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Keywords:

  • epidermal growth factor receptor;
  • head and neck cancer;
  • spheroids;
  • immune cells;
  • PBMC;
  • chemotaxis

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of squamous cell carcinoma of the head and neck (SCCHN). Monoclonal antibodies (mAbs) against EGFR are currently used for therapy of recurrent or metastatic disease; however, their mode of action is not completely understood. To investigate the immunological effects of anti-EGFR mAb, we generated a three-dimensional spheroid model of EGFR-expressing SCCHN and used this model to study the effect of anti-EGFR mAb on leukocyte migration toward tumors. Pretreatment with the blocking anti-EGFR mAb EMD 72000, its F(ab′)2 fragments or an EGFR tyrosine kinase inhibitor led to substantially increased leukocyte infiltration into EGFR overexpressing tumor spheroids, but not into those with low EGFR expression. Nonblocking anti-EGFR mAb or fibroblast-specific mAb did not affect leukocyte infiltration, suggesting that the observed increase in leukocyte infiltration depends on interference with EGFR activation. Using a human cytokine macroarray, we demonstrated that the blockade of EGFR by anti-EGFR mAb in EGFR-overexpressing SCCHN cells leads to differential expression of several cytokines and chemokines, including the chemokine MCP-1/CCL-2. The significant upregulation of MCP-1/CCL2 on exposure to anti-EGFR mAb was confirmed by quantitative PCR and enzyme-linked immunospot analyses. Moreover, blocking anti-MCP-1 antibody inhibited leukocyte migration toward tumor cells induced by anti-EGFR mAb, pointing to an important role of MCP-1/CCL2 in anti-EGFR mAb-induced leukocyte migration. Our findings demonstrate that anti-EGFR mAb induces leukocyte infiltration to tumor spheroids by upregulating chemokine expression. This novel mechanism for anti-EGFR mAb action may contribute to the antitumor effects of anti-EGFR mAb in vivo. © 2009 Wiley-Liss, Inc.

Cellular transformation is associated with aberrant or unregulated expression of growth factors, growth factor receptors and components of their intracellular signaling pathways. Consequently, these molecules are believed to represent key elements involved in the development and progression of cancer.1, 2 The epidermal growth factor receptor (EGFR) is a transmembrane phosphoglycoprotein that has been identified in almost all adult tissues with the exception of hematopoietic cells, and overexpression and constitutive activation of EGFR is a hallmark of several solid human cancers, including squamous cell carcinoma of the head and neck (SCCHN).3–6 Binding of peptide growth factors of the epidermal growth factor (EGF) family to the extracellular EGFR domain induces receptor dimerization and autophosphorylation of its cytoplasmic domain, hereby triggering a complex system of intracellular signals which culminate in the phosphorylation of cellular response proteins and transcription factors.7, 8 These EGFR-mediated events modulate a variety of biological phenomena known to be involved in the expression of the malignant phenotype, such as proliferation, differentiation, apoptosis, adhesion, invasion and angiogenesis.9, 10 Hence, the EGFR is considered to be a primary target for antitumor treatment strategies. As the receptor is located in the tumor cell surface membrane, monoclonal antibodies (mAb) raised against epitopes on the external domain of the human EGFR seem to be suitable agents for antitumor therapy.9, 11, 12 Unconjugated mAb may act either through interference with receptor-ligand interactions, thus blocking EGFR tyrosine kinase activity, or by coating of tumor cells with immunoglobulin (opsonization), which facilitates the recruitment and activation of host immune cells. In a number of in vitro and in vivo models of SCCHN, EGFR mAb have been shown to exert multiple effects, including inhibition of tumor proliferation, induction of terminal differentiation and modulation of chemo- and radiosensitivity.11, 13–15 Previously, we reported on the potential of EGFR mAb to induce antibody-directed cellular cytotoxicity (ADCC) in established cell lines of SCCHN.16 This study, using a multicellular tumor spheroid model of SCCHN, extends the scope of EGFR mAb-mediated effects to include a novel mechanism. Here, we show a substantially increased infiltration of leukocytes into tumor spheroids following pretreatment with anti-EGFR mAb and identify molecular mechanisms responsible for anti-EGFR mAb-mediated infiltration of immune cells into tumors.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell lines and culture conditions

