Dissertations / Theses on the topic 'Orthotopic mouse model'

Le cancer colorectal (CCR) est le troisième cancer le plus fréquent et le deuxième cancer le plus mortel dans le monde. En conséquence, de nouveaux biomarqueurs diagnostiques ainsi qu’une amélioration des traitements actuels sont nécessaires. Les tumeurs se développent dans des microenvironnements complexes où les cellules cancéreuses interagissent et modulent la réponse immunitaire locale pour persister et se multiplier. Les lectines de type C, exprimées notamment par les cellules de l’immunité, régulent la réponse anti-cancéreuse, et donc le développement tumoral. Parmi elles, l'immunorécepteur des cellules dendritiques (DCIR), a été montré comme jouant un rôle immunitaire majeur au cours des maladies infectieuses et auto-immunes. À l’inverse, son rôle dans l'immunité tumorale reste méconnu. L'analyse des données transcriptomiques de deux cohortes de patients atteints de CCR a révélé un lien entre une expression élevée de DCIR et une meilleure survie des patients. Dans ce contexte, l'objectif principal de ma thèse était de déterminer l'impact de DCIR sur le développement du CCR et la réponse immunitaire associée. Dans ce but, j’ai établi un modèle murin préclinique, orthotopique et syngénique du CCR consistant en l'injection intracaecale de cellules tumorales MC38 exprimant la luciférase (MC38-fLuc+) dans des souris C57BL/6. Le suivi de la croissance tumorale par bioluminescence a montré que, malgré l’acquisition initiale de tumeurs solides par toutes les souris, seulement 30% des souris ont développé un CCR progressif et mortel, tandis que les autres animaux ont spontanément rejeté leurs tumeurs. Aucun rejet des tumeurs CCR MC38 n'a été observé en l'absence d'immunité adaptative, ni lors de l'injection de cellules MC38 dans d'autres sites anatomiques. L'immunophénotypage par transcriptomique et cytométrie de flux a révélé que les souris développant des tumeurs progressives présentaient une réponse immunitaire pro-tumorale, définie par une signature caractéristique des lymphocytes T régulateurs, détectable peu après l'implantation tumorale, et par une infiltration de cellules myéloïdes suppressives connues pour favoriser la croissance tumorale. En revanche, les souris rejetant les tumeurs présentaient une signature pro-inflammatoire précoce et un microenvironnement anti-tumoral enrichi en lymphocytes T CD8+. Ainsi, nos résultats démontrent un rôle du microenvironnement spécifique du côlon dans la régulation de l'équilibre entre les réponses immunitaires anti- ou pro-tumorales et souligne l'importance d'utiliser des modèles murins orthotopiques pour les études in vivo. Dans la seconde partie de ma thèse, j’ai utilisé ce modèle murin de CCR pour comparer le développement tumoral dans des souris C57BL/6 de type sauvage ou des souris déficientes pour l’expression de mDcir1 (mDcir1-KO), un homologue murin du DCIR humain. Bien que l'absence de mDCIR1 n'ait aucune incidence sur le pourcentage de souris développant ou rejetant les tumeurs du CCR, nous avons observé que les animaux mDcir1-KO développaient des tumeurs plus importantes que les sauvages En accord avec ce résultat, nous avons constaté une infiltration plus faible de lymphocytes CD8+ cytotoxiques et une activation moindre des lymphocytes T CD4+ et CD8+ (c'est-à-dire T-BET+, CD44haut, CTLA-4+) dans les tumeurs des souris mDcir1-KO par rapport aux souris sauvages. Ainsi, nos données indiquent un rôle protecteur et anti-tumoral de DCIR pendant le développement du CCR, probablement dû à une dérégulation de l'équilibre existant entre la tumeur et la réponse immunitaire. Dans l'ensemble, cette étude ouvre la voie à la mise au point éventuelle de biomolécules pharmacologiques ciblant DCIR pour déclencher une réponse immunitaire anti-tumorale efficace dans le contexte du CCR et au-delà
Colorectal cancer (CRC) is the third most common and second deadliest cancer worldwide accounting for 900.000 deaths in 2018. Consequently, there is a strong need for new biomarkers as well as an improvement of the current treatments. Tumors develop in complex microenvironments where cancer cells constantly crosstalk with, and modulate, the local immune response to persist and replicate. C-type lectins receptors, expressed in particular by immune cells, actively regulate the immune response to cancer cells and, therefore, tumor development. Dendritic cell immunoreceptor (DCIR), a C-type lectin expressed by myeloid cells, has been shown to play a major role in immunity to infectious and