<p>Prostate cancer is the second leading cause of cancer-related death among American men. Prostate tumor cells exhibit significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, variable patient response remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. It was hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy (Interleukin-27 (IL-27)) could treat bone-metastatic prostate cancer and also exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which promotes immune cell recruitment to tumors, while also promoting bone repair. To test this hypothesis, <i>in vivo</i> experiments were performed where syngeneic C57BL/6J mice were implanted intratibially with TRAMP-C2ras-Luc cells able to form tumors in bone. Immunotherapy was administered in the form of intramuscular gene therapy, delivering plasmid DNA encoding a reporter gene (Lucia), or a therapeutic gene (IL-27). Ultrasound was used to aid gene delivery. Various gene delivery methods were tested and optimized through <i>in vivo</i> studies, with microbubbles in combination with ultrasound (sonoporation) emerging as the best method. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging was used to monitor tumor size over time. Combinatorial therapy inhibited tumor growth and improved survival. Further, RNA sequencing and cytokine arrays were used to investigate the mechanisms involved. Microcomputed tomography and differentiation assays indicated that the combination therapy improved bone health by improving osteoblast differentiation and inhibiting osteoclast differentiation. Our conclusion is that a chemo-immunotherapy approach such as the one examined in this work has potential to emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer. This approach should enable a significant reduction in chemotherapy-associated toxicity, improving sensitivity to immunotherapy, and simultaneously improving bone quality.</p>

<p>Sea cucumbers have been found to contain bioactive compounds such as saponin, fucoidan, frondoside, and glycosides that have pharmacological properties like antitumor, antibacterial, anti-inflammation, and antihyperglycemic activity. Although several species of sea cucumbers have been studied and reared for the food and medicinal industries, not much research has been conducted on the species in the waters of the Eastern United States. In this research, physiological and immunological parameters of coelomic fluid from <em>Cucumaria</em> <em>frondosa</em>, <em>Isostychopus</em> <em>badionotus</em>, and <em>Pentacta</em> <em>pygmaea</em> were compared to find the most promising candidate with these properties and pharmacological benefits. We found that <em>C. frondosa</em> was the species with the best immunological and physiological parameters among the three studied. <em>C. frondosa</em> illustrated that its coelomic fluid contains the highest concentrations of cells and lysozymes that had the highest activity. Using <em>C. frondosa</em>’s tissue extracts and coelomic fluid, the ability of the extracts and coelomic fluid to inhibit murine melanoma cells (B16-F10) and modulate T-lymphocytes <em>in vitro</em> were investigated. Although no significant differences were seen statistically, the experiments illustrated that T-lymphocytes were highly activated at higher concentrations (0.001g/uL-0.0002g/uL) for tissue extracts and at lower concentrations (0.000008g/uL) for coelomic fluid. On the other hand, melanoma cells were inhibited highest at lower concentrations (0.000008g/uL-0.0000016/uL). In addition to these studies, the antibacterial activity of <em>C. frondosa</em> extract was tested on ten pathogenic bacterial species. Antibacterial activity of the <em>C. frondosa</em> extract was not seen in this experiment. However, hemolytic activity by compounds present in <em>C. frondosa</em> extracts was seen in blood agars culturing <em>Streptococcus pneumoniae</em> and <em>Enterococcus faecalis</em> in our experiment. Lastly, an <em>in vivo </em>study was conducted to see if <em>C. frondosa</em> extract can modulate stress in Nile tilapia. In our experiment, we observed that <em>C. frondosa</em> extract was able to enhance the activity of one of the parameters, phagocytic capacity significantly. However, we are not able to conclude that <em>C. frondosa</em> extract was able to mitigate chronic stress from the results obtained. Overall, observing the results from the projects, we cannot conclude that <em>C. frondosa</em> extracts illustrated pharmacological properties. Extensive studies are recommended and required to use <em>C. frondosa</em> extract for medicinal purposes. </p>