Tumor cell lines (UD-SCC 4, 5, 6) and autologous fibroblast cultures were previously established from patients with SCCHN.17 In addition, we used the SCCHN lines UM-SCC 14C and 22B established in the laboratory of Dr. Thomas Carey at the University of Michigan (UM) (Ann Arbor, MI), and HLaC 79 isolated by H.-P. Zenner (Tubingen, Germany). Tumor cell lines and fibroblasts were grown in plastic culture flasks (Greiner, Solingen, Germany) under standard conditions (37°C, 5% CO2, fully humidified atmosphere) in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum (both Gibco, Eggenstein, Germany), 2 mM L-glutamine, 50 IU/ml penicillin and 50 μg/ml streptomycin (all ICN, Amsterdam, The Netherlands). EGFR expression on tumor cells was determined as previously described.13 The UD-SCC and UM-SCC cell lines overexpressed EGFR as indicated in Tables I–III, whereas HlaC 79 showed only low EGFR expression and served as a control. Similarly, fibroblasts showed low EGFR expression and served as a negative control in the autologous setting.

Table I. PBMC Infiltration into Tumor (T) or Tumor/Fibroblast (T/F) Spheroids after Treatment with Anti-EGFR Mab EMD 72000
Cell lineEGFR expression (fmol/mg protein)Spheroid typePBMCPBMC + EMD 72000
  • 1

    Degree of PBMC infiltration into spheroids, evaluated by immunohistochemistry. Five degrees were distinguished: negligible (−), weak (1+), good (2+), very good (3+) and extraordinary (4+). The tumor:PBMC ratio was 1:10 if not otherwise indicated (1:1 to 1:20). Results were obtained in ≥3 experiments. Fibroblasts (e.g., SKUD-8) had low EGFR expression (<20 fmol/mg protein).

HLaC 7920T1+11+
T/F1+1+
UD-SCC 61,700T (1:1)1+
T (1:5)1+2+
T (1:10)1+2+
T (1:20)2+3+
T/F (1:10)1+1+/2+
UD-SCC 42,100T1+2+
T/F1+1+/2+
UM-SCC 22B3,600T1+3+
T/Fn.d.n.d.
UD-SCC 57,800T1+3+
T/F1+1+
UM-SCC 14 C8,100T1+4+
T/F1+3+
Table II. PBMC Infiltration into Tumor Spheroids Following Pretreatment with Blocking Anti-EGFR mAb (EMD 72000), Fibroblast Antibody (AS02), Nonblocking EGFR Antibody (E62) or the F(ab′)2 Fragment of EMD 72000 mAb
Cell linePBMCPBMC + AS02PBMC + E62PBMC + anti-EGFR mAbPBMC + F(ab′)2
  • 1

    Degree of PBMC infiltration into spheroids, evaluated by immunohistochemistry. Five degrees were distinguished: negligible (−), weak (1+), good (2+), very good (3+) and extraordinary (4+). Results were obtained in ≥3 experiments.

UD-SCC 61+/2+1−/1+1+2+/3+2+
UM-SCC 14C1+1+1+4+3+
UM-SCC 22B1+/2+1+1+3+3+
Table III. PBMC Infiltration into Tumor Spheroids following Preincubation with Anti-EGFR mAb (EMD 72000) or EGFR Tyrosine Kinase Inhibitor (PD153035)
Cell linePBMCPBMC + PD153035PBMC + anti-EGFR mAbPBMC + PD153035 + anti-EGFR mAb
  • 1

    Degree of PBMC infiltration into spheroids, evaluated by immunohistochemistry. Five degrees were distinguished: negligible (−), weak (1+), good (2+), very good (3+) and extraordinary (4+). Results represent the mean of 2 (UM-SCC 22B) or 4 (UD-SCC 6) experiments.

UD-SCC 61+/2+12+/3+2+/3+3+
UM-SCC 22B1+/2+2+/3+3+4+

Antibodies and reagents

EMD 55900 (E. Merck, Darmstadt, Germany), originally developed as mAb 425 at the Wistar Institute in Philadelphia,18 is a murine IgG 2a mAb directed against the extracellular domain of the human EGFR. EMD 72000 (E. Merck) is a reshaped humanized IgG1 analog of EMD 55900,16 and both antibodies have been shown to effectively interfere with EGFR tyrosine kinase activation.19, 20 The F(ab′)2 fragment of EMD 72000 was obtained by limited proteolysis.21 The diagnostic IgG2b mouse mAb E 62 (E. Merck) also recognizes an epitope on the extracellular domain of EGFR but exerts neither inhibitory nor stimulatory activity. AS02 (Dianova, Hamburg, Germany) is a murine IgG1 mAb that reacts exclusively with human fibroblasts and is directed against a membrane protein on human fibroblasts.22 Lyophilized E 62 was dissolved in sterile water, and AS02 required the removal of sodium acetate by repeated Centricon-30 centrifugation (Amicon, Beverly, MA) prior to in vitro administration. PD 153035 (Parke-Davis, Pfizer, Karlsruhe, Germany) is a specific inhibitor of the EGFR tyrosine kinase.23, 24

