Deciphering the Role of the Anaphylatoxin C3a: A Key Function in Modulating the Tumor Microenvironment

Simple Summary The complement system is activated within the tumor microenvironment and its role in cancer development has raised much attention over the last decade. The aim of our study was to investigate the effects of the C3a anaphylatoxin on tumor cells (B16/F0 melanoma cell line) and two cell components of the tumor microenvironment: macrophages (Raw 264.7 Blue cell line) and mesenchymal stem cells (3T3-L1-like cell line). We showed that C3a plays a crucial role in the tumor microenvironment and may influence the tumor’s fate by modulating the expression of cytokines (including IL-10, TGFβ1), chemokines, Cox-2, and HO-1, and upregulating the oxidative stress response. We also demonstrated that C3a regulates VEGF expression, suggesting a role of the C3a/C3aR axis in angiogenesis. Our results provide novel insights into tumorigenesis and open new therapeutic avenues (C3aR antagonists) in cancer therapy. Abstract The complement system plays a crucial role in cancer development. Our study investigated the role of C3a anaphylatoxin on the tumor microenvironment. Our models consisted of mesenchymal stem cells (MSC-like, 3T3-L1), macrophages (Raw 264.7 Blue, (RB)) and tumor cells (melanoma B16/F0). Recombinant mouse (Mo) C3a (rC3a) was produced in CHO cells transfected with a Mo-IL10-signal peptide-Mo C3a plasmid construct. The effects of rC3a, IFN-γ, TGF-β1, and LPS were tested on the expression of C3, C3aR, PI3K, cytokines, chemokines, transcription factors, antioxidant defense mechanisms, angiogenesis and macrophage polarization (M1/M2). 3T3-L1 expressed the highest levels of C3, while C3aR was expressed more by RB. Interestingly, expression of C3/3T3-L1 and C3aR/RB was markedly upregulated by IFN-γ. rC3a was found to upregulate the expression of anti-inflammatory cytokines (IL-10) on 3T3-L1 and TGF-β1 on RB. rC3a also upregulated the expression of pro-inflammatory cytokines in RB. The expression of CCL-5 increased in 3T3-L1 in response to rC3a. On RB, rC3a did not alter M1/M2 polarization but upregulated the expression of antioxidant defense genes, HO-1, and VEGF. C3/C3a produced mainly by MSC may play a critical role in TME remodeling by stimulating both anti-inflammatory and proangiogenic activities of tumor stromal cells.


Introduction
The complement system is an integral and highly regulated part of the host immune system [1,2]. It bridges innate and the adaptative immunity [3], leads to the removal of pathogens, complements the inflammatory processes (by triggering the release of inflammatory cytokines), and plays a critical role in the responses to different diseases and

qRT-PCR (Sybergreen) Analyses
Total RNA extraction from harvested cell cultures (6-well plates) was performed using a Zymo kit (ZYMO, Catalog R1035). Extracted RNA was stabilized in 200 µL of RNA Shield and 800 µL of lysis buffer, collected and kept at −20 • C. qRT-PCR was performed using the One Step Bioline Sensifast Probe NO-ROX One step Kit (Meridian Bioscience, Cincinati, OH, USA, Bio-76005) and the Syber Green reagent (Lonza, Rockville, MD, USA, Cat.No.50513). The reaction mix contained 1 µL of extracted total RNA, 1.3 µL of primers mix (final primer concentration of 250 nM), and 2.7 µL of enzyme mix. qRT-PCR was performed using a Quantstudio 5 PCR thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). The 2−∆∆CT method has been used as a relative quantification strategy for quantitative real-time polymerase chain reaction data analysis. Moreover, GAPDH was selected as the reference gene to calculate relative gene expressions. Experiments were performed in triplicate. Primer and probe sequences are listed in Table 1.

Statistical Analysis
Results were expressed as mean ± standard error of the mean (SEM). All experiments consisted of three independent biological replicates. Statistical analysis was performed with GraphPad Prism 6 software. Two-way ANOVA tests followed by Bonferroni multiple comparison tests were performed (significance p < 0.05).

