||The mutual interaction between tumor associated macrophages and cancer cells and its impact on pancreatic cancer stemness
||Institute of Basic Medical Sciences
Cancer stem cells
胰臟癌在全世界癌症死亡排名第四位，每年新增病例數目和死亡病例數目幾乎相等，且在台灣發生率正逐年增加。手術切除腫瘤是目前主要的治療方法，但約有90%的患者因診斷較晚已合併遠端轉移、或因嚴重發炎導致組織固化而無法接受手術根除治療，這些患者的五年存活率低於5%，顯示現有疾病的診斷與治療效果有限。研究證實腫瘤相關巨噬細胞的浸潤和癌幹細胞的參與會導致癌細胞增殖、侵犯和轉移，與病患的存活率降低、易復發及喪失對原本有效藥物的反應等結果有關，但目前兩者在胰臟癌所扮演的角色與作用機制尚未清楚。因此我們想要研究胰臟癌細胞與腫瘤浸潤巨噬細胞間的交互作用為何及其對胰臟癌幹細胞活性之影響。首先我們以包含96個胰臟癌病人的組織微陣列進行螢光染色發現腫瘤浸潤巨噬細胞標記C204的表現量和胰臟癌幹細胞標記CD44和CD133呈正相關，且同時高表現CD204和CD44/CD133的病人之存活率是所有組別中最差的，顯示腫瘤浸潤巨噬細胞和胰臟癌幹細胞的存在有密切關係。為了探討兩者間的作用關係，我們將癌細胞與單核球細胞進行共同培養以模擬腫瘤微環境，結果發現胰臟癌細胞中具有癌幹細胞特性的細胞族群會增加，且單核球細胞也因胰臟癌細胞的刺激而分化成腫瘤浸潤巨噬細胞，將胰臟癌幹細胞接種至免疫缺陷老鼠，結果顯示同時接種胰臟癌幹細胞和腫瘤浸潤巨噬細胞的組別會較單獨接種胰臟癌幹細胞的組別形成較大的腫瘤，推論腫瘤浸潤巨噬細胞的存在可以促進胰臟癌幹細胞活性，進一步透過RNA microarray和cytokine protein array的分析，我們認為MIF、IL-8及CCL5可能參與胰臟癌細胞與腫瘤浸潤巨噬細胞間的交互作用，利用MetaCore、即時聚合酶鏈式反應和西方墨點法的結果確認癌細胞會分泌MIF使單核球活化成腫瘤浸潤巨噬細胞，並誘導腫瘤浸潤巨噬細胞分泌IL-8和CCL5去調控胰臟癌幹細胞活性，此一效應可被NF-κB抑制劑所抑制，利用藥物或siRNA抑制MIF的活性可有效減少IL-8和CCL5的產生，進而減少胰臟癌幹細胞和腫瘤浸潤巨噬細胞的族群，此結果也在動物模式上得到驗證，最後分析組織微陣列和病人血清中MIF、IL-8及CCL5的表現量，顯示這3個分子之間呈正相關的關係且與病人預後有關。總結我們的研究發現，腫瘤浸潤巨噬細胞對調控胰臟癌幹細胞扮演一個重要的角色，合併抑制MIF和腫瘤浸潤巨噬細胞的作用能提供治療胰臟癌可針對的標靶。
Pancreatic cancer (PC) is the fourth commonest cause of cancer-related mortality across the world, with incidence equaling mortality. The incidence of PC is gradually increased in Taiwan. Surgery is the primary method to treat patients with PC, but only 10% of the diagnosed patients can be treated by surgical resection. These unresectable cases were divided into two groups on metastasis or locally advanced PC. The five-year survival rate is less than 5%, suggesting the limited in diagnosis and treatment of PC. Recently reports illustrate tumor associated macrophages (TAMs) infiltration in tumor tissue and the existence of cancer stem cells (CSCs) may promote cancer cells proliferation, invasion, and metastasis. Both TAMs and CSCs were associated with poor prognosis. However, the interaction between CSCs and TAMs and the way by which TAMs sustain CSCs mediates PC progression remains to be explored. In this study, we found that CD204-positive TAMs expression related with CD44 and CD133-positive CSCs in tissue microarray containing 96 clinical PC specimens, and coexpression of CSCs and TAMs predicted poor prognosis. Furthermore, we established a coculture system of pancreatic cancer cells and monocytes to monitor how the interplay between CSCs and TAMs accelerates tumor development and progression. The results showed that cancer cells induced TAMs activation via coculture. TAMs promoted cancer stemness and tumorigenesis in vitro and in vivo. On the basis of RNA microarray and cytokine array data, we proposed the interplay between CSCs and TAMs was mediated by secreting MIF, IL-8, and CCL5. We also verified these results by MetaCore, quantitative real-time PCR, and Western blot analysis, and found that CSC growth was regulated in a paracrine manner by TAMs through MIF/IL-8/CCL5 axis; in particular, the inhibition of MIF signaling using a specific inhibitor could suppress cancer stemness and tumour growth. Importantly, the induction of MIF, IL-8, or CCL5 in response to coculture was abolished by NF-κB inhibitor BAY 11-7082. Finally, we confirmed these findings in a cohort of 96 PC patients and determined the clinical significance of MIF/IL-8/CCL5 paracrine signaling on PC progression. Taken together, our results suggest that tumor microenvironment TAMs may play an important role in maintaining cancer stemness. Simultaneous targeting cancer-derived MIF and TAMs is a new therapeutic strategy for PC.
