Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-Mediated Inflammatory Cell Infiltration

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Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant

Protein-1-Mediated Inflammatory Cell Infiltration

Chunmei Piao1,2, Lun Cai1, Shulan Qiu1, Lixin Jia1, Wenchao Song1, Jie Du1,2

1Beijing Anzhen Hospital Affiliated to the Capital Medical University, Beijing 100029, China; 2The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Capital Medical University,

Ministry of Education, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing 100029,

China;

*Running title: C5a promotes metastasis by inflammatory cell infiltration

1To whom correspondence should be addressed: Jie Du, Ph. D, Institute of Heart Lung and Blood Vessel

Diseases, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China. Phone:

+86-10-6445-6030; Fax: +86-10-6445-6095; E-mail: [email protected]

Keywords: Complement 5a; macrophages; metastasis; tumor microenvironment, Monocyte

Chemoattractant Protein-1

Background: It is known that complement system

contributes to tumor progression, but exact mechanism is

still unclear.

Results: Complement 5a enhances tumor metastasis via

monocyte chemoattractant protein-1 mediated

inflammatory cells infiltration.

Conclusion: Complement 5a plays a pro-metastasis role

by establishing inflammatory microenvironment required

for tumor metastasis.

Significance: Our results provide a therapeutic insight for

complement in treatment of malignant tumors.

ABSTRACT

Complement 5a (C5a), a potent immune

mediator generated by complement activation,

promotes tumor growth; however, its role in

tumor metastasis remains unclear. We

demonstrate that C5a contributes to tumor

metastases by modulating tumor inflammation

in hepatic metastases of colon cancer. Colon

cancer cell lines release C5a under serum-free

conditions and C5a levels increase over time in

a murine syngeneic colon cancer hepatic

metastasis model. Furthermore, in the absence

of C5a receptor or upon pharmacological

inhibition of C5a production with an anti-C5

monoclonal antibody, tumor metastasis is

severely impaired. A lack of C5a receptor in

colon cancer metastatic foci reduces the

infiltration of macrophages, neutrophils, and

dendritic cells, and the role for C5a receptor on

these cells were further verified by bone

marrow transplantation experiments.

Moreover, C5a signaling increases the

expression of the chemokine monocyte

http://www.jbc.org/cgi/doi/10.1074/jbc.M114.612622The latest version is at JBC Papers in Press. Published on March 4, 2015 as Manuscript M114.612622

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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chemoattractant protein-1(MCP-1) and the

anti-inflammatory molecules arginase-1,

interleukin 10, and transforming growth factor

beta, but is inversely correlated with the

expression of pro-inflammatory molecules,

which suggests a mechanism for the role of C5a

in the inflammatory microenvironment

required for tumor metastasis. Our results

indicate a new and potentially promising

therapeutic application of complement C5a

inhibitor for the treatment of malignant

tumors.

Malignant tumors are characterized by their

ability to metastasize. Increasing evidence shows

that the development of a supportive

microenvironment in solid tumors plays a critical

role in tumor metastasis(1). In the tumor

microenvironment, inflammatory cells and

molecules influence almost every aspect of cancer

progression, including the tumor cells’ ability to

metastasize(2). Inflammatory immune cells

constitute a substantial proportion of the cells

within the tumor microenvironment and are

associated with tumor malignancy in patients and

animal models of cancer(3). It is clear that the

immune system is a major contributor to

pathogenesis, although the mechanisms of tumor

metastasis are not fully understood. Therefore, a

better understanding of the underlying

immune-mediated pathways involved in tumor

metastasis may identify new targets that could be

manipulated pharmacologically or biologically to

halt disease progression.

As a central contributor to the innate

immune response, complement plays a major role

as a first defense against harmful molecules and

microbes that are unwanted by the host (4-6).

Anaphylatoxins (Complement 3a (C3a),

Complement 4a (C4a), and Complement 5a

(C5a)) are a group of small peptides generated by

complement activation that play important roles

in innate immunity through the initiation and

regulation of inflammatory responses (7,8).

Complement activation contributes to cancer

progression, and complement deposition is

observed in different tumor types (9-11).

However, the function of C5a in tumor metastasis

is controversial(12). In some studies complement

shows an active and beneficial role in the fight

against malignant cells, and

complement-dependent cytotoxicity synergizes

with tumor-directed antibody therapy in tumor

treatment (13,14). Markiewski et al(15)

demonstrated that C5a in the tumor

microenvironment leads to significant tumor

progression in a mouse model of cervical cancer,

which is mediated, in part, by the recruitment of

myeloid-derived suppressor cells (MDSCs).

Furthermore, lung cancer cells can produce

complement C5a, and blocking C5a by

antagonist inhibited tumor growth (16). These

findings suggest that C5a contributes to tumor

growth in the immunosuppressive

microenvironment. Complement activation may

also be linked to angiogenesis. In human colon

cancer, the immune response strongly influences

tumor metastasis(17), and elevated complement

levels in hepatic metastases are observed in colon

cancer patients (18). Sixty percent of patients

with colon cancer develop liver metastasis, which

is responsible for a large percentage of colon

cancer-related deaths (19,20). However, the

function of C5a in hepatic metastasis of

colorectal cancer has not been elucidated.

Therefore, we sought to demonstrate C5a

function with emphasis on the tumor

microenvironment.

In this context, we hypothesized that

complement activation may contribute to the

generation of an inflammatory microenvironment

that favors colon cancer metastasis. Our results

demonstrate that C5a is released and promotes a

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pro-tumor environment through a mechanism

that involves increased inflammatory infiltration,

the production of monocyte chemoattractant

protein-1 (MCP-1) and a reduction in the levels

of immune-modulators. These results provide

new information about the relationship between

complement activation and tumor metastasis,

which could influence the development of future

therapeutic strategies.

