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研究生：	汪法娣 Wang, Fa-Di
論文名稱：	胰腺癌細胞之KRAS突變促進M2腫瘤相關巨噬細胞的極化 KRAS Mutation in PDAC Cells Promotes The Polarization of M2-TAMs
指導教授：	沈延盛 Shan, Yan-Shen
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2024
畢業學年度：	112
語文別：	英文
論文頁數：	68
中文關鍵詞：	胰臟癌、腫瘤相關巨噬細胞, 、腫瘤微環境、活性氧化物質、KRAS 、TGF-β 、AP-1
外文關鍵詞：	Pancreatic ductal adenocarcinoma, Tumor-associated macrophages, Tumor microenvironment, reactive oxygen species, KRAS, TGF-β, AP-1
相關次數：	點閱：68 下載：8
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胰臟癌是全球癌症相關死亡的主要原因之一，診斷後存活率低於一年。而其中胰腺癌佔整體胰臟癌病例中的九成。雖然使用靶向免疫檢查點抑制劑在臨床試驗多種實體癌症中取得了令人鼓舞的結果，但其標靶免疫檢查點的癌症免疫療法對胰臟癌的益處仍然有限。KRAS突變是胰臟癌最主要突變，佔胰臟癌病人約九成，並與較差的總體存活率有關。
帶有KRAS突變的腫瘤細胞與免疫抑制和抗發炎微環境相關。KRAS突變導致細胞分泌調節免疫細胞和巨噬細胞的細胞因子表達量增加。此外，這些細胞因子分泌會產生免疫抑制的環境，進而促使癌細胞逃脫免疫監視。
腫瘤相關巨噬細胞（TAM）是腫瘤微環境（TME）中最豐富的免疫細胞。這些巨噬細胞根據表型和功能可以進行完善的分類。IFN-ɣ、TNF-α、LPS和HMGB1刺激巨噬細胞朝向M1表型發展，並具有抗腫瘤活性。相反的，IL-4、IL-10、IL-13、GC和AMP刺激的M2巨噬細胞具有促腫瘤功能。M2-TAM在胰臟癌發展過程中扮演很重要的角色，促進慢性炎症、癌幹性、纖維增生、免疫抑制、血管生成、侵襲和轉移，而且它們還可能導致胰臟癌的藥物抗性。針對胰臟癌腫瘤微環境中M2-TAM的轉變進行抑制具有顯著潛力可以提高患者的治療效果。
綜合以上，我們假定胰腺癌中KRAS的高活性會促使M2-TAM的分化。我們的初步實驗結果發現U937細胞與PANC-1細胞（KRAS-G12D突變）共培養會導致M2-TAM標記的表達量比與BxPC3（野生型KRAS）進行共培養時來得更高。PANC-1細胞中KRAS的沉默導致共培養系統中U937 M2-TAM水平降低，表明KRAS活性會影響胰腺癌介導的TAM極化。此外，我們從KRAS沉默的PANC-1細胞中收集了調節培養基（conditioned medium），以探討參與在M2-TAM分化的因子。結果顯示，在KRAS沉默的PANC1 調節培養基中轉化生長因子β (TGF-β)的分泌水平顯著降低。我們接下來探討轉化生長因子β在M2發展中的作用。經過轉化生長因子β處理後，U937中M2標記的表現量增加，但這些標記在U937與轉化生長因子β沉默的PANC-1細胞共培養中減少。
活性氧化物質(Reactive oxygen species)眾所周知在M2-TAM分化中有著至關重要的角色。我們的數據顯示，加入轉化生長因子β會增加U937細胞中的活性氧化物質的活性，並在N-乙醯基半胱氨酸(N-acetylcysteine)的處理下抑制U937細胞中的M2極化。我們進一步研究了胰臟癌中KRAS活性與轉化生長因子β表達之間的機轉。KRAS缺陷降低了PANC-1細胞中轉化生長因子β的蛋白和RNA合成。此外，ERK/AP-1路徑可能是KRAS調控轉化生長因子β表達的關鍵機制。ERK和AP-1複合物抑制劑導致PANC-1細胞中轉化生長因子β的含量在調節培養基、蛋白質以及mRNA降低。
在這項研究中，我們證明了胰臟癌中KRAS活性上升會透過釋放促發炎細胞因子之轉化生長因子β促進腫瘤微環境中的M2群體形成。因此，阻斷腫瘤微環境中轉化生長因子β是抑制M2-TAM分化的潛在策略，有望成為有效控制胰臟癌進展的治療方法。

Pancreatic cancer is one of the leading causes of cancer-related deaths worldwide, with a survival rate of less than one year following diagnosis. Specifically, pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of all pancreatic cancer cases. Despite the promising outcomes in the targeted immune checkpoint inhibitors trials across various solid cancers, the application of cancer immunotherapy targeting immune checkpoints provides limited benefits for pancreatic cancer. The KRAS mutation is the predominant mutation found in approximately 90% of pancreatic cancer patients and associated with poor overall survival.
