免疫細胞和發炎性因子是腫瘤微環境中促進乳腺癌進展的重要組成部分。為探討腫瘤微環境中的關鍵分子，我們將低侵入性和高侵入性的MDA-MB-231乳腺癌細胞與人類單核細胞THP-1細胞共同培養，並通過細胞蛋白因子陣列鑑定出差異分泌的細胞因子。我們發現，在共培養條件下，乳腺癌細胞分泌的CSF1誘導了單核細胞中CXCL7的表達，從而促進ROCK2蛋白於單核細胞，使其細胞分化成M2型巨噬細胞，並以FAK與MMP13蛋白增強乳腺癌細胞的轉移和侵襲能力。在異種移植小鼠模型中，CXCL7抗體可以顯著降低腫瘤的生長和遠處轉移。總體而言，我們的研究揭示了，由腫瘤促使單核細胞分泌的新型關鍵分子CXCL7，可改變腫瘤微環境，促進M2巨噬細胞分化和癌細胞轉移、侵襲，均有助於促進乳腺癌的進展和轉移。

Immune cells and inflammatory cytokines are essential components in the tumor microenvironment to promote breast cancer progression. To identify key players in the tumor microenvironment, we applied low and high invasive MDA-MB-231 breast cancer cell lines to co-culture with human monocyte THP-1 cells, and identified differentially secreted cytokines through cytokine array. We found that under co-culturing condition, CSF1, which was secreted by breast cancer cells, induced expression of CXCL7 from THP-1 monocytes, resulting in the promotion of their differentiation into M2-type macrophages by ROCK2 activation and enhanced migration and invasion ability of breast cancer cells via FAK and MMP13. Blocking CXCL7 by antibodies significantly reduced tumor growth and distant metastasis in the xenograft mouse model. Overall, our study unveils a novel key player, CXCL7, secreted by tumor infiltrating monocytes, to alter the tumor microenvironment in favor of M2 macrophage differentiation and cancer cell migration/invasion, both contributing to the promotion of breast cancer progression and metastasis.

摘要 3
Abstract 4
誌謝辭 5
Part I 12
Introduction 13
Materials and Methods 19
Cell culture 19
Co-culture system and cytokine array 20
Enzyme-Linked Immuno Sorbent Assay 20
Migration and invasion assays 21
Flow Cytometry analysis and macrophage differentiation 21
Macrophage suppression assay 22
Transfection assay 22
Quantitative RT-PCR analysis 23
Western blot analysis 23
In vivo xenograft mouse model studies 24
Immunohistochemistry (IHC) analysis 25
Statistical method 25
Results 26
Identification of CXCL7 cytokine in the co-culturing system of monocytes and invasive breast cancer cells 26
CSF1 produced by breast cancer cells induces CXCL7 secretion by monocytes 31
CXCL7 promotes breast cancer cell migration and invasion, which is correlated with activation of FAK-mediated signaling pathway 35
CXCL7 recruits and modulates monocytes to become M2-like macrophages, which is correlated with increase of ROCK2 40
Inhibition of CXCL7 suppresses tumor growth and reduces the incidence of distant metastases in xenograft breast cancer model 45
Expression of CXCL7 and ROCK2 correlated with breast cancer progression and patients' survival 50
Discussion 56
Reference 61
Part II 67
Abstract 68
Introduction 69
Materials and Methods 73
Cell culture and transfections 73
Cell knockout and mutagenesis 74
Nucleotides and reagents 74
Quantitative real-time PCR 75
H2S measurements (lead sulfide method) 76
Modified biotin switch (S-sulfhydration) assay 77
Nuclear/cytosol fractionation 78
Western blot analysis 78
Transwell cell migration and cell invasion assay 79
Cell proliferation assay 79
GSH/GSSG assay 80
Immunofluorescence 80
Endothelial cell tube formation assay 82
IL-1β detection 82
Mouse orthotopic implantation and tail-vein injection model 83
Immunohistochemistry 84
Retrieving RNA-seq data from The Cancer Genome Atlas 84
Results 86
Expression of CTH was upregulated in bone-metastatic PC cells 86
Increased expression of CTH correlated with progression and poor survival in PC 88
CTH promoted PC cell migration and invasion, but not proliferation 98
Knockdown of CTH suppressed cell invasion through inhibition of NF-κΒ-IL-1β-mediated signaling 107
The H2S-mediated sulfhydration of the NF-κB p65 subunit resulted in increased IL-1β production and enhanced cell invasion 118
Knockdown of CTH suppressed tumor growth and reduced the incidence of paraaortic lymph nodes and bone metastases in the mouse orthotopic implantation model 131
Discussion 140
Contributions 143
Reference 145


List of Figures
Part I

Figure 1 28
Figure 2 32
Figure 3 37
Figure 4 42
Figure 5 47
Figure 6 52
Figure 7 55


Supplemental table 1 64
Supplemental table 2 65
Supplemental table 3 66

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