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of June 13, 2013.
This information is current as Reduces In Vivo Tumor Growth
Cancer Cells to Complement Attack and
Factor H Sensitizes Non-Small Cell Lung
Down-Regulation of Human Complement
Montuenga and Ruben Pio
Daniel Ajona, Yi-Fan Hsu, Leticia Corrales, Luis M.
http://www.jimmunol.org/content/178/9/5991
2007; 178:5991-5998; ;J Immunol
References http://www.jimmunol.org/content/178/9/5991.full#ref-list-1
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2007 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
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Down-Regulation of Human Complement Factor H Sensitizes
Non-Small Cell Lung Cancer Cells to Complement Attack
and Reduces In Vivo Tumor Growth
1
Daniel Ajona,* Yi-Fan Hsu,* Leticia Corrales,* Luis M. Montuenga,
2
*
†
and Ruben Pio
2
*
‡
Malignant cells are often resistant to complement activation through the enhanced expression of complement inhibitors. In this
work, we examined the protective role of factor H, CD46, CD55, and CD59 in two non-small cell lung cancer cell lines, H1264 and
A549, upon activation of the classical pathway of complement. Complement was activated with polyclonal Abs raised against each
cell line. After blocking factor H activity with a neutralizing Ab, C3 deposition and C5a release were more efficient. Besides, a
combined inhibition of factor H and CD59 significantly increased complement-mediated lysis. CD46 and CD55 did not show any
effect in the control of complement activation. Factor H expression was knockdown on A549 cells using small interfering RNA. In
vivo growth of factor H-deficient cells in athymic mice was significantly reduced. C3 immunocytochemistry on explanted xeno-
grafts showed an enhanced activation of complement in these cells. Besides, when mice were depleted of complement with cobra
venom factor, growth was recovered, providing further evidence that complement was important in the reduction of in vivo
growth. In conclusion, we show that expression of the complement inhibitor factor H by lung cancer cells can prevent complement
activation and improve tumor development in vivo. This may have important consequences in the efficiency of complement-
mediated immunotherapies. The Journal of Immunology, 2007, 178: 5991–5998.
There are important features that distinguish cancer cells
from their normal counterparts, making them recogniz-
able by the immune system. In fact, cancer cells must
develop mechanisms to avoid immune recognition or activation
(1). The elucidation of these mechanisms may provide ways to
improve cancer immunotherapy. Many mAbs against cancer-asso-
ciated Ags are also able to activate the complement system (2).
Chimerized or humanized mouse mAbs containing the human
IgG1 Fc region are examples of complement-activating mAbs (3).
The Fc regions of membrane-bound Abs interact with the hete-
rooligomeric complex C1q and activate the classical pathway.
Complement activation results in the deposition of C3b, which
leads to the formation of the cytolytic membrane attack complex
(MAC)
3
(3) in a process known as complement-dependent cyto-
toxicity. Complement-dependent cellular cytotoxicity also hap-
pens when C3b is converted to iC3b, which interacts with CR3
(CD11b/CD18) on mononuclear phagocytes (4), NK cells (5), and
lymphocytes (6). Finally, the complement cascade of proteolytic
enzymes releases anaphylatoxins C4a, C3a, and C5a which medi-
ate proinflammatory responses. Despite these powerful effector
mechanisms, tumor cells are usually resistant to complement at-
tack through a variety of protective strategies (7). Membrane-
bound complement regulatory proteins (mCRPs), CD55 (decay-
accelerating factor), CD46 (membrane cofactor protein), and
CD59 contribute to the protection of several tumor cells (8 –10). A
role for soluble complement inhibitors, such as factor H, has also
been suggested (10 –14). Factor H is a 150-kDa glycoprotein
present in human plasma, which inhibits the formation and activity
of the C3 convertase (15–17). Besides, alternative splicing of fac-
tor H mRNA yields a 42-kDa protein, named factor H-like protein
1 (FHL-1), which shares the complement inhibitory activities of
factor H (18, 19). Expression of factor H and/or FHL-1 has been
described in primary tumors and cell lines from different origins
(13, 20 –25). We have recently demonstrated that factor H is fre-
quently expressed in non-small cell lung cancer (NSCLC). Factor
H is also secreted to the extracellular milieu and is able to bind to
lung tumor cell surfaces, inhibiting the activation of the alternative
pathway of complement (14). In the present work, we demonstrate
that factor H expression is important for the control of complement
after activation of the classical pathway in lung cancer cells. First,
we show that two lung cancer cell lines are able to resist the ac-
tivation of complement in vitro by the expression of factor H, but
not by the expression of the mCRP CD46 and CD55. Second,
using a nude mouse xenograft model, we show that factor H down-
regulation sensitizes tumor cells to complement-mediated attack
and reduces tumor growth in vivo.
Materials and Methods
Lung cancer cell lines
H1264 (lung adenocarcinoma) and A549 (bronchoalveolar lung carcinoma)
cell lines were obtained from the American Type Culture Collection. Cells
were grown in RPMI 1640 with L-Glutamax (Invitrogen Life Technologies)
supplemented with 10% FBS, 100 U/ml penicillin, and 100
g/ml streptomycin.