EXPERIMENTAL PROCEDURES Antibodies and reagents-The antibody

against Ki-67, PCNA were from Santa Cruz

Biotechnology (Santa Cruz, CA, USA); the anti-

bodies against F4/80, C5a receptor, and Ly6G

were from Abcam (Cambridge, MA, USA); and

ChemMate TM EnVision System/DAB Detection

Kits were from Dako (Glostrup, Denmark). The

following antibodies were from Biolegend (San

Diego, CA, USA): PerCP/Cy5.5-conjugated

CD45.2, phycoerythrin (PE)-conjugated F4/80,

fluorescein isothiocyanate (FITC)-conjugated

F4/80, FITC-conjugated CD206, FITC-conjugated

CD4, FITC-conjugated CD8, and isotype controls.

Anti-mouse C5 monoclonal antibody (BB5.1) and

the irrelevant IgG control of the same isotype

(MOPC) which are a widely used C5 blocking

antibody and control antibody had been previously

demonstrated for its effectiveness were used as

described previously (21-23). Protein kinase B

(also known as Akt) inhibitor MK-2206 was from

Selleck Chemicals (Huston, TX, USA).

Recombinant mouse C5a was from R&D Systems

(Minneapolis, MN, USA). Mouse C3a and C5a

ELISA Kits were from KeYingMei Technology

Co. Ltd (KYM, Beijing, China).

Cell culture- SL4 colon carcinoma cells were

maintained in DMEM/F12 culture medium as

described(24), HCT116 human colorectal carcinoma

cells and SW480 human colon adenocarcinoma cells

were maintained in IMDM, CT26 mouse colon cancer

cells were maintained in RPMI-1640 medium. Cultures

were supplemented with 10% fetal bovine serum (FBS)

and 100 units/mL each penicillin and streptomycin and

grown under a 5% CO2 at 37°C. All cell lines were

obtained from the American Type Culture Collection

(Rockville, MD, USA).

Animals-C5aR-/- mice, backcrossed onto the

genetic background of C57BL/6 for more than 10

generations, were as described previously (14).

Mice were 8-12 weeks old at the beginning of the

experiments and were matched for age and sex

with wild-type (WT) mice. All mice were housed

under specific pathogen-free conditions at the

Beijing Anzhen Hospital, which is affiliated with

the Capital Medical University, China. All animal

care and experimental protocols complied with the

Animal Management Rule of the Ministry of

Health, People’s Republic of China

(Documentation no. 55, 2001) and the Guide for

the Care and Use of Laboratory Animals published

by the US National Institutes of Health (NIH

Publication no. 85-23, revised 1996) and were

approved by the Institutional Animal Care and Use

and Committee of the Capital Medical University.

Tumor model and administration of anti-C5

mAbs-SL4 colon carcinoma cells were derived

from C57BL/6 mice on the same background as

the C5aR-/- and WT control mice，as described

previously(25). SL4 colon carcinoma cells were

maintained in DMEM/F12 culture medium,

supplemented with 10% FBS in a humidified 37oC

incubator under 5% CO2. For the in vivo hepatic

metastasis model, after anaesthetizing mice, a

transverse incision in the left flank was made,

exposing the spleen, and then 5×105 SL4 tumor

cells in 100 μL DMEM/F12 medium were

intrasplenically injected using a 26-gauge needle.

Hepatic ultrasonography was performed on mice

at day 14 after SL4 injection using a 15-mHz

probe for adults and the VEVO 770 software

package (Visual Sonics). Fourteen days after

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inoculation, mice were sacrificed, and the tissues

were processed as described below. The spleen

and liver were removed, wet spleen and liver

weights were measured, and the incidence of

hepatic metastases was examined. The neutralizing

monoclonal anti-C5 antibody BB5.1 or isotype

control mouse IgG1, MOPC-31C (all at a dose of

1 mg/mouse) administrated as described

previously (23).

Histology and immunohistochemistry-

Paraffin Serial sections of 5 μm thick were

obtained for histologic analysis, and stained with

Hematoxylin&eosin (HE) by standard procedures

(26). Paraffin sections were incubated with the

primary antibody against Ki-67, (1:200) at 4 oC

overnight and then incubated with the Dako

Chem-Mate™ EnVision System (Dako, Glostrup,

Denmark) for 30 min. Images were viewed and

captured using a Nikon Labophot 2 microscope

equipped with a Sony CCD-IRIS/RGB color video

camera attached to a computerized imaging system

and analyzed by Image Pro Plus 3.0. The

expression of Ki67 was calculated as the

proportion of positive area to total tissue area for

all measurements of the sections, using Aperio

(Vista, CA, USA) Full Automatic Digital Slide

Scanning System(27).

Frozen tumor sections (7 μm) and cell slides

were incubated with the primary antibodies against

F4/80 (1:100), C5aR (1:200), or Ly6G (1:200), at

4 oC overnight and then with FITC- or tetra-

methylrhodamine isothiocyanate–conjugated

secondary antibodies (Jackson ImmunoResearch

Laboratories) at room temperature for 1 hr.

Sections were viewed with a confocal laser

scanning microscope (TCS 4D; Leica, Heidelberg,

Germany) and a Nikon Labophot 2 microscope

equipped with a Sony CCD-IRIS/RGB color video

camera(28).

SL4 sh-C5 cells selection- SL4 cells were

transfected with a plasmid expressing C5 shRNA

from Santa Cruz Biotechnology (CA, USA) to

downregulate C5, and puromycin (1μg/ml) were

used for screening the stably transfected cell

colonies. Reduction in C5 was confirmed by

Real-time PCR with primers for C5 shown in

table1.