autoimmune diseases. Yet, the role played by DCIR in tumor immunity remains unknown. Analysis of publicly available transcriptomic data from two cohorts of CRC patients revealed an association between high DCIR gene expression and improved survival of patients. In this context, the principal objective of my PhD thesis was to determine the role played by DCIR in the immune response during CRC development. First, I developed an orthotopic syngeneic pre-clinical CRC mouse model consisting in the intra-caecal injection of engineered MC38 tumor cells expressing firefly luciferase (MC38-fLuc+) in C57BL/6 mice. Monitoring of the tumor growth by bioluminescence revealed that, despite an initial growth of solid tumors in all the mice, only 30% of mice developed a progressive lethal CRC, while the remaining animals spontaneously rejected their solid tumor and survived more than 100 days. No rejection of tumors was observed in the absence of adaptive immunity, nor when MC38-fLuc+ cells were injected in other anatomical locations (i.e., liver and skin). Immunophenotyping by transcriptomic and flow cytometry showed that mice with progressive CRC tumors exhibited a pro-tumor immune response, characterized by a regulatory T cell pattern, discernible shortly post-tumor implantation, as well as myeloid suppressor cells that are well-known to favor tumor growth. By contrast, tumor-rejecting mice presented an early pro-inflammatory response and an anti-tumor microenvironment enriched with CD8+ T cells. Taken together, our results demonstrate a preponderant role of the colon-specific microenvironment in regulating the balance between anti- or pro-tumor immune responses and underline the importance of using orthotopic mouse models for in vivo studies. In a second part of my thesis, we used this CRC mouse model to compare the tumor development in wild-type (WT) C57BL/6 mice or mice deficient for mDcir1 (mDcir1-KO), a murine homologue of human DCIR. While the lack of mDCIR1 has no impact on the percentage of mice developing or rejecting CRC tumors, we observed that mDcir1-KO animals developed bigger tumors than their WT counterparts. In line with this result, we found a lower infiltration of cytotoxic CD8+ and decreased activation of both CD4+ and CD8+ T cells (i.e., T-BET+, CD44high, CTLA-4+) in CRC tumors from mDcir1-KO mice compared to WT mice. Altogether, our data point to a protective and anti-tumor role of DCIR during CRC development, probably due to a dysregulation of the balance existing between the tumor and the immune response. Overall, this study paves the way for the potential future development of pharmacological biomolecules targeting DCIR to trigger an efficient anti-tumor immune response in the context of CRC and beyond

Renal cell carcinoma (RCC) is the third most common genito-urinary cancer. Beyond surgery, few other treatment options for RCC exist and about one-third of the patients who have had nephrectomy develop metastasis subsequently. The treatment of the metastatic disease remains a clinical challenge. Hence, novel therapeutic options are necessary for the patients with metastatic RCC. Immunotherapy is the most common mode of treatment in RCC presently; however, it contributes to a large number of toxic side effects to the patients. The immunotherapeutic regimens currently used to treat metastatic renal cancer are recombinant human interleukin -2 (IL-2) and recombinant human interferon alpha alone or in combination. However, the uses of these high dose cytokines are limited by their toxicity and poor patient response rates. Preclinical studies in animal tumor models of RCC are required to address the newer and effective therapeutic approaches for late stage metastatic RCC. A suitable animal model for studying RCC is lacking. Hence, development of a novel animal model would largely contribute in testing newer therapeutics and combating the metastatic disease. Cyclooxygenases are group of enzymes that catalyze the conversion of arachidonic acid to prostaglandins (PGE2). It comprises of two isoforms Cox-1 and Cox-2. Previous studies have implicated the potential role of Cox-2 in carcinogenesis and the expression of Cox-2 have been reported in colorectal, lung, breast, gastric and esophageal carcinomas. Cox-2 is also highly expressed in RCC and a potential biomarker in RCC. Based on emerging clinical evidences on the role of Cox-2 in several malignancies, we hypothesize that overexpression of Cox-2 in RCC promotes tumor growth and metastasis. Selective Cox-2 inhibitors act by