1-1 Pancreatic cancer...2
1-2 Pancreatic cancer stem cells...2
1-3 Tumor microenvironment in pancreatic cancer...4
1-4 Tumor associated macrophages...5
1-5 Role of TAMs in pancreatic cancer progression...7
1-6 Interplay between CSCs and TAMs...10
1-7 Macrophage migration inhibitory factor...13
1-11 Specific aims...19
Chapter 2：Materials and Methods....20
2-1 Patients and TMA construction....21
2-2 Primary cell culture...21
2-3 Monocyte isolation from human peripheral blood..21
2-4 Cell culture....22
2-5 Sphere formation and CSCs harvest..22
2-6 Lentiviral transduction and stable cell line generation...23
2-7 Flow cytometric analysis and cell sorting (FACS) ..23
2-8 Methyl-thiazol-tetrazolium (MTT) assay..23
2-9 Adhesion assay....24
2-10 Phagocytosis assay....24
2-11 RNA extraction, reverse transcription, and quantitative real-time PCR..24
2-12 Cell lysis and Western blot analysis...25
2-13 Cytokine array...25
2-15 Immunofluorescence staining and measurement..26
2-16 Tumor formation in NOD/SCID or C57BL/6 mice and drug treatment..27
3-1 Clinicopathological characteristics and outcomes...30
3-2 CD44+/CD133+ CSCs or CD204+ TAMs expression in normal and cancer are..30
3-3 CD44+/CD133+ CSCs or CD204+ TAMs expression versus clinicopathological
3-4 Clinicopathological features and expression of CD44+/CD133+ CSCs or CD204+
TAMs versus survival....31
3-5 Coexpression of CD44/CD133 and CD204 associated with poor outcomes in PC..32
3-6 CSCs and TAMs are co-present in pancreatic tumors...33
3-7 Coculture of monocytes with pancreatic cancer cells leads to TAMs activation.33
3-8 CD44+CD133+ cells have high CSCs capacity..34
3-9 TAMs support pancreatic CSCs maintenance and promote tumorigenicity.35
3-10 A paracrine network mediates interaction between pancreatic cancer cells and
3-11 MIF contribute to TAMs activation..36
3-12 MIF trafficking and secretion regulate a paracrine signaling to affect CSCs
3-13 IL-8 and CCL5 cooperate to improve CSCs activities...38
3-14 MIF signaling is a target to suppress pancreatic cancer progression.39
3-15 MIF/IL-8/CCL5 levels correlate with poor prognosis.39
Chapter 4：Discussion and Conclusion....41
Tables and Figures....68
Table 1. Clinicopathological parameters and clinical outcome (n=96) ..68
Table 2. Clinicopathological parameters and expression of CD44, CD133, CD44/CD133, and CD204 (n=96) ...69
Table 3. Multivariate analysis of prognostic factors for overall and disease-free
Table 4. Significant genes of pancreatic cancer cells after coculture with U937 cells by
Table 5. Significant genes of U937 cells after coculture with pancreatic cancer cells by
Figure 1. Expression of CSCs markers CD44 and CD133 in TMA and the corresponding
full sections of PC tissue. ....75
Figure 2. Expression of TAMs marker CD204 in TMA and the corresponding full
sections of PC tissue. ...77
Figure 3. The correlation between CD44+/CD133+ CSCs and CD204+ TAMs in PC.79
Figure 4. CSCs and TAMs are co-present in fresh pancreatic tumors and ascites
Figure 5. Pancreatic cancer cells promote TAMs activation...83
Figure 6. Functional characterizations of pancreatic cancer stem cells. ..86
Figure 7. TAMs enhance CSCs properties..88
Figure 8. Pancreatic cancer cells-monocytes crosstalk via paracrine networks mediate
TAMs activation and cancer stemness..91
Figure 9. The MIF/IL-8/CCL5 axis is involved in microenvironmental paracrine
signalling for regulating monocyte-TAMs differentiation and maintaining
Figure 10. Depletion of MIF decreased the presence of CSCs. .97
Figure 11. Blockage of MIF signaling suppresses pancreatic cancer stemness and
Figure 12. The coexpression of MIF, IL-8, and CCL5 are correlated with pancreatic
cancer patients outcomes. ...101
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