Tumor spheroids and PBMC infiltration

Multicellular tumor spheroids and mixed tumor-fibroblast spheroids were produced in wells of round-bottom 96-well microtiter plates (Greiner) as previously described.17 Briefly, tumor cells were detached from monolayers in mid-log phase with 0.05% trypsin/0.02% EDTA solution (Boehringer, Mannheim, Germany), washed twice in culture medium and transferred to microtiter plates precoated with 1% type VII agarose (Sigma, Munich, Germany) at 6000 cells/0.2 ml culture medium/well on day 0. The formation of tumor-fibroblast spheroids was performed with mixtures of 4,000 tumor cells and 2,000 fibroblasts, respectively. On day 3, established spheroids were exposed to 10 μg/ml mAb (EMD 55900, EMD 72000, E 62, or AS02), 10 μg/ml F(ab′)2 fragment, or 12.5 μM PD 153035. The concentrations of mAbs were determined in previous in vitro experiments and significant tumor growth inhibition was observed at these concentrations after 24–72 hr.13, 16 Successful mAb penetration into spheroids within 24 hr was demonstrated by immunohistochemical detection of murine EMD 55900 mAb (Fig. 1a). Briefly, cryosections of spheroids were incubated with blocking reagent, biotinylated mouse IgG antibody, ABC complex and DAB (Vectastain, Burlingame, CA), and counterstaining was performed with Mayer's hemalaun. Allogeneic or autologous PBMC were obtained by Ficoll-Hypaque density gradient centrifugation (ICN Biomedicals, Meckenheim, Germany) from blood of healthy volunteers and the patients UD-SCC 6 and 8, respectively, and labeled with the fluorescent vital dyes PKH 67-GL (green) and PKH26-GL (red) (Sigma, Munich, Germany), as described.25, 26 Coculture of spheroids with labeled PBMC at the effector: target cell ratio of 10:1, unless stated otherwise, started on day 4. On day 5, spheroids were harvested with a Pasteur pipette, fixed in Zamboni solution, mounted with Tissue-Tek (Sakura Finetek, Torrance, CA) on cork plates, frozen in liquid nitrogen-cooled isopentane, and stored at −80°C until further processing. To evaluate the degree of infiltration, 5 μm sections were cut from each spheroid and fluorescently labeled PBMCs were detected using a fluorescent microscope (Olympus, Hamburg, Germany). Quantification of the degree of PBMC infiltration into tumor spheroids was independently performed by two investigators. Since serial sections have confirmed the regularity of PBMC penetration throughout the multicellular constructs, we confined the descriptive analysis to sections near the spheroid centre. Five degrees of infiltration were distinguished: negligible (−), weak (1+), good (2+), very good (3+) and extraordinary (4+).

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Figure 1. Pretreatment of SCCHN spheroids with blocking anti-EGFR mAb induces leukocyte infiltration. Successful mAb penetration of tumor-spheroids within 24 hr was demonstrated by immunohistochemical detection of murine EMD 55900 in a multicellular tumor spheroid (UM-SCC 14C) (a). Fluorescence microscopy picture of a three-dimensional tumor spheroid (UM-SCC 14C) before (b) and after (c) coincubation with PBMC labeled with the fluorescent vital dye PKH26-GL (red), or after treatment with anti-EGFR mAb EMD 72000 and coincubation with PKH26-GL-labeled PBMC (d). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Immunohistochemistry

To phenotype mononuclear cells infiltrating into tumor spheroids, immunohistochemistry was performed using 5 μm cryosections of the spheroids, which had been pretreated or not with anti-EGFR mAb. Sections were incubated with a blocking reagent, followed by mouse anti-human IgG antibody specific for CD3, CD16 or CD11c (Becton Dickinson, Heidelberg, Germany), and a biotinylated secondary antibody, or biotinylated mouse IgG antibody specific for CD14, followed by ABC complex and DAB (Vectastain, Burlingame, CA). Counterstaining was performed with Mayer's hemalaun. Two investigators (T.R., K.S.) independently assessed the degree of PBMC infiltrations in the sections of the spheroids using a light microscope (Olympus, Hamburg, Germany), using the grading scale described earlier.

