肿瘤相关性巨噬细胞在胃癌进展中的作用及相关机制研究
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摘要
随着人们对肿瘤的研究深入,肿瘤的微环境(micro-environment)在肿瘤复发转移中的作用越来越受到重视。研究发现,在肿瘤的微环境中,有多种免疫细胞募集,并参与了肿瘤免疫,包括巨噬细胞。存在于肿瘤微环境中的巨噬细胞,被称为肿瘤相关性巨噬细胞(Tumor-associated macrophages, TAMs)。
巨噬细胞具有可塑性,在不同的环境中,可以分化为不同的亚群,如M1,M2,其在免疫功能上存在显著差异[1-3]。M1通过经典的激活途径,即通过Toll-like受体(TLRs)与抗原结合后,产生IL-12。IL-12使幼稚的T细胞向Ⅰ型辅助性T细胞(Th1)分化,并使T细胞及NK细胞产生释放IFN-γ。IFN-γ作为第二信号,可进一步激活巨噬细胞并增强巨噬细胞分泌细胞因子及抗原递呈的能力,同时分泌大量促炎因子,具有促进免疫的作用。M2则通过非经典途径激活,具体机制现在仍不清楚。其激活的巨噬细胞分泌的细胞因子具有炎症抑制作用,主要为IL-10, TGF-β等[4,5]。
肿瘤对各种浸润的炎性细胞,具有诱导分化作用(re-education),表现出特定的免疫细胞亚型。在对TAMs的研究发现,其大部分表现为M2亚型,具有与M2相似的分子标记(molecular marker)及细胞因子谱(cytokine profile)[6]。常用的TAMs分子标记如CD163[5,7,8], CD206[9-11]及CD204[12,13]等。TAMs对肿瘤的作用目前仍存在争议。一部分研究发现,TAMs具有抗肿瘤作用(anti-tumor),而多数研究则认为,TAMs可促进肿瘤的侵袭、转移以及促进肿瘤血管新生等,从而促进肿瘤进展(pro-tumor)[14-16]。
胃癌是我国最为常见的肿瘤之一,但目前对于胃癌的微环境中的TAMs相关研究较少。本研究通过收集临床手术病例及标本,制作组织芯片,用免疫组化的方法,以CD163作为TAMs标记,观察胃癌组织中TAMs的浸润情况,研究其与肿瘤的病理分化、临床分期以及预后的关系,以分析其对胃癌进展的作用。
上皮间质化(Epithelial-mesenchymal transition, EMT)是在组织胚胎发育中对形态形成起关键作用的过程。在EMT过程中,上皮细胞失去细胞间的紧密连接及细胞极性,形成具有间质细胞特征的梭形细胞[17]。近年来研究发现,肿瘤的EMT效应是肿瘤进展的重要机制,并受到肿瘤微环境的调控[18,19]。肿瘤细胞发生EMT反应后,细胞形态出现间质化,并且迁移侵袭能力将明显增强。TAMs是肿瘤微环境的重要组成部分,而TAMs对肿瘤的EMT效应研究较少。因此我们通过实验分析TAMs对胃癌的EMT效应,以进一步探讨其对胃癌进展的相关机制。
第一部分肿瘤相关巨噬细胞对胃癌根治术后患者生存的影响
本部分研究主要是探讨肿瘤相关巨噬细胞(Tumor-associated macrophages, TAMs)在胃癌组织中的浸润情况以及其与胃癌患者的临床及病理指标之间的关系,分析其对行根治术后胃癌患者的预后影响。
收集135例于1999年2月—-2005年12月期间在本院普外科胃癌组行D2根治术、达到R0切除的胃腺癌患者病例资料,并将手术切除标本制作组织芯片。通过免疫组化染色,以CD163标记TAMs,通过电脑图像分析系统及图像分析软件,计数阳性细胞个数及细胞密度。统计分析其浸润的细胞密度与患者的临床指标、胃癌的分化程度、以及病理分期的关系；并且进一步分析其对胃癌术后患者生存的影响。
结果发现,胃癌组织中TAMs的细胞密度均明显高于正常胃壁组织,差异有统计学意义(p<0.001)；以胃癌组织中TAMs的细胞密度中位值(M=7.48)将胃癌患者分为高表达组(n=68)及低表达组(n--67),通过χ2检验分析发现胃癌组织中TAMs表达与淋巴结转移相关,高表达组淋巴结转移率较低表达组高,具有统计学意义(P=0.025)。TAMs表达与胃癌的T分期具有一定相关趋势,但无统计学意义,而胃癌患者的年龄、性别、肿瘤的大小及部位、细胞分化程度与胃癌组织TAMs的细胞密度均无相关性。通过COX单因素分析发现,胃癌患者的预后与胃癌的浸润深度、淋巴结转移以及胃癌组织中TAMs的细胞密度相关(P=0.032)。通过Kaplan-Meier生存分析结果发现,胃癌组织中TAMs高表达组的总生存期(OS)短于TAMs低表达组(p=0.028)。TAMs高表达组患者的一年、三年、五年的生存率分别是81%、69%、56%；而低表达组患者的一年、三年、五年的生存率分别是88%、81%、77%。但在多因素分析中显示胃癌组织中TAMs表达不是独立的预后因素。
第二部分THP-1来源的巨噬细胞对胃癌细胞生物学作用的观察研究
本部分实验通过THP-1来源的巨噬细胞模拟TAMs的作用,观察THP-1来源的巨噬细胞对胃癌细胞的细胞形态、增殖潜能以及侵袭能力的影响,进一步探讨TAMs对胃癌进展的相关作用。
通过将THP-1细胞予phorbol myristate acetate (PMA)体外诱导的方法,使其分化为巨噬细胞,并且用transwell小室将胃癌细胞株AGS细胞和GSC-7901细胞分别与该巨噬细胞共培养模拟其相关微环境,观察胃癌细胞生长情况及细胞形态的变化。通过全自动活细胞观测分析系统(Cell-IQ)及cck-8增殖实验分析THP-1来源的巨噬细胞对胃癌细胞的增殖能力的影响；并采用Matrigel体外侵袭实验观察胃癌细胞株AGS及SGC-7901细胞与巨噬细胞共培养及未共培养的情况下体外侵袭能力的变化,同时用Western-blot检测其基质金属蛋白酶(matrix metaloproteinases, MMPs)表达差异进一步说明侵袭能力的改变。
