C3aR and C3 mRNA expression in the TCGA cohort and their association with overall survival
A total of 611 files of normalized mRNA expression data were obtained. These include 539 files of ccRCC patients and 72 files of normal controls. After integration of the mRNA expression data from the same patients (four patients were found to have three files each and one patient was found to have 2 flies), a total of 530 mRNA expression data corresponding to 530 ccRCC patients were obtained. Compared with the normal controls, the expression level of C3aR and C3 mRNA increased significantly in the tumor tissues of ccRCC patients (Figure 1A-B).
To analyze the association of C3aR and C3 mRNA level with overall survival, patients were divided into lower C3aR mRNA and higher C3aR mRNA, lower C3 mRNA and higher C3 mRNA group according to the median level of C3aR and C3 mRNA level, respectively. As shown in Figure 1C-D, patients with higher C3 mRNA level were found to have shorter overall survival while no significant difference in overall survival was observed between patients of lower C3aR mRNA and higher C3aR mRNA.
Results of immunohistochemistry for C3aR and C3c in the tumor tissue of ccRCC patients
Positive immunostaining for C3aR was observed in all the tumor tissue samples. As shown in Figure 2, immunostaining for C3aR could be observed in both infiltrating cells and cancer cells. C3aR was found to be distributed in the plasma membrane, cytoplasm and nucleus in the tumor cells. Compared with the normal renal tubular epithelial cells, the cells from which the tumor cells were believed to derive, the expression level of C3aR in the tumor cells of ccRCC patients increased significantly (Figure 2).
C3aR can only exert its roles when it is activated. To determine whether C3aR in ccRCC cells could be activated, it is necessary to examine whether the ligand for C3aR, C3a, could be produced in the tumor tissues. As no proper antibody against C3a could be found, we examined the presence of C3c, a product of C3 activation in the tumor tissue of ccRCC patients. As shown in Figure 3, C3c was found extensively expressed in the tumor tissue of ccRCC patients. And compared with the normal renal tissues, the expression level of C3c in ccRCC tumor tissues increased markedly.
Association of C3aR and C3c protein level with clinicopathological parameters
The level of C3aR in the tumor cells and C3c in the tumor tissue of each patient were scored and their associations with the baseline clinical and pathological characteristics were examined. As shown in Table1, no significant difference in C3aR expression was observed among patients of different ages and genders. Also, the expression level of C3aR in ccRCC tumor cells was not significantly influenced by tumor stage, tumor size and the presence of diabetes mellitus and hypertension. However, higher tumor cell C3aR level was observed in patients with higher Fuhrman grade and patients with necrosis within the tumor (Table 1). Results of bivariate correlation analysis also showed a significant correlation between the level of C3aR in the tumor cells and tumor grade (r=0.454, p<0.01) and the presence of necrosis in the tumor (r=0.298, p<0.001).
The level of C3c in the tumor tissues was also found to be associated with tumor grade and the presence of necrosis in the tumor tissue.
Results of survival analysis based on C3aR level in the tumor cells and C3c level in the tumor tissue of ccRCC patients
Patients were divided into lower C3aR group and higher C3aR group according to the immunostaining intensity for C3aR in the tumor cells, and lower C3c group and higher C3c group according to the immunostaining intensity for C3c in the tumor tissues. As shown in Figure 4, patients with higher C3aR level in the tumor cells or C3c in tumor tissues were found to have a shorter OS and DSS when compared with patients with lower C3aR or lower C3c. Analysis based on Cox regression revealed that higher C3c level in the tumor tissue is an independent risk factor along with tumor stage and tumor grade for both OS and DSS of ccRCC patients. However, the significance of the association of C3aR level with both OS and DSS disappeared in the multivariable Cox analysis (Table 2 and Table 3).
Association of C3aR and C3c expression with E-Cadherin, Vimentin and Ki-67 in tumor tissues
The upregulation of C3aR in tumor cells and increased activation of C3 (as reflected by increased C3c) in tumor tissues indicate that C3aR signaling is over-activated in the tumor cells of ccRCC patients. Previously, activation of C3aR was reported to be able to induce epithelial-to-mesenchymal transition (EMT) in tubular epithelial cells , the cells from which ccRCC was believed to derive. Besides, activation of C3aR has been reported to promote proliferation of glomerular mesangial cells  and cutaneous squamous cell carcinoma cells . To determine whether C3aR signaling also contributes to EMT and proliferation in ccRCC cells, we further examined the expression of the molecular markers for EMT (E-Cadherin and Vimentin) and cell proliferation (Ki-67) in the tumor specimens from ccRCC patients and associate them with the level of C3aR and C3c. As shown in Figure 5, all the three molecules are expressed by tumor tissues with immunostaining for Ki-67 was only observed in the nucleus. Results of spearman coefficient analysis revealed that the expression level of C3aR and C3c correlated positively with the level of Vimentin (r=0.434, p<0.01; r=0.374, p<0.01; respectively) and the ratio of Ki-67 positive tumor cells (r=0.428, p<0.01; r=0.252, p<0.01; respectively) and negatively with the level of E-Cadherin (r=-0.287, p<0.01; r=-0.179, p=0.042; respectively).