*Division of Oncology, Center for Applied Medical Research,
†
Department of
Histology and Pathology, and
‡
Department of Biochemistry, School of Medicine,
University of Navarra, Pamplona, Spain
Received for publication April 28, 2006. Accepted for publication February 14, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was funded through the UTE Project CIMA, Instituto de Salud Carlos III:
Red Tema´tica de Investigacio´n Cooperativa en Ca´ncer (C03/10), the 2004 –2006
American Association for Cancer Research-Cancer Research and Prevention Foun-
dation Career Development Award in Translational Lung Cancer Research, and Min-
isterio de Educacio´n y Ciencia (SAF-2005-01302).
2
Address correspondence and reprint requests to Dr. Ruben Pio or Dr. Luis M Mon-
tuenga, Oncology Division, CIMA Building, Pio XII, 55, Pamplona 31008, Spain.
E-mail address: rpio@unav.es or lmontuenga@unav.es
3
Abbreviations used in this paper: MAC, membrane attack complex; mCRP, mem-
brane-bound complement regulatory protein; FHL-1, factor H-like protein 1; NSCLC,
non-small cell lung cancer; NHS, normal human serum; HI-NHS, heat-inactivated
NHS; siRNA, small interfering RNA; CVF, cobra venom factor.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
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Sera
Normal human serum (NHS) was used as the source of complement. A
pool of sera from 12 healthy donors was prepared. Heat-inactivated NHS
(HI-NHS) was obtained by incubation of the serum at 56°C for 30 min.
Antibodies
Mouse anti-human factor H mAb OX-24 was prepared and purified as
previously described (14). Mouse anti-human CD46 mAb GB-24 was a gift
from Dr. J. Atkinson (Washington University, St. Louis, MO). Mouse anti-
human CD55 mAbs BRIC 110 and BRIC 216 were purchased from
IBGRL. Rat anti-human CD59 mAb YTH53.1 was purchased from Sero-
tec. Isotype controls MOPC-21 (mouse IgG1) and YTH53.1 (rat IgG2b)
were purchased from Sigma-Aldrich and Abcam, respectively. Polyclonal
antisera against H1264 and A549 cells were prepared by immunizing fe-
male New Zealand White rabbits (Harlan) with whole-cell lysates from
each cell line. The experimental protocols were reviewed and approved by
the Institutional Animal Care and Use Committee of the University of
Navarra. Immunization was performed by three injections of 10
7
cells each
at 15-day intervals. For the first injection, cells were resuspended in 0.5 ml
of PBS (10 mM phosphate and 150 mM NaCl, pH 7.4) and mixed with
CFA (Difco). The mixture was injected intradermally on the back of the
rabbits. Subsequent s.c. and i.m. booster injections were conducted with
cells mixed with IFA (Difco). Sera obtained from the rabbits were incu-
bated at 56°C for 30 min to inactivate complement activity. Antiserum
immunoreactivity against each cell line was confirmed by flow cytometry
using a FACSCalibur from BD Biosciences. CellQuest Pro software (BD
Biosciences) was used for data acquisition and analysis.
Expression of mCRPs
Cells were detached from the culture dishes with 1 mM EDTA, washed
once, and resuspended in binding buffer (PBS containing 1% BSA and
0.1% sodium azide). Cells (1 ⫻10
5
)in50
l of binding buffer were
incubated with the primary mAb for 30 min at 4°C. After three washings,
cells were incubated for 30 min at 4°C with Alexa Fluor 488-conjugated
goat anti-mouse IgG (Molecular Probes) or FITC-conjugated rabbit anti-rat
IgG (Serotec) diluted 1/100 in a total volume of 50
l. Cells were washed
three times and analyzed by flow cytometry after the addition of propidium
iodide. Data were collected as mean fluorescence intensity. Cells incubated
only with the anti-mouse secondary Ab were used as negative control.
Deposition of C3-related fragments
Cells were detached from the culture dishes with 1 mM EDTA, washed
once, and resuspended in veronal buffer (1.8 mM barbital, 3.1 mM barbi-
turic acid, 141 mM NaCl, 0.5 mM MgCl
2
, and 0.15 mM CaCl
2
, pH 7.4).
Cells (2 ⫻10
5
) were incubated for 30 min at 4°C with the specific rabbit
antisera diluted 1/50 in a total volume of 100
l. After three washes, cells
were again resuspended in veronal buffer (75
l) and mixed with 125
lof
NHS diluted 1/5. Cells were incubated in the presence of NHS (final di-
lution, 1/8) for 30 min at 37°C. Deposition of C3 or related fragments was
determined as described previously (14). Briefly, cells were incubated for
30 min at 4°C with a FITC-conjugated goat anti-human complement C3 Ab
(ICN Biomedicals) and analyzed by flow cytometry after the addition of
propidium iodide. To block mCRPs, cells were preincubated, before the
addition of the rabbit antiserum, with the blocking Abs (GB-24 or BRIC
110/BRIC 216) at saturating concentrations (49.2, 7.2, and 2.3
g/ml, re-
spectively) during 30 min at 4°C. To block factor H, NHS (diluted 1/5) was
preincubated for 30 min at 4°C with OX-24 at 0.16 mg/ml.