结果显示,胃癌细胞与THP-1来源的巨噬细胞共培养后,发生了上皮间质化(Epithelial-mesenchymal transition, EMT)反应。细胞形态由原来上皮细胞形态转化成为一种间质细胞形态的梭形细胞。细胞生长分散,细胞间隙增大。与THP-1来源的巨噬细胞共培养后,两种胃癌细胞(AGS和SGC-7901胃癌细胞株)体外侵袭能力均有明显增强。共培养后与未共培养的AGS细胞穿出小室的细胞数分别为308±15和168±4,P<0.01；SGC-7901细胞为433±77和106±11,P<0.05。而这两种胃癌细胞的增殖能力共培养处理后均未增强。肿瘤细胞的侵袭力与其分泌MMPs的能力密切相关。AGS细胞共培养处理后MMP7及MMP9表达明显上调,而SGC-7901细胞共培养后也可见MMP7表达上调,验证了THP-1来源的巨噬细胞增强了胃癌细胞的侵袭力。
实验结果提示TAMs对胃癌细胞具有EMT效应,能增强胃癌细胞的侵袭力,而使胃癌发生进展。
第三部分THP-1来源的巨噬细胞促进胃癌细胞EMT反应的相关机制研究
上一部分的实验表明,THP-1来源的巨噬细胞能使胃癌细胞发生EMT反应,增强胃癌细胞的侵袭能力。本部分实验进一步探讨THP-1来源的巨噬细胞促进胃癌细胞EMT转化的可能相关分子机制及信号通路。
在调控EMT效应的诸多转录因子中,Snail/Slug的作用最为重要。Snail/Slug可直接抑制E-cadherin转录,使细胞发生EMT转化。通过qRT-PCR发现,胃癌细胞中Snail和Slug的基因表达水平在与THP-1来源的巨噬细胞共培养后均有不同程度上调。共培养后,胃癌细胞SGC-7901中Snail的基因表达增加了4倍(P<0.01),而Slug的表达则增加了24倍(P<0.01)；AGS细胞中Slug的表达也上调至对照细胞的1.5倍(P<0.05)。提示,THP-1来源的巨噬细胞可能通过上调胃癌细胞中Snail/Slug的基因表达,而使胃癌细胞发生EMT转化。
GSK-3p具有抑制Snail/Slug基因转录的生物活性,并可在Akt(主要活化形式为p-Akt Ser473)的作用下发生磷酸化(p-GSK-3βser9)而失活。实验通过Western-blot检测发现,用THP-1来源的巨噬细胞共培养处理后,AGS细胞和SGC-7901细胞中p-GSK-3βser9和p-Akt Ser473表达水平与未共培养相比,明显增高；总的GSK-3p和Akt表达则相似,无明显差异。结果提示,THP-1来源的巨噬细胞能上调胃癌细胞Akt/GSK-3p通路的磷酸化水平而使GSK-3p的活性减弱,削弱其对Snail/Slug基因表达的抑制。Akt/GSK-3β/Snail (Slug)轴可能与THP-1来源的巨噬细胞介导的EMT作用相关。
由于共培养体系中巨噬细胞与胃癌细胞无直接接触,巨噬细胞对胃癌细胞的作用主要通过其分泌的可溶性分子而引起的。通过荧光磁珠免疫分析(Fluorescent bead immunoassay)的方法检测发现,THP-1细胞在PMA诱导激活并分化为巨噬细胞后,TNF-a表达量增加了8倍(P=0.056),,而IL-1β和IL-8表达量分别增加了20倍和40倍(P均小于0.01)。这些明显增高的细胞因子可能参与了胃癌细胞的EMT转化,但还需进一步实验证实。
结论
1.胃癌组织中TAMs的细胞密度与胃癌淋巴结转移正相关,并对胃癌术后患者有一定的预后预测价值。
2.THP-1来源的巨噬细胞促使胃癌细胞发生EMT转化,增强了胃癌细胞侵袭力,其对胃癌细胞的EMT效应可能与Akt/GSK-3β/Snail (Slug)通路相关。
3.THP-1细胞激活分化为巨噬细胞后,TNF-α、IL-1β和IL-8表示明显上调,这些细胞因子可能介导了胃癌细胞的EMT效应。
The immune microenvironment play an indispensible role on the recurrence and metastasis of malignant tumor. Several kinds of immune cells participate in the tumor immunity, including macrophages. Massive macrophages are observed infiltrating in a wide variety of tumor organizations, and termed tumor-associated macrophages (TAMs).
Macrophages have plasticity and could differentiate to specific subtype with significant differences immune function to different environments, such as M1and M2phenotype[1-3]. M1activation is through the classic path. Once macrophages conjugate antigen through Toll-like receptors, they produce interleukin12(IL-12). IL-12induces naive T-cell polarization to helper T cell type I (Thl), which induce T cells and NK cells to produce IFN-y. As the secondary signal, IFN-y enhances cytokine production and ability to present antigen of macrophages, which enhances immune reaction. M2activation, however, is through the alternative path, and its mechanisms are still unknown. M2type macrophages produce the cytokines with immune suppression effect, such as