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|Title:||Influence of oxygen level in colorectal tumor progression and response under different radiotherapy dose scheme||Authors:||Tam, Shing Yau||Degree:||Ph.D.||Issue Date:||2019||Abstract:||Colorectal cancer (CRC) has been the third most common cancer worldwide and the most common cancer in Hong Kong with about two-thirds of cases located in rectum or sigmoid colon, which requires radiotherapy as the routine treatment modality except for the uncommon early stages. Metachronous distant metastases are common among CRC patients and threatens overall survival rates despite technological advancements of radiotherapy techniques. Tumor hypoxia, which influences various tumor processes such as autophagy, epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSC) stemness maintainence, has been considered as an important factor of promoting metastatic power and reducing radiosensitivity of individual tumor. However, the exact mechanisms remain elusive. Moreover, the recent development of in vivo hypoxia quantification in animals by fluorescence imaging may allow the study of predictive biomarker of radiosensitivity for the development of individualized radiotherapy based on the knowledge of hypoxia-driven radioresistance. This thesis has two aims. The first aim is to evaluate the dynamics of different autophagy, EMT and stemness maintenance signaling pathways under different oxygen level in CRC cells in vitro. The second aim is to establish an effective method for quantifying the degree of hypoxia in colorectal tumor cells in vivo for predicting the radiosensitivity of individual tumor and evaluating the response under different radiotherapy dose scheme. To achieve the aims, this thesis is divided into three studies. The first study targeted at the dynamics of various oxygen level-driven autophagy pathways of CRC cells. Since autophagy can enable tumor cells to survive under stressful condition such as tumor hypoxia and irradiation, knowledge of the autophagy regulatory mechanisms in tumor hypoxia is vital for implementing effective anti-tumor management strategy. Although several pathways have been proposed to influence autophagy under low oxygen level in past studies, there is a lack of research concerning about the oxygen level other than the severe hypoxia level, chronic autophagy modulation and the interactions between different autophagy pathways. This study evaluated the expression and interaction between multiple key regulators in different oxygen levels and time points. Human colorectal adenocarcinoma HT-29 cells were cultured in 1% or 10% O₂ level and irradiated by 2 Gy with different incubation time. Autophagy key regulators, AMPK, HIFs and JNK were evaluated by western blot. Autophagy key regulator activations of AMPK, HIF-1α, HIF-2α and JNK were observed with different onset time. Both 1% and 10% O₂ level could promote autophagy while irradiation could slightly inhibit autophagy. The results of this study supported prior evaluation of oxygen level and autophagy regulators for improving treatment efficacy and indicated that cells incubated in blood oxygen level (10% O₂) remain an important target for further investigations since tumor cells metastasize by bloodstream. The second study deciphered the influence of oxygen level on EMT induction and stemness maintenance and the character of JNK-mediated pathway in CRC cells. EMT has been proposed as an important process of tumor development leading to metastatic spread and CSC was also suggested to be related with metastasis. Although past studies demonstrated that hypoxia could stimulate the progression of EMT in various cancers, most of the studies investigated HIF-mediated EMT induction and stemness maintenance. Meanwhile, the influence of higher oxygen level such as blood oxygen level (10% O₂) on EMT induction and stemness maintenance and the other possible EMT inducing pathways including JNK-mediated pathway are less studied. From the results of the first study, JNK has found to be a delayed autophagy promoter in chronic incubation of low oxygen level and it has been linked with EMT induction and stemness maintenance in previous reports. Thus, this study aimed to further investigate the role of JNK in EMT induction and stemness maintenance under low oxygen levels. Human colorectal adenocarcinoma cell lines with different epithelial-mesenchymal statuses including HT-29, DLD-1 and SW-480 were investigated in this study. They were cultured in 1% or 10% O2 for 48-hour EMT induction and the key EMT and stemness maintenance markers were evaluated by western blot. EMT inductions and stemness maintenance marker upregulations were confirmed in both 1% and 10% O2 levels among the three cell lines studied. Cell morphological analysis, migration assay and immunofluorescence staining of cytoskeleton provided secondary evidences for EMT inductions. While in the presence of the specific JNK inhibitor SP600125, EMT induction and stemness maintenance marker stimulation was relieved with modulations in different level of EMT markers including E-cadherin, vimentin, Snail and TWIST1. Moreover, another EMT mediator Akt was substantially affected by JNK inhibition. This suggested a novel EMT and autophagy regulation pathway as Akt acting as a downstream target of JNK pathway. The results of this study demonstrated EMT induction and stemness maintenance could be stimulated at blood oxygen level (10% O₂), which has a much higher level than hypoxia (1% O₂). JNK has important roles in these two processes under low oxygen levels and its inhibition could relieve the situation. This indicated a possible strategy in limiting EMT and the subsequent metastasis through JNK inhibition.
