The Roles And Molecular Mechanism Of The CCL2/CCR2 Axis In The Perineural Invasion Of Salivary Adenoid Cystic Carcinoma

Salivary adenoid cystic carcinoma(SACC)is one of the most commonly diagnosed salivary gland malignancies,characterized by a high rate of perineural invasion(PNI).PNI of SACC makes it difficult for surgeons to remove the tumor mass completely.Additionally,SACC is refractory to chemotherapy.Thus it is rather difficult to cure this disease,and the prognosis is pessimistic.Accumulated documents showed that C-C motif chemokine ligand 2(CCL2),also known as monocyte chemotactic protein-1(MCP-1),was highly expressed in multiple malignancies and was also secreted by nerves.It is documented that CCL2 could accelerate the invasion and metastasis of tumor cells after binding its receptor,C-C motif chemokine receptor 2(CCR2).It is reported that CCL2 could also recruit tumor-associated macrophages(TAMs)to the tumor site via the CCL2/CCR2 axis.Then TAMs could promote the progression of the malignancies.However,the roles of CCL2/CCR2 axis and TAMs in the process of PNI in SACC remain to be elucidated.This study consists of the following four parts:1.The expression and clinical value of CCL2/CCR2 axis in SACCObjective: To explore the expression of CCL2,CCR2,CD68,and CD163 in SACC tissues,and to analyze the relationship between the expression of CCL2,CCR2,CD68,CD163 and the PNI of SACC.Methods: Seventy-one SACC tissues and 50 normal salivary gland tissues were collected.The expressions of CCL2,CCR2,CD68,and CD163 were analyzed by immunohistochemical staining.The distribution of TAMs in SACC was analyzed by transmission electron microscopy.The correlations between the expressions of CCL2,CCR2,CD68,CD163 and the PNI,TNM stage,the survival rate were evaluated.Results: The expressions of CCL2,CCR2,CD68,and CD163 in SACC tissues were significantly higher than those in normal salivary tissues.The expressions of CCL2 and CCR2 were significantly correlated with the expressions of CD68 and CD163 in SACC tissues.The results of transmission electron microscope investigation showed high infiltration of TAMs in SACC.The expressions of CCL2,CCR2,CD68,and CD163 were significantly correlated with the PNI,the TNM stage,and the poor prognosis of SACC patients.Conclusion: The high expressions of CCL2 and CCR2 in SACC tissues were significantly correlated with the infiltration of TAMs.CCL2/CCR2 axis and TAMs may play important roles in the PNI of SACC.2.The investigation of the roles of CCL2/CCR2 axis in the interactions between tumor cells and nerve during the process of the PNI in SACCObjective: To investigate the expressions of CCL2 and CCR2 in the tumor cell-dorsal root ganglion(DRG)co-culture system,and to explore whether the CCL2/CCR2 axis could mediate the interactions between tumor cells and DRG.Methods: DRGs were separated from the newborn BALB/c mouse.The DRGs were co-cultured with SACC-83 cells or SACC-LM cells.The expressions of CCL2 or CCR2 in the co-culture system were evaluated by ELISA,q RT-PCR,and Western Blot assays.CCR2 silencing in SACC-83 cells by RNA interference(RNAi)was performed.SACC-83 cells with or without CCR2 silencing were incubated with different concentrations of CCL2.q RT-PCR and Western Blot assays were performed to evaluate the expressions of p-ERK1/2,ERK1/2,MMP-2,and MMP-9 in SACC-83 cells.CCK8,migration,and invasion assays were used to investigate the proliferation,migration,and invasion of SACC-83 cells.The in vitro PNI model which incorporated SACC-83 cells and DRG were established.The effect of the blockade of CCL2/CCR2 axis on the PNI of SACC was investigated using CCR2 antagonist or RNAi techniques.Results: DRG secreted a high level of CCL2 in the conditioned medium,and activated the expression of CCR2 in SACC-83 cells or SACC-LM cells.CCL2/CCR2 axis activated ERK1/2 signaling pathway and promoted the expressions of MMP-2 and MMP-9 in SACC-83 cells,thereby promoting the migration and invasion of SACC-83 cells.Blockade of the CCL2/CCR2 axis by CCR2 antagonist or CCR2 sh RNA significantly inhibited