IL-10, transforming growth factor β (TGF-β)[4,5]. The tumors can re-educated the inflammatory cells to some phenotypes, and the TAMs show the M2subtype molecular markers and cytokine profile [6]. Such as CD163[5,7,8], CD206[9][10,11] and CD204[12,13] are all used as a marker for TAMs in different studies. Still now, it is controversial about the roles on tumors played by TAMs. Even though some studies indicate that TAMs show an anti-tumor role, most researchers believe that TAMs facilitate tumor invasion and metastasis, induce tumor angiogenesis and promote tumor progression, which is a pro-tumor role[14-16].
Studies about the TAMs in the gastric cancer immune microenvironments are relatively rare. Herein, we collected the clinico-pathological key characteristics of gastric carcinoma patients and the operation specimens. The CD163were evaluated as a TAMs marker using immunohistochemishtry enumeration in tissue microarrarys.
The relation between the expression of TAMs and clinico-pathological characteristics was analysed in order to assess the role of TAMs on the progression of gastric cancer.
Epithelial-mesenchymal transition is a process whith play a key role on embryonic development. During the EMT process, epithelial lose the cell-cell conjuction and cell polarity, acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility [17]. Recent studies show that EMT is an important mechanism for the initial step of tumor progression which is regulated by cytokines and other factors derived from the tumor stroma[18,19]. TAMs as a main stromal component, may contribute to the EMT change of cancer cells and initiate tumor progression. So, we investigated the EMT role on human gastric cancer cells by TAMs and the correlate signal pathway, thus to discuss the mechanisms on the progression of gastric cancer initiated by TAMs.
Part I prognostic effects of TAMs for the patients with radical resection of gastric carcinima
This part is to explore the relationship between clinical characteristics and the TAMs infiltrating in tumor sites, and then to evaluate the prognostic prediction of TAMs in gastric cancer patients after radical resection.
Collecting the clinicopathological key characteristics of135gastric carcinoma patients who were underwent D2radical recection (R0resection) at Zhongshan Hospital of Fudan University from February1999to December2005. CD163was stained as the marker of TAMs. The CD163positive macrophages were evaluated using immunohistochemishtry enumeration in tissue microarrarys containing these135patients with the image analysis software. SPSS16.0for statastics.
