| BackgroundColorectal cancer(CRC)is one of the most common malignancies in the world.Macrophages found in close proximity or within tumor masses are indicated as tumor-associated macrophages(TAMs).Studies have found a large number of TAMs were existed in colorectal cancer microenvironment and has been found to play an important role in colon cancer growth,invasion,metastasis and so on.The relationship between TAMs and colorectal cancer is not clear yet.Some clinical studies have reported that more TAMs infiltrated in colorectal cancer,the angiogenesis was significantly increased,the differentiation of tumor cells were worse,lymph node metastasis rate was increased,and the prognosis was worse.While some studies found that TAMs were positively correlated with tumor metastasis,tumor stage and prognosis.Therefore,the relationship between TAMs and colorectal cancer,especially the relationship with CRC liver metastases needs further study.α7 nicotinic acetylcholine receptor(a7nAChR)is expressed on a variety of non-neuronal cells including macrophages.Imbalance expression of a7nAChR leads to tumor formation,progress and so on.Till now,the relationship between the expression of a7nAChR in TAMs and the biological behavior of colorectal cancer after activation of a7nAChR has not been reported yet.AimsTo study the effects of a7nicotinic acetylcholine receptor(a7nAChR)in tumor-associated macrophages on the invasion and migration of colorectal cancer and explore its underlying mechanisms.Methods1.51 cases of CRC tissue specimens were involved to study the relationship between the expression of a7nAChR in CD68 positive cells and the occurrence of liver metastasis and prognosis in patients with colorectal cancer.Immunohistochemical staining was used to detect the expressions of a7nAChR in CRC tissues and CD68 was used to label the TAMs in the same sequential paraffin sections.2.THP-1 monocytes were used and stimulated with PMA to become macrophages(THP-1 derived macrophages,TMs).TAMs were generated through indirect co-culture with CRC cells LoVo and SW620.The expression of a7nAChR in macrophages was down-regulated by siRNA and verified by RT-qPCR and WB.3.TMs and TMα7-/-were indirectly co-cultured with CRC cells LoVo and SW620.Transwell assay was used to detect the invasion and migration of LoVo and SW620 cells induced by TAMs down-regulated by a7nAChR.4.TMs were indirectly co-cultured with LoVo cells and/or stimulated with LPS.The secretion of TNF-a in supernatant was detected by ELISA.5.Western blotting was used to detect the expressions of phosphorylated PI3K,STAT3 and NF-κB in TMs after co-cultured with LoVo cells.Specific inhibitors of the corresponding signaling pathways was used to clarify the role of a7nAChR in TMs-induced CRC cells migration.Results1.CD68 is highly expressed in the tumor stroma,and a7nAChR is mainly expressed on the cell membrane surface.From the IHC results,CD68 is highly expressed and a7nAChR is relatively high expressed in patients with CRC in Dukes A.CD68 is highly expressed while a7nAChR is relatively low expressed in patients with CRC in Dukes D.Combined with the clinicopathological data and 5-year survival analysis,the expression of a7nAChR on the surface of TAMs has no relation with the gender(p = 1.0)and the ages(p = 0.455)of the patients,but related to the metastasis of the tumors.The expression of a7nAChR in TAMs was significantly decreased in cancer patients with Dukes D:13 of 14 patients were less than or equal to Grade 1,and only 1 patient had a7nAChR expression greater than Grade 2;among 37 patients with Dukes A/B/C,TAMs a7nAChR expression was less than or equal to Grade 1 in 20 patients and expression of a7nAChR in 17 patients was greater than or equal to Grade 2(p = 0.010).The 5-year survival rate was also significantly different between the two groups(p =0.020).2.The lentiviral siRNA technology was used to construct a7nAChR knocked down THP-1 cells.RT-qPCR and WB verified that a7nAChR was down-expressed in TMα7-/-cells.3.Transwell results showed that the invasion and migration of LoVo and SW620 cells increased significantly after co-cultured with TAMs low expression of a7nAChR.Cells migration results showed that the numbers of trans-membrane LoVo cells co-cultured with TM or TMα7-/-cells were(42±5.354)and(85.5±9.665),respectively(p=0.007);and the numbers of trans-membrane SW620 cells were(8.25±1.315)and(84.25±10.81),respectively(p= 0.0004).Cells invasion assay results showed that the numbers of trans-membrane LoVo cells cultured with TM or TMα7-/-cells were(85.5±3.797)and(258.8±21.51),respectively(p=0.0002);and the numbers of trans-membrane SW620 cells were(64.25±4.328)and(122.5±6.171),respectively(p=0.0002).The difference was statistically significant.4.TMs were stimulated with LPS and/or co-cultured with LoVo cells and supernatants were collected to determine the TNF-a secretion.ELISA results showed that the secretion of TNF-a in the cell supernatant increased significantly as well.5.The phosphorylation levels of NF-κB p65,STAT3 and PI3K p85 were significantly decreased in TAMs knocked-down with α7nAChR.AG490,the JAK2/STAT3 specific inhibitor,treatment can significantly inhibit the increase of tumor cell migrations induced by α7nAChR knock-down,while Ly294002 and Bay 11-7082 treatment have no similar effects.Conclusions1.The α7nAChR expression in TAMs in CRC tissues are negatively correlated with the tumor cells metastasis and prognosis.Low expression of a7nAChR in TAMs leads to an increased risk of liver metastases and high expression of a7nAChR in TAMs leads to the reduced risk of liver metastases.2.a7nAChR in TAMs inhibits the invasion and migration of CRC cells.3.TAMs upregulate TNF-a secretion through a7nAChR,suggesting that α7nAChR can modulate TAMs to promote pro-inflammatory effects.4.a7nAChR in TAMs can activate NF-κB/STAT3/PI3K signaling pathways.Furthermore,TAMs a7nAChR-mediated tumor cell invasion and metastasis are mainly associated with STAT3 signaling pathway. |
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