Renca cells (ATCC: CRL-2947) were obtained in 2013 from the American Type Culture Collection. The cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS), penicillin (40 U/ml) and streptomycin (40 μg/ml) and were maintained at 37°C in 5% CO₂.

Recombinant C5a was purchased from Hycult Biotech (Plymouth, PA, USA). G418 was purchased from InvivoGen (San Diego, CA, USA). U0126 was purchased from Promega (Madison, WI, USA). PI-103 was purchased from Merck (Darmstadt, Germany). Anti-C5aR antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for phospho-ERK and total ERK, phospho-Akt and total Akt and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Renal cell carcinoma tissue samples were obtained by surgical resection or core needle biopsy from 127 patients in Kumamoto University Hospital, and the usage of those samples for the present study was approved by The Internal Review Board of Kumamoto University Hospital. Immunohistochemistry for C5aR in the RCC samples was performed according to a previously described protocol (9). The relationship between the C5aR expression and baseline demographic data/clinicopathological parameters was analyzed by Fisher’s exact test.

Immunoblotting was performed as previously described (10). Briefly, cell lysates were analyzed by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes (Protran; GE Healthcare Life Sciences, Pittsburgh, PA, USA). After blocking with blocking buffer (1% TBS-T buffer containing 5% bovine serum albumin), the membranes were incubated with the primary antibodies indicated according to the manufacturer’s instructions, followed by incubation with appropriate HRP-conjugated secondary antibodies. The bands were visualized by ECL Plus Western Blotting Detection System (GE Healthcare Life Sciences) according to the manufacturer’s instructions.

pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA). Renca cells were transfected with pCMV-C5aR using the ProFection Mammalian Transfection System (Promega). After 48 h, the medium was replaced with a selection medium supplemented with G418 (400 μg/ml) then cultured for 2 weeks. G418-resistant cells were isolated by limiting dilution and propagated. The cells were subjected to flow cytometric analysis by FACSCan (BD Biosciences, San Jose, CA, USA) as previously described (9) to select cells expressing C5aR (designated as Renca/C5aR cells). Renca cells transfected with the empty plasmid pCMV and resistant to 400 μg/ml G418 were isolated and then designated as Renca/empty cells. For the in vivo study, the Renca-derived cells were injected into the renal subcapsular space in mice according to Shvarts et al (11). This experiment was approved by the Kumamoto University Animal Experiment Committee.

Anoikis assay was performed based on the protocol reported by Berezovskaya et al (12) with minor modifications. Renca-derived cells were dissociated by Accutase (Millipore, Billerica, MA, USA), and then washed with serum-free medium and suspended with medium containing 0.5% FCS. The viable cells were counted using the trypan blue dye exclusion method to confirm that the initial viability of the cells after dissociation by Accutase was >95%. A suspension of 1×10⁶ viable cells was treated with or without 10 nM recombinant C5a (rC5a) then plated in 2.0 μl of serum-free medium in 6-well ultra-low-attachment polystyrene plates (Corning, Tewksbury, MA, USA) and incubated at 37°C in 5% CO₂ overnight. The numbers of the total and viable cells after incubation were counted as described above, and then the percentage of the viable cells was calculated.

Cells (5×10⁴) were seeded on glass coverslips and incubated for 24 h. After serum starvation overnight, the cells were stimulated with 10 nM rC5a for the stated time periods. The cells were then fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 for 5 min, and were incubated with 5 U/ml Alexa 488-phalloidin (Invitrogen Life Technologies, Carlsbad, CA, USA) for 40 min, followed by washing with PBS. Nuclei were counterstained with 1.0 mM TO-PRO^®-3 (Invitrogen Life Technologies) for 15 min. Images were obtained and processed by FluoView 300 laser scanning confocal microscope (Olympus, Melville, NY, USA).

