Complement activation mediates cetuximab inhibition of non-small cell lung cancer tumor growth in vivo
© Hsu et al; licensee BioMed Central Ltd. 2010
Received: 15 January 2010
Accepted: 7 June 2010
Published: 7 June 2010
Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), increases survival in patients with advanced EGFR-positive non-small cell lung cancer when administrated in combination with chemotherapy. In this study, we investigated the role of complement activation in the antitumor mechanism of this therapeutic drug.
EGFR-expressing lung cancer cell lines were able to bind cetuximab and initiate complement activation by the classical pathway, irrespective of the mutational status of EGFR. This activation led to deposition of complement components and increase in complement-mediated cell death. The influence of complement activation on the activity of cetuximab in vivo was evaluated in xenografts of A549 lung cancer cells on nude mice. A549 cells express wild-type EGFR and have a KRAS mutation. Cetuximab activity against A549 xenografts was highly dependent on complement activation, since complement depletion completely abrogated the antitumor efficacy of cetuximab. Moreover, cetuximab activity was significantly higher on A549 cells in which a complement inhibitor, factor H, was genetically downregulated.
We demonstrate for the first time that the in vivo antitumor activity of cetuximab can be associated with a complement-mediated immune response. These results may have important implications for the development of new cetuximab-based therapeutic strategies and for the identification of markers that predict clinical response.
Lung cancer accounts for more than 25% of all cancer deaths in United States . Non-small cell lung cancer (NSCLC) represents about 80% of all lung cancers. Current treatment options consist of surgical resection, platinum-based doublet chemotherapy, and radiation. Unfortunately, despite these therapies, the prognosis remains poor. Recent advances in the understanding of the molecular pathogenesis of the disease have led to the development of molecular targeted therapies for NSCLC . Bevacizumab, a monoclonal antibody to vascular endothelial growth factor, and erlotinib, a small-molecule tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), are targeted agents approved in the treatment of NSCLC . The clinical efficacy of cetuximab, a humanized monoclonal antibody against the extracellular domain of EGFR, has also been evaluated. A randomized phase III trial has recently shown significantly prolonged survival of advanced NSCLC patients who received cetuximab in combination with platinum-based chemotherapy as first-line treatment . Conversely, combinations of gefitinib or erlotinib, EGFR tyrosine kinase inhibitors (TKIs), with standard chemotherapy in advanced NSCLC have failed to show clinical benefit [5–8]. Another remarkable observation is that, in contrast to the evidence for TKI treatment, KRAS mutation status does not appear to be predictive of response to cetuximab in NSCLC [9–11]. These data strongly suggest clinically relevant differences between the mechanisms of action of EGFR-TKIs and cetuximab . In this sense, it has been suggested that immune mechanisms may contribute to the antitumor activity of cetuximab . In particular, cetuximab, alone or in combination with other antibodies, may elicit immunological responses such as antibody-dependent cellular cytotoxicity (ADCC) or complement activation [14–17].
A better understanding of the mechanisms that govern cetuximab antitumor activity is necessary to optimize its therapeutic efficacy and to identify those patients who are going to benefit from the treatment. In the current report we investigated the influence of the activation of complement in the action of cetuximab in an in vivo animal model. We also explored the possibility of enhancing complement activation in an attempt to increase the clinical efficacy of cetuximab.
Lung cancer cell lines
A549 (lung adenocarcinoma), HCC827 (lung adenocarcinoma), and H187 (small-cell lung carcinoma) cell lines were obtained from the American Type Culture Collection. Cells were grown in RPMI 1640 supplemented with 10% Fetalclone III (Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin.
Normal human serum (NHS) was used as the source of complement. A pool of sera from ten healthy donors was prepared. Heat inactivated NHS (HI-NHS) was obtained by incubation of the serum at 56°C for 30 minutes.