We found that TAMs desity was higher in tumor sites than in normal tissues (p<0.001).135patients were devided into high expression group (n=68) and low expression group (n=67) by the midiean of cell density in tumor or in normal tissues, and it was related to tumor depth,lymph node metastasis and TNM stage in normal tissue, but in tumor sites was only related to lymph node stage. The overall suvival time (OS) of high CD163group is shorter than low CD163group (p=0.028) with K-M method, but it was not an independent prognostic factor.
In conclusion, the infiltration level of TAMs in tumor sites was a prognostic parameter for gastric carcinoma, but not an independent factors.
Part II The biological effects of gastric cancer cells initiated by THP-1derived macrophages
THP-1derived macrophages show a similar feature to tumor-associated macrophages, including the cell molucular markers and cytokine profiles. Here we investigated the effect of THP-1derived macrophages exerted on human gastric cancer cell lines AGS and SGC-7901by coculture with a transwell insert.
THP-1cell line cells, after treated with phorbol myristate acetate (PMA), differentiated to macrophages, then were cocultured with AGS cells and SGC-7901cells separately by a transwell inserts. The growing patterns and cell morphology of the gastric cancer cells were observed under a inverted microscope. The cell proliferation assay of gastric cancer cells was assessed by an automated cell imaging and analysis system and CCK-8test. In vitro Matrigel invasion assay was carried out, and the mumber of the gastric cancer cells which penetrate the Matrigel and the8.0μm pore polycarbonate membrane was counted and compared. MMPs (MMP7and MMP9) protein expression in gastric cancer cells cocultured with macrophages or non-cocultured was assessed by Western-blot.
The results indicated that cancer cells cocultured with macrophages show a morphological change. These cells showed loss of epithelial morphology which was seen when they were cultured in growth medium alone, and became a spindle shape, acquired a mesenchymal phenotype, just like the process called Epithelial-mesenchymal transition (EMT). The soluable factors induced by macrophages initiated the responses of gastric cancer cells without the need of cell-cell direct contact. Both the two cancer cell lines cocultured with macrophages have not show enhencement of proliferation, even show slight decrease of proliferation in AGS cells, but they show invasiveness enhancement compared to thoser cultured in growth medium alone. The number of the AGS cells that penetrate the transwell inserts was308±15for cocultured and168±4for non-cocultured, P<0.01;that was433±77and106±11in SGC-7901cells, P<0.05. After cocultured with macrophages,the AGS cells show MMP9expression, but not those non-cocultured. Both AGS and SGC-7901cells increase MMP7expression after cultured with macrophages.
In conclusion, the THP-1derived macrophages could enhance the invasive activities both in the AGS cells and the SGC-7901cells, which verified by the MMPs expression upregulation after coculture.
Part Ⅲ The related mechanisms of EMT effects on cancer cells initiated by THP-1derived macrophages
This part aim was to discuss the related mechanisms of EMT effects on cancer cells initiated by macrophages.
Related gene expression Snail and Slug was measured by qRT-PCR in both cocultured cancer cells and those non-cocultured in order to confirm the effects initiated by the macrophages. Investigate GSK-3β、p-GSK-3βser9、Akt、p-Akt Ser473protein expression in gastric cancer cells cocultured with macrophages or non-cocultured by Western-blot.
qRT-PCR indicated that the gene expression of snail and slug, two vital transcription factors promoting EMT response, was upregulated in AGS cells and SGC-7901cells cocultured with macrophages. Both the two cancer cell lines cocultured with macrophages had no change in the expression of total GSK-3β and Akt, but dramatically promoted the expression of p-GSK-3βSer9and p-Akt Ser473; so did the ratio of p-GSK-3βSer9/total GSK-3β and p-Akt Ser473/total Akt, which could lead to the Snail/Slug gene upregulation. In conclusion, the THP-1derived macrophages could upregulate the snail and slug gene expression in AGS and SGC-7901cells to regulate the EMT effects on the two cancer cell lines possibly through AKT/GSK3β pathway.
The soluable factors induced by macrophages initiated the EMT responses of gastric cancer cells without the need of cell-cell direct contact. So we screened the cytokines induced by THP-1derived macrophages through Fluorescent bead immunoassay. Compared with the THP-1cells without activation, the PMA activated macrophages secreted TNF-a8fold higher (P=0.056), IL-1β20fold higher (P<0.01) and IL-840fold higher (P<0.01). Those cytokines might contribute to the EMT response on gastric cancer cells initiated by the macrophages.
Conclusions
1. the infiltration level of TAMs in tumor sites was a prognostic parameter for gastric carcinoma, but not an independent factors.