Apoptosis analysis- The apoptosis of

formalin-fixed liver sections from WT or C5aR-/-

mice after SL4 cells inoculation, was analyzed by

the TdT-mediated dUTP Nick-End Labeling

(TUNEL) technique from Promega (Madison, WI,

USA).

Macrophage and neutrophil Extraction,

Culture and Treatment-Treated mice were injected

with 1.5 ml of 3.85% thiogycollate 3–5 days

before macrophage isolation, 3-4 hours before

neutrophil isolation. Peritoneal macrophages and

neutrophils were obtained by lavaging the

peritoneal cavity with 5 ml of 10 mM

phosphate-buffered saline (PBS), cells underwent

hemocytometry and plating in 6-well plates at

3×106 cells per well, then culture in DMEM

containing 10% fetal bovine serum. After 4 hrs,

non-adherent cells were removed by changing the

medium (29). For recombinant C5a treatment,

cells were washed with PBS and incubated with

recombinant C5a (10 nM) for 6 hrs.

Flow cytometry-For staining of immune

markers, single-cell suspensions were prepared by

mechanical dispersion and enzymatic digestion of

tumor tissues. Briefly, tumor tissues were cut into

multiple small cubes and digested in an enzyme

mixture containing collagenase type I (0.05

mg/mL) and type IV (0.05 mg/mL), hyaluronidase

(0.025 mg/mL), DNase I (0.01 mg/mL), and

soybean trypsin inhibitor (0.01 mg/mL) for 45 min

at 37 oC. The cell suspension was centrifuged and

preincubated with fragment crystallizable-γ

blocking antibody (anti-mouse CD16/32;

PharMingen, San Diego, CA, USA) to prevent

nonspecific binding. Cell staining involved

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different combinations of fluorochrome-coupled

antibodies to CD45.2, F4/80, CD206, and CD8 for

30 min at 4℃ in the dark. Fluorescence data were

collected using an EPICS XL Flow Cytometer

(Beckman Coulter, Fullerton, CA), and analyzed

using CellQuest. A fluorescence-negative control

was included to determine the level of nonspecific

staining and autofluorescence associated with

subsets of cells in each fluorescence channel (30).

Bone Marrow Transplantation-Bone marrow

was harvested from 6- to 8-week-old WT and

C5aR KO mice by flushing the femurs and tibias

with 2% FBS in phosphate-buffered saline (PBS).

Cells (2×106) were intravenously injected through

the tail vein of lethally irradiated (10 Gy) recipient

mice. Tumor cell implantation was performed by

intrasplenic injection 8 weeks later.

Real-time PCR-Total ventricular RNA was

extracted using TRIzol reagent (Invitrogen,

Carlsbad, CA, USA) according to the

manufacturer’s protocol. PCR amplification was

performed using the iQ5 Real-Time PCR

Detection System (Bio-Rad, Hercules, CA, USA)

with SYBR Green JumpStartTM Taq

ReadyMixTM (Takara, Otsu, Shiga, Japan) and

primers for mouse, MCP-1, Macrophage

Inflammatory Proteins 1 alpha (MIP-1α),

Macrophage Inflammatory Proteins 1 beta

(MIP-1β), Macrophage Inflammatory Proteins 1

gamma (MIP-1γ), Regulated on activation, normal

T cell expressed and secreted (RANTES),

Arginase 1, interleukin 1 beta (IL-1β), interleukin

6 (IL-6), interleukin 10 (IL-10), interleukin 12

(IL12)-p35, IL12-p40, interleukin 23 (IL23)-p19,

transforming growth factor beta1 (TGFβ1), and

Nitric oxide synthase 2 (NOS2) (Table 1). Melting

curve analysis was performed at the end of each

PCR reaction. The housekeeping gene -Tubulin was used as control, and the expression of other

genes was expressed as a ratio to the expression of

-Tubulin.

Chemokine Analysis-To evaluate the

production of chemokines in macrophages and

neutrophils, mouse peritoneal macrophages and

neutrophils were plated at 1×105 per well in

24-well plates and cultured for 6 h. After

incubation, the media was collected and analyzed

using BD LSR Fortessa and FACSDiva software

and BD™ Cytometric Bead Array (CBA) kits (BD

Biosciences, San Jose, CA, USA) to measure

levels of the chemokine keratinocyte

chemoattractant (KC), MCP-1, MIP-1α, MIP-1β,

and RANTES.

Western blotting-Protein extracts were

diluted with loading buffer and separated by

electrophoresis on 10% SDS-polyacrylamide gels

before transfer to nitrocellulose membranes. The

membranes were blocked in Odyssey blocking

buffer (LI-COR Bioscience, Lincoln, NE) at

room temperature for 2 hr, then incubated at 4°C

overnight with primary antibodies: phosphor-p38,

p38, phosphor-Akt, Akt, phosphor-p42/44,

p42/44 or actin (1:1000) (Cell Signaling

Technology, MA, USA). The membranes were

washed and incubated with fluorescent secondary

antibodies (Alexa Fluor 680 or IRDye 800,

Rockland Immunochemicals, PA, US) for 1 hr at

room temperature at 1:5000; blots were analyzed

with the Odyssey infrared imaging system and

Odyssey software(31).

Invasion assay- BD bioCoat matrigel

invasion chamber (BD Biosciences, Bedford,

MA) assays were done in 24-well plates using

polyethylene terephthalate inserts (8-m pores)

with matrigel coating. In this assay, 2105 SL4 were seeded in the upper chamber in serum-free

media and 1 106 macrophage with 5% FBS in the bottom chamber. After 24 hr, noninvading

cells were removed from the upper membrane

surface of the insert using a cotton swab. The

invading cells on the lower membrane were

stained and counted (32).

