Pathological significance of C3aR signaling in the tumor cells of clear cell renal cell carcinoma: a clinical observation

Background Increasing evidences suggest that anaphylotoxin-induced signaling is involved in tumor pathogenesis, but the exact role of C3a/C3aR signaling in clear cell renal cell carcinoma (ccRCC) still remains to be investigated. The aim of the study was to investigate the pathological significance of C3a/C3aR signaling in ccRCC. Methods The expression of C3aR and C3 mRNA in the tumor tissues of ccRCC patients were analyzed by using the data from TCGA database. The expression of C3aR and C3c protein in the tumor tissues of another 129 ccRCC patients were examined by immunohistochemistry. Results Compared with the normal controls, both C3aR and C3 mRNA increased in the tumor tissues. Patients with higher C3 mRNA had shorter survival time. Immunostaining for C3aR and C3c also increased in the tumor tissues when compared with the adjacent normal renal tissues. Higher level of C3aR in the tumor cells and C3c in the tumor tissues were found to be associated with indices reflecting poor prognosis including higher tumor grade, the presence of necrosis in tumor tissues and shorter survival time. Besides, the level of C3aR in the tumor cells and C3c in the tumor tissues were found to correlate with the level of Vimentin, E-Cadherin and the ratio of Ki-67 positive tumor cells. Conclusions These results support the idea that C3aR signaling is over-activated in the tumor cells and may contribute to the progression of ccRCC. Inducing EMT and promoting the proliferation of tumor cells might be among the mechanisms underlying the role of C3aR signaling in ccRCC. production of C3a, the ligand for C3aR, in the tumor tissues. These results suggest the presence of over-activation of C3aR signaling in the tumor cells of ccRCC patients and its involvement in the pathogenesis of ccRCC. In

cell renal cell carcinoma (ccRCC) is the major histological subtype of RCC, comprising more than 70% of all RCC cases [2]. ccRCC is commonly resistant to conventional chemotherapy and radiotherapy. Early surgical resection is the preferred therapy.
However, up to 40% of ccRCC patients with localized disease will eventually suffer a relapse or develop metastatic disease even after nephrectomy [3][4]. In the last two decades, some adjuvant therapies including immunotherapy and target therapy have been introduced to treat patients with metastatic ccRCC. However, these therapies either have limited effect and severe side role or develop resistance quickly. Clinically, improvement in the effect of ccRCC treatment is limited. Therefore, there is an urgent need to found new prognosis marker and therapeutic target for the disease.
The complement system is an effector arm of innate immunity, which is evolved as a safeguard against non-self elements and consists of more than 30 soluble and membranebound plasma proteins. It can be activated mainly through three pathways: the classical, the alternative and the lectin pathways. All the three pathways converge into the generation of C3 convertase, which splits complement component C3 into C3a and C3b.
C3b binding to C3 convertase assembles the C5 convertase that cleaves C5 into C5a and C5b. In combination with C6, C7, C8 and C9, C5b assembles the membrane attack complex C5b-9, which mediates targeted cell lysis. Traditionally, complement activation is believed to be advantageous in tumor control. However, this longstanding dogma is challenged now. Increasing evidences from recent years suggest that complement enhances rather than inhibits tumor growth and metastasis and thus promote tumor progression [5][6]. It has been reported that the role of complement in promoting tumor progression is mediated mainly by anaphylotoxins, the bioactive complement activation products C3a and C5a. Of note, most of the previous studies are based on in vitro or animal experiments. The exact clinical significance and underlying mechanism still remain to be 4 elucidated in specific diseases. Besides, most of the previous reports have been focused on the role of C5a/C5aR, studies about the role of C3a/C3aR in tumors is still limited.
The expression of C5a and its receptor C5aR1 in the tumor tissue of patients with ccRCC has been reported by several studies [7][8][9]. However, to our knowledge, no study has reported the clinical significance of C3a/C3aR signaling in patients with ccRCC. Here, we investigated the expression of C3aR and the production of its ligand in the tumor tissue of ccRCC patients and associated them with various clinicopathological indices.

Patients
Two cohorts of patients were included. The cohort of TCGA patients included 530 ccRCC patients with RNA-seq data available in TCGA database (TCGA-KIRC). All the normalised mRNA expression data and clinical data were obtained from the TCGA website (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga). mRNA expression data of 72 normal controls were also obtained from the database. This cohort included 344 male and 186 female patients with a median age of 61 years (ranging from 26 to 90 years).
The other cohort of patients included 129 patients, who were hospitalized at Department of Urology, Taizhou hospital, Wenzhou medical university from 2004 to 2013. All the patients were histologically confirmed ccRCC after partial or radical nephrectomy with no other malignancy history and no history of anticancer therapy. Patients with mixed histological type and died in a month after surgery were excluded. The patient cohort included 88 males and 41 females with a median age of 58 years (ranging from 24 to 78 years). Among them, 20 patients have lost follow-up. The clinical and pathological data were collected from medical records and follow-up records. The time interval between surgery and the date of death or the last visit was calculated and defined as overall 5 survival (OS). The interval between primary surgery and death from ccRCC or the last follow-up visit was defined as disease-specific survival (DSS). For the analysis of diseasespecific mortality, deaths as a result of other causes were censored. Informed consent has been obtained from all the participants. All research work with human participants was in accordance with the ethical standards of the responsible committee on human experimentation and with the Declaration of Helsinki. The Taizhou Hospital Ethics Committee approved the present study (No. K20191201).