inhibiting PGE2 synthesis and have been shown to retard tumor formation, metastasis, and angiogenesis. They induce apoptosis and inhibit the PGE2 induced immunosupression. Thus, a specific Cox-2 inhibitor like indomethacin or NS398 would be able to inhibit the tumorigenic properties of Cox-2, thereby attenuating tumor growth and dissemination to other organs. In this context, we injected the Cox-2 engineered Renca cell lines (COX2 -ve and COX2 +ve) in the subcapsular space of kidney of a Balbc/Cr mice. This resulted in the tumor growth, which was monitored by bioluminescence imaging (BLI) for a period of three weeks post-inoculation. Metastases were evident in distant organs such as lung, liver, spleen and lymph nodes. This was expressed as luciferase activity per milligram of protein of the particular tissue normalized with the background luciferase activity per milligram of the tissue from control (non-injected mice). Thus, the animal model was established and validated in our preliminary studies. To address the potential role of PGE2 in the tumor microenvironment, tumors were harvested and then processed for assessment by histology and immunohistochemistry. Initial characterization studies included immunohistochemical assessment of tumor vasculature as elucidated by staining with specific markers for lymphatic vessels (LYVE-1), blood endothelium (CD31) and tumor infiltrating macrophages (Cd11b). Macrophage recruitment close to the LYVE-1+ structures were also determined by the double positive events obtained by staining with both CD11b and LYVE-1. A large number of peripheral (18%±2.79), intratumoral (12%±3) and marginal (7%±2.01) LYVE-1+ve structures were found in PGE2 producing tumor than in the control tumor. Although, the frequency of blood vessels in both the tumor types were unaltered, however, an increased vascular area was obtained in the COX2 +ve tumor than in COX2 -ve tumor. There was a significant increase in the frequency of infiltrating macrophages in the peripheral (25%± 3.86), intratumoral (10%± 3.93) and tumor-kidney margin of COX-2 positive tumors (10%± 2.34) than in the COX-2 negative tumors (12% ± 2.36, 0% and 0%) respectively. Frequency of the blood vessels in both the tumors were unaltered, however, a significant increase in the mean fluorescent intensity (MFI) in the peripheral region (4.3±0.1) of COX2 +ve tumor was observed when compared to COX2 -ve tumors (2.3±0.05). These preliminary studies indicate the potential role of PGE2 in promoting tumor vasculature, increased macrophage recruitment within the tumors and tumor-kidney margins. In our initial studies, a significant enrichment of LYVE-1+ve macrophages were observed in the kidney-tumor sections of the COX2 +ve mice, which might indicate that, PGE2 may promote differentiation of the macrophages into a lymphatic phenotype. Thus, this animal model would further help in thorough characterization of other immune infiltrating cells like CD8+ T cells and NK cells and thereby lead us to identify the cause of immune dysregulation in renal cell carcinoma due to RCC derived soluble factors like PGE2 and TGF-beta. Furthermore, treatment with Cox-2 inhibitors like NS-398 should retard tumor growth, metastases, immune cell dysregulation, and tumor vasculature.

This Thesis presents the first steps of the development of a humanized and patient-specific mouse model of multiple myeloma (MM). Novel therapeutic approaches are getting increasingly complex and relevant pre-clinical drug testing is becoming a great challenge. The mouse model presented here features a humanized bone marrow compartment with a humanized bone shell that is able to engraft MM cells and patient-derived hematopoietic stem cells, mimicking myeloma bone disease serving as a unique platform for MM drug testing and discovery.

Preclinical osteosarcoma (OS) models often fail to predict effects of cancer drugs. Two humanized bone models are described which comprise human bone matrix, vascular and hematopoietic components. The first model utilizes ectopic implantation of human bone and rhBMP-7 into mice. Injection of OS cells produces high similarities to the human disease. A second scaffold-based model allows modular orthotopic application. Human osteoblasts seeded into mPCL-CaP scaffolds and human endothelial cells within a biomimetic hydrogel are positioned around the mouse femur. Human CD34+ transplantation allows humanization of the hematopoietic system. Analysis demonstrated that the models allow predictive marker studies of bone malignancies.