Cell Viability
We assessed the viability of 3T3-L1, B16/F0 and Raw 264.7 Blue cells to different sets of stimulation, such as IFN-γ (20 ng/mL), TGF-β1 (20 ng/mL), LPS (1 µg/mL) or CHO F3 clone supernatants (2% and 20%) for 24h. Control (CT) cells were treated with control CHO supernatants (20%). LDH release assay revealed that there was no effect on cell viability compared to the basal release in control cells ( Figure 1). In addition, MTT assay revealed that all treatments used did not affect mitochondrial metabolic activity. . We evaluated cytotoxicity as the ratio of released LDH levels after treatments compared to the maximum LDH release induced by Triton 1% exposure. Values correspond to means ± SEM of three independent replicates. Two-way ANOVA G1781, CytoTox 96 ® Non-Radioactive Cytotoxicity Assay, Promega). We evaluated cytotoxicity as the ratio of released LDH levels after treatments compared to the maximum LDH release induced by Triton 1% exposure. Values correspond to means ± SEM of three independent replicates. Two-way ANOVA followed by a multiple comparison test (Bonferroni's test) were performed. (**** p < 0.0001 as compared to non-stimulated cells (CT)).

C3/C3aR Expression
Next, we aimed to dissect the contribution of inflammatory mediators [34] during tumor development in regulating the expression of the C3 (precursor of C3a) and its receptor C3aR coupled to PI3K signaling. In addition, several pro-inflammatory mediators were tested given that they have been identified as playing a key role in proliferation, angiogenesis, tissue invasion, and metastasis propensity. They include TNF-α, Il-6, IL-1β, but also chemokines (IL-8 (CXCL8), CCL-2, CCL5) [35]. We stimulated all three cell types to study C3aR/C3 regulated expression ( Figure 2). Indeed, inflammation occupies an important position in the establishment of the TME that is decisive for the progression of oncogenesis.
C3aR mRNA expression was observed in all three cell models, while the highest expression (by a factor of 100-fold/B16 melanoma) was detected in Raw 264.7 Blue cells. After 24 h of stimulation by IFN-γ (20 ng/mL), TGF-β1 (20 ng/mL), or LPS (1 µg/mL), Raw 264.7 Blue showed significantly higher expression of C3aR mRNA ( Figure 2A). All treatments also upregulated the expression of C3aR mRNA on 3T3-L1.
C3 mRNA expression was more prominent in 3T3-L1 cells (50-60-fold/B16 melanoma) and was upregulated essentially in response to IFN-γ, a cytokine known to be released by intra-tumoral T cells ( Figure 2B).
PI3K signaling pathway is known to play an essential role in macrophage polarization [36]. It was also demonstrated that C3a alters the function of cancer-associated fibroblasts (CAFs known as MSC in cancer) by binding to its receptor and activating the PI3K/AKT pathway [22]. Both B16/F0 and 3T3-L1 expressed the highest levels of PI3K mRNA and particularly in response to TGF-β1 treatment for 3T3-L1 ( Figure 2C).
In Figure 2D, we observed a strong increase in IL-6 gene expression in all three cell types in response to IFN-γ. LPS had a pro-inflammatory action in 3T3-L1 and Raw Blue. LPS had a pro-inflammatory action in 3T3-L1 and Raw Blue. TNF-α gene expression was increased in response to LPS and IFN-γ treatment in all three cell types ( Figure 2E). TGF-β was only active in 3T3-L1 cells. Regarding chemokine expression ( Figure 2F,G) IFN-γ showed the highest proinflammatory activity in all three cell types.

Recombinant C3a in CHO Expression
Recombinant C3a (rC3a) can be produced in bacteria but may potentially be contaminated by LPS. Hence, we generated different clones of transfected CHO cell lines that express mouse rC3a ( Figure 3). The transfection was performed using a Mouse-IL10sp-Mo-C3a plasmid construct ( Figure 3A). After validating the efficiency of CHO transfection by qRT-PCR and the expression of the Mo-IL10sp-Mo-C3a mRNA ( Figure 3B), we validated the expression of the C3a protein via immunofluorescence using a specific rat anti-mouse C3a antibody (in green) ( Figure 3C). The supernatants from one of the CHO transfected clones obtained by limiting cell dilution, F3, cultured in DMEM in the absence of hygromycin, were used as a source of rC3a and tested on all three cell types at a percentage of 2% and 20%. Conditioned medium was prepared using the control CHO cells and stored until use.