Immunohistochemical staining and analysis
Immunohistochemical staining was performed on formalin-fixed Paraffin sections. Briefly, the sections were deparaffinised in xylene, rehydrated with graded ethanols, autoclaved for antigen repair and treated with 3% hydrogen peroxide solution to inactivate the

Statistical analysis
The data were analyzed with the SPSS software, version 17.0. Mann-Whitney U test was used to compare the difference between two groups while Kruskal-Wallis test was used to compare difference among three groups. The Spearman correlation analysis was used to explore the correlation of C3aR and C3c level (represented by score) with the clinicopathological indices and the level of other molecules. The Kaplan-Meier method and log-rank test were used for survival analysis. The Cox proportional hazards model was used to determine which variables influenced survival. The variables that significantly impacted survival in univariate analyses were included in multivariate analyses. All statistical tests were two tailed, and P values <0.05 were considered significant.

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, 7 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. 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 8 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  (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 9 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 [10], the cells from which ccRCC was believed to derive. Besides, activation of C3aR has been reported to promote proliferation of glomerular mesangial cells [11] and cutaneous squamous cell carcinoma cells [12]. 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

Discussion
In the present study, we explored the clinical and pathological significance of C3aR signaling in ccRCC by examining the expression of C3aR and the production of its ligand in the tumor tissue of ccRCC patients and their association with the disease. C3aR was found increasingly expressed in both mRNA and protein level in the tumor tissue of ccRCC patients. It was found to be distributed mainly in the tumor cells. In the meanwhile, the expression of C3 mRNA and the immunostaining for C3c, a product of C3 activation, were also found to increase, indicating increased production of C3a, the ligand for C3aR, in the tumor tissues. These results suggest the presence of over-activation of C3aR signaling in the tumor cells of ccRCC patients and its involvement in the pathogenesis of ccRCC. In accordance with this, higher C3aR level in the tumor cells and C3c level in the tumor tissues were found to be associated with higher tumor grade and the presence of tumor necrosis, two indices that were thought to be associated with worse prognosis in ccRCC patients [13][14]. Indeed, patients with higher C3aR or C3c level were found to have shorter OS and DSS. And higher C3c level in the tumor tissues was found to be an independent risk factor for worse prognosis. Together, these results strongly suggested that C3aR signaling have important role in the regulation of ccRCC progression and higher C3aR in tumor cells and C3c in the tumor tissues might be used as markers of worse prognosis of ccRCC.
As a member of G protein coupled receptor, C3aR was initially found to be expressed by some immune cells and to play important role in the regulation of inflammation by recruiting and activating inflammatory cells [15]. However, increasing evidence from the last two decades has demonstrated that C3aR is also expressed by many non-immune cells and participates in various physiological and pathological processes. For examples, C3aR signaling was reported to participate in the regulation of eye morphogenesis [16], neural development [17][18], embryonic chick retina [19] and cardiac resident cell [20] regeneration, astroglial cell differentiation [21] and survival [22], diet-induced obesity and metabolic dysfunction [23], and tau hyperphosphorylation in Alzheimer's Disease [24].
Also, C3aR signaling was found to be involved in the induction of inflammatory cytokines [25], proliferation of mesangial [11] and carcinoma cells [12] and EMT of tubular epithelial cells [10]. Recently, C3aR signaling was believed to be an important factor in maintaining cellular homeostasis [26][27].
In the field of oncology, signaling through C3aR has been suggested to promote tumor progression. However, variable results about the role of C3aR signaling in tumor cells have been reported in previous studies. In 2014, Cho et al first reported that tumor cell derived anaphylotoxins (C3a and C5a) promoted proliferation, migration and invasiveness of ovarian, uterine and lung cancer cells [28]. Later, Maurer et al reported that activation of C3aR could stimulate proliferation of medulloblastoma cells [29]. In addition, Fan et al reported that activation of C3aR promoted proliferation, migration and stemness in cutaneous squamous carcinoma [12]. However, in other studies [30][31] Limitations are present in the present study. First, in the analyzing of C3aR and C3 mRNA expression, we only analyzed the data from TCGA database. No further proving work, such as quantitative RT-PCR, was done. Secondly, in the analyzing of C3aR and C3c protein expression, only patients from our hospital were included and the patient population involved is somehow small. Given the high heterogeneity of ccRCC, bias due to both a single center study and a small population may be inevitable.

Conclusions
In summary, for the first time, the present study examined the pathological significance of

Availability of data and materials
All data generated or analyzed during this study are included in this article

Ethical approval and consent to participate
The experimental applications of patient specimens were approved by The Taizhou Hospital Ethics Committee (No. K20191201). Informed consent has been obtained from all the participants.

Consent of publication
All Authors have seen and approved the manuscript and consent publication. Tables   Table 1 Association of C3aR and C3c level with clinicopathological parameters   Variable C3aR level in tumor cells (score) p C3c level in tumor tissue (score) p      in tumor cells and C3c in tumor tissue, patients were divided into lower C3aR group and higher C3aR group, lower C3c group and higher C3c group. Then, the overall survival and Disease-specific survival were compared between the groups.