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Statistical analysis-Results are expressed as

mean ± SEM. Data analysis involved the use of

GraphPad software (GraphPad Prism version 5.00

for Windows). Comparison between groups was

analyzed by one-way ANOVA; p values < 0.05

were considered statistically significant.

RESULTS

Colon cancer cells produce C5a-The

complement system can recognize tumor cells, and

complement deposition is observed in different

types of tumors (9,15). Cells from some tumor

types under serum-free conditions will activate

and release C5a. To determine whether the colon

cancer cell line SL4 releases C5a, we cultured SL4

cells in serum free medium for 48h. Conditioned

medium was collected and C5a and C3a levels

were determined (Fig. 1A). C5a levels were

elevated whereas C3a levels remained constant.

C5a production in other mouse colon cancer cell

lines and in human colon cancer cells were also

high (Fig. 1B, 1C). To verify these results, we

employed the SL4 syngeneic metastasis model of

colon cancer in mice. In this model, metastatic foci

in the liver develop after intrasplenic injection of

colon cancer cells. Our results demonstrate that

levels of C5a in the plasma are increased at 10

days after intrasplenic injection of colon cancer

cells and remain high for at least two additional

weeks, to rule out a non-specific response to the

cell injection itself, injection with mouse NIH3T3

cells did not increase plasma C5a level in mice

(Fig. 1D). To demonstrate C5a originated from

SL4 cells has pro-metastatic effects, C5a was

downregulated by stable transfection of SL4 cells

with a plasmids expressing C5-shRNA,

downregulation of C5 in SL4 cells indeed reduced

tumor metastasis ability (Fig. 2A). These results

support a role for colon cancer cells in promoting

C5a production.

Disruption of host C5aR signaling inhibits

tumor metastasis of colon cancer-C5a acts on

specific receptors on various types of cells

resulting in downstream immunomodulatory

function. The receptor for C5a (C5aR) is normally

expressed on myeloid cells, although its detection

on non-myeloid cells has also been reported in the

literature (33). We hypothesized that C5aR

expression by phagocytes may contribute to the

process of tumor development. To investigate

whether the C5a-C5aR pathway is required for

tumor metastases, we blocked C5a generation in

tumor-bearing wild-type mice using an anti-mouse

C5 monoclonal antibody (mAb; BB5.1)

administered 1 day before the injection of tumor

cells. An irrelevant IgG of the same isotype

(MOPC) was used as a control (22).

Anti-C5-treated mice had impaired hepatic

metastases of colon cancer relative to mice treated

with control IgG (Fig. 2B). These results support

our hypothesis that C5a-C5aR signaling

contributes to the development of hepatic

metastasis by colon cancer cells.

We considered the possibility that the

contribution of C5aR to the metastasis of colon

cancer cells in our model could be explain by

signaling either through C5aR on the injected SL4

cells or through C5aR on host cells. To distinguish

these two possibilities and to further verify the role

of C5a-C5aR signaling in colon cancer metastasis,

we assessed tumor metastasis of SL4 cells injected

into mice deficient in C5aR. Hepatic metastases

from the C5aR-deficient mice had significantly

reduced size as compared to the WT controls (Fig.

3A). To determine if reduced formation of

metastatic liver foci in C5a-R background is due to

increased apoptosis, we examined apoptosis with

TUNEL staining at early time point (day1) after

same amount SL4 cell injection into spleen, there

was no difference in apoptosis in WT and C5aR-/-

(Fig. 3C). These results verify that C5a signaling

through C5aR is involved in colon cancer

metastasis, and also clarify that C5aR expressed

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on host cells, rather than on the SL4 colon

carcinoma cells, is involved.

C5aR deficiency reduces macrophage and

neutrophil infiltration in metastatic foci in the

liver-In the tumor microenvironment,

inflammatory cells and molecules influence almost

every aspect of cancer progression, including the

tumor cells’ ability to metastasize(15,34). To

assess the mechanism of C5a in hepatic metastases

of colon cancer, we examined inflammatory cell

infiltration in tumors of WT and C5aR-/- mice.

Inflammatory cell infiltration in

collagenase-digested tumors was analyzed by flow

cytometry. We excluded PI+ nonviable cells, gated

on CD45+ population cells, and then compared the

relative proportion of helper T lymphocytes (CD4),

cytotoxic T lymphocytes (CD8), macrophages

(F4/80), neutrophils (Ly6G), and dendritic cells

(CD11c) in liver metastatic foci from C5aR-/- and

WT mice. As shown in Fig. 4A, the proportion of

infiltrating CD45+F4/80+ cells in liver metastatic

tumors was significantly reduced in

C5aR-deficient mice compared with WT mice.

Similarly, the proportion of infiltrating

CD45+Ly6G+ cells and CD45+CD11c+ cells in

liver metastatic tumors was significantly reduced

in C5aR-deficient mice compared with WT mice.

There were no significant differences in the

amount of CD4+ or CD8+ T lymphocytes

infiltrated into tumors in C5aR-deficient mice

compared with WT mice (data not shown). We

also assessed the inflammatory cells in the blood

of tumor-bearing mice and did not detect

significant differences in the inflammatory cells in

the blood (Fig. 4B). Thus, the inhibition of tumor

metastasis in C5aR-deficient mice is specifically

associated with reduced infiltration into the tumor

of neutrophils, monocytes and dendritic cells, all

of which are of myeloid origin(35).