Modulation of C3aR and PI3K Expression by Recombinant C3a
rC3a significantly enhanced the expression of its own receptor in Raw 264.7 Blue cells, 3T3-L1 and, to a much lesser extent, in B16/F0 melanoma ( Figure 4). The stimulation by rC3a significantly increased the expression of C3aR~4-fold in 3T3-L1 and~5-fold in Raw264.7 Blue cells ( Figure 4A). This result implies that there is a positive autoregulation loop exerted by rC3a. PI3K mRNA was significantly increased by~4-fold in 3T3-L1 cells but there was no significant difference in Raw 264.7 Blue cells ( Figure 4B). It is important to note that PI3K mRNA level does not reflect the status of its activated phosphorylated form. In Figure 2D, we observed a strong increase in IL-6 gene expression in all three ce types in response to IFN-γ. LPS had a pro-inflammatory action in 3T3-L1 and Raw Blue LPS had a pro-inflammatory action in 3T3-L1 and Raw Blue. TNF-α gene expression wa increased in response to LPS and IFN-γ treatment in all three cell types ( Figure 2E). TGF β was only active in 3T3-L1 cells. Regarding chemokine expression ( Figure 2F,G) IFNshowed the highest proinflammatory activity in all three cell types. . Reported values are means ± SEM of three independent experiments. Two-way ANOVA followed by a multiple comparison tes (Bonnferroni) was used to calculate the p value. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to unstimulated cells (Ct)).

Recombinant C3a in CHO Expression
Recombinant C3a (rC3a) can be produced in bacteria but may potentially be contam inated by LPS. Hence, we generated different clones of transfected CHO cell lines tha express mouse rC3a ( Figure 3). The transfection was performed using a Mouse-IL10sp . Reported values are means ± SEM of three independent experiments. Two-way ANOVA followed by a multiple comparison test (Bonnferroni) was used to calculate the p value. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to unstimulated cells (Ct)).

Modulation of C3aR and PI3K Expression by Recombinant C3a
rC3a significantly enhanced the expression of its own receptor in Raw 264.7 Blue cells, 3T3-L1 and, to a much lesser extent, in B16/F0 melanoma (Figure 4). The stimulation by rC3a significantly increased the expression of C3aR ~4-fold in 3T3-L1 and ~5-fold in

Modulation of Cytokine Expression by Recombinant C3a
To further understand the immunoregulatory role of rC3a, we assessed the expression of genes associated with the regulation of inflammatory responses including TNF-α, NFκB, IL-1β, IL-6, IL-10 and TGF-β1 by qRT-PCR in all three cell types ( Figure 5). Focusing on anti-inflammatory cytokines (associated with pro-tumoral activities), we found that IL-10 expression was more prominent on 3T3 and upregulated by rC3a. With regard to TGF-β1, known for its immune-regulatory and pro-fibrotic activities, it was more expressed by Raw 264.7 Blue cells, particularly in response to rC3a. Pro-inflammatory cytokines (e.g., TNF-α, IL-1β) and the associated transcription factor NFκB were mainly expressed by RAW cells and rC3a upregulated the expression of all three. Interestingly, the pleiotropic cytokine IL-6, mainly expressed by 3T3-L1, was downregulated by rC3a.

Modulation of Cytokine Expression by Recombinant C3a
To further understand the immunoregulatory role of rC3a, we assessed the expression of genes associated with the regulation of inflammatory responses including TNF-α, NFκB, IL-1β, IL-6, IL-10 and TGF-β1 by qRT-PCR in all three cell types ( Figure 5). Focusing on anti-inflammatory cytokines (associated with pro-tumoral activities), we found that IL-10 expression was more prominent on 3T3 and upregulated by rC3a. With regard to TGF-β1, known for its immune-regulatory and pro-fibrotic activities, it was more expressed by Raw 264.7 Blue cells, particularly in response to rC3a. Pro-inflammatory cytokines (e.g., TNF-α, IL-1β) and the associated transcription factor NFκB were mainly expressed by RAW cells and rC3a upregulated the expression of all three. Interestingly, the pleiotropic cytokine IL-6, mainly expressed by 3T3-L1, was downregulated by rC3a.

