The Effect On Gastric Carcinoma Progression Initiated By Tumor-Associated Macrophages And The Related Mechanism

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 carcinimaThis 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 macrophagesTHP-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 macrophagesThis 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. Conclusions1. 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...