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Item Open Access Acute Pathogenesis of Recombinant Vesicular Stomatitis Virus Vaccine Vectors is Linked to Interleukin-1(2011) Athearn, Kathleen ConstanceRecombinant vesicular stomatitis virus (rVSV) is a promising candidate viral vaccine vector for use in humans. VSV is highly immunogenic, pre-existing immunity to VSV is rare, and VSV is able to grow to high titers in cell lines approved for vaccine use. Its potential reactogenicity is a barrier to its use in humans, with small laboratory animals developing fever and losing up to 20% of their pre-immunization body weight in the first four days after administration [1, 2], and the one person to date that has received an experimental rVSV vaccine developed headache, fever, and muscle pain within 12 hours and transient VSV viremia was detected [3]. The underlying cause of these reactions has not yet been studied. Here, we have found that IL-1β and/or IL-1α contributes to rVSV pathology after intramuscular immunization in mice and that IL-1 production is not required for control of rVSV replication in vivo, or for the generation of protective immune responses to VSV antigens. Suppression of IL-1 may be a safe strategy to reduce vector reactogenicity without affecting immunogenicity. Utilizing mice deficient in either ASC or caspase-1, we have also found that production of mature IL-1β in response to rVSV might be independent of inflammasome activation or caspase-1 cleavage. The exact mechanism is yet to be determined, but might depend upon which cell types secrete mature IL-1β after immunization.
Item Open Access Aerobic Training-Induced Host Changes Alter Breast Cancer Cell Phenotypes and Tumor Progression(2015) Glass, OliverA growing number of studies have investigated the role of exercise both during and after a breast cancer diagnosis. Observational data suggests that regular endurance exercise is associated with a 20-50% reduction in cancer-specific mortality in women diagnosed with early stage breast cancer, compared to inactive women; however it is unclear whether there is a differential association across breast cancer subtypes. As a pre-requisite to guide future large phase II/III clinical trials, there is a critical need to confirm the biological plausibility of the exercise association in breast cancer patients as well as elucidate the underlying mechanisms of action via utilization of preclinical models.
In the present study we investigated the systemic effects of prescribed aerobic training in cancer patients and the direct impact on breast cancer cell subtype phenotypes. In order to test the in vivo significance, we interrogated aerobic training effects on breast cancer progression and tumor biology using syngeneic breast cancer mouse models.
Our results suggest that aerobic training may alter the host availability of pro-inflammatory and growth factor cytokines in patients with solid tumors. Modulation of systemic effectors in breast cancer patients compared to controls causes a differential phenotypic response on breast cancer cell subtypes. In vivo, aerobic training has a differential response on breast tumor progression compared to controls that is mediated by Hif1-α and metabolic reprogramming of breast cancer cells.
Item Open Access Antibody-Redirected T-Cell Immunotherapy for Brain Tumors(2014) Choi, Bryan DaehahnThe most common primary malignant brain tumor, glioblastoma, is uniformly fatal. Current therapy provides only incremental benefits in survival and is often incapacitating owing to limits defined by nonspecific toxicity. By contrast, immunotherapy offers a particularly promising approach, and has the theoretical potential to target and eliminate malignant cells with unprecedented specificity. The goal of this dissertation is to apply recombinant technologies to develop a new immune-based therapy for patients with malignant glioma. This work will span the design, production, and preclinical testing of a novel bispecific antibody designed to redirect T cells against a tumor-specific mutant of the epidermal growth factor receptor, EGFRvIII.
Chapters 1 and 2 will provide an overview of broad topics in antitumor immunotherapy and immune biology, with special focus on concepts as they relate to tumors of the central nervous system. In addition, the history and current state of bispecific antibodies, particularly those of the bispecific T-cell engager (BiTE) subclass, as well as their potential role in the treatment of malignant disease, will be considered in detail. Data presented in Chapter 3 will describe our approach to generating novel bispecific tandem single-chain antibody reagents, while experiments in Chapter 4 will demonstrate the capacity of one of these molecules, an EGFRvIII-specific BiTE, to achieve antitumor efficacy both in vitro and in vivo using murine models of glioma. Addressing a major barrier to the translation of immune therapies for cancer, chapter 5 will establish a potential role for BiTEs in overcoming cell-mediated immune suppression associated with malignant disease. Lastly, Chapter 6 and 7 will report on emerging areas of study, including the use of syngeneic, transgenic murine systems, and strategies by which BiTEs may be propelled rapidly into early phase clinical trials.
In summary, separating BiTEs from other available immunotherapeutic approaches, our work in this field suggests that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with glioblastoma. Together, these studies have the potential to improve the clinical management of patients with glioblastoma through the generation of a novel therapeutic.
Item Open Access Auxiliary Wnt3A Signaling in Cell Fate Decisions of C3H10T1/2 Mesenchymal Stem Cells(2011) Rossol-Allison, Jessica K.Activation of Wnt signaling pathways is critical to a variety of developmental events across all animal taxa. These highly evolutionarily conserved pathways are also important in the adult organism for maintaining homeostasis of self-renewing tissues. Because of its role in such important physiological processes, deregulation of Wnt signaling can have severe consequences; indeed, inappropriate activation of this pathway has been implicated in multiple human diseases, including cancer.
Upon binding their cellular receptors, canonical Wnt ligands, like Wnt 3A, stimulate the stabilization, accumulation, and nuclear translocation of a multifunctional cellular protein βcatenin, the consequence of which is induction of βcatenin-dependent transcription. This work describes the identification and characterization of two Wnt3A-stimulated intracellular signaling pathways activated in parallel to βcatenin stabilization: the RhoA pathway and the ERK pathway. These two auxiliary pathways do not affect βcatenin stability, accumulation, or subcellular localization; rather, they modulate βcatenin -dependent transcriptional activity through other mechanisms. As a result of their influence on βcatenin-dependent transcription, these pathways instruct cell fate decisions in C3H10T1/2 mesenchymal stem cells, in particular inhibition of adipogenesis and promotion of osteoblastogenesis.
Expression microarray analysis and biochemical and pharmacological techniques were used to further characterize the two Wnt3A-stimulated auxiliary pathways in C3H10T1/2 cells. Remarkably, each pathway influences βcatenin function via a novel mechanism. In the Wnt3A/RhoA pathway, Wnt3A-stimulated trimeric G proteins activate a RhoA-ROCK-SRF cascade. Activated SRF can cooperate with βcatenin to enhance the induction of Wnt3A target genes, like Ctgf, that also contain SRF binding sites within regulatory elements. In the Wnt3A/ERK pathway, Wnt3A transactivates the EGFR in a concentration-dependent manner, leading ultimately to ERK activation, which interacts with and promotes βcatenin/Tcf4 interaction and enhances induction of βcatenin/Tcf4 target genes.
