The Relationship Between Tumor Assosiated Macrophages And Liver Metastases From Colorectal Carcinoma

[Objective]To observe the number and proportion of tumor associated macrophages and its subtypes(M1 and M2)in colorectal cancers with different metastatic potential,and explore the relationship between these indicators and the metastatic ability to liver.Meanwhile,explore the underlying mechanisms by which TAMs and its subsets influence liver metastasis of colorectal cancers.[Methods]From the year 2000 to 2009,120 colorectal cancer patients who received treatments in Tianjin Medical University Cancer Institute and Hospital were enrolled in this study,and of all,40 cases were stage ? without occurrence or metastasis within 2 years after surgery(low metastatic group),in contrast,40 cases were stage? experienced liver metastasis within 2 years after surgery(median metastatic group),and 40 cases with synchronous liver metastases(high metastatic group).Immunohistochemistry was employed to detect TAMs in colorectal cancer specimens with different metastatic potential,and the number and proportion of M1 and M2 were also detected.Liver metastasis model of colorectal cancer was established in mouse by spleen injection using mouse derived CT-26 cell line.CT-26H5 was screened by spleen-liver circulation.The genes which express differently between CT-26 and CT-26H5 were selected by immunofluorescence quantitative RT-PCR.The expression of M-CSF which was selected by immunofluorescence quantitative RT-PCR was measured in the colorectal cancer specimens with different metastatic potential.Chemotaxis Assay was used to detect the chemotactic ability of M-CSF to macrophage.ELISA was used to detect the effect of supernatant with or without M-CSF blocking on the secretion and differentiation of macrophages.The roles of M-CSF and macrophage in the liver metastasis of colorectal cancer and their interaction were investigated by blocking M-CSF and scavenging macrophages in vivo independently.[Results]1.As the metastatic ability grew,the TAMs were diminished among the three groups,but there were no statistical differences(p>0.05).However,as the metastatic ability grew,the number of M1 was decreased significantly(p<0.05).In contrast,the number of M2 was increased significantly(p<0.05).The ratio of M2/M1 was increased statistically(p<0.05).2.The number of Ml was negatively correlated to lymph node and liver metastasis(p<0.05).The number of M2 was positively correlated to the preoperative serum CEA level,lymph node and liver metastasis and tumor differentiation grade(p<0.05).The ratio of M2/M1 was positively correlated to the preoperative serum CEA level,lymph node and liver metastasis and tumor differentiation grade(p<0.05).3.The liver metastatic model of colorectal cancer was successfully established in mouse.Compared to the original CT-26,the metastatic ability of screened CT-26H5 was significantly enhanced(p<0.05).4.M-CSF was the representative gene of screening,and expressed higher in CT-26H5 than in CT-26(p<0.05),the expression of M-CSF in specimens with different liver metastatic ability made significant difference(p<0.05),which was negatively correlated with M1(p<0.05)and positively correlate to liver metastatic ability and M2(p<0.05).5.M-CSF exerted significant chemotactic ability to macrophage,while the chemotactic macrophages were decreased upon M-CSF blocking(p<0.05).The supernatants from CT-26H5 and CT-26 revealed evident chemotactic ability to macrophage,and this ability was compromised by blocking M-CSF(p<0.05).The supernatant from CT-26H5 showed stronger chemotactic ability to macrophage than that from CT-26,however,there was no significant difference in the number of chemotactic macrophage between the two cell lines after M-CSF blocking(P>0.05).6.M-CSF could induce macrophage to differentiate to M2 type,and the supernatant from CT-26H5 showed stronger ability inducing macrophage to differentiate to M2 type than that from CT-26(p<0.05).However,there was no statistical difference between the two cell lines after M-CSF blocking(P>0.05).7.After M-CSF blocking,the liver metastatic ability of both CT-26H5 and CT-26 was significantly diminished,while compared to CT-26,the diminished extent of CT-26H5 was more evident(p<0.05).8.The liver metastases were decreased significantly after macrophage scavenge(p<0.05).9.Receiving the treatment of M-CSF blocking and macrophage scavenge,liver metastases of CT-26H5 were decreased significantly(p<0.05),similar to the results of the single macrophage scavenge group(P>0.05).[Conclusions]1.The role of TAMs in the development of liver metastasis of colorectal cancer does not depend on the total number of TAMs,but depends on the proportion and number of its subtypes;the difference of M-CSF expression of colorectal cancer cells with different liver metastatic ability was an important reason for the differences of the proportion and number of its subtypes.2.M2 are the dominant macrophages in the colorectal cancer with high liver metastatic ability and promote liver metastasis.Macrophage chemotaxis and M2 differentiation induced by M-CSF were important mechanisms of liver metastasis of colorectal cancer.These views in colorectal cancer have not been reported.