Der adoptive Transfer von T-Zell-Rezeptor (TZR) modifizierten T-Zellen gegen krebsspezifische Antigene ist ein vielversprechender Ansatz in der Immuntherapie. Geeignete Zielmoleküle für diese Therapie sollten wichtig für das Überleben von Krebszellen sein und zudem in ausreichenden Mengen auf der Zelloberfläche exprimiert werden, um von T-Zellen erkannt zu werden. Die Identifizierung dieser Zielmoleküle ist jedoch eine Herausforderung und erfordert eine intensive Charakterisierung, um eine ausreichende Prozessierung und Präsentation auf den Tumorzellen zu validieren. Ziel dieser Arbeit war, HLA-A2-spezifische Neoepitope als Zielmoleküle für adoptive T-Zell-Therapie zu validieren. Dafür wurden erfolgreich Immunantworten in einem humanen transgenen Mausmodell nach Peptidimmunisierung induziert und TZRs mit hoher Affinität isoliert. Trotz einer hohen funktionellen Avidität von H3.3K27M-spezifischen T-Zellen wurde keine Erkennung von Tumorzellen erreicht. Zweitens wurden TZR-transduzierte T-Zellen gegen die häufige Melanommutation Rac1P29S isoliert, welche zytotoxisch gegen Melanomzelllinien waren. Letztlich wurde beobachtetet, dass TZRs mit hoher Affinität gegen gespleißte Kras und Rac2 Epitope, welche durch Proteasom-katalysiertes Peptidspleißen erzeugt wurden, keine Immunantwort gegen endogen exprimierte Mutationen hervorrufen konnten. Daraus lässt sich schließen, dass gespleißte Epitope wahrscheinlich seltener vorkommen als zuvor angenommen und daher möglicherweise irrelevant für die adoptive T-Zelltherapie sind. Diese Daten deuten darauf hin, dass die Auswahl von Zielmolekülen für die adoptive T-Zell-Therapie mit Hilfe reverser Immunologie auf der Grundlage von Bindungsalgorithmen und der Häufigkeit von Mutationen allein nicht ausreicht. Daher sind vor der Isolierung und Charakterisierung von TZRs zusätzliche Strategien wie z.B. die Analyse des MHC-Immunopeptidoms erforderlich, um die Auswahl geeigneter Zielmoleküle für die T-Zelltherapie zu verbessern. / Adoptive transfer of T cell receptor (TCR)-engineered T cells against tumour-specific neoantigens is a promising approach in cancer immunotherapy. Ideally, targeted antigens are crucial for cancer cell survival and are generated in sufficient amounts to be recognised by T cells. However, the identification of ideal targets remains challenging and requires intensive characterisation to validate sufficient antigen processing and presentation by the tumour cells. This thesis focused on the validation of HLA-A2 binding neoepitopes carrying the recurrent cancer mutations H3.3K27M, Rac1P29S, Rac2P29L or KrasG12V as targets for adoptive T cell therapy. After peptide immunisation, immune responses in a human transgenic mouse model were elicited and high-affinity TCRs successfully isolated. Although H3.3K27M-specific T cells showed high functional avidity, no recognition of cells endogenously expressing mutant H3.3 was achieved. Furthermore, a mechanism to target the common melanoma mutation Rac1P29S with a TCR raised against a heterologous mutation with higher peptide-MHC affinity was described. TCR-transduced T cells induced cytotoxicity against Rac1P29S expressing melanoma cell lines. Lastly, high-affinity TCRs specific for mutant Kras and Rac2 spliced epitopes generated by proteasome-catalysed peptide splicing were successfully isolated, however, TCR-transduced T cells did not induce an immune response against endogenously expressed mutant transgenes. The results indicate that spliced epitopes are probably less abundant than previously estimated and therefore may play a minor role in the generation of targets for adoptive T cell therapy. These data suggest that target selection using a reverse immunology approach based on binding algorithms and frequency of mutations alone is not sufficient. Thus, additional strategies to improve the selection of suitable targets such as the analysis of the MHC immunopeptidome are required prior to TCR isolation and characterisation.

This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.

Breast cancer (BC) is one of the deadliest forms of cancers with high incidence and mortality rates, especially in women. Encouragingly, targeted therapies have improved the overall<br>survival and quality of life in patients with various subtypes of BC. Unfortunately, these first-line therapies often fail due to inherent as well as acquired resistance of cancer cells. Treatment evading cancer cells can exhibit systemic dormancy in patients over a long period of time without manifesting any symptoms. In a suitable environment, these undetected disseminated tumor cells can relapse in the form of metastasis. Therefore, it is essential to understand the mechanisms of<br><div>BC recurrence and to develop durable therapeutic interventions to improve patient’s survival. In this dissertation work, we studied fibroblast growth factor receptors (FGFR), as therapeutic targets to treat the recurrence of drug-resistant and immune-dormant BC metastasis. <br></div><div><br></div><div>The HER2 subtype of BC is characterized by the overexpression of human epidermal growth factor receptor 2 (HER2), which drives elevated downstream signaling promoting tumorigenesis. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in which an anti-HER2 antibody targets HER2 overexpressing tumor cells and delivers a highly potent microtubule inhibitor. Using novel models of minimal residual disease (MRD) following T-DM1 treatments, we found that epithelial to mesenchymal transition is a critical process for cells to persist the TDM1 treatments. The upregulation of FGFR1 may facilitate insensitivity to T-DM1. Our data also showed that FGFR1 overexpression in HER2+ tumors leads to a higher incidence of recurrence, and these recurrent tumors show sensitivity towards covalent inhibition of FGFR. <br></div><div><br></div><div>In addition to drug-induced MRD in the primary tumor sites, disseminated tumor cells (DTCs) can demonstrate dormant phenotype via maintaining an equilibrium with immunemediated tumor clearance. Factors affecting such equilibrium may contribute to the recurrence of breast cancers metastasis. We show that such immune-mediated dormancy can be modeled with the 4T07 tumors. These tumors display immune-exclusion phenotypes in metastatic pulmonary organs. The inhibition of FGFR modulates the immune cell compositions of pulmonary organs favoring anti-tumor immunity. However, inhibition of FGFR may also affect T cell receptor downstream signaling, resulting in the inhibition of cytolytic T cell’s function. Finally, we report that combination therapy using the FGFR kinase inhibitor and an immune checkpoint blockade showed effective targeting of metastatic 4T07 tumors. <br></div><div><br></div><div>FGFR signaling as a therapeutic target in various tumors has been an active focus of cancer research. In this dissertation work, we have expanded our understanding of the role of FGFR in the recurrence of drug-resistant breast cancers as well as in the maintenance of an immune evasive microenvironment promoting pulmonary growth of tumors. Moreover, we presented evidence that it is possible to repurpose FGFR targeted therapy alone or in combination with checkpoint blockades to target recurrent metastatic BCs. In the future, our novel models of minimal residual diseases and systemic immune dormancy may act as valuable biological tools to expand our understanding of the minimal residual disease and dormant tumor cells.</div>