These data emphasize the complexity of Wnt signaling and have intriguing implications regarding cross-regulation of the pathway, especially in stem cells. Also, since not all cells are capable of responding to Wnt3A by activation of these auxiliary pathways, this work identifies novel mechanisms that could underlie cell type-specific responses to Wnts and provides mechanistic insight into cellular responses to Wnt concentration gradients. Moreover, this work identifies novel transcriptional mechanisms important for promoting osteogenic cell fate specification, which could ultimately provide new therapeutic targets in disease states with bone loss or ineffective bone formation.
Item Open Access Bench to bedside: A Bispecific Antibody for treating Brain Tumors(2019) Schaller, Teilo HMalignant gliomas are the most common primary brain tumor in adults, with an incidence of five cases per 100,000 persons per year. Grade IV glioblastoma is the most aggressive form and prognosis remains poor despite the current gold-standard first-line treatment – maximal safe resection and combination of radiotherapy with temozolomide chemotherapy – resulting in a median survival of approximately 20 months. Tumor recurrence occurs in virtually all glioblastoma patients, and there currently exists no accepted treatment for these patients. Recent advances in novel directed therapeutics are showing efficacy and have entered clinical trials. This work spans the pre-clinical and clinical development of a bispecific antibody – EGFRvIII:CD3 bi-scFv – for the treatment of malignant gliomas.
Chapter 1 reviews current front-line immunotherapy research in the fields of antibodies, including BiTEs and checkpoint inhibitors, and tumor vaccinations, including peptide and dendritic cell vaccinations. Furthermore, challenges specific to high-grade gliomas as well as opportunities for combination therapies are discussed. Chapter 2 introduces the architecture of the novel bispecific antibody EGFRvIII:CD3 bi-scFv and provides an overview of the molecule’s efficacy in various models. EGFRvIII:CD3 bi-scFv is a truncated antibody with dual specificity. One arm targets the epidermal growth factor receptor mutation variant III (EGFRvIII), a tumor-specific antigen found on glioblastoma. The other arm targets the human CD3 receptor on T cells. As an obligate bispecific antibody, simultaneous binding of both receptors by multiple EGFRvIII:CD3 bi-scFv’s results in the crosslinking of CD3 receptor, activation of T cells, and release of perforin/granzyme which lyses the proximal EGFRvIII-expressing tumor cells. EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma.
Chapter 3 outlines the in-house development of a scalable clinical production process using a WAVE (GE) bioreactor and describes the cGMP-compliant clinical production of EGFRvIII:CD3 bi-scFv. The 250-liter cGMP-production run yielded more than four grams of clinical drug material.
Chapter 4 demonstrates that EGFRvIII:CD3 bi-scFv produced using the cGMP development process is efficacious in both in vitro and in vivo models of glioblastoma. The chapter also describes the approach used to calculate the starting dose for the upcoming first-in-human clinical trial. First-in-human clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal model. However, with the advent of target-specific and highly active immunotherapeutics, both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have provided guidance that recommend determining a safe starting dose based on a minimum anticipated biological effect level (MABEL) approach. In order to establish a first-in-human dose, as advised by the FDA for bispecific antibodies, this work uses a MABEL approach to select a safe starting dose for EGFRvIII:CD3 bi-scFv, based on a combination of in vitro data, in vivo animal studies, and theoretical human receptor occupancy modeling. Using the most conservative approach to the MABEL assessment, a dose of 57.4 ng EGFRvIII:CD3 bi-scFv/kg body weight was selected as a safe starting dose for a first-in-human clinical study.
Chapter 5 describes the pharmacokinetic properties of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, the chapter first describes the development of a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, was used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ~8 minutes and a terminal half-life of ~2.5 hours. These results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and Phase I studies of EGFRvIII:CD3 bi-scFv.
Finally, Chapter 6 provides an outlook of the future development of cancer therapeutics for treating malignant gliomas.
Item Open Access Biochemical Characterization and Genetic Modeling of Glioma-Associated Mutations in Isocitrate Dehydrogenases.(2014) Lopez, Giselle YvetteGliomas are the most common tumors of the central nervous system. Our lab recently identified mutations in IDH1 and IDH2 as occurring frequently in progressive gliomas. We applied a series of biochemical and genetic approaches to explore the roles of the mutations in tumors and generate models for study.
IDH1/2 mutations have the potential to impact a number of metabolic pathways. IDH1/2 convert isocitrate to α-ketoglutarate while simultaneously converting NADP+ to NADPH. To assess changes in metabolism, we completed metabolic profiling and complementary studies in cell lines with and without mutant IDH1 or mutant IDH2. We identified a decrease in hypoxia signaling and a decrease in global 5-hydroxymethylcytosine in cell lines with mutant IDH1/2 .
Having observed mutations in IDH1/2 in a large fraction of progressive gliomas, we asked if the mutations were either 1) advantageous for growth in brain parenchyma, or 2) advantageous in a particular cell-of-origin. Sequencing of a series of metastases to the brain from non-central nervous system tumors identified no mutations in IDH1/2, lending less credence to the first hypothesis. To elucidate whether mutations in IDH1/2 can initiate glioma progression and explore the potential cell-of-origin for progressive gliomas, we generated mice in which we induced expression of mutant IDH2 in different populations of cells in the brain, either alone or in combination with TP53 deletion, another frequently altered gene in progressive gliomas. Mice with broad expression of mutant IDH2 developed hydrocephalus and encephalomalacia early in life, but did not develop tumors. Therefore, we restricted expression, and two brain tumors were identified in mice with both IDH2 mutation and TP53 deletion. While this suggests that both mutations might be required for the development of tumors, this is too small a number to draw significant conclusions. Further research with an expanded cohort of mice, utilization of additional drivers of expression, and further characterization of identified tumors will help in elucidating the role of mutant IDH2 and the cell-of-origin for progressive gliomas.
Item Open Access Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Regulates Dendritic Cells and Myeloid Derived Suppressor Cells Development in the Lymphoma Microenvironment(2016) Huang, WeiCalcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.
We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.
In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.