Modulation of Chemokine Expression by Recombinant C3a
In this experiment, we studied the effect of rC3a on the major chemokines involved in inflammatory status, such as CCL-2, CCL-5 and MSC stem cell function CXCL-12 (CXCL-12, also known as the SDF-1 alpha) ( Figure 6). CCL-5 is a major chemoattractant for lymphocytes (particularly T cells) and we found that rC3a significantly increased the expression of CCL-5 by all three cell types, although the highest level of expression was found for 3T3-L1 ( Figure 6A). The constitutive expression of CXCL-12 ( Figure 6C) was also more prominent for 3T3-L1 MSC-like cells but with a downregulating activity of rC3a. CCL-2 is very modestly expressed by B16/F0 and Raw 264.7 Blue and its level of expression was upregulated by rC3a. rC3a, in contrast, had no effect on CCL-2 expression by 3T3-L1, which expressed the highest level of CCL-2 mRNA compared to Raw 264.7 Blue and B16/F0 ( Figure 6B).  expression of CCL-5 by all three cell types, although the highest level of expression was found for 3T3-L1 ( Figure 6A). The constitutive expression of CXCL-12 ( Figure 6C) was also more prominent for 3T3-L1 MSC-like cells but with a downregulating activity of rC3a. CCL-2 is very modestly expressed by B16/F0 and Raw 264.7 Blue and its level of expression was upregulated by rC3a. rC3a, in contrast, had no effect on CCL-2 expression by 3T3-L1, which expressed the highest level of CCL-2 mRNA compared to Raw 264.7 Blue and B16/F0 ( Figure 6B).

Regulation of COX-2 Expression Involved in Prostaglandin Biosynthesis by Recombinant C3a
Upregulation of Cox-2, a rate-limiting key enzyme in the synthesis of prostaglandins, plays an important role in the control of inflammation associated with cancer. We found that rC3a significantly increased the expression of COX-2 in B16/F0 and 3T3-L1 cells but had no effect on Raw 264.7 Blue cells (Figure 7).

Regulation of COX-2 Expression Involved in Prostaglandin Biosynthesis by Recombinant C3a
Upregulation of Cox-2, a rate-limiting key enzyme in the synthesis of prostaglandins, plays an important role in the control of inflammation associated with cancer. We found that rC3a significantly increased the expression of COX-2 in B16/F0 and 3T3-L1 cells but had no effect on Raw 264.7 Blue cells (Figure 7).

Modulation of Antioxidant Defense System by Recombinant C3a
We examined the effects of rC3a on the regulation of the oxidative stress response, and measured RNA expression of SOD-1-2, catalase, and Nrf2. The upregulated expression of all antioxidant genes was particularly prominent in Raw 264.7 Blue cells ( Figure  8). SOD-2 and catalase expression was also upregulated in B16/F0, while no major regulation of these genes was noted when C3a stimulated 3T3-L1 cells. The results indicated that

Modulation of Antioxidant Defense System by Recombinant C3a
We examined the effects of rC3a on the regulation of the oxidative stress response, and measured RNA expression of SOD-1-2, catalase, and Nrf2. The upregulated expression of all antioxidant genes was particularly prominent in Raw 264.7 Blue cells (Figure 8). SOD-2 and catalase expression was also upregulated in B16/F0, while no major regulation of these genes was noted when C3a stimulated 3T3-L1 cells. The results indicated that C3a may participate in the regulation of oxidative stress (which plays a crucial role in tumorigenesis and metastasis) by increasing the expression of antioxidant systems in macrophages and to a less extent in tumor cells.

Modulation of Antioxidant Defense System by Recombinant C3a
We examined the effects of rC3a on the regulation of the oxidat and measured RNA expression of SOD-1-2, catalase, and Nrf2. The u sion of all antioxidant genes was particularly prominent in Raw 264 8). SOD-2 and catalase expression was also upregulated in B16/F0, wh tion of these genes was noted when C3a stimulated 3T3-L1 cells. The r C3a may participate in the regulation of oxidative stress (which pla tumorigenesis and metastasis) by increasing the expression of antioxid rophages and to a less extent in tumor cells.

Pro-Angiogenic Role of C3a
Angiogenesis has an undeniable role in the aggressiveness of tumor cells and in metastasis [37]. Moreover, it was reported that in a model of ovarian cancer, C3 silencing in cancer cells reduced the microvessel density in tumors [38]. Here, we wanted to see the effect of C3a on VEGF expression, which has a crucial role in promoting angiogenesis (Figure 9). Interestingly, we found that rC3a modulates the angiogenic properties of macrophages (Raw 264.7 Blue), mesenchymal (3T3-L1) and tumor cells (B16/F0) by significantly increasing the expression of VEGF mRNA by these cells (Figure 9A). In addition, rC3a significantly enhances these cells' expression of HO-1 ( Figure 9B). It is important to note that high levels of HO-1 have been particularly associated with tumor-associated macrophages (TAMs) with a polarization program similar to the M2 tumor promoting macrophage phenotype [39]. expression were determined by qRT-PCR. Reported values are means ± SEM of three independ experiments. (** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to untreated cells (Ct)).