C5a release
Cells were processed and treated as described in the previous paragraph.
Release of C5a was quantified in the supernatants using the Human C5a
ELISA Kit (BD Biosciences) according to the manufacturer’s instructions.
Complement-mediated cytotoxicity
Cell lysis was evaluated using the calcein release assay as previously de-
scribed, with slight modifications (26). In brief, 4 ⫻10
6
cells were resus-
pended in 2 ml of veronal buffer containing 2
M calcein-AM (Molecular
Probes). Cells were loaded with calcein at 37°C for 1 h and washed once with
veronal buffer. Aliquots of 2 ⫻10
5
cells were treated with the antisera
and neutralizing Abs as described in the previous section. Besides, the Ab
YTH53.1 was used to block CD59 activity at a saturating concentration of
60
g/ml. After incubation with NHS, cells were pelleted by centrifugation
and supernatants were transferred to a 96-well plate (plate 1). Pelleted cells
were lysed in 55
l of 0.1% Triton X-100 and transferred to a 96-well plate
(plate 2). Fluorescence was measured with excitation at 485 nm and emis-
sion at 520 nm. Calcein release (percent) was calculated as follows: (plate
1 value) ⫻100/(plate 1 value ⫹plate 2 value). The specific release (com-
plement-mediated cytotoxicity) was the calcein release calculated above
minus the calcein release in cells not exposed to complement (cells treated
with HI-NHS).
Vector-based small interfering RNA (siRNA)
Factor H oligonucleotides for siRNA were cloned in the pSHH vector
using the GeneSilencer system (Imgenex). Oligonucleotides were designed
against positions 808 –828 of factor H mRNA (G.I. 31964). A plasmid
containing an irrelevant siRNA with a scramble sequence (AATTCTC
CGAACGTGTCACGT) was used as control. For transfection, A549 cells
(10
6
) were grown in a 100-mm
2
culture plate with DMEM supplemented
with 10% FBS during 48 h. Transfection was performed using a mixture of
10
g of siRNA plasmid and 10
l of Lipofectamine 2000 (Invitrogen Life
Technologies) in 1 ml of
␣
MEM. Cells were incubated at 37°C with the
DNA-Lipofectamine mixture and after 4 h were diluted twice with DMEM
containing 20% FBS. Stable clones were selected in RPMI 1640 sup-
plemented with 10% FBS and 0.5 mg/ml geneticin (Invitrogen Life
Technologies).
Factor H quantification
A polystyrene 96-well plate was coated with 50 ng/well of the anti-factor
H mAb OX-24 (in 50
l of 50 mM sodium bicarbonate, pH 8.3) during 1 h
at room temperature. After washings, the plate was blocked overnight at
4
o
C with blocking buffer: TBS (25 mM Tris and 150 mM NaCl, pH 7.4)
with 1% BSA and 0.1% Tween 20. A volume of 50
l of samples (super-
natants of cells grown in RPMI 1640 without FBS for 48 h) or standards
(factor H ranging from 1.5 to 200 ng/ml) was added and the plate was
incubated for2hatroom temperature. Human factor H was obtained from
Sigma-Aldrich. After washings, a rabbit anti-factor H Ab (1/1000; Serotec)
was added, and after a 30-min incubation at room temperature the assay
was developed using a donkey anti-rabbit Ab coupled to HRP (1/2000;
Amersham Biosciences) and o-phenylenediamine dihydrochloride (Sigma-
Aldrich). The plate was read at 450 nm.
Western blotting
Supernatants of cells grown in RPMI 1640 without FBS for 48 h were
concentrated, and factor H expression was analyzed as described pre-
viously (14).
Northern blotting
RNA purification was achieved with the Ultraspec Total RNA Isolation
Reagent (Biotecx) according to the manufacturer’s instructions. Analysis
for factor H mRNA expression by Northern blotting was performed as
described previously (14).
Cell proliferation assay
Stable A549 siRNA cells (750 cells/well) were seeded in 96-well plates
and cultured in 100
l of RPMI 1640 supplemented with 10% FBS and 0.5
mg/ml geneticin. During 5 days, cell proliferation was determined daily
using a MTT assay (Roche) per the manufacturer’s instructions.
In vivo xenograft studies
A549 cells stably transfected with the siRNA vector (at 80% confluence)
were trypsinized and washed twice with PBS. Six million cells were re-
suspended in 150
l of PBS and injected s.c. on the right flank of 4- to
6-wk-old female athymic nude mice (Harlan). Tumor development was
monitored for ⬃6 wk. Tumors were measured with a caliper and tumor
volumes (V) were calculated using the formula: V(mm
3
)⫽L⫻W
2
, where
Lis the length and Wis the width of the tumor. In a subset of cases, cells
were first preincubated in 3 ml of PBS with 15
l of anti-A549 antiserum
or 15
l of preimmune serum and washed three times before injection. In
these cases, continuous stimulation of complement was performed by in-
tratumoral injection of antiserum 1/5 (or preimmune serum) in 75
lof
PBS every 3 days.