Item Open Access CAR T-cell Immunotherapy for Brain Tumors(2017) Suryadevara, CarterGlioblastoma (GBM) is the most common and deadly primary malignant brain tumor. Despite an aggressive multimodal standard of care, prognoses and patient quality of life remain exceptionally poor, due in part to the non-specific and toxic nature of conventional treatment options. By contrast, adoptive cell transfer of T cells genetically modified to express tumor-specific chimeric antigen receptors (CARs) has emerged as a promising approach to targeting brain tumors, given that T cells have migratory capacity within the brain parenchyma, a mechanism to discriminate between normal and neoplastic tissue, and can develop immunological memory. This work spans the development of an effective CAR T-cell immunotherapy strategy targeting the tumor-specific driver mutation, EGFRvIII, which is expressed exclusively by GBM and other cancers but not normal tissue.
Chapters 1 and 2 provide an overview of GBM and the current clinical standard of care, the role of the immune system as it relates to the development and eradication of cancer, and an introduction to various immunotherapy platforms under active preclinical and clinical investigation. Chapter 3 details the historical context of adoptive T-cell immunotherapy and its evolution to present day, detailing our early proof-of-principle studies that led to the inception of the original research described herein. Data presented in Chapter 4 summarizes our translational objectives in implementing CAR T-cell immunotherapy clinically for patients with newly-diagnosed GBM. Chapter 5 addresses a perennial limitation to the immunotherapy of solid tumors by demonstrating an ability of modified CARs to circumvent intratumoral immunosuppression mediated by regulatory T cells. In Chapter 6, we present data that demonstrate, for the first time, a novel role for host lymphodepletion in cellular immunotherapy delivered directly into the brain. Lastly, Chapter 7 contains concluding remarks on the current state of CAR technology and important future directions.
In summary, our work here demonstrates that CAR T cell immunotherapy 1) has curative potential against highly established, orthotopic and syngeneic murine GBM, 2) can be strategically implemented within the current clinical treatment paradigm for GBM, and 3) can overcome a major mechanism of immunosuppression, demonstrating the versatility of gene-modified T cells for the treatment of malignant brain tumors. Together, these studies have paved way for the rationale design of two phase I clinical trials in patients with newly-diagnosed and recurrent EGFRvIII-positive GBM at Duke University.
Item Open Access Cell-Surface GRP78 and its Antibodies: Pathologic and Therapeutic Roles in Cancer(2010) de Ridder, Gustaaf GregoireThe chaperone protein GRP78 is primarily expressed in the endoplasmic reticulum, but it is also aberrantly expressed on the surface of cells under pathological conditions. One the cell membrane, GRP78 acts as a signaling molecule with unique properties. The amino-terminal domain acts as a growth factor receptor-like protein, while the carboxyl-terminal domain functions as a death-signaling receptor-like protein to extrinsically induce apoptosis. Autoantibodies that react with cell-surface GRP78 on many tumor cell lines occur in the sera of patients with prostate cancer, melanoma, and ovarian cancer. These autoantibodies are a negative prognostic factor in prostate cancer and melanoma, and when purified, stimulate tumor cell proliferation in vitro. It is unclear, however, whether these IgGs are merely a biomarker, or if they actually promote tumor growth in vivo. We immunized C57Bl/6 mice with recombinant GRP78 and then implanted the B16F1 murine melanoma cell line as flank tumors. We employed the antisera from these mice for in vitro cell signaling and proliferation assays. The immunodominant epitope in human cancer patients was well represented in the antibody repertoire of these immunized mice. We observed significantly accelerated tumor growth, as well as shortened survival in GRP78-immunized mice as compared to controls. Furthermore, antisera from these mice, as well as purified anti-GRP78 IgG from similarly immunized mice, stimulate Akt phosphorylation and proliferation in B16F1 and human DM6 melanoma cells in culture. These studies demonstrate a causal link between a humoral response to GRP78 and the progression of cancer in a murine melanoma model. They support the hypothesis that such autoantibodies are involved in the progression of human cancers and are not simply a biomarker. Because GRP78 is present on the surface of many types of cancer cells, this hypothesis has broad clinical and therapeutic implications.
We generated and characterized a panel of monoclonal murine antibodies (mAbs) against GRP78 with the goal of identifying therapeutic candidate IgGs. We developed three stable hybridomas that produce interesting antibodies. The N88 IgG reacts with the NH2-terminal domain and is an agonist. The C38 IgG reacts with the COOH-terminal domain and is an antagonist of NH2-terminal signaling. The C107 IgG binds the COOH-terminal domain and induces apoptosis.
We examined the effect of these three mAbs on the growth of B16 flank tumors. N88 accelerated and C107 slowed tumor growth, while C38 had no net effect. We are currently developing these antibodies and derivatives thereof as therapeutics for melanoma as well as for cancers of the brain, breast, ovary, and prostate. In fact, any tumor cell that over-expresses GRP78 on its surface is a potential therapeutic target for our future studies.
Item Open Access Characterizing and Arresting Bone Marrow T-cell Sequestration in the Setting of Glioblastoma and Other Intracranial Tumors(2020) Chongsathidkiet, PakawatInitiation and maintenance of a productive anti-tumor immune response requires a functional T-cell repertoire. Disruptions to T-cell function contribute to tumor immune escape, and to failure of the anti-tumor immune response in cancer patients. T-cell dysfunction is particularly severe in certain types of cancers such as glioblastoma (GBM), which is the most common primary malignant brain tumor in adults and is extremely lethal. Despite near universal confinement to the intracranial compartment, GBM frequently depletes both the number and function of systemic T-cells. A lack of understanding of the mechanisms underlying T-cell dysfunction poses challenges to the goal of developing appropriate and meaningful therapeutic platforms. Currently available treatments, including immunotherapies, for GBM and other intracranial diseases have proven ineffective in part because of underlying T-cell dysfunction. Thus, there is an unmet need for therapies that effectively address T-cell dysfunction. In this dissertation, we explore bone marrow T-cell sequestration, a novel mode of T-cell dysfunction present in GBM and other intracranial tumors.
Chapter 1 provides a comprehensive review of the epidemiology, clinical manifestation and diagnosis, and current standard of care for GBM. Chapter 2 outlines immunotherapeutic strategies under investigation for GBM. Chapter 3 describes the fading notion of traditional brain immune privilege but provides the current understanding of how the brain remains immunologically distinct. In Chapter 4, we explore bone marrow T-cell sequestration, and how this mechanism is usurped by GBM and other intracranial tumors to prevent anti-tumor efficacy of T-cell based immunotherapeutic modalities. In Chapter 5, we propose β-arrestin 2 (BARR2) depletion as a strategy to overcome bone marrow T-cell sequestration. In summary, this original work provides encouraging insights for the development of strategies to enhance anti-tumor efficacy of T-cell based immunotherapy for GBM, reversal of bone marrow T-cell sequestration.
