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Sensitivity of glioma initiating cells to a monoclonal anti-EGFR antibody therapy under hypoxia
Life Sciences 137 (2015) 74–80
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Sensitivity of glioma initiating cells to a monoclonal anti-EGFR antibody therapy under hypoxia Tatiana Randriarimanana, Alicia Chateau, Béatrice Faivre, Sophie Pinel, Cédric Boura ⁎ a b
Université de Lorraine, CRAN, UMR 7039, Campus Science, BP 70239, 54506 Vandœuvre-lès-Nancy, France CNRS, CRAN, UMR 7039, Vandœuvre-lès-Nancy Cedex 54506, France
a r t i c l e
i n f o
Article history: Received 2 February 2015 Received in revised form 26 June 2015 Accepted 24 July 2015 Available online 1 August 2015 Keywords: Glioma initiating cells Anti-EGFR therapy Hypoxia Stemness Angiogenesis
a b s t r a c t Aims: Glioma initiating cells (GICs) represent a subpopulation of tumor cells endowed with self-renewal and multilineage differentiation capacity but also with innate resistance to cytotoxic agents, a feature likely to pose major clinical challenges towards the complete eradication of minimal residual disease in glioma patients. Materials and methods: In this work, GICs were obtained from two patient-derived high-grade gliomas xenograft model, expressing differently EGFR. GICs were exposed to anti-EGFR monoclonal antibody cetuximab during 48 h in 1% or 21% oxygen tension. Cell viability and self-renewal capacity were then evaluated as well as their angiogenic properties. Key findings: GICs were sensitive to cetuximab only in normoxic condition whatever the EGFR status. Nevertheless, under hypoxia cetuximab was able to decrease the self-renewal capacity as well as the expression of CD133 while expression of GFAP increased. Moreover, cetuximab decreased the effect of GICs on endothelial cell migration under hypoxia. Significance: Consequently, anti-EGFR therapy can be envisaged to target specifically GICs in order to limit the tumor recurrence. © 2015 Elsevier Inc. All rights reserved.
1. Introduction In gliomas, accumulating evidences support the existence of a small subpopulation of cancer cells with properties of cancer stem cells. These cells express stem cell markers and have the ability to self-renewal as well as to differentiate into glial cells [28] In addition, these cancer stem-like cells have a high tumorigenic potential and are resistant to chemotherapy and irradiation, suggesting that these cell properties might be responsible for tumor development, recurrence and metastasis [1,3]. These glioma stem-like cells also called glioma initiating cells (GICs) have the ability to form spheres in vitro under serum free culture conditions [18] and to regenerate a tumor identical to the initial tumor in vivo [29]. However, the lack of specific stem cell markers impedes the isolation and analysis of GICs. For the moment, CD 133 (prominin-1), is the relevant marker for identifying GICs even if neural stem/progenitor cell markers such as nestin, Sox 2 and Musashi-1 have been yet used [2, 28]. One of the most common signaling changes in gliomas is the aberrant expression of various receptor tyrosine kinases as the epidermal growth factor receptor (EGFR). Wild-type EGFR overexpression as
⁎ Corresponding author at: Université de Lorraine, CRAN UMR CNRS 7039, Faculté de Médecine, 9 av. de la forêt de Haye, Bât D, 54505 Vandoeuvre-lès-Nancy, France. E-mail address: [email protected] (C. Boura).
http://dx.doi.org/10.1016/j.lfs.2015.07.024 0024-3205/© 2015 Elsevier Inc. All rights reserved.
well as constitutively activated mutated form of EGFR (EGFRvIII), are observed in more than 50% of patients with glioma and are associated with tumor progression [15]. EGFR inhibition with monoclonal antibodies such as cetuximab is of interest for glioma treatment [14,27]. Indeed, cetuximab inhibits the binding of natural ligands of EGFR such as EGF or TGF-α and is also able to induce the internalization and degradation of the truncated receptor form of EGFRvIII [17]. Investigations of the mechanisms that regulate GIC expansion, maintenance and control of microenvironment are, therefore, essential to better understand glioma pathology and to select effective therapeutics. In fact, GICs are localized preferentially in specific hypoxic regions (hypoxic niche) [26] or closed to endothelial cells (peri-vascular niche) [8]. The involvement of GICs in the response of EGFR therapy in particular depending on oxygenation conditions is important to predict the possible tumor recurrence with this targeted therapy. Indeed, GICs interact with cells of the microenvironment, such as endothelial cells, in order to maintain their stemness properties [33]. Understanding the relationship between GICs and their microenvironment is of major interest to envisage the complete eradication of this cell population. The aim of our study was to investigate in vitro the effect of oxygen level in order to mimic hypoxic or peri-vascular niches on the response of GICs to cetuximab, in particular on their survival and their selfrenewal capacity. Finally, reduced survival and self-renewal capacity of GICs after EGFR inhibition was evaluated on the relationship with endothelial cells by using conditioned media.