Background Novel theranostic options for high-risk non-muscle invasive bladder cancer are urgently needed. This requires a thorough evaluation of experimental approaches in animal models best possibly reflecting human disease before entering clinical studies. Although several bladder cancer xenograft models were used in the literature, the establishment of an orthotopic bladder cancer model in mice remains challenging. Methods Luciferase-transduced UM-UC-3LUCK1 bladder cancer cells were instilled transurethrally via 24G permanent venous catheters into athymic NMRI and BALB/c nude mice as well as into SCID-beige mice. Besides the mouse strain, the pretreatment of the bladder wall (trypsin or poly-L-lysine), tumor cell count (0.5 × 106–5.0 × 106) and tumor cell dwell time in the murine bladder (30 min – 2 h) were varied. Tumors were morphologically and functionally visualized using bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and positron emission tomography (PET). Results Immunodeficiency of the mouse strains was the most important factor influencing cancer cell engraftment, whereas modifying cell count and instillation time allowed fine-tuning of the BLI signal start and duration – both representing the possible treatment period for the evaluation of new therapeutics. Best orthotopic tumor growth was achieved by transurethral instillation of 1.0 × 106 UM-UC-3LUCK1 bladder cancer cells into SCID-beige mice for 2 h after bladder pretreatment with poly-L-lysine. A pilot PET experiment using 68Ga-cetuximab as transurethrally administered radiotracer revealed functional expression of epidermal growth factor receptor as representative molecular characteristic of engrafted cancer cells in the bladder. Conclusions With the optimized protocol in SCID-beige mice an applicable and reliable model of high-risk non-muscle invasive bladder cancer for the development of novel theranostic approaches was established.

Background Novel theranostic options for high-risk non-muscle invasive bladder cancer are urgently needed. This requires a thorough evaluation of experimental approaches in animal models best possibly reflecting human disease before entering clinical studies. Although several bladder cancer xenograft models were used in the literature, the establishment of an orthotopic bladder cancer model in mice remains challenging. Methods Luciferase-transduced UM-UC-3LUCK1 bladder cancer cells were instilled transurethrally via 24G permanent venous catheters into athymic NMRI and BALB/c nude mice as well as into SCID-beige mice. Besides the mouse strain, the pretreatment of the bladder wall (trypsin or poly-L-lysine), tumor cell count (0.5 × 106–5.0 × 106) and tumor cell dwell time in the murine bladder (30 min – 2 h) were varied. Tumors were morphologically and functionally visualized using bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and positron emission tomography (PET). Results Immunodeficiency of the mouse strains was the most important factor influencing cancer cell engraftment, whereas modifying cell count and instillation time allowed fine-tuning of the BLI signal start and duration – both representing the possible treatment period for the evaluation of new therapeutics. Best orthotopic tumor growth was achieved by transurethral instillation of 1.0 × 106 UM-UC-3LUCK1 bladder cancer cells into SCID-beige mice for 2 h after bladder pretreatment with poly-L-lysine. A pilot PET experiment using 68Ga-cetuximab as transurethrally administered radiotracer revealed functional expression of epidermal growth factor receptor as representative molecular characteristic of engrafted cancer cells in the bladder. Conclusions With the optimized protocol in SCID-beige mice an applicable and reliable model of high-risk non-muscle invasive bladder cancer for the development of novel theranostic approaches was established.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, May, 2020
Cataloged from the official PDF of thesis.
Includes bibliographical references (pages 70-75).
Liver metastasis is a major cause of mortality in patients with colorectal cancer (CRC). Immune checkpoint blockade (ICB) therapy has significantly improved overall survival in several cancer types including melanoma and non-small cell lung cancer. However, patients with mismatch repair-proficient (pMMR) metastatic CRC do not respond to ICB therapy. MC38 and CT26 are two of the most commonly used mouse syngeneic CRC cell lines in preclinical studies of colorectal cancer. In most of these preclinical studies, MC38 and CT26 are implanted under the skin in the hind flank of the mice where they grow into subcutaneous tumors. Several studies have shown that these subcutaneous MC38 or CT26 tumors respond very well to ICB treatment. However, MC38 and CT26 have been reported previously to be pMMR CRC cell lines, indicating that these subcutaneous tumor mouse models do not recapitulate the clinical reality well. In this thesis, we show that when pMMR CRC cell lines are implanted orthotopically in the liver as liver metastasis, the resultant liver metastases are unresponsive to ICB, which recapitulates the clinical reality that patients with pMMR metastatic CRC do not respond to ICB treatment. We further show that when treated with ICB, these orthotopic pMMR CRC liver metastasis mouse models have poor infiltration and activation of key immune cells and significantly decreased activity of key pathways that are critical for the efficacy of ICB. We also evaluated several strategies aimed at overcoming the inefficacy of ICB in these pMMR CRC liver metastasis mouse models. We found that radiation therapy was able to overcome inefficacy of ICB in the pMMR CRC liver metastasis mouse model with moderately low tumor mutational load. We also found that antibody-peptide epitope conjugates (APECs) were able to increase the efficacy of ICB in the pMMR CRC liver metastasis mouse model with very low tumor mutational load. Our results demonstrate that by implanting pMMR CRC cell lines in a relevant tissue site such as in the liver to model CRC liver metastasis, we can more accurately recapitulate the clinical efficacy of therapies such as ICB.