cDNA macroarrays

The EGFR overexpressing cell line UM-SCC 14C was either treated with EMD 72000 mAb for 24 hr or left untreated. mRNA was isolated from tumor cells and labeled with 33P during subsequent cDNA synthesis. The labeled probes were hybridized to a human cytokine expression array (R&D, Wiesbaden, Germany) containing ∼356 double-spotted elements. Filters were washed, exposed to a storage phosphor screen and scanned by a PhosphorImage Analyzer (Raytest, Schwelm, Germany). AIDA image analysis software (version 3.52.046 Raytest Isotopenmeßgeräte GmbH, Schwelm, Germany) was used to evaluate expression of the spotted elements by quantifying the hybridization signal. The most consistent results were obtained by normalization to the median value of all spots. To identify differentially expressed genes two criteria were consistently applied: (i) spots had to have a signal intensity exceeding the background by a factor of three and (ii) genes with more than 2.5-fold change in expression on mAb treatment were considered as differentially expressed.

Quantitative real-time RT-PCR

Total RNA of tumor cells was extracted (TRIzol reagent, Invitrogen, Carlsbad, CA), DNase-treated, and reverse transcribed as described previously.27, 28 Complementary DNA was quantitatively analyzed for the expression of MCP-1/CCL-2 by the fluorogenic 5′-nuclease PCR assay as reported.27, 28 Specific primers and probes were obtained from Applied Biosystems (Foster City, CA). Gene-specific PCR products were continuously measured during 40 cycles with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Probes for the internal positive control (ribosomal 18S RNA) were associated with the VIC reporter. Target gene expression was normalized between different samples based on the values of ribosomal 18S RNA expression. Dilution series of plasmid DNA were used for quantification of target gene-specific mRNA expression.

Enzyme-linked immunospot assay for MCP-1

The enzyme-linked immunospot (ELISPOT) assay was performed in 96-well plates with polystyrole inserts (Nunc Inc., Naperville, IL). An anti-MCP-1/CCL-2 mAb (R&D) was used for detection of cellular protein production. Six thousand tumor cells/well were plated and coincubated with anti-EGFR antibody for 24 and 48 hr. Afterward, biotinylated anti-human-MCP-1 mAb (R&D) was used, followed by streptavidin-HRP (R&D) and BM blue POD substrate (Boehringer, Mannheim, Germany). The spots were independently counted by two investigators (K.S., T.K.H.). The assay reproducibility was controlled using PBMC obtained from a normal donor, cryopreserved in a series of vials and tested each time the assay was performed after stimulation with phorbol 12-myristate 13-acetate (PMA; 1 ng/ml) and ionomycin (1 μM; both from Sigma).

MCP-1 blocking and transwell migration assays

Tumor spheroids (UD-SCC 6 and UM-SCC 14C) were incubated for 24 hr with anti-EGFR mAb (10 μg/ml EMD 72000) +/− neutralizing anti-MCP-1 mAb (1 μg/ml, R&D) for 6 hr and subsequently with PKH26-GL-labeled PBMCs at the ratio of 10:1 for 24–72 hr. Spheroids were harvested with a Pasteur pipette, fixed in Zamboni solution, mounted with Tissue-Tek on cork plates, frozen in liquid nitrogen-cooled isopentane, cryosectioned and analyzed by fluorescent microscopy as described earlier.

For quantification of the effect of MCP-1 blockade on anti-EGFR mAb-induced PBMC migration, transwell experiments were performed. Migration of PBMCs was assayed in 24-well cell-culture chambers using inserts with 3-μm pore membranes as previously described.29 The lower chamber contained tumor monolayers (18,000 cells of UD-SCC 6, UM-SCC 14C, or UM-SCC 22B) with either medium alone, or media supplemented with anti-EGFR mAb or media with anti-EGFR mAb and neutralizing anti-MCP-1 mAb for the same time intervals as for the treatment of spheroids described above. Afterward, the upper chamber was inserted containing PBMC at the ratio of 10:1. After 72 hr, cells in the lower chamber were fixed with methanol for 20 min at 4°C, stained with anti-CD2 mAb (DAKO, Glostrup, Denmark) and counterstained with Vectastain ABC Kit (Vector, Burlingame, CA). Spots were counted under a light microscope in at least 5 high power fields. All assays were performed 3 times in triplicate wells.