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Fig. 1. EGFR expression of glioma tumor models and of the derived GICs. EGFR expression of our different glioma derived tumor xenografts (called TCG2, 3, 4, 9 and 11) was analyzed by Western blotting (a). EGFR (green) of neurospheres derived from TCG3 and TCG9 was observed by fluorescence imaging (b) (scale bar represents 200 μm). Effect of cetuximab on the expression of phosphorylated EGFR of GIC3 and GIC9 (c) was analyzed by Western blotting after 48 h of culture in presence, or not, of cetuximab (20 μg/mL) and in different conditions of oxygenation (21% or 1% O2), β-actin expression was used as control for equal loading of proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2. Material and methods 2.1. Glioma initiating cell culture and treatments Tissue fragments of TCG3 or TCG9, two patient-derived high-grade glioma xenograft model [25], were subjected to mechanical homogenization followed by enzymatic digestion as previously described [9]. Primary tumor cell cultures were maintained in DMEM/F12 medium (Invitrogen, France) containing B27 supplement (Invitrogen, France), 20 ng/mL of human recombinant epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After one week of culture in hydrophobic flasks at 37 °C with 5% CO2 in a humidified atmosphere, spheres were obtained. GICs obtained from TCG3 or TCG9 were named GIC3 and GIC9 respectively, later in the manuscript. GICs were dissociated from spheres using Accumax (Millipore, France) and seeded at 3.104 cells/mL in 6 well plates or 25 cm2 flasks following the experiment. GICs were then exposed to Cetuximab (Merck Serrano) at 20 μg/mL and/or hypoxia (1% O2) in a hypoxic chamber (Hypoxystation, Don Whitley Scientific, UK) during 48 h.
2.2. Protein expression analysis For analysis of EGFR, pEGFR, CD133 and glial fibrillary acidic protein (GFAP) expression, Western blotting was realized as previously described [22]. Briefly, protein aliquots (40 μg) were denatured in the Laemmli buffer, containing ß mercaptoethanol and before being resolved in SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred onto PVDF membranes (Amersham Biosciences, Orsay, France). After blocking the PVDF membrane with Tris basebuffered saline prepared with 0.1% (v/v) Tween-20 containing 5% (w/
Fig. 2. Effect of cetuximab on sphere formation. The neurospheres were visualized by contrast phase microscopy (a) after 48 h of culture in presence, or not, of cetuximab (20 μg/mL) and in different conditions of oxygenation (21% or 1% O2) and sphere diameter (b) was measured with NIS element software (Nikon, japan). Scale bar represents 100 μm.
v) bovine serum albumin, the following primary antibodies against human EGFR (#2232, Cell Signaling, 1:1000 dilution), phosphorylated EGFR at Tyr1068 (pEGFR) (#2236, Cell Signaling, 1:1000 dilution), CD133 (# 130-090-422, Miltenyi Biotec, 1:500 dilution), GFAP (#12389, Cell Signaling, 1:1000 dilution) and ß-actin (#4970, Cell Signaling, 1:1000 dilution) were incubated overnight at 4 °C. Subsequently, immuno-reactive proteins were visualized using the enhanced chemiluminescence procedure (Pierce, USA). Quantification of relative band densities was performed using a densitometer (LAS Imager Fujifilm) and ß-actin was used as internal control. Images of neurospheres allow one to visualize the expression of EGFR by using a fluorescence macroscope (Nikon AZ100, Japan).
2.3. Glioma initiating cell viability The viability of GICs was evaluated by cell count after trypan-blue exclusion and MTT assay. GICs were seeded in 6 well plates at a density of 9.104 cells/well. For trypan-blue exclusion assay, cells were dissociated from sphere using a 0.025% trypsin–EDTA solution (Sigma, France) for 5 min at 37 °C and resuspended in complete medium. Cells
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were then stained with 0.4% trypan-blue solution and viable cells were counted twice using a Malassez hemocytometer. Cell viability was also determined by monitoring the mitochondrial activity using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (MTT, Sigma). At the set time, 0.5 μmol/L MTT was added into each well. Following a 3 h incubation at 37 °C, formazan crystals resulting from MTT metabolization were solubilized and cells were lysed by adding 400 μL of DMSO into each well. The absorbance of the resulting solution was measured at 570 nm using wells without cells as blank (Multiskan Ascent microplate photometer, Thermo Scientific, France).
2.4. Clonogenic assay After exposition of GICs to cetuximab and hypoxia, a soft agar anchorage independent clonogenic growth assay was done. Briefly, GICs (1 × 104) were suspended in 1 mL of DMEM/F12 containing 0.3% agar (Invitrogen, France). The cell suspension was then added on top of a presolidified 0.5% agar in 6 well plates. After 15 days of incubation, colonies were incubated with MTT solution as previously described and colonies larger than 50 μm in diameter were quantified using GelCount™ (Oxford Optronix, UK). Each experiment was repeated twice, in triplicate.