<p dir="ltr">Metastatic breast cancer (BC) remains a dauting therapeutic challenge due to the heterogeneity and cellular plasticity that exists. Because of these, BC resistance to targeted therapies and immune checkpoint blockade (ICB) present major challenges in the clinical setting. As a result, incomplete clearance of BC during a therapeutic regimen can lead to the persistence of minimal residual disease (MRD) which greatly contributes to tumor relapse. Here we develop a powerful in vivo model of lung metastasis in which we can achieve robust pulmonary tumor regression in response to the fibroblast growth factor receptor (FGFR) inhibitor, pemigatinib.</p><p dir="ltr">To enhance the efficacy of ICB, tumors must first be converted from an immune “cold” environment to an immune “hot” environment. Using our in vivo model of lung metastasis, we demonstrated that pemigatinib can significantly increase the presence of infiltrating T-cells into the lungs while suppressing the presence of MDSCs both locally in the lungs and systemically. Taken together, pemigatinib is an ideal candidate to prime these immune “cold” tumors for combination with ICB.</p><p dir="ltr">Upon establishment of MRD by pemigatinib in our in vivo model we observe upregulation of an alternate growth factor receptor, platelet-derived growth factor receptor (PDGFR). Functionally, upon FGFR inhibition, there is increased response to pulmonary fibroblast derived PDGF ligand, fueling survival of MRD. We demonstrated that knockdown of PDGFR significantly delayed tumor growth reinitiation in an in vitro 3D culture following pemigatinib as well as delayed tumor relapse in our pulmonary metastasis model.</p><p dir="ltr">To limit cellular plasticity and reduce survival of MRD, we propose a novel dual-targeted approach utilizing pemigatinib, in conjunction with inhibition of DNMT1 using the reversible inhibitor GSK3484862. We used our in vivo model of lung metastasis after treatment with pemigatinib as a model of cellular plasticity to targeted therapy. This combination therapy prevented growth factor plasticity and delayed tumor recurrence. Through prevention of PDGFR upregulation induced by pemigatinib.</p><p dir="ltr">In the present dissertation works, our study demonstrates pemigatinib’s robust ability to increase infiltrating T-cells in addition to its strong antitumor effects on pulmonary tumors. Despite the robust effects of pemigatinib, acquired mechanism of resistance through upregulation of PDGFR allows survival of MRD and are supported by PDGF secreting fibroblasts. Using an approach of limiting cellular plasticity through DNA methylation inhibition combined with pemigatinib, we achieved a more durable therapeutic response. Our findings underscore the significance of understanding adaptive responses to targeted therapies and provide a tangible therapeutic strategy to prolong treatment response in metastatic breast cancer.</p>

<p>  </p> <p>The phosphatases of regenerating liver are a subfamily of protein tyrosine phosphatases that consist of PRL1, PRL2 and PRL3. The overexpression of PRLs promote cell proliferation, migration and invasion and contribute to tumorigenesis and metastasis to aggravate survival outcome. Although there is increasing interest in understanding the implication of these phosphatases in tumor development, currently, limited knowledge is available about their mechanism of action and the efficacy of PRL inhibition in <em>in vivo</em> tumor models, the tumor extrinsic role of PRLs that allow them to impact tumor development, as well as <em>in vivo</em> physiological function of PRLs that could implicate them in diseases other than cancer. The work presented here aims to address these limitations.</p> <p><br></p>