Depletion of complement
Depletion of complement in nude mice was achieved by i.p. injection of 5
g of cobra venom factor (CVF; Aczon) in 100
l of PBS at 28, 24, and
4 h before injection of tumor cells. This regimen of injections has been
previously described (27). In control mice, i.p. injections of 100
lofPBS
were performed. To avoid complement recovery during the experiment,
periodically 5
g of CVF injections was administered every 3 days. Com-
plement depletion was monitored by quantification of C3 in mouse sera.
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Quantification of C3 was conducted following the protocol described
above for factor H. Plates were coated with goat anti-mouse C3 (1/1000;
Cappel Laboratories), serum samples were loaded at different dilutions, and
the assay was developed using a goat anti-mouse C3 coupled to HRP
(1/10000, Cappel Laboratories). Percentage of C3 depletion was calculated
using the levels of C3 in serum before treatment with CVF as reference.
Statistical analysis
Data were analyzed by Student’s ttest. A p⬍0.05 was considered to be
statistically significant.
Results
Expression of mCRPs in H1264 and A549 lung cancer cells
In a previous study, we have shown that H1264 and A549 cells are
able to produce and bind the complement regulator factor H (14).
We now studied the presence of the membrane-bound complement
inhibitors CD46, CD55, and CD59 in both lung cancer cell lines.
Flow cytometry analysis revealed the expression of the three com-
plement regulators in the two cell lines. Expression of CD46 was
similar in H1264 and A549 cells, while expression of CD55 was
higher in H1264 cells and expression of CD59 was higher in A549
cells (Fig. 1).
Activation of the classical pathway of complement on H1264
and A549 cells
Human NSCLC cells are highly resistant to the activation of the
classical pathway of complement as compared with normal cells
(28). Factor H, CD46, and CD55 are complement regulators that
inhibit C3 convertases, key enzymes in the activation cascade of
complement. We evaluated whether the resistance on H1264 and
A549 NSCLC cells was related to the activity of any of these
regulators. The classical pathway of complement was activated
with specific antisera generated in rabbits against whole-cell ex-
tracts of both cell lines. The activity of each regulator was blocked
with specific mAbs (OX-24 for factor H/FHL-1, GB-24 for CD46,
and BRIC 110/216 for CD55). C3 convertase activity was evalu-
ated by flow cytometry with an Ab that recognizes C3 and C3-
derived fragments. Fig. 2 shows C3 deposition after activation of
the classical pathway with NHS (diluted 1/8) in the presence or
FIGURE 1. Cell membrane expression of mCRPs CD46, CD55, and
CD59 in H1264 (A) and A549 (B) lung cancer cell lines assessed by flow
cytometry and measured as the increase in intensity in the green channel.
Incubation of cells without the primary Ab was used as negative control.
FIGURE 2. Deposition of C3 and C3-related fragments after stimula-
tion of the classical pathway of complement in H1264 (A,C, and E) and
A549 cells (B,D, and F). C3 deposition was determined by flow cytometry
using a polyclonal Ab that recognizes C3 and C3-related fragments. Dep-
osition was analyzed after incubation of the cells with NHS (diluted 1/8),
HI-NHS, or NHS after blocking factor H (Aand B), CD46 (Cand D), or
CD55 (Eand F) with the corresponding neutralizing Abs (OX-24, GB-24,
or BRIC110/226, respectively). Deposition is evidenced by an increase in
intensity in the green channel.
FIGURE 3. Anaphylatoxin C5a release (nanograms per milliliter) after
stimulation of the classical pathway of complement in H1264 (A) and A549
cells (B). C5a was measured in the medium by ELISA after incubation of
the cells with NHS (diluted 1/8) or NHS after blocking factor H, CD46, or
CD55 with specific neutralizing Abs (OX-24, GB-24, or BRIC110/226,
respectively). The graphs show mean and SD of three independent exper-
iments. Incubation with MOPC-21 was used as an isotype control for the
neutralizing Abs. Statistical significance of each treatment when compared
with NHS is indicated. ⴱⴱ,p⬍0.01; ⴱⴱⴱ,p⬍0.001; n.s., Not significant.
5993The Journal of Immunology
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absence of neutralizing Abs. Blockade of either factor H or CD55
slightly increased C3 deposition on both H1264 and A549 cell
membranes, suggesting that the activities of factor H and CD55
protect these lung cancer cells against the deposition of C3-related
fragments. Inhibition of CD46 increased C3 deposition on A549
cells, but not on H1264 cells. In the activation cascade, C3 con-
vertase activity leads to the formation and activation of C5 con-
vertases, which cleave C5 in two fragments: cell-bound C5b and
anaphylatoxin C5a. We evaluated the ability of factor H, CD46,
and CD55 to control the C5a production on H1264 and A549 lung
cancer cells upon activation of the classical pathway of comple-
ment. Incubation of H1264 and A549 cells in the presence of NHS
(1/8) produced a moderate increase in the release of C5a (Fig. 3).