EGFR mRNA expression
RNA was purified from cells using the Ultraspec Total RNA Isolation Reagent (Biotecx). RNA was reverse transcribed and the expression of human EGFR mRNA was analyzed by PCR using the following primers: sense 5'-GGACGACGTGGTGGATGCCG-3', antisense 5'-GGCGCCTGTGGGGTCTGAGC-3'. GAPDH was used as an internal control. Primers for GAPDH mRNA amplification were: sense 5'-ACTTTGTCAAGCTCATTTCC-3', antisense 5'-CACAGGGTACTTTATTGATG-3'. PCR conditions were: 1 cycle of 2 min at 95°C, followed by 30 cycles of 30 sec at 95°C, 30 sec at 55°C, and 30 sec at 72°C, and finishing with 10 min at 72°C.
Human KRAS codon 12 mutations were assessed by sequencing. Genomic DNA was subjected to PCR amplification with the following set of intronic primers: sense 5'-CGATACACGTCTGCAGTCAA-3', antisense 5'-GGTCCTGCACCAGTAATATGC-3'. The PCR products were sequenced using the Big Dye Terminator V1.1 Cycle Sequencing Kit (Applied Biosystems) according to the protocol supplied by the manufacturer.
A polystyrene 96-well plate was coated with 30 to 2000 ng of antibody per well in 100 μl of 50 mM sodium bicarbonate (pH 8.3) during one hour at room temperature. After washing, the plate was blocked overnight at 4°C with Tris-buffered saline (TBS) containing 1% bovine serum albumin, and 0.1% Tween 20. After washing, normal human serum, used as the source of C1q, was added in 100 μl of veronal buffer [1.8 mM barbital, 3.1 mM barbituric acid, 141 mM sodium chloride, 0.5 mM MgCl2 and 0.15 mM CaCl2 (pH 7.4)] and incubated for 30 min at 37°C. The plate was washed and the assay was developed with a rabbit anti-human C1q antibody (1:500; Dako), a goat anti-rabbit antibody coupled to horseradish peroxidase (1:1,000; Sigma-Aldrich) and O-phenylenediamine dihydrochloride (Sigma-Aldrich). A human IgG1 antibody (Sigma-Aldrich) was used as a positive control. The anti-factor H monoclonal antibody OX24, and heat inactivated NHS (56°C for 30 minutes) were used as negative controls. Cetuximab was kindly provided by Merck KGaA. OX24 was obtained as previously described .
Binding of cetuximab and deposition of complement components
Cells were detached from culture dishes with trypsin/EDTA (Lonza), washed once, and resuspended in veronal buffer. Cells (2 × 105) were mixed and incubated for 15 min at 37°C with NHS (diluted 1:5) and cetuximab (40 μg/ml). After washing, cells were incubated for 30 min at 4°C with the following antibodies: fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG (1:100; Sigma-Aldrich), rabbit anti-human C1q (1:100; Dako), FITC-conjugated goat anti-human C3 (1:100, ICN Biomedicals), or mouse anti-human C5b-9 (1:100; Dako). Secondary antibodies were goat anti-rabbit IgG labeled with Alexa-Fluor 488 (1:100; Invitrogen), or FITC-conjugated goat anti-mouse IgG (1:100; Invitrogen). Cells were analyzed by flow cytometry.
Complement-mediated cell death
Complement-mediated cell death is associated with DNA fragmentation . DNA fragmentation was evaluated using a method previously described . In brief, 7 × 105 cells were resuspended in 0.2 ml of RPMI medium containing 30% NHS (or HI-NHS), and treated with 40 μg/ml of cetuximab for 24 hours at 37°C. Afterwards, cells were pelleted and fixed in 2 ml of cold 70% ethanol for 60 min at 4°C. Cells were centrifuged, washed twice with PBS, resuspended in 0.5 ml of PBS, and incubated with 10 μl of 1 mg/ml RNase A (Sigma) during 1 hour at 37°C. Finally, 5 μl of 1 mg/ml 7-aminoactinomycin D (Sigma) were added and, after incubation in the dark for 15 min at room temperature, cells were analyzed by flow cytometry. The percentage of DNA giving fluorescence below the G1/G0 peak was taken as measure of cell death.