Pro-Angiogenic Role of C3a
Angiogenesis has an undeniable role in the aggressiveness of tumor cells and in m tastasis [37]. Moreover, it was reported that in a model of ovarian cancer, C3 silencing cancer cells reduced the microvessel density in tumors [38]. Here, we wanted to see effect of C3a on VEGF expression, which has a crucial role in promoting angiogene ( Figure 9). Interestingly, we found that rC3a modulates the angiogenic properties of m rophages (Raw 264.7 Blue), mesenchymal (3T3-L1) and tumor cells (B16/F0) by sign cantly increasing the expression of VEGF mRNA by these cells (Figure 9A). In additi rC3a significantly enhances these cells' expression of HO-1 ( Figure 9B). It is importan note that high levels of HO-1 have been particularly associated with tumor-associa macrophages (TAMs) with a polarization program similar to the M2 tumor promot macrophage phenotype [39].

Expression of C3a and C3ar in Human Melanoma
Lastly, we used immunohistochemistry to investigate the expression of C3a a C3aR in a case of human melanoma ( Figure 10A-F) and in a benign case of human n vocelllular naevus ( Figure 10G,H). In melanoma, both C3a and C3aR expressions predo inated in the perivascular areas. C3aR+ cells had a macrophage-like morphology with abundant cytoplasm and a small, round nucleus. Interestingly, we observed that C3a calized closely to C3aR+ cells within the perivascular bed ( Figure 10E,F, arrowhead) addition, we detected a small subset of tumor cells that express C3a, but not C3aR. Wit the benign naevus, C3a and C3aR were not expressed ( Figure 10G,H).

Expression of C3a and C3aR in Human Melanoma
Lastly, we used immunohistochemistry to investigate the expression of C3a and C3aR in a case of human melanoma ( Figure 10A-F) and in a benign case of human naevocelllular naevus ( Figure 10G,H). In melanoma, both C3a and C3aR expressions predominated in the perivascular areas. C3aR+ cells had a macrophage-like morphology with an abundant cytoplasm and a small, round nucleus. Interestingly, we observed that C3a localized closely to C3aR+ cells within the perivascular bed ( Figure 10E,F, arrowhead). In addition, we detected a small subset of tumor cells that express C3a, but not C3aR. Within the benign naevus, C3a and C3aR were not expressed ( Figure 10G,H).  D,F,H). Using serial tissue sections, we were able to identify cells single or double st for both markers. For example, the arrow points to a C3aR+/C3a− cell and the arrowhea C3aR+/C3a+ perivascular cells (macrophage-like) in melanoma. Brown background staining detected due to the presence of the melanin pigment in melanoma. The benign naevus reveal C3a/C3aR expression. Images are shown at ×100 (A,B), ×200 (C,D) and ×400 (E-H) magnificatio addition, we confirmed that, in malignant melanoma, the majority of C3a and C3aR cells coized with CD68 and CD163 macrophages within the perivascular areas ( Figure 11A-D). In add few CD248/endosialin + MSC were observed within the vascular walls ( Figure 11E). To a less tent, C3a cells colocalized with SOX10 (nuclear staining) melanoma tumor cells ( Figure 11F). N bly, double-staining experiments-for example, those using peroxidase-conjugated antibody DAB brown color development-were not conclusive due to the intrinsic presence of brown nin pigment within the melanoma cells and melanophages.  D,F,H). Using serial tissue sections, we were able to identify cells single or double stained for both markers. For example, the arrow points to a C3aR+/C3a− cell and the arrowheads to C3aR+/C3a+ perivascular cells (macrophage-like) in melanoma. Brown background staining was detected due to the presence of the melanin pigment in melanoma. The benign naevus revealed no C3a/C3aR expression. Images are shown at ×100 (A,B), ×200 (C,D) and ×400 (E-H) magnification. In addition, we confirmed that, in malignant melanoma, the majority of C3a and C3aR cells colocalized with CD68 and CD163 macrophages within the perivascular areas ( Figure 11A-D). In addition, few CD248/endosialin + MSC were observed within the vascular walls ( Figure 11E). To a lesser extent, C3a cells colocalized with SOX10 (nuclear staining) melanoma tumor cells ( Figure 11F). Notably, double-staining experiments-for example, those using peroxidase-conjugated antibody and DAB brown color development-were not conclusive due to the intrinsic presence of brown melanin pigment within the melanoma cells and melanophages.