2.5. Angiogenesis assays Conditioned media were harvested by centrifugation at 350 g during 10 min of culture media obtained from GIC3 and GIC9 exposed or not to cetuximab and hypoxia. Angiogenesis properties of GICs were then evaluated by two in vitro assays performed on Human Umbilical Vein Endothelial Cells (HUVECs) obtained and cultured as we have previously described [16]. 2.5.1. Matrigel™ assay HUVECs were plated (90,000 cells/cm2) onto a 24-well plate precoated with Matrigel™ (BD Biosciences, France). After 1 h, media were removed and replaced by control or conditioned media (CM) and HUVECs were cultured during 24 h before being fixed with 4% paraformaldehyde. Network formation was analyzed after phalloidin– sulforhodamine staining (Fluoprobes®, Interchim, France). Photomicrographs of the whole culture surface were taken (Nikon AZ100, Digital Sight DS-Qi1Mc camera, Nikon, France). Endothelial cell density was calculated with NIS Element Software and represented as the area fraction in relation to the whole field. The total additive length of all cellular structures including all branches and the number of junctions was quantified with Angiosys Software (TCS Cellworks, UK). 2.5.2. In vitro wound assay In vitro wound assay for quantification of endothelial cell motility was performed using HUVEC cells in cultures. Briefly, 1.5 × 105 cells/ well were seeded in 24 well plates. A 1-mm wide linear wound was made across the center of each well with a micropipette tip. For each well five areas along the length of the wound were chosen randomly for photography under phase contrast microscopy. After photography, the cells were incubated at 37 °C and allowed to migrate. Photographs of the exact wound areas chosen on day 0 were again taken at 16 h. Photographs taken at the various time points were used for analyses of cell motility and morphometric quantification of the width of the wound by using NIS element software (Nikon, Japan). Experiments were repeated three times, in triplicate. 2.6. Statistical analysis All results were given as mean ± standard error of the mean (SEM) was used. Non-parametric Mann–Whitney test was employed to determine the statistical significance with a limit set to p b 0.05 using GraphPad Prism 5 (GraphPad Software, US) versus cells exposed to cetuximab. 3. Results 3.1. Effect of cetuximab on EGFR expression of GICs Effect of cetuximab on expression of EGFR was evaluated by Western blotting (Fig. 1). GICs in culture differently expressed EGFR. This expression was in accordance with the expression of total EGFR evaluated on the tumor extracts (Fig. 1a). Indeed, GIC3 expressed higher total EGFR than GIC9 (Fig. 1b and c). EGFRvIII form can be detected by Western blotting due to its lower molecular mass of 145 kDa as compared to wild-type EGFR (175 kDa), but no expression of EGFRvIII was detected on our experiments. It can be observed that cetuximab did not modify the expression of total EGFR for GIC3 and GIC9. Concerning the expression of phosphorylated EGFR (Fig. 1c), pEGFR was detected for GIC3 more strongly than for GIC9 and, as expected, cetuximab decreased pEGFR expression whatever the oxygen condition. 3.2. Effect of cetuximab on sphere formation and viability of GICs
Fig. 3. Effect of cetuximab on viability of GICs. Cell number was evaluated by cell counting (a) after trypan blue exclusion and metabolic activity by MTT assay (b). The values were reported to untreated controls for GIC3 and GIC9.
Sphere formation was observed by phase contrast microscopy after 48 h under the different conditions of culture (Fig. 2). GIC3 formed
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Fig. 4. Effect of cetuximab on CD133 and GFAP expression and self-renewal capacity of GICs. Expression of CD133 and GFAP (a) was evaluated by Western blotting on GIC3 and GIC9 protein extracts. Colonies formed after 15 days of culture of GIC3 under hypoxia (b) allow evaluating the self-renewal capacity by colonies counting (c). The relative expression of CD133 (d) and GFAP (e) was quantified from blots and the values were normalized by actin expression and normoxia.
rapidly numerous large spheres in basal condition contrary to GIC9 (Fig. 2a). Cetuximab was not able to prevent the formation of spheres for GICs but can decrease significantly the diameter of GIC spheres (Fig. 2b). Indeed, hypoxia increased significantly the size of spheres for GICs (30% of increase); nevertheless cetuximab is able to reduce it significantly at 12% and 20% for GIC3 and GIC9, respectively. The sensitivity of GICs to cetuximab was assessed by cell counting (Fig. 3a) and MTT assay (Fig. 3b). Cetuximab decreased significantly the viability of GIC3 cells in normoxia of approximately 37 and 31% evaluated by cell counting and MTT assay, respectively. The sensitivity of GIC9 for cetuximab was also important in normoxia, and despite a lower EGFR expression, the cell number was significantly decreased at 40%, but the decrease of MTT value was only at 19%. In hypoxia condition, cell viability was increased but cetuximab had no effect on the cell viability whatever the tumor origin.
differentiated cells) using Western blotting and their self-renewal capacity by using forming colony assay (Fig. 4). Cetuximab decreased the expression of CD133 for GIC3 and GIC9 but CD133 was detected more weakly for GIC9 whatever the culture condition (Fig. 4a). Even if hypoxia increased the expression of CD133, cetuximab was able to counteract this overexpression. Oppositely, the expression of GFAP increased after exposure to cetuximab. The number of colonies of GIC3 was significantly increased after a short period of 48 h under hypoxia of approximately 100%; nevertheless the same period of treatment with cetuximab was able to inhibit this effect (Fig. 4b and c). No colonies for GIC9 were observed whatever the conditions in the semi-solid medium.
3.4. Effect of cetuximab on the angiogenic potential of GICs 3.3. Effect of cetuximab on CD 133 and GFAP expression and self-renewal capacity of GICs The effect of cetuximab to affect the stem properties of GICs was evaluated by the expression of CD133 and GFAP (marker of
In order to evaluate the effect of cetuximab and hypoxia on the behavior of GICs on their microenvironment in particular on endothelial cells, conditioned media were harvested and put on endothelial cells. Angiogenesis was evaluated by Matrigel™ assay (Fig. 5) and endothelial cell migration assay (Fig. 6).
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The culture of endothelial cells on Matrigel™ allowed the formation of an endothelial network and the quantification of angiogenic parameters such as the number of junctions (Fig. 5a), of tubules (Fig. 5b) and the length of tubules (Fig. 5c). Nevertheless even hypoxia seemed to increase the angiogenic properties in particular for GIC3; no significant differences were observed on Matrigel whatever the cell origin or the culture conditions.
Fig. 6. Effect of cetuximab and hypoxia on the capacity of GICs to induce the migration of endothelial cells. A wound was made on endothelial cell monolayers and the distance of cell migration after 16 h of contact with the different conditioned media was measured.
Migration of endothelial cells represents another mechanism involved in angiogenesis. Hypoxia induced an increase of the migration of endothelial cells cultured with GIC conditioned media (Fig. 6). Cetuximab inhibited the migration of endothelial cells significantly of approximately 60% in hypoxia. In normoxia, the effect of cetuximab is different following cell origin. Indeed, strangely cetuximab induced an increase of endothelial cell migration for GIC9.