To verify that the infiltrating cells express

C5aR, we performed double immunofluorescence

staining with antibodies against C5aR and F4/80,

Ly6G (Fig. 5). Overlap was observed between

C5aR and each of these myeloid markers, thus

confirming that the infiltrating myeloid cells

express C5aR.

Bone marrow-derived cells facilitate tumor

metastasis in C5aR deficient mice-Our results

suggest that C5aR affects the infiltration of

myeloid cells into metastatic foci in the liver,

which implies a bone marrow origin. To determine

whether cells of bone marrow origin contribute to

hepatic metastasis of colorectal cancer, we

performed bone marrow(BM) transplantation and

created C5aR-chimeric mice(23). Two months

after BM transplantation, mice were inoculated

with SL4 colon cancer cells. As shown as Fig. 5,

the tumor size was dependent on the genotype of

the cells that were transplanted. Transplantation of

bone marrow from C5aR-deficient mice (KOBM)

conferred a reduced size of the hepatic metastases

of colorectal cancer as compared to bone marrow

from WT mice (WTBM) for irradiated recipient

mice of WT or C5aR-/- genotype Fig. 6. Thus,

these results verify that the inflammatory cells

infiltrating into the tumor originate from the bone

marrow, and that C5a facilitates their recruitment

to the tumor site.

Blockade of C5aR reduces MCP-1 release in

macrophage-We postulated that the strong

chemoattractant activity of C5a(36) may be

derived, in part, from the activation of chemokines.

Monocyte chemoattractant protein-1

(MCP-1)/CCL2 has potent monocyte chemotactic

activity and is responsible for the recruitment of

immunosuppressive macrophages that promote

tumor growth (37,38). Therefore, we determined

whether C5a regulates inflammatory cell

infiltration by promoting the expression of MCP-1

or other chemokines in hepatic metastatic foci of

colon cancer. The expression of MCP-1 mRNA

was markedly decreased in metastatic foci in

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C5aR-deficient mice, though there were no

significant differences in the mRNA expression of

other chemokines tested (Fig. 7A). To further

support these findings, we assessed protein levels

by CBA assay for macrophages and neutrophils

isolated from mice. MCP-1 was highly expressed

in macrophages. The amount of MCP-1 was not

statistically different between WT and C5aR-/-

mice; however, the addition of 50 nM C5a to the

culture medium for 6 hours caused a statistical

increase in the levels of MCP-1, Macrophage

Inflammatory Proteins 1 alpha (MIP-1 and Macrophage Inflammatory Proteins 1 beta

(MIP-1 in WT cells but not in C5aR-/-

macrophages (Fig. 7B). To gain mechanistic

insights into a functional link between C5a/C5aR

mediated signaling and inflammatory molecules,

we examined C5a/C5aR mediated inflammatory

signaling, namely, Mitogen-activated protein

kinases (MAPK) and Phosphoinositide 3-kinase

(PI3K)-AKT(39), C5a/C5aR-activated AKT signal

increased the expression of inflammatory

molecules, which was blocked by Akt inhibition

(MK-2206) (Fig. 7C). These results suggest that

the levels of MCP-1 and other chemokines

produced by macrophages may be dependent on

C5a signaling through C5aR.

C5aR deficiency enhances the expression of

immune stimulatory genes and reduces metastases

ability-To assess the effect of C5aR in the immune

response to tumor metastasis; we evaluated the

expression of several immune-related molecules in

the tumor microenvironment. Total RNA was

extracted from metastatic foci of colon cancer in

the livers of C5aR-/- and WT mice. Real-time

PCR was performed for 8 immune associated

genes. The expression of anti-inflammatory NOS2

and IL-23 was significantly increased in the

metastatic foci of colon cancer in tumors of the

C5aR-deficient mice (Fig. 8A). Furthermore, the

expression of pro-inflammatory Arg1, TGF and

IL-10 was significantly decreased in

C5aR-deficient mice (Fig. 8B), while the

expression of IL-1 and IL-12 p40 and p35 was essentially unchanged. To establish a causal role

for C5a -stimulated chemokines from macrophage

in the liver metastasis of colon cancer cells, we

performed in vitro invasion assays using matrigel

-loaded transwell chambers. We found

C5a-stimulated WT macrophage increased SL4

cell invasion in vitro, compared with

non-stimulated WT macrophage, and importantly,

C5a failed to cause SL4 cell invasion when C5aR

was deficient in macrophage (Fig. 8C). These

results are consistent with a role for C5a in the

generation of an immunosuppressive tumor

microenvironment.

DISCUSSION

Tumor metastasis is a complex event that

requires interactions between tumor cells and the

surrounding stroma(40). Inflammatory cells and

molecules influence almost every aspect of this

process (2,41). Moreover, for colorectal cancer,

the prognosis is closely associated with the

presence of hepatic and other metastases(42). In

this study, we have demonstrated the contribution

of complement C5a to hepatic metastasis. We

demonstrated that colon cancer cell lines can

generate C5a and that C5a levels increase upon

tumor progression. C5aR deficiency in a murine

colon cancer model reduces hepatic metastasis, as

well as inflammatory cell infiltration.

Macrophage-derived MCP-1 and other cytokines

are decreased in C5aR-deficient mice, suggesting a

pathway for the associated levels of inflammatory

cells infiltration. Furthermore, using bone marrow

transplantation experiments we showed that bone

marrow derived inflammatory cells contribute to

tumor metastasis and that this contribution is

dependent on C5aR.

C5a is a potent pro-inflammatory mediator of

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inflammation and infection and is widely

understood to be able to both promote and

exacerbate tumor growth for premalignant skin

lesions (15,16). Our demonstration of its role in

metastasis extends its function to advanced,

invasive tumors. Colorectal carcinomas are well

characterized with regard to the expression of

membrane-bound complement regulators (43-45).