Statistical analysis

Unpaired two-tailed Student's t test and linear regression were used for statistical analysis using GraphPad InStat version 3.01 and Prism 4, GraphPad software (San Diego, CA). The p values less than 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Anti-EGFR mAb EMD 72000 promotes leukocyte infiltration into tumor spheroids

Coincubation of tumor spheroids with allogeneic PBMC led to minor spontaneous infiltration of immune cells into multicellular constructs (Fig. 1c). This was a consistent observation for tumor or mixed tumor-fibroblast spheroids for all of the studied SCCHN cell lines (n = 6). Increasing the number of PBMCs merely increased the accumulation of PBMC on the spheroid surface but did not enhance infiltration of PBMCs into the spheroids. In contrast, preincubation of spheroids with EGFR mAb for 24 hr resulted in their marked infiltration by PBMCs (Table I, Fig. 1d). PBMC infiltration after anti-EGFR pretreatment was less pronounced in mixed tumor-fibroblast spheroids compared to those made with tumor cells alone (Fig. 2, Table I). In cases of mixed tumor-fibroblast constructs separate compartments were often formed within the spheroid, e.g., UD-SCC 6 cells formed a distinct core of fibroblasts surrounded by tumor cells. In these spheroids, PBMC infiltration was most pronounced in the tumor cell compartment (Fig. 2). No differences in the ability to infiltrate spheroids were observed between allogeneic and autologous immune cells, and between PBMC from healthy volunteers or tumor patients, respectively (data not shown).

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Figure 2. Leukocyte infiltration into a tumor/fibroblast spheroid is directional toward tumor cells. Fluorescence microscopy of a three-dimensional autologous UD-SCC 6 tumor/fibroblast spheroid pretreated with anti-EGFR mAb EMD 72000 and after coincubation with PBMC stained with the fluorescent vital dye PKH67-GL (green). Fibroblasts are stained with PKH26-GL (red) and tumor cells are unstained. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Anti-EGFR mAb-induced leukocyte infiltration depends on blockade of EGFR

To obtain further insights into the mechanism of anti-EGFR mAb-induced leukocyte infiltration/migration toward tumor cells, we compared PBMC infiltration into tumor spheroids of SCCHN cell lines with different levels of EGFR expression (n = 6; 20–8100 fmol/mg EGFR), after treatment with EMD 72000. Anti-EGFR mAb induced PBMC infiltration was most prominent in spheroids derived from the cell line with the highest EGFR expression (UM-SCC 14C), and an increase in leukocyte infiltration after anti-EGFR mAb exposure was also observed for other cell lines with high EGFR expression (UD-SCC 4/5/6/UM-SCC 22B) (Table I, Fig. 3). By contrast, induction of PBMC infiltration was almost absent in spheroids derived from the low EGFR-expressing HLaC 79 cells (Table I, Fig. 3). A significant correlation was observed between EGFR protein expression of the cell lines and the intensity of PBMC infiltration after anti-EGFR mAb treatment (r = 0.9065, p < 0.05), suggesting that PBMC infiltration into tumor spheroids induced by anti-EGFR mAb is directly related to EGFR expression on the tumor cell surface.

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Figure 3. Anti-EGFR mAb-induced PBMC infiltration correlates with EGFR expression on tumor spheroids (r = 0.9065, p = 0.0127). Each dot represents an individual cell line (UD-SCC 4/5/6, UM-SCC 14C/22B, HLac79) and a mean of ≥3 experiments. The y-axis represents the degree of infiltration with PKH26-GL-stained PBMCs into tumor spheroids, on a scale from 0 to 4.

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To investigate whether inhibition of EGFR signaling is required for the enhanced PBMC infiltration into tumor spheroids after incubation with anti-EGFR mAb, the effect of preincubation of SSCHN spheroids with various compounds interfering or not with EGFR signaling was evaluated. The F(ab′)2 fragment of mAb EMD 72000 (Table II) and the EGFR tyrosine kinase inhibitor PD153035 (Table III), agents that are known to interfere with EGFR activation and receptor mediated signal transduction, respectively, proved to be as efficient in inducing PBMC infiltration as the complete EMD 72000 mAb. In contrast, the fibroblast-specific mAb AS02 and the nonblocking EGFR mAb E62 had no effect on the infiltration of PBMC into either tumor or tumor-fibroblast spheroids (Table II). These results suggested that anti-EGFR mAb induced PBMC infiltration depends on interference with EGFR activation.