Item Open Access Characterizing Bladder Adaptive Immune Responses to Uropathogenic Escherichia coli Infections(2012) Chan, Cheryl Yuen YuThe mammalian urinary bladder is a highly specialized organ that must be able to withstand considerable amounts of osmotic pressure at its mucosal surface, in addition to maintaining an impenetrable barrier against potential pathogens. The lower urinary tract's virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected with uropathogenic E.coli (UPEC) in combination with clinical observations that antibody responses following bladder infections are not detectable, suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells. This mast cell-dependent phenomenon involves localized production of IL-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of mast cells orchestrating the early innate immune responses in the bladder during infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of mast cell-maintained immune privilege.
Interestingly, though the bladder is not capable of initiating an effective adaptive immune response during bladder infections, we have generated data showing that it was possible to circumvent the immune limitations of the bladder to provoke a strong adaptive and protective immune response by vaccinating against UPEC at an alternate mucosal site. We reasoned that by immunizing the nasal regions of mice with a vaccine formulation comprising of FimH adhesin, a highly conserved adhesive moiety of type 1 fimbriae expressed on UPEC, and an effective mucosal adjuvant we would evoke protective immunity against UPEC infections. We found that a FimH vaccine coupled with either a mast cell activating adjuvant c48/80 or CpG oligodeoxynucleotide, a TLR9 agonist, evoked high levels of FimH specific IgG antibody in the serum and IgA in the urine of immunized mice. We also observed that following UPEC challenge, these FimH/adjuvant immunized mice exhibited significantly reduced bacterial load in the bladders compared to mice challenged with just FimH. These studies reveal that immunization of nasal regions with a FimH vaccine is an effective strategy to overcome the limitation in adaptive immunity observed in the bladder.
Item Open Access Developing a Senomorphic Treatment Strategy in Osteoarthritis(2022) Chen, Yu-HsiuOsteoarthritis (OA) is the most common form of arthritis in the population worldwide, resulting in significant disabilities. Currently, no treatments can prevent or reverse the development of OA. Cellular senescence has been identified as a major contributing factor to OA. Therefore, a therapy targeting senescence could be an effective treatment for OA. Several strategies have been proposed to target senescence in OA, including neutralizing agents for the senescence-associated phenotypes (SASPs), senolytics for eliminating senescent cells, and senomorphics for modifying the senescence phenotype. In fact, a senolytic, UBX0101, showed a protective effect for post-traumatic OA (PTOA) development in mouse models by reducing both OA histological grading and OA related pain. However, it failed to meet the primary endpoint of relieving symptoms in a clinical phase 2 trial. The failure of the human trial may be related to a high placebo response rate from the control group or, more likely, the heterogeneous phenotypes involved in human OA disease differ from the PTOA in the mouse model. The lack of available senescent-specific biomarkers, which could be used to refine the phenotype of the subject enrollment or to monitor the occurrence of senescence presents a challenge to evaluate a trial successfully. Alternatively, senolytics may be detrimental in tissue with a higher proportion of senescent cells. After treatment, the remaining cells may not be able to maintain the integrity of the cartilage. Therefore, in the present study, we investigated the association of cellular senescence with OA disease severity, identified a biomarker dipeptidyl peptidase-4 (DPP4) for chondrocyte senescence and OA progression, and proposed a senomorphic treatment using chromobox 4 (CBX4) for modulating cell function of the replicative senescent model WI-38 cells and human osteoarthritic chondrocytes.First, we investigated the association of OA disease severity in human knee joints with the percentage of cells expressing senescence-associated β-galactosidase activity (SA-β-gal) and p16. We assessed three regions within the tibial plateau of the knee corresponding to a gradient of disease severities in tissue procured from human medial-dominant OA. We found that SA-β-gal and p16 were positively correlated with OA severity. Our result suggested cellular senescence could be involved in OA progression, and targeting senescence could be a promising treatment for OA. Subsequently, we identified Dipeptidyl-peptidase 4 (DPP4, also known as CD26) as a potential biomarker for OA senescence. We isolated chondrocytes from knee OA cartilage and determined the association of DPP4 expression with senescence markers, SA-β-gal and p16, by flow-cytometry. We also compared the expression of anabolic and catabolic genes, senescence-related genes, and senescence-associated secretory phenotypes (SASPs) in DPP4+ and DPP4- cells, isolated by two different methods: fluorescence-activated cell sorting and magnetic-activated cell sorting. Additionally, we quantified soluble DPP4 in synovial fluid (SF) and assessed its association with radiographic knee OA. DPP4 expression was associated with higher SA-β-gal, p16 expression, senescence-related gene and catabolic gene expression, SASPs secretion, and lower anabolic gene expression in chondrocytes. In addition, SF DPP4 was significantly associated with radiographic knee OA progression (β=4.657 p=0.015). Next, we identified the senomorphic effect of CBX4 in WI-38 cells. We first observed the decrease of CBX4 protein expression and increased senescence markers and gene expression during WI-38 serial culture. We next evaluated the presence of senescence markers and expression of senescence-related genes in the CBX4 activation and CBX4 knockdown compared to controls in pre-senescent WI-38. Compared to the control group, knockdown of CBX4 increased cellular senescence, whereas activation of CBX4 decreased senescence in the pre-senescent WI-38 cells. Based on these results, we identified that CBX4 regulates replicative senescence in WI-38 cells and functions as a senomorphic and potential anti-senescence target. Additionally, we explored the mechanisms of senescence regulation from CBX4 domains by using CBX4 mutated lentiviral particles and compared them with CBX4 wildtype in WI-38 cells. CDM, SIMs, and C-box domains are all involved in the regulation of senescence by CBX4; where CDM is mainly involved in cell cycle regulation, SIMs are involved in the cell proliferation, DNA damage repair, and SASPs secretion, and C-box is related to cell proliferation and SASPs secretion. Taken together, CBX4 is a multi-functional protein, and these mutants elucidated the different non-overlapping functions in senescence regulation. Finally, we identified the CBX4 senomorphic effect in osteoarthritic chondrocytes by comparing CBX4 wildtype and control transduced cells. Compared to control, CBX4 overexpressed chondrocytes demonstrated lower DPP4 expression and higher proliferation marker EdU. Overall, our study demonstrated that cellular senescence is positively correlated with OA disease severity, identified DPP4 as a potential biomarker for cellular senescence in OA, and explored CBX4 as a potential senomorphic treatment in human WI-38 fibroblasts and OA chondrocytes.