The third study evaluated of the influence of oxygen level on colorectal tumor irradiation response under different radiotherapy dose scheme. As hypoxia has been long regarded as an important factor of radiotherapy failure and correlated with low radiosensitivity, accurate in vivo quantification of oxygen level of individual tumor may indicate the radiosensitivity of individual tumor and thus allow development of individualized radiotherapy dose scheme. Carbonic anhydrase IX (CAIX), which is a tumor-specific enzyme closely related to HIF pathway, was chosen as the indicator of oxygen level of individual tumor. This study utilized the radvanced technique of hypoxia detection through CAIX fluorescence tomography to study the impact of different oxygen level on radiosensitivity of CRC cells in vivo and tried to predict the biological response of colorectal tumor under different radiotherapy dose schemes through studying the intratumor oxygen level. Firstly, in vitro clonogenic assay for investigating the impact of different oxygen level on radiosensitivity of CRC cells was also conducted. Then, HT-29-Red-FLuc (PerkinElmer, Inc.) cells were subcutaneously injected into 6-8 weeks old male nude mice for tumor development. Fluorescence scanning of CAIX expression was conducted after sufficient tumor growth and the tumors were then irradiated by 10 Gy or 15 Gy of radiation. The post-irradiation monitoring of tumor volume changes was then used to evaluate the relationship between CAIX level and irradiation response. The results of clonogenic assay showed that HT-29 cells incubated at 10% were the most radiosensitive while those incubated at 1% were slightly less radiosensitive than 21%. The results of fluorescence scanning demonstrated a feasible method for in vivo quantification of intratumor oxygen level. Although there was no significant correlation found between CAIX level and irradiation response, low CAIX levels were generally found among tumors with good irradiation response in both dose schemes. The results indicated that CAIX may act as a limited predictive biomarker of radiosensitivity and additional biomarkers such as angiogenesis and necrosis biomarkers were suggested for predicting radiosensitivity of individual tumor and the subsequent development of individualized radiotherapy dose scheme. Conclusively, this thesis demonstrated that the blood oxygen level (10% O₂) could be a prominent mediator of autophagy, EMT induction and stemness maintenance apart from the pathological hypoxia level of 1% O₂. Among the studied pathways, JNK-mediated pathway has found to be an important regulator of these processes especially in the chronic EMT induction and stemness maintenance and in 10% O2. Therefore, these significant findings provide a possible future direction in limiting EMT and stemness regulation and the subsequent cancer metastasis through JNK inhibition. The in vivo study of the influence of hypoxia on radiosensitivity of individual tumor demonstrated a feasible method for in vivo quantification of intratumor oxygen level through fluorescence scanning of CAIX expression. Collectively, these findings improve the knowledge of oxygen level-driven pathways in these important tumor development processes and provide hints for improving tumor control and treatment scheme.
|Subjects:||Hong Kong Polytechnic University -- Dissertations
Colon (Anatomy) -- Cancer -- Treatment
|Pages:||xxiii, 238 pages : color illustrations|
|Appears in Collections:||Thesis|
View full-text via https://theses.lib.polyu.edu.hk/handle/200/10219
Citations as of May 15, 2022
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