Anti-EGFR mAb-induced leukocyte infiltrates consist of T-cells, macrophages, dendritic cells and natural killer-cells

To characterize the immune cell infiltrates in tumor spheroids after incubation with anti-EGFR mAb, immunohistochemical analyses were performed. Results of the immunohistochemical analyses demonstrated that the infiltrates consisted of T-cells (CD3+; Fig. 4), macrophages (CD14+; Fig. 4), dendritic cells (DCs; CD11c+) and natural killer cells (NK; CD16+) (not shown).

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Figure 4. Characterization of subsets of infiltrating mononuclear cells in SCCHN spheroids. Representative immunohistochemical data show infiltration with T-cells (CD3+, left) or monocytes/macrophages (CD14+, right), in cryosections from tumor spheroids (UM-SCC 14C) left untreated (upper panels) or pretreated with the anti-EGFR mAb EMD 72000 (lower panels) before coincubation with PBMCs. Data are representative for 3 of 5 tumor cell lines tested. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Blockade of EGFR upregulates MCP-1/CCL2 expression in tumor cells

To elucidate the molecular mechanisms underlying the enhanced leukocyte infiltration after preincubation of tumor cells with blocking anti-EGFR mAb, we investigated gene expression changes after blocking anti-EGFR mAb treatment of tumor cells, using a human cytokine array. Blockade of EGFR with EMD 72000 in the EGFR-overexpressing SCCHN line UM-SCC 14 led to differential expression of several genes including cytokines and chemokines, e.g., EphA7 (x11,91), MMP-9 (x8,36), iNOS (x7,69), FGF-7 (x4,60), MMP-10 (x3,51) and MCP-1 (x2,50) and Tie-2 (x0,38), DR-6 (x0,35), R-Cadherin (x0,29), MIG (x0,25), VEGF-B (x0,24), MIP-1alpha (x0,24) and PARC (x0,17).

One of the genes upregulated after anti-EGFR mAb treatment was the monocyte chemoattractant protein-1 (MCP-1/CCL2), a chemokine known to induce directional migration of macrophages, T cells and NK cells,30–32 thus representing a promising candidate for chemotactic modulation of leukocyte infiltration to tumor spheroids. To confirm the regulation of MCP-1/CCL-2 by blocking anti-EGFR mAb, we examined MCP-1 mRNA expression by quantitative real-time RT-PCR, and MCP-1 protein secretion by ELISPOT analysis in SCCHN cells with or without pretreatment with blocking anti-EGFR mAb (EMD 72000). Quantitative real-time RT-PCR analysis confirmed the upregulation of MCP-1/CCL-2 mRNA in UM-SCC 14 cells pretreated with anti-EGFR mAb (data not shown). Furthermore, ELISPOT analysis showed a significant increase in MCP-1 protein production in EGFR overexpressing SCCHN cell lines after EMD 72000 treatment (Fig. 5a): MCP-1 production was increased 3-fold in UD-SCC 6 cells (p < 0.05), 3,9-fold in UM-SCC 14C cells (p < 0.05) and 3,6-fold in UM-SCC 22B cells (p < 0.05).

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Figure 5. Anti-EGFR mAb EMD 72000 induces MCP-1/CCL2 production in SCCHN cell lines and blockade of MCP-1/CCL2 inhibits anti-EGFR mAb-induced PBMC migration. (a) MCP-1 secretion was measured in ELISPOT assays. The y-axis shows the mean number of spots per well counted in ELISPOT assays. Bars represent mean + SEM of 3 independent experiments performed with each cell line (UD-SCC 6, UM-SCC 14C, UM-SCC 22B); *p < 0.05, **p < 0.01. (b) Transwell migration assays were performed with SCCHN cells in medium alone (white bars) or medium + anti-EGFR mAb (EMD 72000, gray bars) or medium + anti-EGFR mAb + neutralizing MCP-1 mAb (black bars). Bars represent the mean + SEM (n = 3) number of CD2+ cells among 1,000 tumor cells in the lower chamber (i.e., migrated toward the tumor monolayer in the lower chamber). Statistics were obtained when values of neighbored columns were compared with *p < 0.05, **p < 0.01.