the PNI of SACC-83 cells.Conclusion: CCL2 from nerve could activate the expression of CCR2 in SACC-83 cells.The CCL2/CCR2 axis could activate the ERK1/2 signaling pathway and up-regulate the expressions of MMP2 and MMP-9 in SACC-83 cells,then promote the PNI of SACC.3.The investigation of the roles and mechanism of the CCL2/CCR2 axis in the interactions of SACC-83 cells and TAMsObjective: To explore the roles and mechanism of the CCL2/CCR2 axis in the interactions of SACC cells and TAMs,and to evaluate the roles of TAMs in the PNI of SACC.Methods: THP-1 cells were induced by 13-12-phorbol myristate acetate(PMA,100 n M,24 h)to differentiate to be macrophages.SACC-83 cells were co-cultured with macrophages using Transwell co-culture system to simulate the interactions between SACC cells and TAMs.The expressions of CCL2 or CCR2 in the co-culture system were analyzed by ELISA,q RTPCR,and Western Blot.CCL2 silencing in SACC-83 cells was performed.The expressions of CCL2 or CCR2 in SACC-83 cells with or without CCL2 silencing were investigated by q RT-PCR and Western Blot.The effect of SACC-83 cell conditioned medium on the migration of TAMs was evaluated by Transwell migration assays.CCR2 specific antagonist RS504393(50 ng/m L)was used to block the expression of CCR2 in TAMs.The effect of blockade of the CCL2/CCR2 axis on the migration of macrophages was analyzed.Macrophages were induced with SACC-83 cell conditioned medium for 48 h to obtain TAMs.The effect of blockade of the CCL2/CCR2 axis on the expressions of M2 TAMs surface markers CD163 and CD206 were detected by flow cytometry.The effect of blockade of the CCL2/CCR2 axis on the expressions of TNF-?,IL-1?,Arg1,IL-10,and GDNF in TAMs were analyzed by q RT-PCR and Western Blot.SACC-83 cells were induced by TAMs conditioned medium,and the expressions of p-RET and RET in SACC-83 cells were detected by Western Blot 48 h later.GDNF neutralizing antibody(GDNF Nab,2 ?g/m L)was used to remove GDNF in the conditioned medium of TAMs,and RS504393(50 ng/m L)was used to block the expression of CCR2 in TAMs.Western Blot was used to evaluate the expressions of p-RET and RET in SACC-83 cells.RET antagonist pyrazolo pyrimidine-1(PYP1,5 ?M)was used to block the RET signaling in SACC-83 cells.And RS504393(50 ng/m L)was used to block the expression of CCR2 in TAMs.Then CCK8,scratch wound healing,and Transwell assays were performed to analyze the proliferation,migration,and invasion of SACC-83 cells.Results: SACC-83 cells derived CCL2 activated the expression of CCR2 in TAMs.The CCL2/CCR2 axis promoted the expressions of CD163,CD206,Arg-1,IL-10,and GDNF in TAMs,but inhibited the expressions of TNF-? and IL-1?.TAMs promoted the proliferation,migration,and invasion of SACC-83 cells through the GDNF/ RET pathway.Conclusion: SACC cells may recruit TAMs through the CCL2/CCR2 axis.The CCL2/CCR2 axis contributed to the "M2 phenotype polarization" of TAMs,and promoted the expression of GDNF in TAMs.Meanwhile,TAMs promoted the proliferation,migration,and invasion of SACC cells through GDNF/RET pathway.4.The investigation of the potential of using CCR2 antagonist in the treatment of the PNI of SACCObjective: To investigate whether the application of CCR2 antagonist could inhibit the PNI of SACC in the xenograft tumor model.Methods: SACC-83 cells were inoculated adjacent to the sciatic nerve in nude mice to establish the in vivo model of PNI.One week after the inoculation of tumor cells,nude mice were administered orally with RS504393(30 mg/kg)every three days for four weeks.The animals were sacrificed five weeks later after the inoculation of SACC-83 cells.The volume of tumor,the dysfunction of sciatic nerve,and the swelling of sciatic nerve were evaluated.Results: The application of CCR2 antagonist significantly inhibited the volume of tumor,the dysfunction of sciatic nerve,and the swelling of sciatic nerve in nude mice.Conclusion: The application of CCR2 antagonist could inhibit the PNI of SACC.Targeting CCL2/CCR2 axis by CCR2 antagonist may provide a new option for the treatment of the PNI of SACC.