Cell proliferation assay
Cells (2,000 A459 cells/well or 4,000 HCC827 cells/well) were seeded in 96-well plates in RPMI medium supplemented with 10% FBS. Twenty-four hours later, cells were treated with cetuximab at different concentrations, and incubated for another 24 hours. Ten microliters of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at 5 g/l (Sigma) was added to each well and incubated for 4 hours at 37°C. Later, 100 μl of dimethyl sulfoxide was added to each well to dissolve the MTT-formazan crystals. Absorbance was measured at 540 nm, with a reference filter at 690 nm, using a microplate reader.
Care of the animals was in accord with our institution guidelines. A549 cells (15 × 106) were mixed 1:1 with Growth Factor Reduced Matrigel Matrix (BD Biosciences), and injected subcutaneously on the right flank of 4-6 week old female athymic nude mice (Harlan Laboratories, Italy). Athymic mice are immunodeficient and cannot develop a complete adaptive immune response, but have complement and NK cell activities. Tumor growth was measured every 2-3 days. Tumor volume was calculated using the formula: Volume = length × width2 × 0.5. Tumors were allowed to reach about 200-250 mm3 before randomization. When indicated, complement was depleted with cobra venom factor (CVF), as previously described .
Xenografts were harvested, fixed in buffered formalin, paraffin-embedded and sectioned (5 μm thick). Slides were deparaffinized, blocked with normal rabbit serum (1:20 dilution), and incubated with a goat anti-mouse C3 (1:500; Santa Cruz Biotechnology). Afterwards, slides were washed and incubated with a rabbit anti-goat IgG coupled to Alexa-Fluor 488 (1:500; Invitrogen). Slides were washed, mounted, and analyzed in an Olympus fluorescence microscope.
Data were analyzed by Student's t-test. A p value of less than 0.05 was considered as statistically significant.
Results and Discussion
It would be interesting to evaluate the immune mechanisms through which complement activation is able to control tumor growth. A direct effect of complement on the target cells may be accompanied by the recruitment and activation of cellular effectors such as lymphokine activated killer (LAK) cells and natural killer (NK) cells. In addition, the combination of cetuximab with modifiers of the immune response may be considered an attractive therapeutic approach to enhance the clinical efficacy of cetuximab against lung, colorectal, and head and neck carcinomas. For example, clinical trials are underway using cetuximab in combination with β-glucans to treat colorectal cancer. β-glucans are polysaccharides that collaborate with complement-activating antibodies in the elimination of tumors . Cetuximab may also be combined with other monoclonal antibodies to potentiate complement activation . Modifications in the Fc-region of the monoclonal antibody can also be attempted to increase the ability of the antibody's activation of complement .
In an unselected population, cetuximab treatment, as many other biological agents, only benefits a small percentage of NSCLC patients . The body of knowledge that has paved the way for more targeted use of cetuximab has been recently reviewed . Many groups are also focusing their research on markers of response (or resistance) to cetuximab. A more rational use of EGFR-targeted agents, such as cetuximab, should provide benefits for patients and health-care providers alike by sparing patients unnecessary treatment and allowing better use of health-care resources . However, at present, no molecular biomarker is available to predict response to cetuximab in NSCLC. Based on our results, polymorphisms or other genetic traits that modulate the immune function may condition the activity of cetuximab, serving as markers to predict clinical response.
There is a pressing need to elucidate the molecular mechanisms involved in cetuximab activity in order to improve its clinical efficacy and to better select patients who would benefit from cetuximab treatment. In this paper we demonstrate that a complement-mediated immune response participates in the inhibition of tumor cell growth by cetuximab in vivo. This observation may have important clinical implications in the development of new cetuximab-based therapeutic strategies and in the identification of markers that predict clinical response.
This work was supported by UTE project CIMA, Spanish Ministry of Science and Innovation [SAF2005-01302], Spanish Ministry of Health [FIS-PI080923], Red Temática de Investigación Cooperativa en Cáncer [RTICC, RD06/0020/0066], Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation & European Regional Development Fund (ERDF) "Una manera de hacer Europa". Cetuximab was kindly provided by Merck KGaA.
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