2. the THP-1derived macrophages induced the EMT effects on the gastric cancer cells and enhanced their invasive activities, possibly through AKT/GSK3P/Snail (Slug) axis.
3. TNF-α IL-1β and IL-8were upregulated after THP-1cells activated to macrophages, and might contribute to the EMT response on gastric cancer cells
巨噬细胞具有可塑性,在不同的环境中,可以分化为不同的亚群,如M1,M2,其在免疫功能上存在显著差异[1-3]。M1通过经典的激活途径,即通过Toll-like受体(TLRs)与抗原结合后,产生IL-12。IL-12使幼稚的T细胞向Ⅰ型辅助性T细胞(Th1)分化,并使T细胞及NK细胞产生释放IFN-γ。IFN-γ作为第二信号,可进一步激活巨噬细胞并增强巨噬细胞分泌细胞因子及抗原递呈的能力,同时分泌大量促炎因子,具有促进免疫的作用。M2则通过非经典途径激活,具体机制现在仍不清楚。其激活的巨噬细胞分泌的细胞因子具有炎症抑制作用,主要为IL-10, TGF-β等[4,5]。
肿瘤对各种浸润的炎性细胞,具有诱导分化作用(re-education),表现出特定的免疫细胞亚型。在对TAMs的研究发现,其大部分表现为M2亚型,具有与M2相似的分子标记(molecular marker)及细胞因子谱(cytokine profile)[6]。常用的TAMs分子标记如CD163[5,7,8], CD206[9-11]及CD204[12,13]等。TAMs对肿瘤的作用目前仍存在争议。一部分研究发现,TAMs具有抗肿瘤作用(anti-tumor),而多数研究则认为,TAMs可促进肿瘤的侵袭、转移以及促进肿瘤血管新生等,从而促进肿瘤进展(pro-tumor)[14-16]。
胃癌是我国最为常见的肿瘤之一,但目前对于胃癌的微环境中的TAMs相关研究较少。本研究通过收集临床手术病例及标本,制作组织芯片,用免疫组化的方法,以CD163作为TAMs标记,观察胃癌组织中TAMs的浸润情况,研究其与肿瘤的病理分化、临床分期以及预后的关系,以分析其对胃癌进展的作用。
上皮间质化(Epithelial-mesenchymal transition, EMT)是在组织胚胎发育中对形态形成起关键作用的过程。在EMT过程中,上皮细胞失去细胞间的紧密连接及细胞极性,形成具有间质细胞特征的梭形细胞[17]。近年来研究发现,肿瘤的EMT效应是肿瘤进展的重要机制,并受到肿瘤微环境的调控[18,19]。肿瘤细胞发生EMT反应后,细胞形态出现间质化,并且迁移侵袭能力将明显增强。TAMs是肿瘤微环境的重要组成部分,而TAMs对肿瘤的EMT效应研究较少。因此我们通过实验分析TAMs对胃癌的EMT效应,以进一步探讨其对胃癌进展的相关机制。
第一部分肿瘤相关巨噬细胞对胃癌根治术后患者生存的影响
本部分研究主要是探讨肿瘤相关巨噬细胞(Tumor-associated macrophages, TAMs)在胃癌组织中的浸润情况以及其与胃癌患者的临床及病理指标之间的关系,分析其对行根治术后胃癌患者的预后影响。
收集135例于1999年2月—-2005年12月期间在本院普外科胃癌组行D2根治术、达到R0切除的胃腺癌患者病例资料,并将手术切除标本制作组织芯片。通过免疫组化染色,以CD163标记TAMs,通过电脑图像分析系统及图像分析软件,计数阳性细胞个数及细胞密度。统计分析其浸润的细胞密度与患者的临床指标、胃癌的分化程度、以及病理分期的关系；并且进一步分析其对胃癌术后患者生存的影响。
结果发现,胃癌组织中TAMs的细胞密度均明显高于正常胃壁组织,差异有统计学意义(p<0.001)；以胃癌组织中TAMs的细胞密度中位值(M=7.48)将胃癌患者分为高表达组(n=68)及低表达组(n--67),通过χ2检验分析发现胃癌组织中TAMs表达与淋巴结转移相关,高表达组淋巴结转移率较低表达组高,具有统计学意义(P=0.025)。TAMs表达与胃癌的T分期具有一定相关趋势,但无统计学意义,而胃癌患者的年龄、性别、肿瘤的大小及部位、细胞分化程度与胃癌组织TAMs的细胞密度均无相关性。通过COX单因素分析发现,胃癌患者的预后与胃癌的浸润深度、淋巴结转移以及胃癌组织中TAMs的细胞密度相关(P=0.032)。通过Kaplan-Meier生存分析结果发现,胃癌组织中TAMs高表达组的总生存期(OS)短于TAMs低表达组(p=0.028)。TAMs高表达组患者的一年、三年、五年的生存率分别是81%、69%、56%；而低表达组患者的一年、三年、五年的生存率分别是88%、81%、77%。但在多因素分析中显示胃癌组织中TAMs表达不是独立的预后因素。
第二部分THP-1来源的巨噬细胞对胃癌细胞生物学作用的观察研究
本部分实验通过THP-1来源的巨噬细胞模拟TAMs的作用,观察THP-1来源的巨噬细胞对胃癌细胞的细胞形态、增殖潜能以及侵袭能力的影响,进一步探讨TAMs对胃癌进展的相关作用。
通过将THP-1细胞予phorbol myristate acetate (PMA)体外诱导的方法,使其分化为巨噬细胞,并且用transwell小室将胃癌细胞株AGS细胞和GSC-7901细胞分别与该巨噬细胞共培养模拟其相关微环境,观察胃癌细胞生长情况及细胞形态的变化。通过全自动活细胞观测分析系统(Cell-IQ)及cck-8增殖实验分析THP-1来源的巨噬细胞对胃癌细胞的增殖能力的影响；并采用Matrigel体外侵袭实验观察胃癌细胞株AGS及SGC-7901细胞与巨噬细胞共培养及未共培养的情况下体外侵袭能力的变化,同时用Western-blot检测其基质金属蛋白酶(matrix metaloproteinases, MMPs)表达差异进一步说明侵袭能力的改变。
结果显示,胃癌细胞与THP-1来源的巨噬细胞共培养后,发生了上皮间质化(Epithelial-mesenchymal transition, EMT)反应。细胞形态由原来上皮细胞形态转化成为一种间质细胞形态的梭形细胞。细胞生长分散,细胞间隙增大。与THP-1来源的巨噬细胞共培养后,两种胃癌细胞(AGS和SGC-7901胃癌细胞株)体外侵袭能力均有明显增强。共培养后与未共培养的AGS细胞穿出小室的细胞数分别为308±15和168±4,P<0.01；SGC-7901细胞为433±77和106±11,P<0.05。而这两种胃癌细胞的增殖能力共培养处理后均未增强。肿瘤细胞的侵袭力与其分泌MMPs的能力密切相关。AGS细胞共培养处理后MMP7及MMP9表达明显上调,而SGC-7901细胞共培养后也可见MMP7表达上调,验证了THP-1来源的巨噬细胞增强了胃癌细胞的侵袭力。