We demonstrated that colorectal cancer cells

generate C5a in the absence of serum and that

injection of these cells into mice leads to C5a

production in metastatic tumors. Furthermore,

using a mouse model of tumor metastasis in which

we inoculated SL4 malignant cells intrasplenically

into mice, we showed that either antibody

neutralization of C5 and consequently C5a

production or C5aR deficiency leads to reduced

tumor metastasis. Our results showed that even

though loss of C5aR significantly reduced liver

metastasis but it does not completely abrogate

liver metastasis, this suggest that C5a-C5aR axis is

necessary but not sufficient for liver metastasis,

and other events such as senescence(46),

angiogenesis(47) are also required for liver

metastasis.

These experiments collectively suggest that

C5aR signaling promotes metastasis of SL4

tumors.

The enhanced infiltration of inflammatory

cells in C5aR-deficient mice suggests the

possibility the C5a has immunomodulatory

functions in tumor metastasis. Several studies with

animal experimental models, as well as studies in

humans, have demonstrated a crucial function for

inflammatory cells in tumor immunity (24,48,49).

Infiltration of leukocytes and macrophages are

molecular signatures linked to a poor prognosis in

cancer patients(50). Therefore, our findings that

C5aR-deficient mice display reduce infiltration of

macrophages and neutrophils in liver metastatic

tumors suggest that C5aR deficiency can delay

tumor metastasis by promoting

immunosurveillance function in the tumor

microenvironment. One of the important

mechanisms used by malignant tumors to suppress

the immune response to tumor antigens is

abnormal myelopoiesis, as well as the recruitment

of myelomonocytic cells to the tumor site and

peripheral lymphoid organs. In neoplasias,

macrophages and neutrophils are recruited into the

tumor from the peripheral circulation by

chemokines. Our observation that MCP-1

expression is increased in metastatic foci of colon

cancer in the liver provides a mechanism to

explain the reduced infiltration and is consistent

with previous work that demonstrates a direct

correlation between the expression of MCP-1 and

macrophage infiltration in human breast cancer

tissues(37).

We have demonstrated an

immunosuppressive capacity of C5a in our model.

Immunosuppression by complement C5a has also

been reported in a cervical cancer model(15), for

which the generation of C5a in the tumor

microenvironment enhances tumor growth by the

recruitment of MDSCs and the suppression of the

antitumor T cell-mediated response, including the

expression of key immunosuppressive molecules

within tumors(15). These results are in full

agreement with our studies. We found that the

expression of anti-inflammatory molecules ARG1,

IL-10, and TGF- was down-regulated; whereas the expression of the key pro-inflammatory

molecules NOS2 and IL-23 was up-regulated.

Furthermore we found macrophage stimulated

with C5a promote invasion ability of SL4 colon

cancer cells.

In conclusion, our results further support the

role of complement C5a in tumor metastasis.

Further studies are needed to extend our findings

to other metastatic models of cancers. The findings

reported here not only introduce a new

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complement-mediated mechanism of

tumor-dependent immunosuppression, but also

provide preliminary evidence of the potential

utility of complement inhibition as a therapeutic

option in anticancer therapy. Given that

complement inhibition overrides tumor-dependent

immunosuppression, this therapeutic approach

may also hold promise as a supplement to

antitumor vaccines.

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Acknowledgments: We would like to thank Cui Wei, Wenmei Zhang, Taotao Li, Congcong Zhang and

Sa Liu for their technical assistance.

FOOTNOTES

*This work was supported by grants from the Chinese High Technology Research and Development Program

(2012AA02A201), the National Natural Science Foundation of China (81301797, 31090363, 81430050), and the

Beijing Natural Science Foundation (7122026).

1To whom correspondence should be addressed: Jie Du, Ph. D, Institute of Heart Lung and Blood Vessel Diseases,

Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China. Phone: +86-10-6445-6030; Fax:

+86-10-6445-6095; E-mail: [email protected]

2The abbreviations used are: MCP-1: monocyte chemoattractant protein-1, MIP-1α: Macrophage Inflammatory

Proteins 1 alpha, MIP-1β: Macrophage Inflammatory Proteins 1 beta, RANTES: Regulated on activation, normal T

cell expressed and secreted, IL-1β: interleukin 1 beta, IL-6: interleukin 6, IL-10: interleukin 10, IL-12: interleukin

12, IL-23: interleukin 23, TGFβ: transforming growth factor beta, Arg-1: Arginase 1, Nitric oxide synthase 2: NOS2.

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FIGURE LEGENDS

Figure 1. C5a production by colon cancer cell lines and plasma from mice with hepatic metastasis of colon cancer.

A. Secretion of C5a and C3a by colon cancer SL4 cells after 48 hr incubation in serum-free medium. C5a and C3a

levels were determined by ELISA. Data represent the mean±SEM from three independent experiments. *p < 0.05

vs. time zero. B. Secretion of C5a by mice colon cancer SL4 or CT26 cells after 48 hr incubation in serum-free

medium. C. Secretion of C5a by human colon cancer HCT116 or SW480 cells after 48 h incubation in serum-free

medium. D. Plasma C5a levels over a time course after intrasplenic injection of SL4 colon carcinoma cells and

non-transformed NIH3T3 cells (5105) into mice.