by William Wee Teck Ho.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering

Objectives: To establish a mouse orthotopic bladder cancer model with consistent tumor-take rate. This orthotopic model was subsequently used to evaluate small animal imaging techniques and investigate new therapeutic agents for bladder cancer treatment.
Materials and Methods: Different orthotopic implantation techniques have been tested. MBT-2 cells and syngeneic C3H/He mice were used in all experiments. Chemical bladder pre-treatment with different agents (saline, hydrochloric acid, trypsin and poly-L-lysine) and different concentration of instilled tumor cells (1 x 10⁶ or 2 x 10⁶) were investigated. In the second part of the experiment, trans-abdominal micro-ultrasound imaging (MUI) technique was investigated and validated. Bladder tumor growths were monitored with longitudinal measurement. Mice were killed at every MUI session. Bladder tumor volumes were measured and correlated with gross stereomicroscopy. Using the optimized orthotopic bladder cancer model, targeted contrast enhanced micro-ultrasound imaging has been investigated. VEGFR2 targeted contrast agent was prepared and injected intravenously before imaging sessions. The intra-tumoral perfusion, VEGFR2 expression and blood volume in real time were quantified. Contrast enhanced MUI was performed on Days 14 and 21. The feasibility of targeted contrast enhanced micro-ultrasound imaging was confirmed. After the establishment of orthotopic model and in vivo molecular imaging techniques, this robust platform was used for investigating new treatment agent in localized bladder cancer. Tumor-bearing mice were randomized into control and sunitinibtreated (40 mg/kg) groups. Tumor volume, intra-tumoral perfusion, and in vivo VEGFR2 expression were measured using a targeted contrast-enhanced micro-ultrasound imaging system. The effects of sunitinib malate on angiogenesis and cellular proliferation were measured by CD31 and Ki-67 immunohistochemistry. The clinical outcomes including total bladder weight, tumor stage, and survival were evaluated.
Results: A consistent tumor take-rate of over 90% was achieved by using poly-L-lysine pretreatment with 2 x 10⁶ MBT-2 cells in all of the experiments. MUI identified all tumors that were present on final histology. Measurements of tumor size by MUI and gross microscopy had a high correlation coefficient (r = 0.97). Measurements of intra-tumoral perfusion and in vivo VEGFR2 expression were also proved to be feasible. After the technical refinement and modification, complete measurements could be performed in all mice (n = 10) at 2 consecutive imaging sessions. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. This is the first report of applying targeted contrast enhanced MUI in orthotopic bladder cancer model. Finally, sunitinib was found to have significant tumor growth inhibition in both in vitro and in vivo experiments. In the orthotopic model, tumors in sunitinib-treated mice had reduced tumor volume and stage, lower proliferation index and micro-vessel density. Sunitinib prolonged survival in tumor-bearing mice as compared to control group.
Conclusions: The development of reliable orthotopic animal models assists in the discovery of novel therapeutic agents. The establishment in the methods of implantation with improved tumor-take rate and the advances in imaging technology form the important foundation of basic research in bladder cancer. Trans-abdominal MUI is proven to be a valuable tool for translational studies involving orthotopic mouse bladder cancer models. Furthermore, the first report of the application of targeted contrast enhanced MUI in deep-seated tumor in bladder has been published. It enables investigators to monitor tumor angiogenesis and vascular changes after treatment. It will be useful for direct, noninvasive, in vivo evaluation of anti-angiogenesis therapeutic agents. The preclinical study has demonstrated the activities of a new class of targeted therapy against localized bladder cancer in an orthotopic mouse model. Sunitinib inhibits tumor growth and thus decreases the tumor burden and prolongs survival compared with placebo. These results provide a rationale for future clinical trials using VEGFR-targeted treatments of localized bladder cancer in the neo-adjuvant and adjuvant settings.
Chan, Shu Yin Eddie.
Thesis (M.D) Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 189-212).