实验结果提示TAMs对胃癌细胞具有EMT效应,能增强胃癌细胞的侵袭力,而使胃癌发生进展。
第三部分THP-1来源的巨噬细胞促进胃癌细胞EMT反应的相关机制研究
上一部分的实验表明,THP-1来源的巨噬细胞能使胃癌细胞发生EMT反应,增强胃癌细胞的侵袭能力。本部分实验进一步探讨THP-1来源的巨噬细胞促进胃癌细胞EMT转化的可能相关分子机制及信号通路。
在调控EMT效应的诸多转录因子中,Snail/Slug的作用最为重要。Snail/Slug可直接抑制E-cadherin转录,使细胞发生EMT转化。通过qRT-PCR发现,胃癌细胞中Snail和Slug的基因表达水平在与THP-1来源的巨噬细胞共培养后均有不同程度上调。共培养后,胃癌细胞SGC-7901中Snail的基因表达增加了4倍(P<0.01),而Slug的表达则增加了24倍(P<0.01)；AGS细胞中Slug的表达也上调至对照细胞的1.5倍(P<0.05)。提示,THP-1来源的巨噬细胞可能通过上调胃癌细胞中Snail/Slug的基因表达,而使胃癌细胞发生EMT转化。
GSK-3p具有抑制Snail/Slug基因转录的生物活性,并可在Akt(主要活化形式为p-Akt Ser473)的作用下发生磷酸化(p-GSK-3βser9)而失活。实验通过Western-blot检测发现,用THP-1来源的巨噬细胞共培养处理后,AGS细胞和SGC-7901细胞中p-GSK-3βser9和p-Akt Ser473表达水平与未共培养相比,明显增高；总的GSK-3p和Akt表达则相似,无明显差异。结果提示,THP-1来源的巨噬细胞能上调胃癌细胞Akt/GSK-3p通路的磷酸化水平而使GSK-3p的活性减弱,削弱其对Snail/Slug基因表达的抑制。Akt/GSK-3β/Snail (Slug)轴可能与THP-1来源的巨噬细胞介导的EMT作用相关。
由于共培养体系中巨噬细胞与胃癌细胞无直接接触,巨噬细胞对胃癌细胞的作用主要通过其分泌的可溶性分子而引起的。通过荧光磁珠免疫分析(Fluorescent bead immunoassay)的方法检测发现,THP-1细胞在PMA诱导激活并分化为巨噬细胞后,TNF-a表达量增加了8倍(P=0.056),,而IL-1β和IL-8表达量分别增加了20倍和40倍(P均小于0.01)。这些明显增高的细胞因子可能参与了胃癌细胞的EMT转化,但还需进一步实验证实。
结论
1.胃癌组织中TAMs的细胞密度与胃癌淋巴结转移正相关,并对胃癌术后患者有一定的预后预测价值。
2.THP-1来源的巨噬细胞促使胃癌细胞发生EMT转化,增强了胃癌细胞侵袭力,其对胃癌细胞的EMT效应可能与Akt/GSK-3β/Snail (Slug)通路相关。
3.THP-1细胞激活分化为巨噬细胞后,TNF-α、IL-1β和IL-8表示明显上调,这些细胞因子可能介导了胃癌细胞的EMT效应。
The immune microenvironment play an indispensible role on the recurrence and metastasis of malignant tumor. Several kinds of immune cells participate in the tumor immunity, including macrophages. Massive macrophages are observed infiltrating in a wide variety of tumor organizations, and termed tumor-associated macrophages (TAMs).
Macrophages have plasticity and could differentiate to specific subtype with significant differences immune function to different environments, such as M1and M2phenotype[1-3]. M1activation is through the classic path. Once macrophages conjugate antigen through Toll-like receptors, they produce interleukin12(IL-12). IL-12induces naive T-cell polarization to helper T cell type I (Thl), which induce T cells and NK cells to produce IFN-y. As the secondary signal, IFN-y enhances cytokine production and ability to present antigen of macrophages, which enhances immune reaction. M2activation, however, is through the alternative path, and its mechanisms are still unknown. M2type macrophages produce the cytokines with immune suppression effect, such as IL-10, transforming growth factor β (TGF-β)[4,5]. The tumors can re-educated the inflammatory cells to some phenotypes, and the TAMs show the M2subtype molecular markers and cytokine profile [6]. Such as CD163[5,7,8], CD206[9][10,11] and CD204[12,13] are all used as a marker for TAMs in different studies. Still now, it is controversial about the roles on tumors played by TAMs. Even though some studies indicate that TAMs show an anti-tumor role, most researchers believe that TAMs facilitate tumor invasion and metastasis, induce tumor angiogenesis and promote tumor progression, which is a pro-tumor role[14-16].