4. Discussion
Fig. 5. Effect of cetuximab and hypoxia on the capacity of GICs to induce the formation of endothelial network. Endothelial cells were cultured on Matrigel™ in presence of conditioned media obtained from GIC and junction number (a), tubule number (b) as well as the total tubule length (c) were quantified by AngioSys software (TCS Cellworks, UK).
Recently, it has been demonstrated that a specific cancer cell population possesses stemness properties such as tumor initiation, selfrenewal capacity and multilineage differentiation [28]. This tumor initiating cells could induce resistance to radiotherapy or chemotherapy and could be involved in tumor recurrence [2]. In this study, we evaluated for the first time, to the best of our knowledge, the in vitro response of GICs to anti-EGFR monoclonal antibody therapy such as cetuximab depending on the oxygenation level. We envisaged also the benefit of EGFR inhibition on the relationship between this cancer specific cell population and endothelial cells. In our GIC models with different EGFR status, the capacity of GICs to form spheres in vitro was corroborated with EGFR expression as previously demonstrated by Mazzoleni et al. As expected, cetuximab decreased the activation of EGFR; however the dependence to EGFR signal seems to be less important for GICs under hypoxia. It has been recently demonstrated that GICs are preferentially localized in the tumors in two specific regions extremely different in terms of oxygen level, the hypoxic and the peri-vascular niches [5,8]. The response of GICs to cetuximab could be largely dependent of the conditions encountered in their microenvironment as oxygen tension. We showed that oxygenation tension modified the sensitivity of GICs to cetuximab. Indeed, hypoxia inhibited the sensitivity of GICs to cetuximab probably by the induction of cell survival pathways such as HIF signaling pathway [19]. Moreover, cetuximab could change the activation of NF-kappa B (nuclear factor kappa B) which is a direct modulator of HIF-1 alpha expression as demonstrated by van Uden et al. [32]. In GICs, it seems that the up-regulation of the HIF2α sub-unit is specifically involved in the response to hypoxia [20]. Mazzoleni et al. [21] hypothesized that in tumors different subtypes of GICs could express more or less EGFR and could explain the tumor relapse after EGFR therapy. Cetuximab is well known to inhibit cell survival/proliferation signaling pathways [22]. Nevertheless, it is interesting to note that cetuximab was relatively
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efficient in normoxia even on GICs expressing weakly EGFR as also shown previously by Clark et al. [10]. Our two GIC models did not express EGFR variant III, which is a mutated form of EGFR activated constitutively, and can be involved in the resistance mechanism to cetuximab [31]. 30% of glioma expresses the active mutant EGFRvIII protein [12] and the mutation promotes glioma growth and vascularization through NF-Kappa B signaling [6]. It would be interesting to evaluate the self-renewal of GICs expressing EGFRvIII to cetuximab due to the ability of cetuximab to decrease strongly the in vitro activity of EGFRvIII [17]. Hypoxia promoted the expansion of GICs probably by selecting CD133 positive cells as previously described by Soeda et al. [30]. A recent work of Eimer et al. [11] showed that EGFR inhibition using erlotinib decreased cell proliferation without altering self-renewal capacity. The decrease of CD133 expression and the increase of GFAP observed in our study under hypoxia with the cetuximab could lead to an early differentiation of GICs and enhance the effectiveness of conventional therapies used concomitantly as the radiotherapy or chemotherapy in particular in hypoxic regions. Nevertheless, Clark and colleagues show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members enabling GIC proliferation, and therefore simultaneous blockade of multiple HER family members may be required for more efficacious GBM therapy [10]. The establishment of new blood vessels in the tumors (neoangiogenesis) is due to the release of molecular signals (growth factors) by tumor cells with a strong involvement of GICs that leads to the tumor growth [4,7]. In our models, cetuximab did not prevent the endothelial network formation induced by GICs but the EGFR inhibition of GICs decreased strongly endothelial cell migration under hypoxia. Cetuximab is known to modify the release of growth factors by cancer cells [16]. Nevertheless, it will be interesting to evaluate in our models the release of growth factors such as TGFβ which is known to modulate endothelial cell migration [13] or PDGF which is strongly involved in glioma progression [24]. As endothelial cell migration is essential to neoangiogenesis [23], the use of cetuximab could enhance the effect of anti-angiogenic therapies such as bevacizumab or cilengitide. Moreover, in limiting the attractiveness of GICs for endothelial cells, cetuximab could promote GIC differentiation. Indeed, the cell–cell communication involving GICs and endothelial cells is very important to maintain the stemness properties of GICs [33].
5. Conclusion Taking into account all our results, EGFR inhibition by using cetuximab is efficient on GICs in a high level of oxygenation as those found in the peri-vascular niches but can also affect their self-renewal capacity in hypoxic regions. Moreover, cetuximab could alter the relationship between GICs and the endothelial cells and enhance the therapeutic potential of anti-angiogenic therapeutic agents. Nevertheless, further investigations using in vivo preclinical models will be necessary to elucidate the real impact of anti-EGFR therapies on the relationship between GICs and endothelial cells and their involvement on the recurrence phenomena.
Conflicts of interest The authors declare that they have no conflict of interest.
Acknowledgment This study was supported by grants from the “Université de Lorraine”, the “Région Lorraine” and the French “Ligue Nationale contre le Cancer”.
