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Anti-epidermal growth factor receptor (EGFR) antibodies overcome resistance of ovarian cancer cells to targeted therapy and natural cytotoxicity.

Gottschalk N, Kimmig R, Lang S, Singh M, Brandau S - Int J Mol Sci (2012)

Bottom Line: Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs.ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1.Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, University of Duisburg-Essen, 45147 Essen, Germany; E-Mails: nina.gottschalk@uk-essen.de (N.G.); stephan.lang@uk-essen.de (S.L.) ; Department of Gynecology and Obstetrics, University of Duisburg-Essen, 45157 Essen, Germany; E-Mail: rainer.kimmig@uk-essen.de.

ABSTRACT
The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.

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Related in: MedlinePlus

NK activity in the presence of monocytes, PstS-1, TKI and Cetuximab and correlation to MHC class I-expression. NK cells and monocytes of healthy donors were isolated by density gradient centrifugation and purified by MACS. NK cells were co-cultured with monocytes (5 × 105 cells/well in 1:1 cell-ratio) and stimulated with PstS-1 (10 μg/mL). After 24 h Cetuximab (1 μg/mL) and TKI (100 nM) were added. NK cytotoxicity was assessed by CD107a degranulation assay after FITC-coupled staining with anti-CD107a and isotype control and gating on NK cells. Means and standard errors of three to six independent experiments are shown. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated. (a) Natural cytolytic activity of unstimulated NK cells (NK) against EGFR-positive and -negative ovarian cancer cells. (b) ADCC: CD107a-expression of NK cells co-incubated with ovarian cancer cells in the presence of Cetuximab (Cet) and TKI (Erlo); (c) MHC class I-expression of ovarian cells after staining with PE-coupled anti-MHC-class I and isotype control (Iso) analyzed by flow cytometry; (d) Effect of monocytes on NK-cytotoxicity in the presence or absence of Cetuximab and TKI; (e) Impact of PstS-1 on monocytes-stimulated NK cells (NK + Mo). CD107a-expression of monocytes-stimulated NK cells is moderately but significantly enhanced by PstS-1 in the presence of Cetuximab and TKI (39.3% ± 6.3%) compared to absence of PstS-1 (34.9% ± 6.8%).
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f4-ijms-13-12000: NK activity in the presence of monocytes, PstS-1, TKI and Cetuximab and correlation to MHC class I-expression. NK cells and monocytes of healthy donors were isolated by density gradient centrifugation and purified by MACS. NK cells were co-cultured with monocytes (5 × 105 cells/well in 1:1 cell-ratio) and stimulated with PstS-1 (10 μg/mL). After 24 h Cetuximab (1 μg/mL) and TKI (100 nM) were added. NK cytotoxicity was assessed by CD107a degranulation assay after FITC-coupled staining with anti-CD107a and isotype control and gating on NK cells. Means and standard errors of three to six independent experiments are shown. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated. (a) Natural cytolytic activity of unstimulated NK cells (NK) against EGFR-positive and -negative ovarian cancer cells. (b) ADCC: CD107a-expression of NK cells co-incubated with ovarian cancer cells in the presence of Cetuximab (Cet) and TKI (Erlo); (c) MHC class I-expression of ovarian cells after staining with PE-coupled anti-MHC-class I and isotype control (Iso) analyzed by flow cytometry; (d) Effect of monocytes on NK-cytotoxicity in the presence or absence of Cetuximab and TKI; (e) Impact of PstS-1 on monocytes-stimulated NK cells (NK + Mo). CD107a-expression of monocytes-stimulated NK cells is moderately but significantly enhanced by PstS-1 in the presence of Cetuximab and TKI (39.3% ± 6.3%) compared to absence of PstS-1 (34.9% ± 6.8%).

Mentions: As shown in Figure 4a degranulation of unstimulated NK cells against different EGFR-positive ovarian cancer cells (IGROV-1, ASC, SKOV-3) was only minimal, while the EGFR-negative cell line A2780 induced higher natural NK-cytotoxicity. This innate NK-resistance of EGFR-positive ovarian cancer cells could by overridden by the addition of Cetuximab (1 μg/mL) (NK + Cet) via an ADCC-mechanism, which resulted in a significant enhancement of NK-degranulation (Figure 4b: IGROV-1: p ≤ 0.01, ASC: p ≤ 0.01). This ADCC-effect was not impaired by addition of TKI (Erlo) in a concentration of 100 nM (Figure 4b: IGROV-1: p ≤ 0.01, ASC: p ≤ 0.01). This is of particular importance as related compounds as Dasatinib and Nilotinib negatively affected NK-functions [20]. However, Erlotinib, Gefitinib and Vandetanib could also not enhance susceptibility to NK-cytotoxicity. Further studies showed that in our collection of ovarian cancer cells the extent of NK-susceptibility was correlated to the expression level of MHC class I-molecules, as is illustrated in Figure 4c. While SKOV-3, IGROV-1 and ASC displayed high expression of MHC class I, A2780 expressed lower levels of MHC class I. In contrast, the level of the NKG2D-ligand MICA did not correlate to NK-cytotoxicity (data not shown). In addition, coincubation of TKI for the duration of CD107a-assay (6 h) altered neither MICA- nor MHC-expression (data not shown). As expected, NK-cytotoxicity against the EGFR-negative line A2780 could not be further enhanced by the addition of Cetuximab or TKI.