Studies about the TAMs in the gastric cancer immune microenvironments are relatively rare. Herein, we collected the clinico-pathological key characteristics of gastric carcinoma patients and the operation specimens. The CD163were evaluated as a TAMs marker using immunohistochemishtry enumeration in tissue microarrarys.
The relation between the expression of TAMs and clinico-pathological characteristics was analysed in order to assess the role of TAMs on the progression of gastric cancer.
Epithelial-mesenchymal transition is a process whith play a key role on embryonic development. During the EMT process, epithelial lose the cell-cell conjuction and cell polarity, acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility [17]. Recent studies show that EMT is an important mechanism for the initial step of tumor progression which is regulated by cytokines and other factors derived from the tumor stroma[18,19]. TAMs as a main stromal component, may contribute to the EMT change of cancer cells and initiate tumor progression. So, we investigated the EMT role on human gastric cancer cells by TAMs and the correlate signal pathway, thus to discuss the mechanisms on the progression of gastric cancer initiated by TAMs.
Part I prognostic effects of TAMs for the patients with radical resection of gastric carcinima
This part is to explore the relationship between clinical characteristics and the TAMs infiltrating in tumor sites, and then to evaluate the prognostic prediction of TAMs in gastric cancer patients after radical resection.
Collecting the clinicopathological key characteristics of135gastric carcinoma patients who were underwent D2radical recection (R0resection) at Zhongshan Hospital of Fudan University from February1999to December2005. CD163was stained as the marker of TAMs. The CD163positive macrophages were evaluated using immunohistochemishtry enumeration in tissue microarrarys containing these135patients with the image analysis software. SPSS16.0for statastics.
We found that TAMs desity was higher in tumor sites than in normal tissues (p<0.001).135patients were devided into high expression group (n=68) and low expression group (n=67) by the midiean of cell density in tumor or in normal tissues, and it was related to tumor depth,lymph node metastasis and TNM stage in normal tissue, but in tumor sites was only related to lymph node stage. The overall suvival time (OS) of high CD163group is shorter than low CD163group (p=0.028) with K-M method, but it was not an independent prognostic factor.