Tumors carrying with the KRAS mutation are linked to an immunosuppressive and anti-inflammatory microenvironment. The presence of the KRAS mutation results in elevated expression of cytokines that regulate immune cells and macrophages. Moreover, the secretion of these cytokines creates an immunosuppressive environment, facilitating cancer cells to escape from immune surveillance.
Tumor-associated macrophages (TAMs) are the most abundant immune cells within the tumor microenvironment (TME). These TAMS are well-classified depending on their phenotype and function. Interferon (IFN)-ɣ, Tumor necrosis factor (TNF)-α, Lipopolysaccharide (LPS), and High mobility group box 1 (HMGB1) stimulate the transformation of macrophages towards a M1 phenotype, exhibiting anti-tumor activity. Conversely, M2 macrophages induced by Interleukin (IL)-4, IL-10, IL-13, GC, and AMP, demonstrate pro-tumor functionality. M2-TAMs play a critical role in the development of PDAC by promoting chronic inflammation, cancer stemness, desmoplasia, immune suppression, angiogenesis, invasion, metastasis, and contribute to drug resistance. Addressing transformation of M2-TAMs within pancreatic TME hold significant potential to improve patients’ outcomes.
Collectively, we hypothesize that high KRAS activity in PDAC promotes the differentiation of M2-TAMs. Our preliminary data revealed that co-culturing U937 cells with PANC-1 cells (with KRAS-G12D mutation) resulted in an elevated expression of M2-TAM markers compared to co-culture with BxPC3 cells (wild-type KRAS). Knockdown of KRAS expression in PANC-1 cells led to a reduction in U937 M2-TAM levels within the co-culture system, suggesting that KRAS activity is implicated in the polarization of TAMs mediated by PDAC.
Furthermore, we collected conditioned medium (CM) from PANC-1 cells with KRAS knockdown to investigate the factors which are involved in M2-TAM differentiation. Our finding indicated that the secretion levels of transforming growth factor beta (TGF-β) were significantly decreased in the CM derived from KRAS knockdown PANC-1 cells. Subsequently, we investigated the role of TGF-β in M2-TAM development. The expression of M2 markers inU937 cells increased after TGF-β treatment, while a decrease was observed when co-cultured with PANC-1 cells with TGF-β knockdown.
It is well-known that reactive oxygen species (ROS) play an essential role in M2-TAM differentiation. Our data showed that ROS activity raised after the introduction of TGF-β in U937 cells. Additionally, the treatment of N-acetylcysteine (NAC) suppressed M2 polarization in U937 cells. We further investigated the mechanism between KRAS activity and TGF-β expression in pancreatic cancer. Both the protein and RNA synthesis of TGF-β were reduced in the PANC-1 in the absence of KRAS. Moreover, ERK/AP1 pathway appears to a critical mechanism in the regulation of TGF-β expression by KRAS. Inhibitors targeting ERK and AP-1 complex resulted in a decrease in TGF-β expression at the levels of conditioned medium, mRNA, and protein expression in PANC-1.
In this study, we have demonstrated that increased KRAS activity in pancreatic cancer promotes M2 population in TME through the secretion of inflammatory cytokine TGF-β. Consequently, blocking TGF-β within the TME emerges as a potential strategy to suppress M2-TAM differentiation, which could be a promising approach for effectively controlling the progression of pancreatic cancer.