Figure 2. C5a originated from SL4 cells facilitates tumor metastasis of colon cancer. A. Gross tumor of colon

cancer in spleen 14 days after intrasplenic injection of SL4 colon carcinoma cells (5105) and SL4 sh-C5 cells into

mice (n=4). The mean±SEM of C5 mRNA expression in SL4 cells and SL4 sh-C5 cells are quantified by

Real-time PCR (right panel), **p < 0.01 vs. sh-cont. B. The mean±SEM of tumor weights and tumor area % of

liver tissue are quantified (bottom panel), *p < 0.05, ***p < 0.01 vs. sh-Cont. B. Gross hepatic metastases of colon

cancer 14 days after intrasplenic injection of SL4 colon carcinoma cells (5105). Mice were pretreated with control

antibody (Cont IgG) or C5 antibody (C5 Ab) (n=4). The mean±SEM of tumor weights (upper panel) and tumor

area % of liver tissue (bottom panel). ***p < 0.001, *p < 0.05 vs. control antibody.

Figure 3. Host C5a receptor deficiency inhibits tumor metastasis. A. Gross hepatic metastases of colon cancer 14

days after intrasplenic injection of SL4 colon carcinoma cells (5105) into wild-type (WT) and C5aR-deficient mice

(upper panel) (n=5). HE staining shows metastatic foci of liver colon cancer 14 days after intrasplenic injection of

SL4 colon carcinoma cells (5×105) into WT and C5aR-/- mice. (n=5) (lower panel). The mean±SEM of tumor

weights (upper panel) and tumor area % of liver tissue (lower panel) is quantified. **p < 0.01 vs. WT. B.

Immunohistochemical analysis of Ki-67 expression after injection of SL4 colon carcinoma cells into WT and

C5aR-/- mice is shown (n=6). C. TUNNEL staining shows apoptosis of liver colon cancer 1 day after intrasplenic

injection of SL4 (5105) into WT and C5aR-/- mice (n=4).

Figure 4. C5aR deficiency decreases inflammatory cell infiltration in hepatic metastases of SL4 colon cancer. A.

CD4+T lymphocytes (CD45+CD3+CD4+) and CD8+ T lymphocytes (CD45+CD3+CD8+) (left panel); and monocytes

(CD45+CD11b+), neutrophils (CD45+Ly6G+), and dendritic cells (CD45+CD11C+) (right panel) were quantified by

flow cytometry analyses of blood from WT and C5aR-/- mice. Data represent the mean ± SEM for n=5 mice per

group. B. Macrophages (CD45+CD11b+), neutrophils (CD45+Ly6G+), and dendritic cells (CD45+CD11C+), were

detected by flow cytometry analyses of hepatic metastases of SL4 colon cancer in WT and C5aR-deficient mice.

Data represent the mean±SEM for n=5 mice per group. *, p＜0.05, **, P＜0.01 vs. WT mice.

Figure 5. C5aR protein expression in tumor infiltrating macrophages and neutrophils. A. Double-color

immunofluorescence analyses of macrophages and C5aR expression in metastatic foci in the liver from WT and

C5aR-/- mice. The sections were immunostained using a combination of anti-F4/80 and anti- C5aR antibodies.

Bars=25μm. B. Double-color immunofluorescence analyses of neutrophils and C5aR expression in metastatic foci in

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the liver from WT and C5aR-/- mice. The sections were immunostained using a combination of anti-Ly6G and anti-

C5aR antibodies. Bars=50 μm.

Figure 6. Bone marrow of C5aR-deficient mice confers a reduced size of hepatic metastases of colon cancer A.

Gross hepatic metastases of colon cancer in bone marrow chimeric mice. WT or C5aR-deficient (KO) mice were

grafted with either WT or KO bone marrow cells. Tumor samples were collected 14 days after intrasplenic injection

of SL4 colon carcinoma cells (5×105) (n=4). B. Quantification of tumor weights of colon cancer from various bone

marrow chimeric mice (mean±SEM of 4 mice in each group). *, p＜0.05, **, P＜0.01 vs. WT.

Figure 7. Effect of C5a on macrophage chemokine release in SL4 cells. A. mRNA levels of MCP-1, MIP-1,

MIP-1MIP-2, or RANTES were detected by real-time PCR from hepatic metastases of SL4 cells in WT or

C5aR-/- mice. Results represent mean±SEM of expression normalized to GAPDH and vs. WT mice. **p< 0.01 vs.

WT. B. Protein levels of chemokines, including KC, MCP-1, MIP-1, MIP-1, or RANTES were detected by

CBA assay of macrophages or neutrophils from WT and C5aR-/- mice pretreated with PBS or 50nM recombinant

C5a, subtracted the basal level at time zero. Data represent mean±SEM. *, p＜0.05 vs. WT. C. Protein levels of

phospho-Akt, phospho-p38, phosphor-p42/44, total Akt, total p38, total p42/44, or actin were detected by Western

Blot of macrophages from WT and C5aR-/- mice pretreated with PBS or 50nM recombinant C5a (left panel);

Protein levels of MCP-1 were detected by CBA assay of macrophages from WT and C5aR-/- mice pretreated with

MK-2206 (1μM) for 30 min, then treated PBS or 50nM recombinant C5a (right panel). Data represent

mean±SEM. **p< 0.01, *, p＜0.05 vs. WT control.

Figure 8. C5a receptor deficiency inhibits tumor immunosuppressive capacity in metastases of colon cancer. A.

mRNA levels of NOS, IL-23, IL-1, or IL-12p40 were measured by real-time PCR of hepatic metastases of SL4

colon cancer in WT and C5aR-deficient mice. B. mRNA levels of Arg1, TGF-, IL-10 or IL-12p35 were measured

by real-time PCR of hepatic metastases of SL4 cells in WT and C5aR-deficient mice. Data represent the mean ±

SEM for n=5 mice per group. *p＜0.05 vs. WT mice. C. Invasion assay of SL4 cells were performed with

macrophages from WT and C5aR-/- mice pretreated with PBS or 50nM recombinant C5a. Data represent

mean±SEM *, p＜0.05 vs. WT control.

