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. 2012 Sep 15;189(6):2985-94.
doi: 10.4049/jimmunol.1200846. Epub 2012 Aug 22.

Opposing roles for complement component C5a in tumor progression and the tumor microenvironment

Lacey Gunn  1 , Chuanlin Ding, Min Liu, Yunfeng Ma, Chunjian Qi, Yihua Cai, Xiaoling Hu, Deep Aggarwal, Huang-Ge Zhang, Jun Yan
Affiliations

Opposing roles for complement component C5a in tumor progression and the tumor microenvironment

Lacey Gunn et al. J Immunol. .

Abstract

Promoting complement (C) activation may enhance immunological mechanisms of anti-tumor Abs for tumor destruction. However, C activation components, such as C5a, trigger inflammation, which can promote tumor growth. We addressed the role of C5a on tumor growth by transfecting both human carcinoma and murine lymphoma with mouse C5a. In vitro growth kinetics of C5a, control vector, or parental cells revealed no significant differences. Tumor-bearing mice with C5a-transfected xenografted tumor cells had significantly less tumor burden as compared with control vector tumors. NK cells and macrophages infiltrated C5a-expressing tumors with significantly greater frequency, whereas vascular endothelial growth factor, arginase, and TNF-α production were significantly less. Tumor-bearing mice with high C5a-producing syngeneic lymphoma cells had significantly accelerated tumor progression with more Gr-1+CD11b+ myeloid cells in the spleen and overall decreased CD4+ and CD8+ T cells in the tumor, tumor-draining lymph nodes, and the spleen. In contrast, tumor-bearing mice with low C5a-producing lymphoma cells had a significantly reduced tumor burden with increased IFN-γ-producing CD4+ and CD8+ T cells in the spleen and tumor-draining lymph nodes. These studies suggest concentration of local C5a within the tumor microenvironment is critical in determining its role in tumor progression.