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References [1] T.R. Alves, F.R. Lima, S.A. Kahn, D. Lobo, L.G. Dubois, R. Soletti, H. Borges, V.M. Neto, Glioblastoma cells: a heterogeneous and fatal tumor interacting with the parenchyma, Life Sci. 89 (2011) 532–539. [2] S. Bao, Q. Wu, Z. Li, S. Sathornsumetee, H. Wang, R.E. McLendon, A.B. Hjelmeland, J.N. Rich, Targeting cancer stem cells through L1CAM suppresses glioma growth, Cancer Res. 68 (2008) 6043–6048. [3] S. Bao, Q. Wu, R.E. McLendon, Y. Hao, Q. Shi, A.B. Hjelmeland, M.W. Dewhirst, D.D. Bigner, J.N. Rich, Glioma stem cells promote radioresistance by preferential activation of the DNA damage response, Nature 444 (2006) 756–760. [4] S. Bao, Q. Wu, S. Sathornsumetee, Y. Hao, Z. Li, A.B. Hjelmeland, Q. Shi, R.E. McLendon, D.D. Bigner, J.N. Rich, Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor, Cancer Res. 66 (2006) 7843–7848. [5] E.E. Bar, Glioblastoma, cancer stem cells and hypoxia, Brain Pathol. 21 (2011) 119–129. [6] R. Bonavia, M.M. Inda, S. Vandenberg, S.Y. Cheng, M. Nagane, P. Hadwiger, P. Tan, D.W. Sah, W.K. Cavenee, F.B. Furnari, EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway, Oncogene 31 (2012) 4054–4066. [7] J.M. Butler, H. Kobayashi, S. Rafii, Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiocrine factors, Nat. Rev. Cancer 10 (2010) 138–146. [8] C. Calabrese, H. Poppleton, M. Kocak, T.L. Hogg, C. Fuller, B. Hamner, E.Y. Oh, M.W. Gaber, D. Finklestein, M. Allen, A. Frank, I.T. Bayazitov, S.S. Zakharenko, A. Gajjar, A. Davidoff, R.J. Gilbertson, A perivascular niche for brain tumor stem cells, Cancer Cell 11 (2007) 69–82. [9] S.A. Choi, K.C. Wang, J.H. Phi, J.Y. Lee, C.K. Park, S.H. Park, S.K. Kim, A distinct subpopulation within CD133 positive brain tumor cells shares characteristics with endothelial progenitor cells, Cancer Lett. 324 (2012) 221–230. [10] P.A. Clark, M. Iida, D.M. Treisman, H. Kalluri, S. Ezhilan, M. Zorniak, D.L. Wheeler, J.S. Kuo, Activation of multiple ERBB family receptors mediates glioblastoma cancer stem-like cell resistance to EGFR-targeted inhibition, Neoplasia 14 (2012) 420–428. [11] S. Eimer, F. Dugay, K. Airiau, T. Avril, V. Quillien, M.A. Belaud-Rotureau, F. Belloc, Cyclopamine cooperates with EGFR inhibition to deplete stem-like cancer cells in glioblastoma-derived spheroid cultures, Neuro Oncol. 14 (2012) 1441–1451. [12] Faulkner C, Palmer A, Williams H, Wragg C, Haynes HR, White P, DeSouza RM, Williams M, Hopkins K, Kurian KM, 2015. EGFR and EGFRvIII analysis in glioblastoma as therapeutic biomarkers. Br. J. Neurosurg. 29, 23–29. [13] K. Giehl, A. Menke, Moving on: molecular mechanisms in TGFβ-induced epithelial cell migration, Signal Transduct. 6 (2006) 355–364. [14] B. Hasselbalch, U. Lassen, S. Hansen, M. Holmberg, M. Sørensen, M. Kosteljanetz, H. Broholm, M.T. Stockhausen, H.S. Poulsen, Cetuximab, bevacizumab, and irinotecan for patients with primary glioblastoma and progression after radiation therapy and temozolomide: a phase II trial, Neuro Oncol. 12 (2010) 508–516. [15] Hoelzinger DB, Mariani L, Weis J, Woyke T, Berens TJ, McDonough WS, Sloan A, Coons SW, Berens ME, 2005. Gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets. Neoplasia 7, 7–16. [16] V. Jouan-Hureaux, C. Boura, J.L. Merlin, B. Faivre, Modulation of endothelial cell network formation in vitro by molecular signaling of head and neck squamous cell carcinoma (HNSCC) exposed to cetuximab, Microvasc. Res. 83 (2012) 131–137. [17] B. Jutten, L. Dubois, Y. Li, H. Aerts, B.G. Wouters, P. Lambin, J. Theys, G. Lammering, Binding of cetuximab to the EGFRvIII deletion mutant and its biological consequences in malignant glioma cells, Radiother. Oncol. 92 (2009) 393–398. [18] J. Lee, S. Kotliarova, Y. Kotliarov, A. Li, Q. Su, N.M. Donin, S. Pastorino, B.W. Purow, N. Christopher, W. Zhang, J.K. Park, H.A. Fine, Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines, Cancer Cell 9 (2006) 391–403. [19] X. Li, Y. Lu, K. Liang, T. Pan, J. Mendelsohn, Z. Fan, Requirement of hypoxia-inducible factor-1alpha down-regulation in mediating the antitumor activity of the antiepidermal growth factor receptor monoclonal antibody cetuximab, Mol. Cancer Ther. 7 (2008) 1207–1217. [20] Z. Li, S. Bao, Q. Wu, H. Wang, C. Eyler, S. Sathornsumetee, Q. Shi, Y. Cao, J. Lathia, R.E. McLendon, A.B. Hjelmeland, J.N. Rich, Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells, Cancer Cell 15 (2009) 501–513. [21] S. Mazzoleni, L.S. Politi, M. Pala, M. Cominelli, A. Franzin, L. Sergi Sergi, A. Falini, M. De Palma, A. Bulfone, P.L. Poliani, R. Galli, Epidermal growth factor receptor expression identifies functionally and molecularly distinct tumor-initiating cells in human glioblastoma multiforme and is required for gliomagenesis, Cancer Res. 70 (2010) 7500–7513. [22] J. Mriouah, C. Boura, S. Pinel, A.S. Chretien, A. Fifre, J.L. Merlin, B. Faivre, Cellular response to cetuximab in PTEN-silenced head and neck squamous cell carcinoma cell line, Int. J. Oncol. 37 (2010) 1555–1563. [23] J. Mriouah, C. Boura, M. Thomassin, T. Bastogne, D. Dumas, B. Faivre, M. BarberiHeyob, Tumor vascular responses to antivascular and antiangiogenic strategies: looking for suitable models, Trends Biotechnol. 30 (2012) 649–658. [24] I. Nazarenko, S.M. Hede, X. He, A. Hedrén, J. Thompson, M.S. Lindström, M. Nistér, PDGF and PDGF receptors in glioma, Ups. J. Med. Sci. 117 (2012) 99–112. [25] S. Pinel, J. Mriouah, M. Vandamme, A. Chateau, F. Plénat, E. Guérin, L. Taillandier, V. Bernier-Chastagner, J.L. Merlin, P. Chastagner, Synergistic antitumor effect between gefitinib and fractionated irradiation in anaplastic oligodendrogliomas cannot be predicted by the Egfr signaling activity, PLoS One 8 (2013) e68333. [26] F. Pistollato, S. Abbadi, E. Rampazzo, L. Persano, A. Della Puppa, C. Frasson, E. Sarto, R. Scienza, D. D'avella, G. Basso, Intratumoral hypoxic gradient drives stem cells distribution and MGMT expression in glioblastoma, Stem Cells 28 (2010) 851–862.