Inhibition of factor H by the neutralizing mAb OX-24 increased
significantly the release of C5a in H1264 (from 15.8 ⫾3.2 ng/ml
to 42.3 ⫾3.2 ng/ml, p⬍0.001) and A549 cells (from 12.3 ⫾2.8
ng/ml to 48.7 ⫾8.1 ng/ml, p⫽0.002) (Fig. 3). When the same
experiment was conducted in the presence of MOPC-21, an iso-
type control, C5a release was not affected. Neither neutralization
of CD46 nor neutralization of CD55 affected C5a release after
activation of the classical pathway of complement. Therefore, the
three complement regulators are able to control the deposition of
C3-related fragments on H1264 and A549 cell membranes, but
only neutralization of factor H triggers an increase in C5 conver-
tase activity. These data suggest that factor H protects H1264 and
A549 cells after activation of the classical pathway of complement.
Complement-mediated cytotoxicity on H1264 and A549 cells
Among other immunological implications, activation of the clas-
sical pathway of complement may ultimately lead to complement-
mediated cytotoxicity. We tested whether the inhibition of factor
H, CD46, or CD55 may increase this cytotoxicity. H1264 and
A549 cells were incubated in the presence of NHS with and with-
out neutralizing Abs against the three complement regulators. Cy-
totoxicity was determined as calcein release using HI-NHS to dif-
ferentiate complement-mediated release from nonspecific release.
After activation of the classical pathway, blockade of factor H
increased cytotoxicity, although this effect did not reach statistical
FIGURE 4. Complement-mediated cytotoxicity after stimulation of the classical pathway of complement in H1264 (A,C, and E) and A549 (B,D, and
F) cells. Cells were incubated with NHS (diluted 1/8), HI-NHS, or NHS after blocking factor H (Aand B), CD46 (Cand D), CD55 (Eand F), or CD59
(A–F) with neutralizing Abs (OX-24, GB-24, BRIC110/226, or YTH53.1, respectively). MOPC-21 was used as an isotype control. Lysis was measured with
a calcein release assay and percentage of complement-mediated lysis was calculated by subtracting the percentage of lysis obtained after incubation with
HI-NHS. Graphs show mean and SD of three independent experiments. Aand B, If not indicated, statistical analysis was performed between the corre-
sponding treatment and the treatment with NHS alone. ⴱ,p⬍0.05; ⴱⴱ,p⬍0.01; ⴱⴱⴱ,p⬍0.001; n.s., Not significant.
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significance (Fig. 4, Aand B). In contrast, neutralization of CD59,
with mAb YTH53.1, significantly increased complement-mediated
cytotoxicity in both H1264 (from 13.1% ⫾11.5 to 52.2% ⫾11.8;
p⫽0.015) and A549 cells (from 16.0% ⫾8.6 to 66.5% ⫾6.2;
p⫽0.001) (Fig. 4, Aand B). CD59 is a complement regulator that
prevents the formation of the lytic membrane attack complex
(MAC). Interestingly, simultaneous neutralization of factor H and
CD59 increased lysis to 85.7 ⫾14.6% in H1264 cells and to
83.4 ⫾6.0% in A549 cells (Fig. 4, Aand B). In both cell lines,
cytotoxicity was significantly higher than that obtained by neutral-
ization of CD59 alone (H1264: p⫽0.036; A549: p⫽0.028).
Isotype controls for OX-24 and YTH53.1 did not affect cytotox-
icity. YTH53.1 did not induced C5a release, ruling out an activa-
tion of complement by this Ab that may account for the increase in
lysis. Our data suggest that there is an increase of the activity of the
classical pathway of complement in H1264 and A549 cells after
neutralization of factor H, but this does not trigger cell lysis due to
the presence of CD59. Neither inhibition of CD46 nor inhibition of
CD55, alone or in combination with inhibition of CD59, had any
effect on cell lysis after activation of the classical pathway of com-
plement (Fig. 4, C–F).
Role of factor H in the protection of lung cancer cells in vivo
Neutralization of factor H increases C3 fragment deposition and
C5a release after complement activation. Therefore, factor H ac-
tivity may be relevant for the growth of tumors in vivo. To eval-
uate this hypothesis, we blocked the expression of factor H in
A549 cells and grew them in athymic mice as s.c. xenografts.
Athymic mice are immunodeficient and cannot develop a complete
adaptive immune response, but have normal complement activity.
First, we generated an A549 stable clone in which expression of
factor H/FHL-1 was inhibited by siRNA (clone named FH-siRNA
A549). A549 cells stably transfected with an irrelevant siRNA
were used as control (clone named control-siRNA A549). The
gene expression knockdown was confirmed by Northern blot and
Western blot analyses (Fig. 5, Aand B). More than 90% inhibition,
measured by ELISA in the serum-free conditioned medium, was
achieved (24.2 ⫾0.8 pg/
l in FH-siRNA A549 vs 365.0 ⫾101.5
pg/
l in control-siRNA A549). Control-siRNA and FH-siRNA
A549 cells showed identical proliferation rates in vitro, ruling out
any effect of factor H down-regulation on in vitro growth (Fig.