博士
國立中正大學
分子生物研究所
104
Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor. Even with the current standard of care involving surgery and combined radio-chemotherapy, the median survival duration of patients with GBM remains about 15 months. Recent reports of the presence of the JC virus (JCV) DNA and early protein in human malignant glioma and glioblastoma cells indicate that human glioblastoma cells may be susceptible to JCV as well as JCV virus-like particles (JC VLP) infection. Our unpublished data demonstrated that the JC VLPs were able to deliver a GFP reporter plasmid into the human glioblastoma, U87-MG, cells for expression in vitro and in vivo and were able to carry a suicide gene administered via tail vein injection to inhibit the growth of the subcutaneous U87 tumors in mice. In order to simulate the biological characteristics and treatments of brain tumor clinically, we used the intratumoral injection of JC VLPs carrying a herpes simplex virus thymidine kinase suicide gene (tk-VLPs) combined with ganciclovir (GCV) to treat orthotopic human glioblastoma xenografts, inoculated intracranially with U87 stable cell lines expressing GFP and iRFP dual fluorescent genes (U87-L-iRFP), in athymic nude mice. Besides, fluorescence molecular tomographic imaging system (FMT) was employed to monitor the status of gene therapy in vivo. The tk-VLPs were stereotactically implanted in the right brain of a nude mouse concurrently with or one week after intracranial inoculation of U87-L-iRFP cells, resulting in lower fluorescence concentration in tumor measured from FMT during the experiment and finally prolonged progression-free survival of nude mice in combination with GCV treatment. The presence and location of tumors detected by FMT correlated well with those of macroscopic and microscopic tumors histopathologically. These results demonstrate that the JC VLPs carrying a suicide gene combined with GCV can be used to treat human glioblastomas in an orthotopic nude mouse model. FMT allows us to monitor the tumor response of human GBM stably expressing iRFP in live mice during treatment with tk-VLP/GCV and potentially optimize gene therapy.

碩士
國立中正大學
生物醫學研究所
101
Urothelial carcinoma is the most common tumor type in the urinary system, it has high recurrent rate after surgery. After transurethral resection of bladder tumor, it will combine treatment with Bacillus Calmette-Guerin (BCG) or mitomycin C to prevent the recurrence. However, the BCG treatment caused severe side effects such as hepatitis, abscess or pneumonitis. Thus, in this study we want to use the traditional chinese medicine Guei-Chih-Fu-Ling-Wan (桂枝茯苓丸, GFW) to investigate whether it will improve the treatment of bladder tumor. First, we established the orthotopic bladder cancer model to investigate the efficiency of GFW treatment and compared the efficacy with BCG, and mitomycin C in vivo. We found that GFW could inhibit the tumor growth in mouse bladder, and the efficacy was similar with BCG and mitomycin C, but did not show sever side effect such as hematuria. Next, we investigated the inhibitory mechanisms of GFW toward urothelial carcinoma. In vitro, cell survival were gradually decreased after GFW treatment and with dose dependent under 1.5mg/ml, however, most cell died when GFW dose were greater than 1.5mg/ml. And, the underlying mechanism of cell death after GFW treatment is through autophagy and necrosis pathway at the low and high dosage of GFW, respectively. Summarized our above data, in vivo, we established the orthotopic bladder cancer model and found that GFW inhibited the mouse bladder tumor growth orthotopically and with minor side effect compared with BCG and mitomycin C. After treatment with GFW, the cell performed cytotoxcicty through autophagy in lower dose but cell necrosis at higher dose.

碩士
國立成功大學
環境醫學研究所
103
Breast cancer is the most common cancer in modern women. Although there are currently a variety of therapeutic methods for the treatment of breast cancer, severe problems still exist due to serious side effects triggered by clinical drugs and less anti-cancer activity in triple negative breast cancer. In this study, triple negative breast cancer cell line was applied to investigate the anti-cancer effects of new histone deacetylase inhibitor, YCW-3 alone or in combination with topoisomerase inhibitor, etoposide and to determine the mechanisms of these effects in vitro and in vivo. Firstly, we confirm that YCW-3 is a potent pan-HDAC inhibitor in which expressed a higher capability to inhibit HDACs and superior pharmacokinetics profile than traditional HDACi. Combination treatment with YCW-3 and etoposide resulted a synergistic cytotoxicity effect and induced remarkable programmed cell death in breast cancer cell line. The combined treatment enhanced significantly DNA damage, quantified by comment assay, when compared to YCW-3 or etoposide treatment alone. From a mechanistic insight, YCW-3 inhibited the expression level of the crucial DNA repair protein, DNA-PK. The effect of YCW-3 on the degradation of DNA-PK protein could be through the ubiquitin proteasome pathway, and the proteasomal degradation of DNA-PK protein was suggested by enhancing the association of DNA-PK with the ubiquitin E3 ligase RNF144A.Thus, our results demonstrated that the combination of YCW-3 and etoposide exerts strong cytotoxic effects in breast cancer and deserve further clinical-related investigation.