80
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[27] B.J. Shin, J.K. Burkhardt, H.A. Riina, J.A. Boockvar, Superselective intra-arterial cerebral infusion of novel agents after blood–brain disruption for the treatment of recurrent glioblastoma multiforme: a technical case series, Neurosurg. Clin. N. Am. 23 (2012) 323–329. [28] S.K. Singh, I.D. Clarke, M. Terasaki, V.E. Bonn, C. Hawkins, J. Squire, P.B. Dirks, Identification of a cancer stem cell in human brain tumors, Cancer Res. 63 (2003) 5821–5828. [29] S.K. Singh, C. Hawkins, I.D. Clarke, J.A. Squire, J. Bayani, T. Hide, R.M. Henkelman, M.D. Cusimano, P.B. Dirks, Identification of human brain tumour initiating cells, Nature 432 (2004) 396–401. [30] A. Soeda, M. Park, D. Lee, A. Mintz, A. Androutsellis-Theotokis, R.D. McKay, J. Engh, T. Iwama, T. Kunisada, A.B. Kassam, I.F. Pollack, D.M. Park, Hypoxia promotes
expansion of the CD133-positive glioma stem cells through activation of HIF1alpha, Oncogene 28 (2009) 3949–3959. [31] J.C. Sok, F.M. Coppelli, S.M. Thomas, M.N. Lango, S. Xi, J.L. Hunt, M.L. Freilino, M.W. Graner, C.J. Wikstrand, D.D. Bigner, W.E. Gooding, F.B. Furnari, J.R. Grandis, Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targeting, Clin. Cancer Res. 12 (2006) 5064–5073. [32] P. van Uden, N.S. Kenneth, S. Rocha, Regulation of hypoxia-inducible factor-1alpha by NF-kappaB, Biochem. J. 412 (2008) 477–484. [33] T.S. Zhu, M.A. Costello, C.E. Talsma, C.G. Flack, J.G. Crowley, L.L. Hamm, X. He, S.L. Hervey-Jumper, J.A. Heth, K.M. Muraszko, F. DiMeco, A.L. Vescovi, X. Fan, Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells, Cancer Res. 71 (2011) 6061–6072.
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Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Sensitivity of glioma initiating cells to a monoclonal anti-EGFR antibody therapy under hypoxia Tatiana Randriarimanana, Alicia Chateau, Béatrice Faivre, Sophie Pinel, Cédric Boura ⁎ a b
Université de Lorraine, CRAN, UMR 7039, Campus Science, BP 70239, 54506 Vandœuvre-lès-Nancy, France CNRS, CRAN, UMR 7039, Vandœuvre-lès-Nancy Cedex 54506, France
a r t i c l e
i n f o
Article history: Received 2 February 2015 Received in revised form 26 June 2015 Accepted 24 July 2015 Available online 1 August 2015 Keywords: Glioma initiating cells Anti-EGFR therapy Hypoxia Stemness Angiogenesis
a b s t r a c t Aims: Glioma initiating cells (GICs) represent a subpopulation of tumor cells endowed with self-renewal and multilineage differentiation capacity but also with innate resistance to cytotoxic agents, a feature likely to pose major clinical challenges towards the complete eradication of minimal residual disease in glioma patients. Materials and methods: In this work, GICs were obtained from two patient-derived high-grade gliomas xenograft model, expressing differently EGFR. GICs were exposed to anti-EGFR monoclonal antibody cetuximab during 48 h in 1% or 21% oxygen tension. Cell viability and self-renewal capacity were then evaluated as well as their angiogenic properties. Key findings: GICs were sensitive to cetuximab only in normoxic condition whatever the EGFR status. Nevertheless, under hypoxia cetuximab was able to decrease the self-renewal capacity as well as the expression of CD133 while expression of GFAP increased. Moreover, cetuximab decreased the effect of GICs on endothelial cell migration under hypoxia. Significance: Consequently, anti-EGFR therapy can be envisaged to target specifically GICs in order to limit the tumor recurrence. © 2015 Elsevier Inc. All rights reserved.
