595 vectors were selected for in vivo ascites model study of OVCARJ cell with high expression. Delayed and acute toxicity in animal models showed that radiation nephropathy was the cause of body weight loss. Biodistribution studies showed that kidney was the major uptake organ. L-lysine can reduce kidney uptake for 213Bi-PAI2, but no significant differences were found. A single ip injection of 213Bi-C595 or 213Bi-PAI2 can inhibit ascites growth, whereas, 213Bi-Herceptin can inhibit breast cancer growth in a nude mice model. Conclusion: 213Bi labelled targeting vectors can specifically target ovarian cancer cells in vitro and inhibit tumor growth in vivo. These ACs may be useful agents for the treatment of ovarian cancer at the minimum residual disease stage.

Afin de mieux comprendre la physiopathologie et de mieux caractériser les différents types de tumeurs thyroïdiennes, notre travail s’inscrit dans un projet de recherche visant à identifier des biomarqueurs (signatures moléculaires) et à développer des outils permettant le diagnostic, le pronostic et le traitement de ces tumeurs. Afin d’arriver à cet objectif, les profils d’expression de différents types de tumeurs thyroïdiennes sont étudiés, ainsi que leurs modèles in vitro. L’un des mécanismes physiopathologiques rencontré dans le développement de tumeurs est la dérégulation ou l’expression aberrante de microARN. Il a, par ailleurs, été rapporté que cette famille d’ARN pourrait fournir de bons classifieurs moléculaires. Nous nous sommes dès lors demandés quelle pouvait être l’implication de ces microARN dans le développement des tumeurs thyroïdiennes, et s’ils pouvaient fournir un bon outil de classification. <p><p>Ainsi, dans la première partie de cette thèse, nous avons caractérisé les profils d’expression miRNA d’une tumeur bénigne différenciée, l’adénome autonome hyperfonctionnel. Afin d’aller au-delà d’une simple description des régulations miRNA observées, nous avons également étudié les profils d’expression ARNm. Au travers d’algorithmes de prédiction de cibles ARNm de miRNA, nous avons pu proposer des hypothèses quant à la signification des régulations observées. De plus, en utilisant un test fonctionnel luciférase, nous avons montré que deux miRNA (hsa-miR-144 et hsa-miR-451) peuvent lier le 3’UTR et réprimer l’expression d’une phosphodiestérase (PDE4D), mettant ainsi en évidence un mécanisme de rétrocontrôle négatif du niveau d’AMPc dans ce type de tumeur. <p><p>Outre les adénomes autonomes, nous avons également étudié les profils d’expression des microARN dans les adénomes et carcinomes folliculaires, afin de mieux comprendre leurs physiopathologies et d’étudier le pouvoir discriminant des miRNA pour la distinction de ces deux types de tumeurs. Nos résultats, encore partiels, remettent en question l’actuelle classification des adénomes et carcinomes folliculaires en deux groupes distincts. Nos données vont plutôt vers un continuum de tumeurs appartenant à une classe unique mais présentant des stades d’évolution différents. <p><p>Afin de pouvoir étudier plus en profondeur les mécanismes tumoraux, il est nécessaire d’avoir à sa disposition de bons modèles in vitro. Nous avons étudié la représentativité des cultures primaires de thyrocytes humains sains et de lignées cellulaires thyroïdiennes humaines cancéreuses pour l’étude des microARN dans les tumeurs thyroïdiennes. Nos résultats indiquent que les longs temps de stimulation des cultures primaires par la TSH ou l’EGF/sérum ne mimiquent respectivement pas les adénomes autonomes ou les carcinomes papillaires, comme c’est le cas au niveau des ARNm. Par contre, il apparaît clairement que les lignées cellulaires, du point de vue des miRNA, ont toutes évolué vers un phénotype commun, dédifférencié. Elles représentent dès lors un bon modèle pour l’étude des cancers thyroïdiens anaplasiques.<p><p>Nos résultats nous ont également amenés à nous poser des questions sur la stabilité des miRNA. Nous avons ainsi entrepris d’en étudier la demi-vie. Nous avons mis au point une stratégie expérimentale permettant d’en étudier la stabilité dans des conditions les plus physiologiques possibles. Ceci nous a permis d’obtenir un atlas de demi-vies des miRNA dans la lignée cellulaire BCPAP. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Synthetic biology enables the engineering of interactions between living medicines to overcome the specific limitations of monotherapies. A major challenge facing tumor-antigen targeting therapies like chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. In contrast, certain strains of bacteria are gaining recognition as a new class of antigen-agnostic cell therapy due to their selective growth within the immunosuppressive niche of the solid tumor microenvironment (TME). In response, this dissertation aims to pair the cytotoxicity of CAR-T cells with the antigen-independent specificity of tumor-colonizing bacteria to create a new strategy for solid tumor recognition. Here, we reprogram the probiotic strain of E. coli Nissle 1917 to release synthetic CAR targets and human chemokines directly within the solid tumor core. To enable universal targeting, we design synthetic targets to bind ubiquitous components of the TME and broadly tag tumor tissue for CAR-mediated lysis. We demonstrate that these targets robustly coat the surface of cancer cell lines and lead to effective killing by CAR-T cells across various cancer types. We additionally show that injected probiotics selectively grow within the tumor core and maintain target production ¬ in situ – leading to therapeutic efficacy across multiple genetically distinct tumor models. Within this dissertation, we also reveal that intratumoral bacteria provide natural adjuvant effects that serve to activate and increase the effector functions of CAR-T cells in vivo. However, we discover that this can lead to early T cell exhaustion and terminal effector differentiation. To mitigate the counterproductive effects of overstimulation, we generate a new probiotic strain with reduced inflammatory properties that significantly improves CAR-T cell phenotype – leading to enhanced therapeutic benefit in a human model of leukemia. We conclude by discussing the numerous avenues available to optimize cross-Kingdom signaling and to ultimately leverage the full therapeutic benefit of combined cell therapies for future translation. Altogether, this dissertation highlights the potential of the probiotic-guided CAR-T cell (ProCAR) platform to address the critical roadblock of identifying suitable CAR targets by providing an antigen in situ that is orthogonal to both healthy tissue and tumor genetics – and, in turn, aims to establish the foundation for engineered communities of living medicines.

