過去研究發現惡性腫瘤細胞的上皮-間質轉化(epithelial-mesenchymal transition)及間質-上皮轉化 (mesenchymal-epithelial transition)與癌症的轉移有關。經由系統生物學方法分析不同時間點的微陣列(microarray)資料，我們發現骨髓間質幹細胞(bone marrow-derived mesenchymal stem cell)可誘導CD133+/CD83+肺癌腫瘤幹細胞(cancer stem cells) 轉移；而這些細胞轉移至遠端後，骨髓間質幹細胞可經由LIF-LIFR/p-ERK/pS727-STAT3訊息傳導路徑幫助腫瘤細胞進行間質-上皮轉化以獲得增殖及建立前轉移微環境(pre-metastatic niche)的能力。
另一方面，具有腫瘤向性的骨髓間質幹細胞移動至原位腫瘤時，可經由活化IL-6/IL6R/pY705-STAT3訊息傳導路徑，使CD151+/CD38+肺癌細胞經由上皮-間質轉化增強腫瘤幹細胞特性並進行轉移，同時CD133+/CD83+肺癌腫瘤幹細胞於所建立之前轉移利基(premetastatic niche)內的化學趨化物(chemo-attractants)可吸引CD151+/CD38+腫瘤細胞移動至遠端轉移處。總結來說，STAT3分子上不同位置(Tyrosine 705 和 Serine 727)產生磷酸化時，可調控腫瘤幹細胞的上皮-間質轉化與間質-上皮轉化過程，促使腫瘤細胞完成轉移。

Previous research has demonstrated that Epithelial–mesenchymal transition (EMT)/mesenchymal–epithelial transition (MET) processes are a driving force of cancer metastasis. We utilized systems-biology approaches to examine bone marrow-derived mesenchymal stem cell (BM-MSC)-driven lung cancer metastasis models. Microarray time-series data were analyzed, which revealed that BM-MSC-induced signaling triggered early dissemination of CD133+/CD83+ cancer stem cells (CSCs) from the primary tumor site shortly after STAT3 activation. These CSCs, when in the secondary metastatic site, switched from the migratory (mesenchymal) to the proliferative (epithelial) phenotype under the influence of active LIF/LIFR/p-ERK/pS727-STAT3 signaling. At the same time, LIF-LIFR/p-ERK/pS727-STAT3 signaling also promoted the formation of pre-metastatic niche. Then, tumor-tropic BM-MSCs circulated to the primary site and endowed CD151+/CD38+ cells with EMT-associated CSC properties through IL6R/pY705-STAT3 signaling. These CD151+/CD38+ cells were then drawn by chemo-attractants at the pre-metastatic niche and migrated to the secondary site. In summary, STAT3 phosphorylation at tyrosine 705 and serine 727 differentially regulates the EMT–MET switch within distinct molecular subtypes of CSCs to complete the metastatic process.

中文摘要 1
英文摘要(Abstract) 2
縮寫(Abbreviations) 3
致謝 5
目錄 7

Chapter 1. Introduction 10
1.1 Cancer metastasis 10
1.2 The epithelial-mesenchymal transition program 10
1.3 The role of bone marrow-derived mesenchymal stem cells in bone metastasis 11

Chapter 2. Material and Methods 13
2.1 Isolation and characterization of human BM-MSCs 13
2.2 Human samples and IHC analysis 14
2.3 Functional fractionation of cancer cells by invasion assays 15
2.4 Luciferase reporter assay 17
2.5 Western blotting 17
2.6 Sphere-forming culture 17
2.7 Microarray data collection and analysis 18
2.8 Quantitative PCR 18
2.9 Cytokine array 19
2.10 Animal experiments 20
2.11 The bone homing assay 20
2.12 An experimental model of bone metastasis by human lung cancer cells 20
2.13 Isolation of SCDCs from long bones 21
2.14 Data source 22
2.15 Microarray analysis 22

Chapter 3. Results 25
3.1 Isolation and characterization of human BM-MSCs 25
3.2 Identification of a gene expression signature linked to early dissemination 25
3.3 Inspection of the constructed BM-MSC-treated protein–protein interaction (PPI) network by systems biology approaches 30
3.4 BM-MSCs promote mesenchymal-epithelial transition (MET) in HM CD133+/CD83+ early-response cells through activation of the LIFR/p-ERK/pS727-STAT3 signaling pathway 31
3.5 BM-MSCs help LM CD151+/CD38+ late-response cells acquire CSC properties through activation of the IL6R/pY705-STAT3 signaling pathway 33
3.6 BM-MSCs elicit epithelial-mesenchymal transition (EMT) in LM CD151+/CD38+ late-response cells through the IL6R/pY705-STAT3 signaling pathway 34
3.7 Elevated expression of LIFR and phosphorylation of S727-STAT3 are correlated with disease progression and distant metastasis in patients with lung cancer 35
3.8 Heterogeneous CSC subtypes undergo pY705-STAT3-elicited EMT and pS727-STAT3-elicited MET to achieve metastatic colonization in a manner regulated by BM-MSCs in vivo 36

Chapter 4. Discussion and conclusion 39

Chapter 5. Figures and supplementary data 44
Figure 1 Identification of a gene expression signature linked to early dissemination 44
Figure 2 Inspection of the constructed PPI network in BM-MSC-treated cells by systems biology approaches 46
Figure 3 CD133+/CD83+ early-response (HM20) cells undergo STAT3/pS727-elicited MET to achieve metastatic colonization in a manner regulated by BM-MSCs 48
Figure 4 CD151+/CD38+ late-response (LM) cells gain CSC properties through BM-MSCs-elicited activation of STAT3/pY705 signaling 51
Figure 5 BM-MSCs prompted CD151+/CD38+ late-response (LM) cells to undergo STAT3/pY705-elicited EMT to achieve tumor heterogeneity and phenotypic plasticity 53
Figure 6 Clinical significance of IL6R, pY705-STAT3, LIFR and pS727-STAT3 in patients with lung cancer: Elevated expression of LIFR and phosphorylated S727-STAT3 is associated with clinically advanced cancer 55
Figure 7 Heterogeneous CSC subtypes undergo pY705-STAT3-elicited EMT and pS727-STAT3-elicited MET to achieve metastatic colonization in a manner regulated by MSCs in vivo 57
Figure 8 A model depicting our proposed mechanism wherein under the influence of BM-MSCs heterogeneous CD151+/CD38+ late-response and CD133+/CD83+ early-response CSC subtypes undergo STAT3/pY705-elicited EMT and STAT3/pS727-elicited MET, respectively, to form metastatic colonies in the secondary site 59
Supplementary Figure 1 Isolation and characterization of human BM-MSCs 60
Supplementary Figure 2 Identification of a gene expression signature linked to early dissemination using CL1-0 and CL1-5 cells 62
Supplementary Figure 3 Identification of a gene expression signature associated with early cancer dissemination using A549, H1299, H460 and H322 lung cancer cells 63
Supplementary Figure 4 Flow chart describing the construction of the MSCs-treated and Control intracellular PPI network 64
Supplementary Figure 5 The in vivo bone homing assay 66

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