Item Open Access Developing Strategies to Target Glioblastoma Stemness and Immunosuppression(2023) Sun, Michael ABrain tumor-initiating cells (BTICs) drive tumor progression, immunosuppression, and resistance to treatments, posing formidable challenges to advancing effective treatments against glioblastoma (GBM). In this dissertation, we demonstrate that clemastine, an over-the-counter drug for treating hay fever and allergy symptoms, effectively attenuated the stemness and suppressed the propagation of primary BTIC cultures bearing PDGFRA amplification. These effects on BTICs were accompanied by altered gene expression profiling indicative of their more differentiated states, resonating with the activity of clemastine in promoting the differentiation of normal oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes. Functional assays for pharmacological targets of clemastine revealed that the Emopamil Binding Protein (EBP), an enzyme in the cholesterol biosynthesis pathway, is a target that mediates the suppressive effects of clemastine. Consistently, we showed that a neural stem cell-derived mouse glioma model displaying predominantly proneural features was similarly susceptible to clemastine treatment in vitro and in vivo. Surprisingly, we discovered that EBP protein is essential for BTIC propagation and stemness properties, and revealed a potential lipid-independent function of EBP in regulating epigenetic programming. Collectively, this original work identifies pathways indispensible for maintaining the stemness and progenitor features of GBMs, uncovers BTIC dependency on EBP, and suggests that non-oncology, low-toxicity drugs with OPC differentiation-promoting activity can be repurposed to target GBM stemness and aid in their treatment.Another key strategy extensively pursued for treating GBMs focuses on targeting endolysosomes, mainly on the basis that the intact function of these subcellular organelles is crucial for tumor cell autophagy and survival. Through gene expression analyses and cell type abundance estimation in GBMs, we showed that genes associated with the endolysosomal machinery are more prominently featured in non-tumor cells in GBMs than in the tumor cells themselves, and that tumor-associated macrophages represent the primary immune cell type that contributes to this phenomenon. Further analyses uncovered an enrichment of endolysosomal pathway genes in immunosuppressive and pro-tumorigenic macrophages, such as M2-like macrophages or those associated with worse prognosis in glioma patients, but not in those linked to inflammation and anti-tumorigenic properties. Specifically, genes critical to the hydrolysis function of endolysosomes, including progranulin and cathepsins, were among the most positively correlated with immunosuppressive macrophages, and elevated expression of these genes is associated with worse patient survival in GBMs. Together, these results implicate the hydrolysis function of endolysosomes in shaping the immunosuppressive microenvironment of GBM. We propose that targeting endolysosomes, in addition to its detrimental effects on tumor cells, can be leveraged for modulating immunosuppression to render GBMs more amendable to immunotherapies.
Item Open Access Development of Novel Antibody-Based Immunotherapies Targeting Human Chondroitin Sulfate Proteoglycan 4(2018) Yu, XinChondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human CSPG4 antibodies or antibody fragments. In addition, the efficacy of cytotoxic monotherapies, such as the CSPG4-targeting immunotoxins (ITs), is limited by hyperactive anti-apoptotic pathways prevalent in tumor cells. Therefore, there is a need to discover novel, fully human antibodies for CSPG4-targeting immunotherapies and to develop new strategies that sensitize resistant CSPG4-expressing tumor cells to IT therapies.
To discover fully human antibodies that can be developed into potential CSPG4-targeting therapeutics, my first aim is to develop novel human single-chain variable fragments (scFvs) with high binding affinity and specificity to the CSPG4 antigen. Affinity maturation was performed on a novel, fully human anti-CSPG4 scFv using the random mutagenesis approach. A yeast display library was constructed for the mutant clones, and screened using a modified whole-cell panning method followed by fluorescence-activated cell sorting (FACS). After six rounds of panning and sorting, the top seven mutant scFvs were isolated and their binding affinities were characterized by flow cytometry and surface plasmon resonance. These mutant clones were highly specific to the CSPG4 antigen, and displayed nanomolar to picomolar binding affinities. While each of them harbored only two to six amino acid substitutions, they represented approximately 270-3000-fold improvement in affinity compared to the parental clone. These affinity-matured scFvs can be potentially developed into diagnostic or therapeutic agents for evaluation and treatment of CSPG4-expressing tumors.
To facilitate the screening of scFv libraries targeting CSPG4, my second aim is to develop a cell-based fluorescent assay for high-throughput analysis of antibody affinity (KD) in the nanomolar range. In this method, fluorescently labelled antibodies were added to antigen-positive and antigen-negative cell lines fixed on 96-well plates. The fluorescent signals from nonspecific binding to negative control cell lines is subtracted from the specific binding to the antigen-positive cell lines. The results confirmed that the KD values obtained using this method were comparable with values obtained by the conventional flow cytometry and radioactive (I125) scatchard assays. This demonstrates that the cell-based fluorescent method allows for accurate and efficient identification of therapeutically relevant leads.
Finally, to improve the efficacy of ITs targeting CSPG4, especially in the IT-resistant tumor cells, my third aim is to evaluate a multi-pathway therapy that combines anti-CSPG4 ITs and small molecule Bcl-2 inhibitors. To enhance sensitivity of cancer cells to ITs, we combined ITs (9.2.27-PE38KDEL or Mel-14-PE38KDEL) targeting CSPG4 with a Bcl-2 inhibitor (ABT-737, ABT-263, or ABT-199) against patient-derived glioblastoma xenografts, melanoma cell lines, and breast cancer cell lines. Results from the in vitro cytotoxicity assays demonstrated that the addition of the ABT compounds, specifically ABT-737, sensitized all three tumors to the IT treatment, and in some cases improved the IC50 values of 9.2.27-PE38KDEL by over 1000-fold. Mechanistic studies using 9.2.27-PE38KDEL and ABT-737 revealed that the rate of IT internalization and the efficiency of cleaved exotoxin accumulation in the cytosol correlated with the enhanced sensitivity of the tumor cells to the combination treatment. Furthermore, the synergistic effect of 9.2.27-PE38KDEL and ABT-737 combination therapy was confirmed in an orthotopic GBM xenograft model and a model of melanoma metastasized to the brain. For the first time, our study compares the efficacy of ABT-737 and 9.2.27-PE38KDEL combination therapy in GBM and a different brain metastases model, providing insights into overcoming IT resistance in brain tumors.