1. Introduction In gliomas, accumulating evidences support the existence of a small subpopulation of cancer cells with properties of cancer stem cells. These cells express stem cell markers and have the ability to self-renewal as well as to differentiate into glial cells [28] In addition, these cancer stem-like cells have a high tumorigenic potential and are resistant to chemotherapy and irradiation, suggesting that these cell properties might be responsible for tumor development, recurrence and metastasis [1,3]. These glioma stem-like cells also called glioma initiating cells (GICs) have the ability to form spheres in vitro under serum free culture conditions [18] and to regenerate a tumor identical to the initial tumor in vivo [29]. However, the lack of specific stem cell markers impedes the isolation and analysis of GICs. For the moment, CD 133 (prominin-1), is the relevant marker for identifying GICs even if neural stem/progenitor cell markers such as nestin, Sox 2 and Musashi-1 have been yet used [2, 28]. One of the most common signaling changes in gliomas is the aberrant expression of various receptor tyrosine kinases as the epidermal growth factor receptor (EGFR). Wild-type EGFR overexpression as
⁎ Corresponding author at: Université de Lorraine, CRAN UMR CNRS 7039, Faculté de Médecine, 9 av. de la forêt de Haye, Bât D, 54505 Vandoeuvre-lès-Nancy, France. E-mail address: [email protected] (C. Boura).
http://dx.doi.org/10.1016/j.lfs.2015.07.024 0024-3205/© 2015 Elsevier Inc. All rights reserved.
well as constitutively activated mutated form of EGFR (EGFRvIII), are observed in more than 50% of patients with glioma and are associated with tumor progression [15]. EGFR inhibition with monoclonal antibodies such as cetuximab is of interest for glioma treatment [14,27]. Indeed, cetuximab inhibits the binding of natural ligands of EGFR such as EGF or TGF-α and is also able to induce the internalization and degradation of the truncated receptor form of EGFRvIII [17]. Investigations of the mechanisms that regulate GIC expansion, maintenance and control of microenvironment are, therefore, essential to better understand glioma pathology and to select effective therapeutics. In fact, GICs are localized preferentially in specific hypoxic regions (hypoxic niche) [26] or closed to endothelial cells (peri-vascular niche) [8]. The involvement of GICs in the response of EGFR therapy in particular depending on oxygenation conditions is important to predict the possible tumor recurrence with this targeted therapy. Indeed, GICs interact with cells of the microenvironment, such as endothelial cells, in order to maintain their stemness properties [33]. Understanding the relationship between GICs and their microenvironment is of major interest to envisage the complete eradication of this cell population. The aim of our study was to investigate in vitro the effect of oxygen level in order to mimic hypoxic or peri-vascular niches on the response of GICs to cetuximab, in particular on their survival and their selfrenewal capacity. Finally, reduced survival and self-renewal capacity of GICs after EGFR inhibition was evaluated on the relationship with endothelial cells by using conditioned media.
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Fig. 1. EGFR expression of glioma tumor models and of the derived GICs. EGFR expression of our different glioma derived tumor xenografts (called TCG2, 3, 4, 9 and 11) was analyzed by Western blotting (a). EGFR (green) of neurospheres derived from TCG3 and TCG9 was observed by fluorescence imaging (b) (scale bar represents 200 μm). Effect of cetuximab on the expression of phosphorylated EGFR of GIC3 and GIC9 (c) was analyzed by Western blotting after 48 h of culture in presence, or not, of cetuximab (20 μg/mL) and in different conditions of oxygenation (21% or 1% O2), β-actin expression was used as control for equal loading of proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2. Material and methods 2.1. Glioma initiating cell culture and treatments Tissue fragments of TCG3 or TCG9, two patient-derived high-grade glioma xenograft model [25], were subjected to mechanical homogenization followed by enzymatic digestion as previously described [9]. Primary tumor cell cultures were maintained in DMEM/F12 medium (Invitrogen, France) containing B27 supplement (Invitrogen, France), 20 ng/mL of human recombinant epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After one week of culture in hydrophobic flasks at 37 °C with 5% CO2 in a humidified atmosphere, spheres were obtained. GICs obtained from TCG3 or TCG9 were named GIC3 and GIC9 respectively, later in the manuscript. GICs were dissociated from spheres using Accumax (Millipore, France) and seeded at 3.104 cells/mL in 6 well plates or 25 cm2 flasks following the experiment. GICs were then exposed to Cetuximab (Merck Serrano) at 20 μg/mL and/or hypoxia (1% O2) in a hypoxic chamber (Hypoxystation, Don Whitley Scientific, UK) during 48 h.
2.2. Protein expression analysis For analysis of EGFR, pEGFR, CD133 and glial fibrillary acidic protein (GFAP) expression, Western blotting was realized as previously described [22]. Briefly, protein aliquots (40 μg) were denatured in the Laemmli buffer, containing ß mercaptoethanol and before being resolved in SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred onto PVDF membranes (Amersham Biosciences, Orsay, France). After blocking the PVDF membrane with Tris basebuffered saline prepared with 0.1% (v/v) Tween-20 containing 5% (w/
Fig. 2. Effect of cetuximab on sphere formation. The neurospheres were visualized by contrast phase microscopy (a) after 48 h of culture in presence, or not, of cetuximab (20 μg/mL) and in different conditions of oxygenation (21% or 1% O2) and sphere diameter (b) was measured with NIS element software (Nikon, japan). Scale bar represents 100 μm.
v) bovine serum albumin, the following primary antibodies against human EGFR (#2232, Cell Signaling, 1:1000 dilution), phosphorylated EGFR at Tyr1068 (pEGFR) (#2236, Cell Signaling, 1:1000 dilution), CD133 (# 130-090-422, Miltenyi Biotec, 1:500 dilution), GFAP (#12389, Cell Signaling, 1:1000 dilution) and ß-actin (#4970, Cell Signaling, 1:1000 dilution) were incubated overnight at 4 °C. Subsequently, immuno-reactive proteins were visualized using the enhanced chemiluminescence procedure (Pierce, USA). Quantification of relative band densities was performed using a densitometer (LAS Imager Fujifilm) and ß-actin was used as internal control. Images of neurospheres allow one to visualize the expression of EGFR by using a fluorescence macroscope (Nikon AZ100, Japan).