5C). Xenograft tumor growth in vivo was then evaluated in athy-
mic mice. We injected 10 mice with FH-siRNA cells and 10 mice
with control-siRNA A549 cells. Tumor growth was monitored at
least twice a week, starting at day 19 postinjection. One mouse in
the control group had to be euthanized at the beginning of the
experiment due to its rapid weight loss and debilitation. After 28
days postinjection, xenografts formed by FH-siRNA A549 cells
were significantly smaller than those formed by control-siRNA
A549 cells (Fig. 6A). At day 35, mean tumor volumes were 722 ⫾
110 mm
3
for control cells and 393 ⫾185 mm
3
for factor H-defi-
cient cells ( p⬍0.001; Fig. 6A). The experiment was repeated
once with very similar results. To verify the role of complement in
this growth inhibition, deposition of mouse C3 fragments on ex-
planted xenograft tumors was evaluated by immunohistochemical
analysis. In all cases, FH-siRNA tumor cells showed higher levels
of C3 staining when compared with those of control-siRNA tumor
cells (Fig. 6B). The contribution of complement in the reduction of
tumor growth in vivo was further confirmed by the generation of
complement-deficient mice with i.p. injections of CVF. CVF is a
protein that forms stable C3/C5 convertases in mammalian serum
with an elevated half-life (29). The activity of these convertases
triggers an uncontrollable activation of the complement system,
resulting in complement depletion. In our experimental conditions,
FIGURE 5. Down-regulation of factor H expression in A549 cells after
stable transfection with factor H siRNA molecules. The expression of fac-
tor H was evaluated in A549 cells, a stable clone transfected with factor H
siRNA (FH-siRNA A549), and a stable clone transfected with a scramble
siRNA (control-siRNA A549). A, Northern blot analysis was conducted
with 15 ng of total RNA per sample. Detection of GAPDH mRNA was
used to ensure equal loading and RNA integrity. B, Western blot analysis
was performed with serum-free conditioned medium (10
g of total pro-
tein). Four nanograms of purified factor H was used as positive control. C,
FH-siRNA A549 cells and control-siRNA A549 cells showed the same
growth rate in vitro as determined by a MTT assay. The graph shows mean
and SD for an experiment conducted in sixtuplicate and corresponds to an
example of two independent experiments.
FIGURE 6. Effect of factor H expression on A549 in vivo xenograft
growth. A, Tumor volume of A549 cells stably transfected with a specific
FH-siRNA (n⫽10) or a control-siRNA (n⫽9) grown on athymic mice.
Tumor volume is expressed as mean ⫾SD. After 26 days postinjection,
significant differences were observed between both groups. ⴱⴱ,p⬍0.01;
ⴱⴱⴱ,p⬍0.001. B, Representative examples of C3 deposition on xenograft
tumors from A549 cells stably transfected with a FH-siRNA or a control-
siRNA. C3 deposition was determined by immunocytochemistry using an
anti-mouse C3 Ab.
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serum C3 levels were reduced to ⬍10% and, if CVF was not
injected periodically, C3 recovered to 50 and 100% after 5 and 7
days, respectively (data not shown). To maintain a continuous de-
pletion of serum in our experiments, CVF was injected every 3
days throughout the experiment. We inoculated FH-siRNA A549
cells in both PBS- and CVF-treated athymic mice (nine mice per
group). Tumor volume was monitored twice or three times a week
for 40 days. Significant differences were observed between CVF-
treated mice and control mice from day 24 to the end of the ex-
periment (Fig. 7). At day 40 after injection, the mean tumor vol-
ume was 591 ⫾100 mm
3
in mice treated with CVF and 282 ⫾89
mm
3
in mice treated with PBS ( p⬍0.001). This experiment was
repeated once with similar results. Therefore, the growth rate of
FH-siRNA A549 cells in complement-deficient mice is recovered,
which strongly supports the role of complement activation in the
reduction of FH-siRNA A549 cell growth.
Finally, to strengthen the activation of the classical pathway of
complement (athymic mice would likely be unable to elicit an
appropriate Ab-mediated response), FH-siRNA A549 cells pre-
treated with anti-A549 antiserum were used. We injected these
cells into four mice and monitored xenograft growth for 46 days.
Continuous stimulation of the classical pathway of complement
was achieved by intratumoral injection of antiserum every 3 days.
Tumor growth was measured twice or three times a week. Four
mice in which FH-siRNA A549 cells were treated with the corre-
sponding rabbit preimmune serum were used as control. From the
beginning of the experiment, xenografts of FH-siRNA A549 cells
treated with antiserum had a significantly smaller volume than FH-
siRNA A549 cells treated with preimmune serum (Fig. 8). At day
46 postinjection, mean tumor volumes of FH-siRNA A549 cells
treated with antiserum or treated with preimmune serum were
108 ⫾42 mm
3
and 422 ⫾220 mm
3
, respectively ( p⫽0.03).