ABSTRACT .......................................................................................................................... I 
中文摘要 ............................................................................................................................. III 
ACKNOWLEDGMENT .................................................................................................... V 
CONTENTS ..................................................................................................................... VII 
I. ABBREVIATIONS .......................................................................................................... 1 
II. INTRODUCTION .............................................................................................................................. 2 
1. Pancreatic Cancer ........................................................................................................................... 2 
1.1 Pancreatic Cancer Overview .................................................................................................... 2 
1.2 KRAS Mutation in Pancreatic Cancer .................................................................................... 3 
2. Tumor Microenvironment (TME) .............................................................................................. 4 
2.1 Pancreatic Cancer Tumor Microenvironment ................................................................... 4 
2.2 Tumor-Associated Macrophages............................................................................................ 4 
2.3 Therapy on targeting M2-TAMs ............................................................................................. 5 
3. Transforming Growth Factors Beta (TGF-β) .......................................................................... 6 
3.1 The Role of Transforming Growth Factors Beta (TGF-β) in Tumor Microenvironment .............................................................................................................................. 6 
3.2 The Role of Transforming Growth Factors Beta (TGF-β) in Pancreatic Cancer ...... 6 
4. Reactive Oxygen Species (ROS) ................................................................................................ 7 
4.1 TGF-β regulated Reactive Oxygen Species (ROS) in Pancreatic Cancer .................. 7 
4.2 Role of Reactive Oxygen Species (ROS) in Macrophages Polarizations .................. 7 
5. Research Motive ............................................................................................................................. 8 
5.1 Significant ....................................................................................................................................... 8 
5.2 Specific Aims .................................................................................................................................. 8 
III. MATERIAL AND METHODS ....................................................................................................... 9 
Cell Culture ............................................................................................................................................ 9 
Lentivirus Transfection ...................................................................................................................... 9 
Transformation ..................................................................................................................................... 9 
Plasmid Purification ......................................................................................................................... 10 
Transfection KRAS-G12D Plasmid .............................................................................................. 11 
Co-culture U937 with Pancreatic Cancer Cells ...................................................................... 11 
72Hr Co-culture Conditioned Medium Treatment .............................................................. 11 Inhibitor Treatment .......................................................................................................................... 12 
Cell Proliferation Quantification.................................................................................................. 12 
Total Protein Extraction .................................................................................................................. 12 
Cytoplasm and Nucleus isolation ............................................................................................... 12 
Western Blot ....................................................................................................................................... 13 
RNA Extraction .................................................................................................................................. 13 
Reverse Transcriptase Polymerase Chain Reaction .............................................................. 14 
Real-Time Polymerase Chain Reaction ..................................................................................... 14 
ROS Quantification .......................................................................................................................... 14 
Statistical Analysis ............................................................................................................................ 15 
IV. RESULTS .......................................................................................................................................... 16 
KRAS expression is associated with poor overall survival and recurrent free survival of pancreatic cancer patients. ...................................................................................................... 16 
PANC-1 cells promote higher M2-TAM markers mRNA expression after 72 hours of 
co-culture. ........................................................................................................................................... 16 
Lentivirus transfection to knockdown KRAS expression in PANC-1 cells decreased 
expression of M2-TAMs markers. ............................................................................................... 16 
Co-culture with PANC-1 shKRAS conditioned medium decreased M2-TAMs 
differentiation. ................................................................................................................................... 17 
PANC-1 knockdown cell lines secreted higher TGF-β in a conditioned medium .... 18 
Patients with high KRAS and high TGF-β mRNA expression have poorer overall 
survival based on the TCGA dataset. ......................................................................................... 18 
TGF-β had a potential role in M2-TAMs differentiation. .................................................... 18 
PANC-1 knockdown TGF-β resulted in decreased M2-TAMs markers mRNA 
expression. .......................................................................................................................................... 19 
GW788388 treatment decreases M2-TAMs markers in U937 cell lines ...................... 19 
The addition of TGF-β increased ROS expression levels in U937 cells. ........................ 20 
KRAS regulates TGF-β expression in PANC-1 via MMP-2 ................................................. 21 
AP-1 complex plays a role in regulating TGF-β via KRAS/ERK pathways in PANC-1 
cells. ....................................................................................................................................................... 21 
V. DISCUSSION .................................................................................................................................. 23 
VI. REFERENCES ................................................................................................................................. 27 
VII. FIGURES ........................................................................................................................................ 31 
VIII. APPENDIX ................................................................................................................................... 54 
I. List of primary antibodies in this study ................................................................................. 54 
II. List of secondary antibodies used in this study. ............................................................... 55 
III. List of primers used in this study. ......................................................................................... 56 
IX. CURRICULUM VITAE .................................................................................................................. 57
                                    

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