In conclusion, I discovered novel human scFvs with high binding affinities to CSPG4, developed a cell-based fluorescent method for accurate and efficient affinity analysis of antibodies, and investigated combination immunotherapies that utilized Bcl-2 inhibitors to sensitize tumor cells to treatment by CSPG4-targeting ITs. The results from these studies helped to facilitate the development of novel antibody-based immunotherapies and combination immunotherapies for CSPG4-expressing tumors.
Item Open Access Developmentally Regulated Antigens for Immunologic Targeting of Molecular Subtypes of Medulloblastoma(2015) Pham, ChristinaMedulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. The heterogeneity of MB molecular subtypes as well as the failure of standard therapies to treat metastatic or recurrent disease necessitates more potent targeted approaches that minimize collateral toxicity. Immunotherapy presents a promising strategy by specifically targeting cancer cells and to date, there have been few successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting the proliferation and differentiation of progenitor cell populations within the developing cerebellum. To test the developing cerebellum as a source of tumor rejection antigens, we adapted two animal models of MB recapitulating human Sonic Hedgehog (SHH) and Group 3 tumors for immunotherapeutic evaluation. Immunologic characterization of these murine models revealed subtype-specific differences in the tumor microenvironment and a differential response to immune checkpoint blockade. We used total embryonic RNA from the developing mouse cerebellum (P5) to generate antigen-specific T cells and confirmed the immunogenicity of targeting developmentally regulated antigens in vitro. Developmental antigen-specific T cells produced high levels of Th1-type cytokines in response to two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells did not show any cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens conferred a significant survival benefit and long term cures in intracranial treatment models of SHH and Group 3 tumor bearing animals. We additionally tested whether the enrichment of select developmental antigens through the exclusion of normal brain transcripts would potentiate antitumor responses in both animal models. Finally, we evaluated the relevance of targeting fetal antigens across human MB subtypes. Our studies demonstrate that developmental antigens can safely target multiple MB subtypes and can be further refined to preferentially target individual subgroups. Further studies targeting immunogenic developmental antigens and leveraging this strategy with specific immune modulatory interventions represent a novel approach at utilizing patient molecular classification information to mediate safe and effective immunotherapy.
Item Open Access Dietary Carbohydrate Restriction Slows Prostate Tumor Growth(2008) Mavropoulos, John ChristakisGlucose metabolism remains an intensely explored topic of cancer biology since the initial discoveries of Otto Warburg nearly 80 years ago. Many solid tumors metabolize glucose primarily to lactate despite the availability of oxygen, revealing a dependence on glycolysis that may serve as a basis for targeted therapy. In particular, a diet devoid of carbohydrate may minimize the growth capabilities of glucose-dependent cancers. As our interests lie in prostate cancer, we examined whether a ketogenic diet devoid of carbohydrate (NCKD) would reduce the growth rate of tumors derived from human prostate cancer cell lines in a murine xenograft model.
Our initial experiments utilized the LAPC-4 cell line, a human androgen-sensitive prostate cancer cell line, in a SCID-mouse xenograft model to determine the effects of an NCKD on tumor growth and animal survival relative to two other diets: (1.) a Western-type diet (WD) reflecting consumptions patterns of men diagnosed with prostate-cancer in the Western world and (2.) a low-fat diet (LFD) representing the present standard of care. Following this study, we conducted a second study utilizing a different human prostate cancer cell line (LNCaP) in order to assess whether our initial observations were robust across multiple prostate cancer tumor models and to also further explore the molecular underpinnings of our observations. Both studies revealed the NCKD leads to a reduction in tumor growth rate and greater overall mouse survival relative to the WD. In addition, the NCKD was equivalent in these parameters to the LFD. We also observed key associations between survival and extent of urinary ketosis as well as favorable changes in insulin and insulin-like growth factor-1 (IGF-1) and gene expression that would be predictive of prolonged survival in mice consuming the NCKD.
We believe these data provide compelling evidence to consider a potential therapeutic role for dietary carbohydrate restriction in prostate cancer. We hope these results ultimately serve as a basis to conduct future clinical trials assessing whether dietary carbohydrate restriction, either alone or in combination with more conventional therapies, provides clinicians with an additional weapon against prostate cancer.
Item Open Access Driving Brain Tumorigenesis: Generation and Biological Characterization of a Mutant IDH1 Mouse Model(2014) Pirozzi, Christopher JamesDespite decades worth of research, glioblastoma remains one of the most lethal cancers. The identification of IDH1 as a major cancer gene in glioblastoma provides an exceptional opportunity for improving our understanding, diagnostics, and treatment of this disease. In addition to mutations in IDH1, recent studies from our laboratory have characterized the genetic landscape of gliomas and have shown the cooperation between IDH1 mutations and other oncogenic alterations such at TP53 mutations. Normally, IDH1 functions in the oxidative decarboxylation of isocitrate to α–ketoglutarate, however the mutant form confers neomorphic enzymatic activity by producing 2–hydroxyglutarate, an oncometabolite responsible for aberrant methylation in IDH1–mutated tumors, among other mutant IDH1–mediated phenotypes. To determine the role of mutant IDH1 in vivo, we generated a conditional knock–in mouse model. This genetically faithful system is both biologically and clinically relevant and will promote the understanding of mutant IDH1–mediated tumorigenesis while offering a route for therapeutic targeting.
We observed that broad expression of mutant IDH1 throughout the brain leads to hydrocephalus in 80% of animals. In assessing the earliest effects of mutant IDH1 on the brain, we determined mutant IDH1 confers a decrease in the proliferative cells of the subventricular zone of the lateral ventricle, the area which houses the neural stem cells in embryonic and adult animals. Additionally, a perturbation to the normal neural stem cell niche was observed in these animals. Combined, this data suggests that mutant IDH1 may be affecting the signaling pathways involved in differentiation in this population of cells. In vivo and in vitro studies will further elucidate mutant IDH1's effects on the differentiation patterns of neural stem cells expressing mutant IDH1.
To express mutant IDH1 in a more restricted manner and harness spatiotemporal control, we crossed mutant animals to a Nestin–CreERT2 strain of mouse that permits expression of floxed alleles upon treatment with tamoxifen. Animals were sacrificed at the onset of symptoms or at 1–year of age. We observed the development of both low– and high–grade gliomas in approximately 15–percent of E18.5 tamoxifen–treated animals. All tumors were found in a TP53–deleted background with mutant IDH1 being detected in only those tumors with the mutant allele. Lastly, to decrease the latency and increase the penetrance of tumor formation, an orthotopic intracranial injection model was generated to allow for visualization of tumor formation and development, as well as investigation of therapeutic modalities. The models generated and the knowledge gained from these studies will offer an understanding of the biological effects of the most common mutations found in the astrocytic subset of gliomas, bringing us strides closer to determining mechanisms and therapeutic targets for IDH1–mutated cancers.