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Table

Table 1. Sequences of primers used in real-time PCR

Primer Forward Reverse

MCP-1 CAG GTC CCT GTC ATG CTT CT GTC AGC ACA GAC CTC TCT CT

MIP-1 CCA AGT CTT CTC AGC GCC ATA GAT GAA TTG GCG TGG AAT CTT C

MIP-1 TGC TCG TGG CTG CCT TCT CTG CCG GGA GGT GTA AGA GA

MIP-1 CCCTCTCCTTCCTCATTCTTACA AGTCTTGAAAGCCCATGTGAAA

RANTES ACC ATG AAG ATC TCT GCA GC TGA ACC CAC TTC TTC TCT GG

Arginase 1 CCTGAAGGAACTGAAAGGAAAG TTGGCAGATATGCAGGGAGT

IL-1β GCCCATCCTCTGTGACTCAT AGGCCACAGGTATTTTGTCG

IL-6 GCTACCAAACTGGATATAATCAGGA CCAGGTAGCTATGGTACTCCAGAA

IL-10 CCAAGCCTTATCGGAAATGA TTTTCACAGGGGAGAAATCG

IL12-p35 CCATCAGCAGATCATTCTAGACAA CGCCATTATGATTCAGAGACTG

IL12-p40 GATTCAGACTCCAGGGGACA TGGTTAGCTTCTGAGGACACATC

IL23-p19 TCCCTACTAGGACTCAGCCAAC TGGGCATCTGTTGGGTCT

TGFβ1 TTGCTTCAGCTCCACAGAGA TGGTTGTAGAGGGCAAGGAC

NOS2 GGGCTGTCACGGAGATCA CCATGATGGTCACATTCTGC

Tubulin TCTAACCCGTTGCTATCATGC GCCATGTTCCAGGCAGTAG

C5-1 GCCAAGAAGAACGCTGCAAA TCGCTGCTCACAGGTTTCAT

C5-2 GCAGACGAAAGGAGTTCCCA TTTGGGGAGGTGGGTTAGGA

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1

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tota

l w

eig

ht(

g)

liver

wit

h t

um

or

0

**

Co

nt

60

20

40

Cont WT C5aR-/-

tum

or

area

%

of

liv

er t

issu

e

0

**

B

WT

C5a

R-/

-

HE Staining

Ki-

67

Cont WT C5aR-/-

Ki6

7p

osi

tive

are

a(%

)

6

2

4

0

8 *

Co

nt

Fig. 3

WT C5aR-/-Cont

C

WT

TU

NE

L

C5aR -/- NS

WT C5aR -/-

1

2

0

TU

NE

L p

osi

tive

cel

l(%

)

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WT

C5a

R-/

-W

TC

5aR

-/-

A

BC5aRLy6G DAPI merge

C5aRF4/80 DAPI merge

Fig. 4

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CD

3e+

cells

in b

loo

d(%

)

CD4 CD8

CD

45+

cells

in b

loo

d(%

)

100

40

20

0

CD11b Ly6G

60

80

WTC5aR-/-

80

40

20

0

60

WTC5aR-/-

CD11c

A

B

CD

45+

cells

in b

loo

d(%

)

100

10

5

0

60

80

15

70

90

F4/80 Ly6G CD11c

F4/80

Ly6G

CD

45

81.30%

70.54%

13.54%

9.04%

2.64% 1.54%

WT C5aR -/-

CD11c

WTC5aR-/-**

*

*

Fig. 5

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WTBM-> WT

KOBM -> KO

KOBM -> WT

WTBM-> KO

Bone marrow transplantation

A

B

WTBM-> WT KOBM -> KO KOBM -> WT WTBM-> KO

3

1

2

tota

l w

eig

ht(

g)

liver

wit

h t

um

or

0

**5

4

*

Fig. 6

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Ap

rote

in (

pg

/ml)

450

300

150

0

*

*

*

macrophage neutrophil

WT

WT+C5a

KO+C5aKO

B

3

1

2

mR

NA

leve

l(f

old

s o

f co

ntr

ol)

0

4

**

WTC5aR-/-

C

p-Akt

Akt

p-P38

P38

p-Erk1/2

actin

WT C5aR -/-C5a(min) 0 15 30 0 15 30

MC

P-1

(p

g/m

l)

150

100

50

0

WT

Cont

MK-2206+C5a

C5aR -/-

C5a**

Fig. 7

Erk1/2

*

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0.75

0.25

0.50

mR

NA

leve

l(f

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s o

f co

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ol)

0

1.00

1.25

mR

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s o

f co

ntr

ol)

3.00

1.00

2.00

0

4.00

5.00

NOS2 IL-1β IL-23 p19 IL-12 p40

Arg-1 IL-10 IL-12 p35 TGF-β

WTC5aR-/-

WTC5aR-/-

* * *NS

*

*

NS

NS

A

B

Fig. 8

C

Inva

sio

n a

ssay

(fo

lds

of

WT

)

0

WT Macrophage

C5aR -/-Macrophage

3

6

9 *ContC5a 50nM

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Jia, Wenchao Song and Jie DuChunmei Piao, Lun Cai, Shulan Qiu, Lixin  Inflammatory Cell InfiltrationChemoattractant Protein-1-MediatedMetastases of Colon Cancer via Monocyte Complement 5a Enhances HepaticCell Biology:

published online March 4, 2015J. Biol. Chem. 

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