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MCP-1/CCL-2 upregulation in tumor cells contributes to anti-EGFR mAb-induced leukocyte migration

Next, we tested the functional relevance of MCP-1/CCL-2 in leukocyte infiltration induced after pretreatment of SSCHN cells with anti-EGFR mAb. For this, tumor spheroids made with EGFR overexpressing SCCHN cell lines (UD-SCC 6 and UM-SCC 14C) were treated with anti-EGFR mAb in the presence or absence of blocking anti-MCP-1 mAb, and the infiltration of fluorescently labeled PBMCs was assessed. Treatment with neutralizing MCP-1 mAb markedly suppressed anti-EGFR mAb-induced PBMC infiltrations (data not shown). Next, we performed a transwell chemotaxis assay to quantify the effect of the MCP-1 blockade on the anti-EGFR antibody-induced PBMC migration toward SCCHN cells (Fig. 5b). In agreement with the results of the experiments performed using the 3D tumor spheroid model system, results of the transwell chemotaxis assays showed that pretreatment of SCCHN cells with anti-EGFR mAb significantly induced the migration of CD2+ cells toward tumor cells (Fig. 5b), whereas addition of the neutralizing anti-MCP-1 mAb resulted in a significant inhibition of anti-EGFR mAb-induced PBMC migration (UD-SCC 6, p < 0.05, UM-SCC 14C, p < 0.05, UM-SCC 22B, p = 0.06, Fig. 5b). These results confirm the functional relevance of MCP-1 in anti-EGFR induced leukocyte chemotaxis.

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Overexpression of EGFR has been reported in 80–90% of SCCHN, and upregulation of EGFR represents an early event in SCCHN carcinogenesis.3 EGFR is a transmembrane receptor tyrosine kinase which belongs to the ErbB family and triggers downstream signaling pathways consisting of multilayered and cross-connected networks.11 EGFR in SCCHN is constitutively activated, due to autocrine production of TGF-α by these cells.33 A major signaling pathway of EGFR is the Ras-Raf-MAPK-ERK pathway, which controls important cellular functions including proliferation, migration and survival.11

The frequency and a high level of expression of EGFR in SCCHN and the important role of this receptor in signal transduction make it an excellent target for antibody-directed therapy. Blocking anti-EGFR antibodies and small molecular inhibitors of EGFR are emerging as promising new therapies to be used in conjunction with existing conventional therapies for the treatment of a spectrum of EGFR+ solid tumors including SCCHN.34 Recently, the anti-EGFR mAb C225 (Cetuximab, Erbitux®) was approved by FDA/EMEA for treatment of SCCHN,35 and several other anti-EGFR antibodies are currently undergoing clinical testing, including EMD 72000.15, 19, 36–38 Several reports have described antitumor activity of anti-EGFR mAb using in vitro and in vivo models.13, 15, 39 These effects are mainly attributable to the interference with ligand/receptor interactions resulting in tumor cell deprivation of proliferative stimuli or survival signals, and induction of terminal differentiation.14, 15, 40 In addition, anti-EGFR-mAb-coated tumor cells can recruit and activate cytotoxic host effector cells and, related to this mechanism, ADCC is believed to play an important role in cancer immunotherapy with the unconjugated antibody.16, 41, 42 However, the exact mode of action of the antibody, in particular with respect to its effect on immune cells, is not completely understood. Here, we demonstrate that the blocking anti-EGFR mAb EMD 72000 enhances leukocyte infiltration into SCCHN via induction of the chemokine MCP-1/CCL2 in tumor cells, representing a novel mechanism of action for anti-EGFR mAb.

At the site of tumor growth, tissue injury and nonspecific inflammation in addition to specific antitumor responses are believed to induce infiltration of immune cells. In particular, the microenvironment of SCCHN arising within the oral, nasal or laryngeal mucosa is infiltrated with inflammatory cells and rich in soluble factors these cells produce.43 The presence of immune cells, primarily T lymphocytes and DCs but also B cells and plasma cells, NK cells, macrophages and eosinophils influences the initiation, promotion and progression of tumors which arise in this microenvironment.43 Hence, immune surveillance plays a crucial role in the control of tumor progression. However, tumor cells have developed multiple strategies to evade this control either by escaping the host immune system, or by actively corrupting the host antitumor response via several distinct mechanisms.43 These successful escape strategies are seen in a majority of tumor cases, and they have profound implications for immune-mediated therapies as well as for patient survival.