Item Open Access Elucidating the Molecular Architecture of Cartilage by Proteomics(2015) Hsueh, MingFengArticular cartilage is a highly specialized avascular tissue and consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. The chondrocyte morphology and extracellular matrix properties vary with the depth of cartilage. Some past studies have defined the zonal distribution of a broad range of cartilage proteins in different layers. Based on the variations within each layer, the extracellular matrix can be further distinguished to pericellular, territorial and interterritorial regions. However, most of these studies used guanidine-HCl extraction that leaves an unextracted residual with a substantial amount of collagen. The high abundance of anionic polysaccharide molecules from cartilage adversely affects the chromatographic separation. Scatter oriented chondrocytes only account for the small proportion of the whole tissue protein extraction. However, the density of the cell varies with depth of cartilage as well. Moreover, the physiological status may also altered the extracellular matrix properties. Therefore, a comprehensive strategy to solve all these difficulties are necessary to elucidate the molecular structure of cartilage.
In this study, we used quantitative and qualitative proteomic analysis to investigate various cartilage tissue processing protocols. We established a method for removing chondrocytes from cartilage sections that minimized matrix protein loss. Quantitative and qualitative proteomic analyses were used to evaluate different cartilage extraction methodologies. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. The different extraction methods yielded different protein profiles. For instance, an overwhelming number of collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction.
Subsequently we applied these methods to extract cartilage sections from different cartilage layers (superficial, intermediate and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic). We also utilized lase capture microscopy (LCM) to harvest cartilage sample from individual subregions (territorial and interterritorial regions). The results suggested that there is more unique proteins existed in the superficial layer. By removing the chondrocytes, we were able to identify more extracellular matrix proteins. The phenotyping of cartilage subregions provided the chance to precisely localize the protein distribution, such as clusterin protein. We observed that the guanidine-HCl extractability (guanidine-HCl/ guanidine-HCl + in situ digestion extracts) of cartilage proteins. Proteoglycans showed high extractability while collagen and non-collagenous proteins had lower extractability. We also observed that the extractability might differ with depth of cartilage and also disease states might alter the characters as well.
Laser capture microscopy provides us the access to the cartilage subregions in which only few studies have investigated because of the difficulties to separate them. We established the proteomic analysis compatible-protocol to prepare the cartilage section for LCM application. The results showed that most of the proteoglycans and other proteins were enriched in the interterritorial regions. Type III and VI collagens, and fibrillin-1 were enriched in the territorial regions. We demonstrated that this distribution difference also varied with depth of cartilage. The difference of protein abundance between subregions might be altered because of disease states.
Last we were looking for the post-transliational modification existed in these subregions of cartilage. Deamidation is one of the modification without the enzyme involved. Previous studies have showed that deamidation may accumulated in the tissue with low turnover rate. Our proteomic analysis results suggests that abundance of deamidated peptides also varied in different layers and subregions of cartilage.
We have developed the monoclonal antibody based immunoassay to quantify the deamidated cartilage oligomeric matrix protein within cartilage tissue from different joints (hip and knee) and disease states (healthy, para-lesion, and remote lesion). The results suggests that the highest concentration of deamidated COMP was identified in arthritic hip cartilage.
The results of this study generated several reliable protocols to perform cartilage matrix proteomic analysis and provided data on the cartilage matrix proteome, without confounding by intracellular proteins and an overwhelming abundance of collagens. The discovery results elucidated the molecular architecture of cartilage tissue at different joint sites and disease states. The similarities among these cartilages suggested a constitutive role of some proteins such as collagen, prolargin, biglycan and decorin. Differences in abundance or distribution patterns, for other proteins such as for cartilage oligomaric matrix protein, aggrecan and hyaluronan and proteoglycan link protein, point to intriguing biological difference by joint site and disease state. Decellularization and a combination of extraction methodologies provides a holistic approach in characterizing the cartilage extracellular matrix. Guanidine-HCl extractability is an important marker to characterize the statue of cartilage; however it has not been fully understand. The protein distributions in matrix subregions may also serve as an index to characterize the metabolic status of cartilage in different disease states. A large sample cohort will be necessary to elucidate these characters.
Item Open Access Enhancing Cisplatin Delivery and Anti-tumor Efficacy Using Hyperthermia(2013) Landon, Chelsea DawnMild hyperthermia (39°C-43°C) has numerous therapeutic benefits as an adjuvant therapy in the treatment of a variety of tumor types. Hyperthermia increases tumor blood flow and vascular permeability, promoting drug delivery and tumor oxygenation. Hyperthermia enhances the uptake and efficacy of numerous chemotherapeutic agents, including cisplatin, resulting in increased cytotoxicity. In addition to these biological responses, hyperthermia can be used as a drug-release trigger for temperature-sensitive nanoparticles, resulting in an improved and more targeted drug delivery system. Cisplatin was chosen because 1) it shows broad spectrum activity against a wide range of heatable cancers (i.e., those in sites such as the pancreas, colon and rectum, cervix and bladder, and 2) the same hyperthermic temperatures that enable temperature-sensitive lipsome-drug release also enhance cisplatin-induced cytotoxicity.
The role of hyperthermia in enhancing cisplatin delivery and cytotoxicity was investigated at both the cellular and tissue levels. While hyperthermia treatment is applicable to a variety of tumor types, the focus of this work was on bladder cancer. The synergistic effects of hyperthermia and cisplatin were investigated, along with the role of copper transport protein 1 (Ctr1) in this process. In addition, cisplatin was encapsulated within temperature-sensitive liposomes, which were used in combination with hyperthermia for targeted drug delivery. These studies demonstrated that the combination of cisplatin and hyperthermia improved drug delivery, and potentially anti-tumor efficacy, and that targeted delivery was enhanced through incorporation of temperature-sensitive liposomes. As many current methods for administering bladder hyperthermia have drawbacks, such as invasiveness and regional heating, the final aim of this study was to develop and test a less-invasive and more focused preclinical bladder heating device in a rat model.