In conclusion, the infiltration level of TAMs in tumor sites was a prognostic parameter for gastric carcinoma, but not an independent factors.
Part II The biological effects of gastric cancer cells initiated by THP-1derived macrophages
THP-1derived macrophages show a similar feature to tumor-associated macrophages, including the cell molucular markers and cytokine profiles. Here we investigated the effect of THP-1derived macrophages exerted on human gastric cancer cell lines AGS and SGC-7901by coculture with a transwell insert.
THP-1cell line cells, after treated with phorbol myristate acetate (PMA), differentiated to macrophages, then were cocultured with AGS cells and SGC-7901cells separately by a transwell inserts. The growing patterns and cell morphology of the gastric cancer cells were observed under a inverted microscope. The cell proliferation assay of gastric cancer cells was assessed by an automated cell imaging and analysis system and CCK-8test. In vitro Matrigel invasion assay was carried out, and the mumber of the gastric cancer cells which penetrate the Matrigel and the8.0μm pore polycarbonate membrane was counted and compared. MMPs (MMP7and MMP9) protein expression in gastric cancer cells cocultured with macrophages or non-cocultured was assessed by Western-blot.
The results indicated that cancer cells cocultured with macrophages show a morphological change. These cells showed loss of epithelial morphology which was seen when they were cultured in growth medium alone, and became a spindle shape, acquired a mesenchymal phenotype, just like the process called Epithelial-mesenchymal transition (EMT). The soluable factors induced by macrophages initiated the responses of gastric cancer cells without the need of cell-cell direct contact. Both the two cancer cell lines cocultured with macrophages have not show enhencement of proliferation, even show slight decrease of proliferation in AGS cells, but they show invasiveness enhancement compared to thoser cultured in growth medium alone. The number of the AGS cells that penetrate the transwell inserts was308±15for cocultured and168±4for non-cocultured, P<0.01;that was433±77and106±11in SGC-7901cells, P<0.05. After cocultured with macrophages,the AGS cells show MMP9expression, but not those non-cocultured. Both AGS and SGC-7901cells increase MMP7expression after cultured with macrophages.
In conclusion, the THP-1derived macrophages could enhance the invasive activities both in the AGS cells and the SGC-7901cells, which verified by the MMPs expression upregulation after coculture.
Part Ⅲ The related mechanisms of EMT effects on cancer cells initiated by THP-1derived macrophages
This part aim was to discuss the related mechanisms of EMT effects on cancer cells initiated by macrophages.
Related gene expression Snail and Slug was measured by qRT-PCR in both cocultured cancer cells and those non-cocultured in order to confirm the effects initiated by the macrophages. Investigate GSK-3β、p-GSK-3βser9、Akt、p-Akt Ser473protein expression in gastric cancer cells cocultured with macrophages or non-cocultured by Western-blot.
qRT-PCR indicated that the gene expression of snail and slug, two vital transcription factors promoting EMT response, was upregulated in AGS cells and SGC-7901cells cocultured with macrophages. Both the two cancer cell lines cocultured with macrophages had no change in the expression of total GSK-3β and Akt, but dramatically promoted the expression of p-GSK-3βSer9and p-Akt Ser473; so did the ratio of p-GSK-3βSer9/total GSK-3β and p-Akt Ser473/total Akt, which could lead to the Snail/Slug gene upregulation. In conclusion, the THP-1derived macrophages could upregulate the snail and slug gene expression in AGS and SGC-7901cells to regulate the EMT effects on the two cancer cell lines possibly through AKT/GSK3β pathway.
The soluable factors induced by macrophages initiated the EMT responses of gastric cancer cells without the need of cell-cell direct contact. So we screened the cytokines induced by THP-1derived macrophages through Fluorescent bead immunoassay. Compared with the THP-1cells without activation, the PMA activated macrophages secreted TNF-a8fold higher (P=0.056), IL-1β20fold higher (P<0.01) and IL-840fold higher (P<0.01). Those cytokines might contribute to the EMT response on gastric cancer cells initiated by the macrophages.
Conclusions
1. the infiltration level of TAMs in tumor sites was a prognostic parameter for gastric carcinoma, but not an independent factors.
2. the THP-1derived macrophages induced the EMT effects on the gastric cancer cells and enhanced their invasive activities, possibly through AKT/GSK3P/Snail (Slug) axis.
3. TNF-α IL-1β and IL-8were upregulated after THP-1cells activated to macrophages, and might contribute to the EMT response on gastric cancer cells
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