Our results showed that blocking of EGFR signaling with anti-EGFR mAb induced directional leukocyte migration. These findings suggest that constitutive activation of EGFR in SCCHN might suppress infiltration of leukocytes into the tumor, thus contributing to tumor evasion from host immune responses. This concept is in line with recent findings showing that activation of the EGFR signaling pathway regulate small cytokine-like chemotactic proteins called chemokines, which direct the directional migration of leukocytes under homeostatic or pathologic conditions.44–47

The direct involvement of EGFR in anti-EGFR mAb-induced leukocyte migration into SCCHN spheroids was supported by our observation showing a significant correlation between anti-EGFR mAb-induced PBMC migration and EGFR expression in SCCHN spheroids. Moreover, agents interfering with EGFR activation such as the F(ab′)2 fragment of blocking anti-EGFR mAb or PD153035, an EGFR tyrosine kinase inhibitor, promoted leukocyte infiltration, whereas the nonblocking EGFR antibody or the fibroblast-specific antibody AS02 had no effects on leukocyte infiltration into SCCHN spheroids. Altogether, these findings strongly suggest that enhancement of leukocyte infiltration into SCCHN spheroids after treatment with blocking anti-EGFR mAb depends on interference with the constitutive activation of the EGFR pathway.

Gene expression analysis by cDNA macroarray showed that anti-EGFR treatment resulted in the de-regulation of several metalloproteinases, cytokines and chemokines in SCCHN cells. In line with these findings, it has been previously shown that mAbs against EGFR and small molecule EGFR inhibitors modulate the expression of several genes linked to cancer progression.11 Our gene expression study revealed alterations in the expression levels of several potential chemotactic factors after anti-EGFR mAb treatment of SCCHN cells, including pulmonary and activation regulated chemokine (PARC/CCL18) and monocyte chemoattractant protein-1 (MCP1/CCL2). PARC/CCL18 has been proposed to have an antitumor effect in gastric cancer,48 and it may attract lymphocytes toward tumor cells; however, its role in host-tumor interactions is unclear. MCP-1/CCL2 was originally identified in the culture supernatants of human tumor lines49 as a chemokine that recruited mononuclear phagocytes into tumors.30 MCP-1 also influences NK cells31 and T lymphocytes,32 and its expression has been reported in a wide range of tumor types.50 Recently, Pastore et al.45 demonstrated that blockade of EGFR signaling induces the expression of several chemokines including MCP1/CCL-2 in cultured human keratinocytes and in murine skin. Our results showing that anti-EGFR mAb induces MCP-1/CCL2 mRNA and protein expression in SCCHN spheroids as well as cell lines are in agreement with these findings. The upregulation of MCP-1/CCL2 after anti-EGFR mAb treatment was accompanied by significantly enhanced migration of several leukocyte subsets into tumor spheroids. Further, anti-MCP1 neutralizing antibody significantly reduced anti-EGFR mAb-induced PBMC infiltration into tumor spheroids and inhibited anti-EGFR mAb-induced leukocyte migration toward SCCHN monolayers, demonstrating a functional role for MCP-1/CCL2 in inducing the directional migration of leukocytes toward tumor cells. These results extend our understanding of the role of EGFR signaling in the complex interaction of solid tumors and host antitumor immune responses.

The role of MCP-1/CCL2 in cancer progression is controversial,51 reflecting redundancy and/or multiple effects this and other chemokines can have in the tumor microenvironment. MCP-1/CCL2 is frequently expressed by cancer cells and may also promote cancer progression by promoting angiogenesis or by its direct effect on CCR2-expressing tumor cells.51 On the other hand, it has been shown that MCP-1/CCL2 can suppress tumor growth by enhancing antitumor activities of macrophages and it also attracts T cells or NK cells that are potentially cytotoxic for the tumors.51 We have previously shown that leukocytes infiltrating anti-EGFR mAb-coated SCCHN spheroids have significant cytotoxicity,16 indicating that leukocytes attracted to the tumor may contribute to the antitumor effect of anti-EGFR mAb.

Taken together, our findings demonstrate that blocking anti-EGFR mAb induces leukocyte infiltration into tumor spheroids by upregulating chemokine expression, and this effect is directly related to interference with EGFR activation. This novel mechanism for anti-EGFR mAb action may in part be responsible for the antitumor effects of anti-EGFR mAb in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by grants from the Research Committee at the University of Düsseldorf to T.K.H. and A.M., and the German Cancer Foundation, Deutsche Krebshilfe/Mildred Scheel Stiftung to T.K.H. and H.B.

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  3. Material and methods
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  5. Discussion
  6. Acknowledgements
  7. References
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