BioCoat Matrigel invasion chambers were utilized (24-well plates, 8-μm pores; BD Biosciences) as previously described (9). Renca-derived cells (5×10⁴) were suspended in serum-free RPMI-1640, which were then seeded into the upper chamber. RPMI-1640 supplemented with either 10 nM rC5a or carrier solution (PBS) was placed in the lower chamber. PI-103, U0126 or carrier control (DMSO) was added to the medium in both the upper and lower well at the indicated concentrations when appropriate. The numbers of cells that migrated through the membrane were counted in 4 microscopic fields (x20 magnification) per membrane. The average was calculated from triplicate samples, and statistical analyses were performed by two-tailed t-tests.

First, we analyzed C5aR expression in 127 primary RCC specimens using surgically removed or needle biopsy samples. Overall, the C5aR expression was observed in 78 out of 127 samples (61.4%). This sample cohort consisted of 97 RCCs without metastasis and 30 mRCC (Fig. 1). As shown in Table I, there was no significant difference between the C5aR-positive and -negative group in regards to age, gender and histological subtype. Regarding Fuhrman grade, although the grade 3 population was quite low (n=6) in this sample cohort, C5aR-positive tumors tended to exhibit a higher grade than the C5aR-negative tumors (G1: 21.8 vs. 46.9%, G2: 69.2 vs. 49.0%). Regarding T staging, the C5aR-positive group contained significantly less T1 tumors (P=0.0056) and more T3 tumors (P=0.0283) than the C5aR-negative group. As for N staging, more C5aR-negative tumors were without lymph node metastasis (N0) than the C5aR-positive tumors (98.0 vs. 87.2%) with marginal significance (P=0.0497). Distant metastasis was more frequently observed in the C5aR-positive group than in the C5aR-negative group (37.2 vs. 2%). Of note, 96.7% (29/30) of the patients with metastatic disease showed C5aR expression at their primary sites. With regards to microscopic invasion, more C5aR-positive tumors manifested microvascular invasion than C5aR-negative tumors (29.0 vs. 6.3%). These results suggest that C5aR expression contributes to local invasion and distant metastasis of renal cell carcinoma.

To study the biological role of the C5a-C5aR axis in renal cell carcinoma, we employed Renca cells since these cells are the established model for studying renal cell carcinoma metastasis (11,13). Interestingly, both western blotting and flow cytometric analysis showed that C5aR was not expressed in Renca cells (data not shown). Such an observation in cancer cell lines, which does not seemingly reflect the characteristics in clinical specimen, was described and discussed in our previous study, suggesting that C5aR expression was lost during the process of cell line establishment from primary culture of cancer cells in order to prioritize the expression of other essential proteins for clonal development in the context of 2-dimensional culture (9). To establish renal cell carcinoma cells expressing C5aR, C5aR cDNA was stably introduced into Renca cells, and clones expressing C5aR were isolated and propagated. Cells expressing C5aR on their cell surface, confirmed by flow cytometric analysis (Fig. 2A), were selected and designated as Renca/C5aR cells. Cells stably transfected with the empty plasmid were designated as Renca/empty cells. The original purpose of establishing Renca/C5aR cells was to study if C5aR can enhance the metastatic behavior of renal carcinoma cells in a syngeneic orthotopic murine model using BALB/c mice since Renca cells are known to establish multiple lung metastasis in this model (11). However, the Renca/C5aR cells, which were stimulated by recombinant C5a and then injected into the renal subcapsular space, did not exhibit any increase in either the number or the size of lung metastatic nests compared to the Renca/empty cells (data not shown). This may be due to the difficulty of attaining sustained C5aR activation by recombinant C5a until establishing lung metastasis, considering the stability of C5a and the period required for tumor cell implantation into the lungs after subcapsular injection. Because of such technical difficulty of the in vivo study to analyze the biological role of C5aR expression in renal cell carcinoma metastasis, we analyzed the effect of C5aR on crucial steps of cancer metastasis in vitro instead. Anoikis is a subtype of apoptosis induced upon cell detachment from the extracellular matrix, which is an indispensable step for metastasis (14). To analyze whether renal carcinoma cells are able to acquire resistance to anoikis by C5a-C5aR axis activation, Renca-derived cells were treated with or without C5a and then cultured in suspension on ultra low-attachment plates and a number of viable cells was assessed. Fig. 2B shows that the percentage of Renca/C5aR cell survival in suspension culture was slightly higher than that of the Renca/empty cells, which was not significantly enhanced by C5a treatment. This result suggests that, although C5aR expression itself may have a marginal effect on cell survival in an adhesion-independent condition, it is unlikely that anoikis plays an important role in the metastasis of C5aR-expressing renal carcinoma cells.