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Figures

Figure 1
Figure 1. In vivo growth of SKOV-3 tumor cells in SCID mice
(A) In vivo growth of SKOV-3 C5a and controls revealed a significant reduction in tumor growth of SKOV-3 C5a. Following s.c. injection of SCID mice with SKOV-3 tumor cell lines (n=20, 16, 8 for SKOV-3 C5a, CV, WT; respectively), tumor growth was monitored by measuring two perpendicular diameters every 2–4 days. *p<0.05, **p<0.01, ***p<0.001. (B) Tumor weight measurement when all animals were sacrificed. **p<0.01, ***p<0.001.
Figure 2
Figure 2. Enhanced innate immune cell infiltration in SKOV-3 cells expressing C5a
(A) Percentage of Gr-1+CD11b+ cells in SKOV-3 C5a tumor and SKOV-3 CV tumor was not significantly altered. (B) Increased percentage of DX5+CD11b+ NK cells were found to infiltrate SKOV-3 C5a tumors in vivo, as seen by flow cytometry and IF staining. *p<0.05. (C) Increased percentage of F4/80+CD11b+ macrophages in SKOV-3 C5a tumors as determined by flow cytometry and IF staining. *p<0.05, **p<0.01. Representative data from SKOV-3 C5a (n=10) and SKOV-3 CV (n=9) tumors are shown. Bar, 50 μm.
Figure 3
Figure 3. The altered tumor microenvironment by C5a
(A) Total tumor samples were collected and RNAs were extracted. qRT-PCR data revealed the downregulation of VEGF, arginase, and TNF-α mRNA levels in C5a expressing tumors. *p<0.05, **p<0.01. (B) Sorted CD11b+ innate immune cells and CD11b− tumor cells were performed for qRT-PCR analysis. Data indicate that VEGF mRNA level is significantly decreased in C5a expressing tumor cells while the CD11b+ cells sorted from SKOV-3 C5a have significantly lower levels of iNOS mRNA. *p<0.05 (C) The SKOV-3 C5a infiltrating F4/80+ macrophages expressed significantly lower levels of arginase mRNA. *p<0.05.
Figure 4
Figure 4. C5a promotes cytotoxicity of SKOV-3 tumor cells while Gr-1+CD11b+ cells from SKOV-3 C5a tumors are significantly less immunosuppressive
(A) SKOV-3 C5a and CV tumor cells were cultured overnight, and the following day non-adherent leukocytes from naïve SCID mice as effector cells were added at a ratio of 20:1 (E:T). After 16 hours of co-culture, percent of cytotoxicity was calculated (n=6). Data indicate that effector cells kill significantly more SKOV-3 C5a cells than SKOV-3 CV cells. **p<0.01. (B) Similarly, purified NK cells from naïve SCID mice were added to SKOV-3 C5a or CV tumor cells in vitro, and percent of cytotoxicity was determined following 24 hr co-culture. *p<0.05. (C) SKOV-3 tumor cells were co-cultured with non-adherent leukocytes as effector cells (20:1) in the presence or absence of Gr-1+CD11b+ cells sorted from CV or C5a-transfected tumor (1:1), or without effectors but with sorted Gr-1+CD11b+ cells from tumor. The innate leukocytes demonstrated effective cytotoxicity of SKOV-3 tumor cells and cytotoxicity was significantly decreased in the presence of Gr-1+CD11b+ cells sorted from tumors. However, Gr-1+CD11b+ cells from SKOV-3 C5a tumors were significantly less suppressive. **p<0.01. (D) Cytospin and stain of the Gr-1+CD11b+ cells sorted from the SKOV-3 tumors. Images were acquired at 20X and 40X magnification.
Figure 5
Figure 5. C5a-expressing lymphoma cells have significantly enhanced tumor progression
(A) Wildtype C57Bl/6 mice were injected with C5a-expressing lymphoma RMA-3CF4 cells or RMA CVA1 cells (n=10) and tumor growth was recorded. Data are shown as mean±s.e.m. ***p<0.001. (B) Spleen, TDLN, and tumor from tumor-bearing mice were prepared for single cell suspensions. Cells were then stained with Gr-1, CD11b, F4/80, or NK1.1. Representative dot plots and summarized data are shown. (C) Cells were stimulated with PMA/ionomycin and surface stained with CD8 and IFN-γ intracellularly. Representative dot plots (cells were gated on the CD8+ T cells), summarized IFN-γ-producing CD8+ T cells, and total CD8+ T cells are shown. (D) Cells were stimulated with PMA/ionomycin and surface stained with CD4 and IFN-γ intracellularly. Representative dot plots (cells were gated on the CD4+ T cells), summarized IFN-γ-producing CD4+ T cells, and total CD4+ T cells are shown.
Figure 6
Figure 6. Low C5a-expressing lymphoma cells have significantly decreased tumor progression
(A) C5a levels of RMA cells transfected with C5a or CV. Data indicate that RMA 3CF4 clone secreted higher level of C5a than RMA 1474 clone. (B) Wildtype C57Bl/6 mice were injected with low C5a-expressing lymphoma RMA-1474 cells (n=10) or RMA CVA1 cells (n=15) and tumor growth was recorded. Data are shown as mean±s.e.m. *p<0.05. (C) Cells from spleen and tumor from tumor-bearing mice were stained with Gr-1 and CD11b. Representative dot plots and summarized data are shown. (D) Cells were stimulated with PMA/ionomycin and surface stained with CD8 and IFN-γ intracellularly. Representative dot plots (cells were gated on the CD8+ T cells), summarized IFN-γ-producing CD8+ T cells, and total CD8+ T cells are shown. (D) Cells were stimulated with PMA/ionomycin and surface stained with CD4 and IFN-γ intracellularly. Representative dot plots (cells were gated on the CD4+ T cells), summarized IFN-γ-producing CD4+ T cells, and total CD4+ T cells are shown.
Figure 7
Figure 7. C5a regulates CD4 T cell differentiation
Peritoneal macrophages were stimulated with varying concentrations of C5a (0–500 ng/ml) for 24 h and then co-cultured with naïve CD4 OVA Tg T cells in the presence of OVA for 3 days. Cells were then stained intracellularly with IFN-γ (A) and IL-17A (B). For Treg induction assay, macrophages were co-cultured with naïve CD4 OVA Tg T cells in the presence of OVA with varying concentrations of C5a (0–500 ng/ml) for 4 days. Cells were stained intracellularly with Foxp3 (C). Representative dot plots and summarized data are shown. Cells were gated on the CD4+ T cells. Data are representative of at least three independent experiments.
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