Hyperthermia sensitizes cells to the cytotoxic effects of the commonly used chemotherapeutic agent cisplatin by increasing drug accumulation and subsequent platinum-DNA adduct formation. However, the molecular mechanisms underlying this enhancement remain unclear. Understanding the fundamental mechanisms involved in the synergistic interaction is necessary to increase the therapeutic benefits of this combination in the clinic. The synergism between the anti-cancer benefits of cisplatin and the drug delivery benefits of hyperthermia may offer a novel and more effective treatment for many cancer patients. We hypothesized that hyperthermia increases cisplatin accumulation and efficacy in part by modulating the function of Ctr1, a major regulator of cellular cisplatin uptake. To test this hypothesis, we examined the significance of Ctr1 during combined hyperthermia and cisplatin therapies and assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerization in enhancing cisplatin cytotoxicity. We observed increased Ctr1 multimerization following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C), suggesting that this may be a mechanism for increased cisplatin uptake in heat-treated cells. The impact of increased Ctr1 multimerization was evaluated by measuring platinum accumulation in wild-type (WT) and Ctr1-/- cells. WT cells contained greater levels of platinum compared to Ctr1-/- cells. A further increase in platinum was observed following hyperthermia treatment, but only in the WT cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT cells with a dose-modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells. Our data suggest that heat increases Ctr1 activity by increasing multimerization, resulting in enhanced drug accumulation. Although we recognize that the effect of heat on cells is multi-factorial, our results support the hypothesis that Ctr1 is, in part, involved in the synergistic interaction observed with cisplatin and hyperthermia treatment.
In addition to assessing cisplatin delivery at the cellular level, we evaluated cisplatin delivery at the tissue level, using novel cisplatin-loaded temperature-sensitive liposomes. We hypothesized that delivering cisplatin encapsulated in liposomes under hyperthermic conditions would improve the pharmacokinetic profiles of cisplatin, increase drug delivery to the tumor, decrease normal tissue toxicity, and enhance the anti-tumor activity of cisplatin. We successfully prepared temperature-sensitive liposomes loaded with cisplatin and demonstrated that heat (42°C) sensitizes cisplatin-resistant cells to the cytotoxic effects of cisplatin in vitro.
Decreased toxicity was observed in animals treated with the cisplatin liposome (± heat) compared to the free drug treatments. A pharmacokinetic study of cisplatin-loaded temperature-sensitive liposomes and free drug was performed in tumor-bearing mice under normothermic and hyperthermic conditions. Cisplatin half-life in plasma was increased following liposome treatment compared to free cisplatin, and cisplatin delivery to the tumors was greatest in mice that received liposomal cisplatin under hyperthermia. These initial in vivo data demonstrate the potential effectiveness of this cisplatin-loaded liposome formulation in the treatment of certain types of cancer. To assess the anti-cancer efficacy of the liposome treatment, a tumor growth delay study was conducted and demonstrated equivalent efficacy for the cisplatin-loaded temperature-sensitive liposome compared to free drug.
In addition to the liposome work, we developed and evaluated a novel heating device for the bladder. Despite the evidence that hyperthermia is an effective adjuvant treatment strategy, current clinical heating devices are inadequate, warranting the development of a new and improved system. We induced hyperthermia using ferromagnetic nanoparticles and an alternating magnetic field device developed by Actium Biosystems. Initial preclinical studies in a rat model demonstrated preferential bladder heating. However, our preliminary studies show severe toxicity with the direct instillation of the nanoparticles in the bladder, and further studies are needed to potentially modify the nanoparticle coating, the catheterization procedure, as well as to develop a different animal model.
Item Open Access Enhancing Dendritic Cell Migration to Drive Antitumor Responses(2017) Batich, Kristen AnneThe histologic subtypes of malignant glial neoplasms range from anaplastic astrocytoma to the most deadly World Health Organization (WHO) Grade IV glioblastoma (GBM), the most common primary brain tumor in adults. Over the past 40 years, only modest advancements in the treatment of GBM tumors have been reached. Current therapies are predominantly for palliative endpoints rather than curative, although some treatment modalities have been shown to extend survival in particular cases. Patients undergoing current standard of care therapy, including surgical resection, radiation therapy, and chemotherapy, have a median survival of 12-15 months, with less than 25% of patients surviving up to two years and fewer than 10% surviving up to five years. A variety of factors contribute to standard treatment failure, including highly invasive tumor grade at the time of diagnosis, the intrinsic resistance of glioma cells to radiation therapy, the frequent impracticality of maximal tumor resection of eloquent cortical structures, and the fragile intolerance of healthy brain for cytotoxic therapies. Treatment with immunotherapy is a potential answer to the aforementioned problems, as the immune system can be harnessed and educated to license rather potent antitumor responses in a highly specific and safe fashion. One of the most promising vehicles for immunotherapy is the use of dendritic cells, which are professional antigen-presenting cells that are highly effective in the processing of foreign antigens and the education of soon-to-be activated T cells against established tumors. The work outlined in this dissertation encompasses the potential of dendritic cell therapy, the current limitations of reaching full efficacy with this platform, and the recent efforts employed to overcome such barriers. This work spans the characterization and preclinical testing of utilizing protein antigens such as tetanus-diphtheria toxoid to pre-condition the injection site prior to dendritic cell vaccination against established tumors expressing tumor-specific antigens.
Chapter 1 comprises an overview of the current standard therapies for malignant brain tumors. Chapters 2 and 3 provide a review of immunotherapy for malignant gliomas in the setting of preclinical animal models and discuss issues relevant to the efficacy of dendritic cell vaccines for targeting of GBM. Chapters 4 provides the rationale, methodology, and results of research to improve the lymph node homing and immunogenicity of tumor antigen-specific dendritic cell vaccines in mouse models and in patients with newly diagnosed GBM. Chapter 5 delineates the interactions discovered through efforts in Chapter 4 that comprise protein antigen-specific CD4+ T cell responses to induced chemokines and how these interactions result in increased dendritic cell migration and antitumor responses. Lastly, Chapter 6 discusses the future utility of migration of DC vaccines as a surrogate for antitumor responses and clinical outcomes.
This dissertation comprises original research as well as figures and illustrations from previously published material used to exemplify distinct concepts in immunotherapy for cancer. These published examples were reproduced with permission in accordance with journal and publisher policies described in the Appendix.
In summary, this work 1) identifies inefficient lymph node homing of peripherally administered dendritic cells as one of the glaring barriers to effective dendritic cell immunotherapy, 2) provides answers to overcome this limitation with the use of readily available pre-conditioning recall antigens, 3) has opened up a new line of investigation for interaction between recall responses and host chemokines to activate immune responses against a separate antigen, and 4) provides future prospects of utilizing chemokines as adjuvants for additional immunotherapies targeting aggressive tumors. Together, these studies hold great promise to improve the responses in patients with GBM.