碩士
國立成功大學
環境醫學研究所
106
Breast cancer is the most common cancer in women. Although the death rate has dropped recent years, the incidence is increasing because of the change of modern life style. There are several treatments for breast cancer, but problems still exist due to serious side effects triggered by clinical drugs and less anti-cancer activity in triple negative breast cancer. In this study, triple negative breast cancer cell line 4T1-Luc was applied to investigate the anti-cancer effects of ionizing radiation (IR) alone, folate receptors’ antagonist, methotrexate alone or in combination and to determine the mechanisms of these effects in vitro and in vivo. In our current results, we confirm that methotrexate did replace the site of folate receptors which highly express in breast cancer cell and have cytotoxic effect, combined treatment increased the therapeutic efficacy which resulted in a synergistic effect also induced remarkable autophagy, type II programmed cell death in breast cancer cell line, apparent apoptosis and necrosis through long time period. Furthermore, we focus on a novel target SRC-3, which over express in breast cancer cell line. We suggest that the metabolic enzyme PFKFB4 down regulate the high expression level of SRC-3 and was significantly decreased in 4T1-Luc cells treated with combined treatment. In conclusion, our results demonstrated that the combination of methotrexate and radiation exerts strong cytotoxic effects in breast cancer and deserve further clinical-related investigation.

碩士
國立清華大學
生醫工程與環境科學系
99
Introduction: Angiogenesis plays a critical role in tumor growth. Integrin αVβ3 is a kind of cell adhesion molecule (CAM) that overexpresses on active angiogenic endothelium and glioblastoma cells. The arginine-glycin-aspartic acid (RGD) peptide is recognized to possess specific affinity with integrin αVβ3. The blood-brain barrier (BBB) is an obstacle for drugs for treating and diagnosing CNS diseases. This study utilized focused ultrasound (FUS) of low-energy burst wave in the presence of microbubbles to transiently disrupt the BBB to increase the radiotracer 131I-E[c(RGDyK)]2 delivery to brain tumor. Methods: U87MG-fLuc glioma-bearing nude mice were prepared. The radiotracer, 131I-E[c(RGDyK)]2 was prepared by iodogen method. Bioluminescence imaging (BLI) and magnetic resonance imaging (MRI) were utilized to measure tumor areas and volumes of the mice. Nano-SPECT/CT imaging was performed after administering the radiotracer into the glioma-bearing mice after sonication with focused ultrasound. Finally, quantitative autoradiography was applied to double confirm the results from fused SPECT and MRI. Results: 131I-E[c(RGDyK)]2 was obtained in high radiolabeled yield (RCP > 95%). There was a good correlation (R2 = 0.97) between BLI and MRI for monitoring the tumor volumes. The tumor sizes of experimental group and control group were controlled in ranges of 6.534±3.7 and 6.09±4.54 mm3, respectively. By SPECT/CT and MRI analyses, there was 2.03±0.32 fold increase of the tumor uptake comparing FUS (4.97±0.49 %ID/ml) with W/O FUS (2.41±0.48 %ID/ml) at 0.5 h postinjection. The tumor uptake of the FUS treated mice (2.02±0.25 %ID/ml) was found to reach 4.57±2.04 fold increase compared to that of W/O FUS mice (0.44±0.02 %ID/ml) at 24 h postinjection. Autoradiography showed that the accumulation of the radiotracer in the brain tumor of the FUS group (4.890±0.510 %ID/ml) was 2.682±0.279 fold greater than that of the W/O FUS group (1.820±0.031 %ID/ml) at 0.5 h postinjection, which was similar to SPECT/CT and MRI analysis. Conclusion: This study demonstrated that the focused ultrasound technique to disrupt BBB could enhance and probably prolong the tumor uptake of 131I-E[c(RGDyK)]2.