It is known that the chemoattractant C5a causes actin rearrangement and stimulates the migration of leukocytes (15,16). We previously showed that cancer cells can exploit this mechanism to acquire the ability of migration and invasion by activation of aberrantly expressed C5aR using bile duct carcinoma cells (9). To test the hypothesis that C5aR expressed in renal cell carcinoma facilitates actin reorganization by C5a stimulation as well, the effect of C5aR activation on actin rearrangement in Renca cells was analyzed by F-actin immunofluorescent staining. Without C5a stimulation, both Renca/empty and Renca/C5aR cells showed staining of cortical F-actin bundles at the borders of the cells (Fig. 3A and E). As early as 15 min after C5a stimulation, Renca/C5aR cells revealed membrane ruffling formation at the periphery of the cell clusters with reduced F-actin bundles at the cell-cell border (Fig. 3F). Thirty minutes after stimulation, the cell-cell contact became more loosened and some cells started to manifest lamellipodia formation (Fig. 3G). This was followed by marked change in cell shape such as stretched morphology and dissociation of cell clusters accompanied by stress fiber formation (Fig. 3H). In contrast, Renca/empty cells did not show any significant changes in both cytoskeleton and cellular morphology during observation despite C5a stimulation (Fig. 3A–D). These results suggest that C5a elicits cytoskeletal rearrangement and cellular morphological change in renal carcinoma cells via C5aR, leading to their dissociation and scattering.

C5a is known to induce ERK (5,17) and PI3K activation (6,18) in inflammatory/immune cells in a C5aR-dependent manner, leading to manifestation of a variety of biological phenomena including leukocyte migration (19). Since ERK and PI3K are crucial mediators of extracellular stimuli-induced actin reorganization (20,21) which was observed in Fig. 3, we investigated if C5a provokes ERK and PI3K activation in C5aR-expressing renal carcinoma cells. As shown in Fig. 4A, Renca/C5aR cells showed a robust increase in ERK and Akt, substrate of PI3K, phosphorylation by C5a stimuli, whereas such phosphorylation did not occur in the Renca-empty cells. This result indicates that the C5a-C5aR axis can activate ERK and PI3K kinase pathways in renal carcinoma cells. We previously reported that the C5a-C5aR axis enhances cancer cell invasion both in vitro and in vivo (9). To test if this is also the case in renal carcinoma cells, we performed an invasion assay using a Matrigel-coated Boyden chamber. Fig. 4B shows that C5a stimuli elicited invasion of the Renca/C5aR cells, but not the Renca-empty cells, in the Matrigel-coated boyden chamber, indicating that the C5a-C5aR axis can enhance invasion of renal cell carcinoma as well. In addition, treatment with either an MEK inhibitor U0126 or a PI3K inhibitor PI-103 significantly diminished Renca cell invasion promoted by the C5a-C5aR axis (Fig. 4C). This result indicates that the ERK and PI3K pathways are indispensable for C5a-triggered C5aR-expressing renal carcinoma cell invasion. All things considered, these results suggest that the C5a-C5aR axis elicits renal carcinoma cell metastasis by triggering actin reorganization and invasion induced by ERK and PI3K pathway activation.