Discussion
A new function of complement in cancer has emerged, mainly through the actio the anaphylatoxins C3a/C5a and their capacity to modulate immune cell and MSC fu tions.
Clinical observations and animal studies showed the critical role of C3a in suppor tumor growth: C3 (and C3a cleavage product) produced by CD8 + T cells may prom tumor progression by inhibiting these cells' IL-10 production [27].
In contrast, other reports found that mice lacking C3 or C5aR showed decreased mor growth [14]. Additionally, cancer treated with C3a, C3aR and/or C5aR inhibi showed less resistance to therapy or had restricted metastasis [29,32,40], arguing that may have pro-tumorigenic activities, yet poorly characterized mechanisms.
Here, we demonstrated that different stimulatory paradigms present in the str of tumors and mobilized either via innate immune PRRs (i.e., TLR4 agonist LPS), adap immunity (i.e., IFN-γ, produced by T cells) and via tumor growth factor (exemplified profibrotic TGF-β1), regulated the expression of C3 as well as C3aR and downstream P signaling molecule.
Most of all, we found that the C3a/C3aR axis involved principally and, respectiv 3T3-L1 on one hand and Raw 264.7 Blue cells on the other, arguing for the capacit MSC-derived C3a to influence macrophage behavior via C3aR.
Unexpectedly, we found that IFN-γ, a cytokine expressed by infiltrating tumor cells and which is generally thought to protect against tumor expansion, markedly creased both C3 (precursor of C3a) and C3aR expression levels. It will be importan