When the same experiment was conducted with control-siRNA
A549 cells, no differences in growth were observed between cells
treated with antiserum and cells treated with preimmune serum
(data not shown). All of these data suggest that factor H protects
A549 lung tumor xenografts against complement activation in vivo
and inhibition of factor H expression increases complement re-
sponse against these cells.
Discussion
Lung cancer is the leading cause of cancer death worldwide. De-
spite the important advances in surgery, radiotherapy, and chemo-
therapy, the 5-year survival rates for lung cancer are ⬍15% and
have not changed significantly over the past two decades. New
anticancer treatments have been recently proposed that are based
on mAbs targeted to tumor-associated Ags. These Abs, among
other mechanisms, can initiate complement-dependent cell lysis
(2). To make the most of this strategy, the interaction between the
complement system and the tumor cell needs to be clarified. Upon
activation of the classical pathway of complement, lung cancer
cells show a high resistance to complement-mediated cytotoxicity
compared with normal respiratory epithelial cells (28). Immuno-
histochemical analysis has also revealed that lung tumors have
minimal deposition of C3b and apparently lack activation of the
lytic membrane attack complex (30). In the present study, we dem-
onstrate that the complement inhibitor factor H, and/or its alterna-
tive splice form FHL-1, plays an important role in the resistance of
H1264 and A549 lung cancer cells against activation of the clas-
sical pathway of complement. Both H1264 and A549 cells express
factor H, secrete this protein to the extracellular milieu, and are
able to bind it to their cell membranes (14).
In our work, we have first confirmed that NSCLC cells are
highly resistant to complement. The antisera against H1264 and
A549 cells were highly immunoreactive and allowed a powerful
stimulation of the classical pathway of complement. In fact, we
observed extensive C3 deposition in both H1264 and A549 cell
lines after the stimulation. However, despite the strong initial com-
plement activation, the percentage of complement-mediated lysis
remained low (⬃15%), suggesting the presence of highly efficient
complement inhibitors. This fact was previously observed by
Varsano et al. (28) using lung cancer cells ChaGo K-1 and H596
preincubated with anti-carcinoembryonic Abs. Intense C3 deposi-
tion in the absence of subsequent complement activation may in-
dicate the presence of inactive C3 fragments on the cell membrane
due to the action of C3 convertase inhibitors, such as the mCRPs
CD46 and CD55. However, Varsano et al. (28) observed that neu-
tralizing Abs anti-CD46 and anti-CD55 were entirely ineffective in
increasing the susceptibility of the lung cancer cells to comple-
ment. Interestingly, the neutralization of the same inhibitors in-
creased significantly the level of complement-mediated lysis in
normal nasal epithelial cells (28), showing a different behavior
from malignant cells. Our present data suggest that factor H may
be responsible for this inhibitory activity in lung cancer cells. Neu-
tralization of factor H increased C3 deposition moderately and
triggered a significant increase of the C5 convertase activity, as
determined by C5a release. These data suggest a real augment of
active C3b deposition when factor H is blocked. In contrast, we
FIGURE 7. Effect of the down-regulation of factor H expression on the
growth of A549 xenograft tumors on athymic mice depleted of serum C3.
Ten mice were treated with CVF before inoculation of FH-siRNA A549
cells and every 3 days throughout the experiment. Cells grown in 10 mice
treated with PBS were used as control. Tumor volume is expressed as
mean ⫾SD. From day 24 postinjection, significant differences were observed
between both animal groups. ⴱ,p⬍0.05; ⴱⴱ,p⬍0.01; ⴱⴱⴱ,p⬍0.001.
FIGURE 8. Effect of the down-regulation of factor H expression on the
growth of A549 xenograft tumors after activation of the classical pathway
of complement. The graph represents tumor volume (mean ⫾SD) of FH-
siRNA A549 cells injected into athymic mice after preincubation of the
cells with A549 antiserum or preimmune serum (four mice per group).
Intratumoral injections of specific antiserum or preimmune serum were
administered every 3 days throughout the experiment. ⴱ,p⬍0.05; n.s., Not
significant.
5996 COMPLEMENT INHIBITION BY FACTOR H IN LUNG CANCER
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have also confirmed that neither CD46 nor CD55 has any effect on
the control of complement activation. The expression of these
inhibitors in H1264 and A549 cells was high, but their neutraliza-
tion moderately increased C3 deposition and had no effect on C5
convertase activity and complement-mediated cytotoxicity. Inter-
estingly, despite the increase of complement activity after factor H
inhibition, complement-mediated cytotoxicity did not augment
significantly. Only when we neutralized simultaneously factor H
and CD59, an inhibitor of the MAC formation, cell lysis increased,
both in H1264 and A549 cells. As previously reported, blockade of
CD59 alone also increased complement-mediated cytotoxicity
(28). We conclude that both factor H and CD59 play a major role
in the protection of H1264 and A549 lung cancer cells against
complement activity.