Nearly 80% of children treated for cancer are expected to survive, but not without cost. Survivors face unprecedented challenges associated with long-term consequences of treatment, also called late effects. Approximately half of children treated for cancer are at risk for experiencing cognitive late effects, which typically emerge several years post diagnosis. The nature and extent of cognitive late effects appear to be developmental and related to patient, disease, and treatment variables. However, the relationships between these variables is not well understood because there have been few prospective and longitudinal studies that report on the contributions of these variables over time. This dissertation examined the effects of patient, disease, and treatment variables, as well as their interactions over time on neurocognitive functioning in childhood cancer survivors. It comprises part of a large prospective, randomized clinical trial designed to examine changes in cognitive function over three years as a function of different levels of monitoring of school-based intervention based on individual educational plans (IEPs). This dissertation uniquely contributed a new measure (the Treatment Intensity Rating Scale) that was used to systematically classify treatment severity across different types of cancer and cancer treatments. Participants included 61 children ages 7 to 12 years at enrollment who were two to five years from completion of treatment for a brain tumor, leukemia, or lymphoma. Participants received yearly neuropsychological evaluations for a follow-up period of 3 years. Results of these evaluations were used to develop IEPs. Participants were randomized to have their IEPs monitored on a quarterly or annual basis for the duration of the study. Contrary to the progressive decline in neurocognitive functioning that is typically anticipated in pediatric cancer survivors, analyses revealed relative stability of performance on neurocognitive measures over time. Higher neurocognitive performance was noted in children whose IEPs were monitored more frequently versus less frequently. Results also supported gender-specific risk for late effects, with lower performance on select neurocognitive measures in females compared to males. Results of this study provide encouraging evidence of the positive effects of school-based interventions and their close monitoring. This has important implications for quality of life as these children survive well beyond childhood into adulthood.

This study examined the relationship between executive and psychosocial functioning in 45 children and adolescents age 6 to 17 years who had been treated for a brain tumor. Executive functioning deficits can profoundly impact an adult's ability to function successfully in life. The purpose of the study was to evaluate the potential impact of executive functioning deficits on the day-to-day functioning in a pediatric population. The domains of executive functioning assessed included cognitive flexibility, conceptual thinking, sustained attention, and response inhibition. Psychosocial functioning was assessed using both parent and child report. Several significant relationships were found for adolescents ages 15 and older, with effect sizes ranging from medium to large. In particular, cognitive flexibility and conceptual thinking were significantly related to parent report of depression and adaptive functioning. Fewer significant relationships with smaller effect sizes were found for younger children. The results may reflect the developmental emergence of executive functioning abilities and late effects of executive functioning deficits upon psychosocial functioning. The correlational design of this study precludes definitive statements regarding the temporal nature of the relationship. Additional research, including longitudinal research and replicatory studies, will be needed to further investigate the developmental consequences of executive functioning impairment.

Eighty-two patients with breast cancer, twenty-seven patients with nasopharyngeal carcinoma and sixty-five patients with rheumatoid arthritis in this study were selected based on voluntary, randomization and double blind grouping criteria. / In nasopharyngeal carcinoma patients, the decrease in percentage and the absolute count of T lymphocytes in the TCM group was significantly lower than those in the placebo group. Besides, the decrease of the absolute count of T helper and T suppressor in the TCM group was significantly lower than that in the placebo group (all p &lt; 0.05). The decrease may be due to radiotherapy. However, there was no significant difference in plasma sIL-2R and soluble tumor necrosis factor receptor 2 (sTNFR2) between the TCM group and the placebo group. / In rheumatoid arthritis patients, there was no significant difference in plasma. C-reactive protein (CRP), in the percentage, absolute count, and the ratio of CD4+/CD8+/NK/B lymphocytes between the TCM group and the placebo group. / Results showed that the absolute count of T helper lymphocytes (CD4+), the ratio of T helper lymphocytes (CD4+)/T suppressor and cytotoxic lymphocytes (CD8+), and the percentage and the absolute count of B lymphocytes were significantly elevated in the patients with breast cancer after taking Yunzhi-Danshen capsules, while plasma soluble interleukin-2 receptor (sIL-2R) concentration was significantly decreased (all p &lt; 0.05). / This study shows that the selected traditional Chinese medicine have determinable immunomodulatory effects in patients with cancer and rheumatoid arthritis. (Abstract shortened by UMI.) / Traditional Chinese medicine (TCM) has been used to treat chronic diseases and tumor allegedly by immunomodulatory mechanisms. Breast cancer and nasopharyngeal cancer are prevalent carcinoma diseases in Hong Kong. The immune system of such patients could be adversely affected during the course of conventional chemotherapy or radiotherapy. Rheumatoid arthritis is an inflammatory autoimmune disease of the joints. The aim of this study was to assess the immunomodulatory effects of TCM Yunzhi-Danshen in auxiliary treatment of both kinds of cancer patients, and Lingzhi (Ganoderma Lucidum)-San Miao San ( Atractylodes lancea, Phellodendron amurense and Achyranthes bidentata B1) in supplementation treatment of patients with rheumatoid arthritis. / by Bao Yixi. / "July 2005." / Adviser: Wai-Kei Lam. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0166. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 150-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.