Anti-epidermal growth factor receptor (EGFR) antibodies overcome resistance of ovarian cancer cells to targeted therapy and natural cytotoxicity.

Gottschalk N, Kimmig R, Lang S, Singh M, Brandau S - Int J Mol Sci (2012)

NK activity in the presence of monocytes, PstS-1, TKI and Cetuximab and correlation to MHC class I-expression. NK cells and monocytes of healthy donors were isolated by density gradient centrifugation and purified by MACS. NK cells were co-cultured with monocytes (5 × 105 cells/well in 1:1 cell-ratio) and stimulated with PstS-1 (10 μg/mL). After 24 h Cetuximab (1 μg/mL) and TKI (100 nM) were added. NK cytotoxicity was assessed by CD107a degranulation assay after FITC-coupled staining with anti-CD107a and isotype control and gating on NK cells. Means and standard errors of three to six independent experiments are shown. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated. (a) Natural cytolytic activity of unstimulated NK cells (NK) against EGFR-positive and -negative ovarian cancer cells. (b) ADCC: CD107a-expression of NK cells co-incubated with ovarian cancer cells in the presence of Cetuximab (Cet) and TKI (Erlo); (c) MHC class I-expression of ovarian cells after staining with PE-coupled anti-MHC-class I and isotype control (Iso) analyzed by flow cytometry; (d) Effect of monocytes on NK-cytotoxicity in the presence or absence of Cetuximab and TKI; (e) Impact of PstS-1 on monocytes-stimulated NK cells (NK + Mo). CD107a-expression of monocytes-stimulated NK cells is moderately but significantly enhanced by PstS-1 in the presence of Cetuximab and TKI (39.3% ± 6.3%) compared to absence of PstS-1 (34.9% ± 6.8%).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472788&req=5

f4-ijms-13-12000: NK activity in the presence of monocytes, PstS-1, TKI and Cetuximab and correlation to MHC class I-expression. NK cells and monocytes of healthy donors were isolated by density gradient centrifugation and purified by MACS. NK cells were co-cultured with monocytes (5 × 105 cells/well in 1:1 cell-ratio) and stimulated with PstS-1 (10 μg/mL). After 24 h Cetuximab (1 μg/mL) and TKI (100 nM) were added. NK cytotoxicity was assessed by CD107a degranulation assay after FITC-coupled staining with anti-CD107a and isotype control and gating on NK cells. Means and standard errors of three to six independent experiments are shown. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated. (a) Natural cytolytic activity of unstimulated NK cells (NK) against EGFR-positive and -negative ovarian cancer cells. (b) ADCC: CD107a-expression of NK cells co-incubated with ovarian cancer cells in the presence of Cetuximab (Cet) and TKI (Erlo); (c) MHC class I-expression of ovarian cells after staining with PE-coupled anti-MHC-class I and isotype control (Iso) analyzed by flow cytometry; (d) Effect of monocytes on NK-cytotoxicity in the presence or absence of Cetuximab and TKI; (e) Impact of PstS-1 on monocytes-stimulated NK cells (NK + Mo). CD107a-expression of monocytes-stimulated NK cells is moderately but significantly enhanced by PstS-1 in the presence of Cetuximab and TKI (39.3% ± 6.3%) compared to absence of PstS-1 (34.9% ± 6.8%).
Mentions: As shown in Figure 4a degranulation of unstimulated NK cells against different EGFR-positive ovarian cancer cells (IGROV-1, ASC, SKOV-3) was only minimal, while the EGFR-negative cell line A2780 induced higher natural NK-cytotoxicity. This innate NK-resistance of EGFR-positive ovarian cancer cells could by overridden by the addition of Cetuximab (1 μg/mL) (NK + Cet) via an ADCC-mechanism, which resulted in a significant enhancement of NK-degranulation (Figure 4b: IGROV-1: p ≤ 0.01, ASC: p ≤ 0.01). This ADCC-effect was not impaired by addition of TKI (Erlo) in a concentration of 100 nM (Figure 4b: IGROV-1: p ≤ 0.01, ASC: p ≤ 0.01). This is of particular importance as related compounds as Dasatinib and Nilotinib negatively affected NK-functions [20]. However, Erlotinib, Gefitinib and Vandetanib could also not enhance susceptibility to NK-cytotoxicity. Further studies showed that in our collection of ovarian cancer cells the extent of NK-susceptibility was correlated to the expression level of MHC class I-molecules, as is illustrated in Figure 4c. While SKOV-3, IGROV-1 and ASC displayed high expression of MHC class I, A2780 expressed lower levels of MHC class I. In contrast, the level of the NKG2D-ligand MICA did not correlate to NK-cytotoxicity (data not shown). In addition, coincubation of TKI for the duration of CD107a-assay (6 h) altered neither MICA- nor MHC-expression (data not shown). As expected, NK-cytotoxicity against the EGFR-negative line A2780 could not be further enhanced by the addition of Cetuximab or TKI.

Bottom Line: Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs.ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1.Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, University of Duisburg-Essen, 45147 Essen, Germany; E-Mails: nina.gottschalk@uk-essen.de (N.G.); stephan.lang@uk-essen.de (S.L.) ; Department of Gynecology and Obstetrics, University of Duisburg-Essen, 45157 Essen, Germany; E-Mail: rainer.kimmig@uk-essen.de.

ABSTRACT
The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.

Show MeSH
Related in: MedlinePlus