More importantly, we show that expression of factor H by A549
lung cancer cells is critical for A549 tumor growth in vivo. The
resistance against complement activation in vivo confirms the in
vitro results and underlines the importance of modulating comple-
ment activity on lung cancer cells to improve Ab-based immuno-
therapies. The in vivo experiments were conducted with A549
cells stably transfected with a siRNA specific for factor H or an
irrelevant siRNA. Xenografts from factor H-deficient cells were
significantly smaller than those from control cells. We ruled out a
direct effect of factor H on A549 cell growth rate in vitro. Our
results with CVF, along with the high C3 deposition observed in
xenografts from factor H-deficient A549 cells, strongly suggests
that the effect on tumor growth was mediated by an increase in
complement activation on the lung cancer cells. Besides, in com-
plement-depleted mice, factor H-deficient A549 cell growth was
comparable to that of normal A549 cells, suggesting that the com-
plement response played a critical role in the tumor growth reduc-
tion. This may be considered surprising since factor H inhibition in
vitro was not able to increase significantly complement-mediated
lysis. However, it has to be remembered that, upon activation, the
complement system has several potential effects. First, deposition
of C3b on the cell surface leads to the formation of MAC,
disrupting the membrane’s integrity and causing lysis. Second,
deposition of complement components has an important role in
fostering opsonization. Finally, during complement activation,
powerful anaphylatoxins are released which promote inflammation
by stimulating histamine release and attracting phagocytic cells to
the area of activation (31). In an in vivo setting, the control of C3
deposition by factor H may prevent the establishment of relevant
immune responses against the tumor, independent of the formation
of MAC. In a syngeneic mouse model of metastatic lymphoma,
inhibition of MAC-mediated lysis by expression of CD59 did not
hinder the efficacy of Ig2a and IgM mAbs specific for the gangli-
oside GD2, indicating that both complement-dependent cellular
cytotoxicity and Ab-dependent cellular cytotoxicity operate in
vivo (32). It is also important to note that human factor H and
murine factor H share high structural and functional similarities
and that short consensus repeats 18 –20 of human factor H are able
to bind to murine C3b (33). These results suggest that human fac-
tor H should be able to interact with murine complement. Using
factor H-deficient mouse serum, we have observed a reduction in
the deposition of murine C3 on human H1264 and A549 cells
when increasing concentrations of human factor H were added
(our unpublished data). Therefore, human factor H is able to in-
teract with murine complement and inhibit its activity. This is not
the case for CD59, since human CD59 is not able to regulate the
murine complement system (34). The incapacity of human CD59
to inhibit the formation of MAC on A549 cells injected into mice
may also help to explain the profound impact of factor H in the in
vivo model. A question that arises from the in vivo experiments is
the relevance of the endogenous murine factor H in the protection
against complement by human lung cancer cells. Factor H is pro-
duced at high levels in mice and should be able to interact with
tumor cells in a similar way to endogenously produced human
factor H. However, based on our results, we can conclude that the
expression of human factor H, but not the mere presence of murine
factor H, protects human tumor cells against complement activa-
tion. It has been previously suggested that factor H/FHL-1 pro-
duction by tumor cells causes a high accumulation of this protein
in the tumor microenvironment, which would favor its protected
role (25). Further investigation is warranted to clarify this inter-
esting observation.
Given the numerous genetic and epigenetic changes associated
with carcinogenesis, it is clear that tumor cells express many neo-
antigens that may be recognized by the immune system (1). This
is the basis of the immune surveillance hypothesis, which proposes
that the immune system surveys the body for these tumor-associ-
ated Ags, eliminating many or most tumors. A corollary to this
hypothesis is that tumor cells in progressive cancers develop active
mechanisms to escape immune recognition or resist immune at-
tack. Although there is not irrefutable evidence for the existence of
an effective immune surveillance, a wealth of published data sup-
port the role of the immune system as a primary defense against
neoplasia and the importance of the protective mechanisms devel-
oped by the tumors (35, 36). Based on these results and on our
previous studies (14), we propose that lung cancer cells may de-
velop a protective mechanism against complement attack by ex-
pressing and binding factor H to their cell membranes. Several
studies have also suggested the importance of factor H in the
protection of other tumor cells against complement activation
(12, 13, 25, 37). For the first time, we demonstrate the impor-
tance of factor H expression for the protection of cancer cells in
an in vivo model. Hopefully, these results will help to elucidate
the mechanisms used by lung tumor cells to avoid complement
activity and will assist in the design of more efficient comple-
ment-mediated immunotherapies.
Acknowledgments
We thank Dr. John Atkinson (Washington University, St. Louis, MO) for
providing the anti-human CD46 mAb GB-24. We greatly appreciate Dr.
Juan J. Lasarte and Elena Ciordia for their help with the production of Abs
and Amaya Lavin and Paz Zamora for technical assistance.
Disclosures
The authors have no financial conflict of interest.
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