Discussion
A new function of complement in cancer has emerged, mainly through the action of the anaphylatoxins C3a/C5a and their capacity to modulate immune cell and MSC functions.
Clinical observations and animal studies showed the critical role of C3a in supporting tumor growth: C3 (and C3a cleavage product) produced by CD8 + T cells may promote tumor progression by inhibiting these cells' IL-10 production [27].
In contrast, other reports found that mice lacking C3 or C5aR showed decreased tumor growth [14]. Additionally, cancer treated with C3a, C3aR and/or C5aR inhibitors showed less resistance to therapy or had restricted metastasis [29,32,40], arguing that C3a may have pro-tumorigenic activities, yet poorly characterized mechanisms.
Here, we demonstrated that different stimulatory paradigms present in the stroma of tumors and mobilized either via innate immune PRRs (i.e., TLR4 agonist LPS), adaptive immunity (i.e., IFN-γ, produced by T cells) and via tumor growth factor (exemplified by profibrotic TGF-β1), regulated the expression of C3 as well as C3aR and downstream PI3K signaling molecule.
Most of all, we found that the C3a/C3aR axis involved principally and, respectively, 3T3-L1 on one hand and Raw 264.7 Blue cells on the other, arguing for the capacity of MSC-derived C3a to influence macrophage behavior via C3aR.
Unexpectedly, we found that IFN-γ, a cytokine expressed by infiltrating tumoral T cells and which is generally thought to protect against tumor expansion, markedly increased both C3 (precursor of C3a) and C3aR expression levels. It will be important to better address the phenotype of infiltrating T cells producing IFN-γ which may be differentiating into immunoregulatory Th2 or Treg phenotypes while infiltrating tumor beds. The capacity of C3a to upregulate the expression of TGF-β1 and IL-10, respectively, by Raw 264.7 Blue cells and 3T3-L1 is interesting and may represent a plausible link between C3a, polarized Treg differentiation, and immunoparalysis, given that both cytokines control lymphocyte adaptative immune functions. T cells infiltrate tumors in response to chemokines and, indeed, we found that C3a was able to upregulate CCL-5 but not CCL-2 (macrophage chemokine) via 3T3-L1 cells. CCL-5 is a chemokine involved in T cell chemotaxis. We also tested but failed to validate the capacity of C3a to regulate the expression of the chemokine CXCL-12, also known as a stromal growth factor (SDF-1).
Focusing on macrophages, it has been reported that C3a controls TAM's polarization through C3a-C3aR-PI3Kγ signaling pathway. Mechanistically, it was shown that C3a could modulate TAMs into an M2 phenotype through the C3a-C3aR-PI3Kγ pathway [41]. Using our Raw cell model, we were not able to reach conclusive interpretations regarding the capacity of rC3a to modulate M2 versus M1 phenotypes. Our study was limited to a subset of cell markers, and a more comprehensive study needs to be performed. However, our data clearly indicated that C3a upregulated the expression of two major proinflammatory cytokines, namely TNF-α and IL-1β. This result has to be correlated with increased expression of their transcription factor, NFκB, due to rC3a exposure. High reactive oxygen species (ROS) levels play an important role as a cancer stimulator, inducing a wide range of mutations through DNA damage. Oxidative stress occurs in cells when the generation of ROS overwhelms the cell natural antioxidant defenses. We found that rC3a enhanced intracellular ROS generation in Raw 264.7 Blue cells but this was accompanied by elevated expression of anti-oxidant defense molecules such as SOD1/2, and catalase. We also found that rC3a enhanced the expression of heme-detoxification molecule HO-1 in Raw 264.7 Blue macrophages.
The C3a-C3aR-PI3K pathway was demonstrated to have an important role in activating CAFs (representing the population of intra-tumoral MSC) leading to lung metastasis in a breast cancer model [22]. Angiogenesis is a key event in cancer progression [42]. It is highly regulated by cancer and stromal cells.
The role of anaphylatoxins in angiogenesis is poorly studied. C3 has shown an antiangiogenic role in a model of retinopathy of prematurity (ROP): it was found that mice deficient in C3 displayed increased neovascularization in this model and in the in vivo Matrigel plug assay [43]. In contrast, both C3a and C5a promote choroidal neovascularization in an age-related macular degeneration. In this study, we demonstrated that C3a affects angiogenesis by regulating the essential vascular endothelial growth factor (VEGF) expression by 3T3-L1 cells. It will be important to ascertain whether the C3a-C3aR axis may also regulate the production of other pro-angiogenic factors. We and others have recently found a strong link between high levels of C3aR+ TAM and VEGF expression in human and mouse models of cancer [21,29]. Indeed, Davidson and colleagues reported that the lack of a functional C3aR leads to a reduction in the B16 tumor mass and is associated with an increase in non-differentiated pro-inflammatory monocytes [29]. These findings support the critical role of C3a in monocyte recruitment and differentiation to anti-inflammatory macrophages. C3aR expression was also confirmed in the study that used the MN/MCA1 fibrosarcoma or 3-MCA-derived tumor models. The authors demonstrated that C3aR was mainly expressed by TAM [31]. These authors reported an upregulation of M1-like markers (CD11c, major histocompatibility complex class II (MHC-II), CD80, and CD86) and a reduction in the frequency of CD206+ (an M2-like marker) TAMs in C3aR-deficient mice. The frequency of infiltrating CD4+ T lymphocytes was significantly increased in C3aR-deficient mice to mediate a more robust adaptive immune response against cancer [31] and in light of the observation that the number of CD8+ infiltrating T cells was also higher in C3aR KO [29]. Thus, our results using model cell lines and the aforementioned mouse C3aR KO models argue for a major capacity of C3a/C3aR signaling to control immune cell infiltration and anti-tumor responses.
Lastly, we showed through immunohistochemistry that C3a and C3aR were mainly expressed and colocalized within the perivascular areas in a human melanoma tumor case. C3a and C3aR were totally absent from our benign case of human naevocelllular naevus. The perivascular preferential localization of both markers in melanoma supports our in vitro results arguing for a potential role of C3a/C3aR axis in angiogenesis and with the critical role of TAM in this process. Yet, the exact phenotype of those C3a+/C3aR+ perivascular cells is still to be demonstrated, in spite of their macrophage-like morphology. In addition, we observed that a subset of melanoma cells expresses C3a, suggesting a potential role of tumor cells in macrophage recruitment within the tumor microenvironment. Further morphological investigations are required to better characterize C3a and C3aR expressions in a much larger panel of human melanomas and to identify the differential phenotype of the C3a+ and C3aR+ cells.

Conclusions
In the present study, we show that the anaphylatoxin C3a plays a crucial role in the modulation of TME. It exerts a pro-(including the chemotaxis of T cells) and antiinflammatory action depending on the cell types. These two effects may be interconnected in the case of melanoma (B16/F0) and transform TME into an immunosuppressive setting. A better understanding of the mechanism of action, the signaling pathways involved in modulating the TME with the identification of antagonists, will provide new breakthroughs in cancer therapy.