2.3. Glioma initiating cell viability The viability of GICs was evaluated by cell count after trypan-blue exclusion and MTT assay. GICs were seeded in 6 well plates at a density of 9.104 cells/well. For trypan-blue exclusion assay, cells were dissociated from sphere using a 0.025% trypsin–EDTA solution (Sigma, France) for 5 min at 37 °C and resuspended in complete medium. Cells
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were then stained with 0.4% trypan-blue solution and viable cells were counted twice using a Malassez hemocytometer. Cell viability was also determined by monitoring the mitochondrial activity using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (MTT, Sigma). At the set time, 0.5 μmol/L MTT was added into each well. Following a 3 h incubation at 37 °C, formazan crystals resulting from MTT metabolization were solubilized and cells were lysed by adding 400 μL of DMSO into each well. The absorbance of the resulting solution was measured at 570 nm using wells without cells as blank (Multiskan Ascent microplate photometer, Thermo Scientific, France).
2.4. Clonogenic assay After exposition of GICs to cetuximab and hypoxia, a soft agar anchorage independent clonogenic growth assay was done. Briefly, GICs (1 × 104) were suspended in 1 mL of DMEM/F12 containing 0.3% agar (Invitrogen, France). The cell suspension was then added on top of a presolidified 0.5% agar in 6 well plates. After 15 days of incubation, colonies were incubated with MTT solution as previously described and colonies larger than 50 μm in diameter were quantified using GelCount™ (Oxford Optronix, UK). Each experiment was repeated twice, in triplicate.
2.5. Angiogenesis assays Conditioned media were harvested by centrifugation at 350 g during 10 min of culture media obtained from GIC3 and GIC9 exposed or not to cetuximab and hypoxia. Angiogenesis properties of GICs were then evaluated by two in vitro assays performed on Human Umbilical Vein Endothelial Cells (HUVECs) obtained and cultured as we have previously described [16]. 2.5.1. Matrigel™ assay HUVECs were plated (90,000 cells/cm2) onto a 24-well plate precoated with Matrigel™ (BD Biosciences, France). After 1 h, media were removed and replaced by control or conditioned media (CM) and HUVECs were cultured during 24 h before being fixed with 4% paraformaldehyde. Network formation was analyzed after phalloidin– sulforhodamine staining (Fluoprobes®, Interchim, France). Photomicrographs of the whole culture surface were taken (Nikon AZ100, Digital Sight DS-Qi1Mc camera, Nikon, France). Endothelial cell density was calculated with NIS Element Software and represented as the area fraction in relation to the whole field. The total additive length of all cellular structures including all branches and the number of junctions was quantified with Angiosys Software (TCS Cellworks, UK). 2.5.2. In vitro wound assay In vitro wound assay for quantification of endothelial cell motility was performed using HUVEC cells in cultures. Briefly, 1.5 × 105 cells/ well were seeded in 24 well plates. A 1-mm wide linear wound was made across the center of each well with a micropipette tip. For each well five areas along the length of the wound were chosen randomly for photography under phase contrast microscopy. After photography, the cells were incubated at 37 °C and allowed to migrate. Photographs of the exact wound areas chosen on day 0 were again taken at 16 h. Photographs taken at the various time points were used for analyses of cell motility and morphometric quantification of the width of the wound by using NIS element software (Nikon, Japan). Experiments were repeated three times, in triplicate. 2.6. Statistical analysis All results were given as mean ± standard error of the mean (SEM) was used. Non-parametric Mann–Whitney test was employed to determine the statistical significance with a limit set to p b 0.05 using GraphPad Prism 5 (GraphPad Software, US) versus cells exposed to cetuximab. 3. Results 3.1. Effect of cetuximab on EGFR expression of GICs Effect of cetuximab on expression of EGFR was evaluated by Western blotting (Fig. 1). GICs in culture differently expressed EGFR. This expression was in accordance with the expression of total EGFR evaluated on the tumor extracts (Fig. 1a). Indeed, GIC3 expressed higher total EGFR than GIC9 (Fig. 1b and c). EGFRvIII form can be detected by Western blotting due to its lower molecular mass of 145 kDa as compared to wild-type EGFR (175 kDa), but no expression of EGFRvIII was detected on our experiments. It can be observed that cetuximab did not modify the expression of total EGFR for GIC3 and GIC9. Concerning the expression of phosphorylated EGFR (Fig. 1c), pEGFR was detected for GIC3 more strongly than for GIC9 and, as expected, cetuximab decreased pEGFR expression whatever the oxygen condition. 3.2. Effect of cetuximab on sphere formation and viability of GICs
Fig. 3. Effect of cetuximab on viability of GICs. Cell number was evaluated by cell counting (a) after trypan blue exclusion and metabolic activity by MTT assay (b). The values were reported to untreated controls for GIC3 and GIC9.
Sphere formation was observed by phase contrast microscopy after 48 h under the different conditions of culture (Fig. 2). GIC3 formed
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Fig. 4. Effect of cetuximab on CD133 and GFAP expression and self-renewal capacity of GICs. Expression of CD133 and GFAP (a) was evaluated by Western blotting on GIC3 and GIC9 protein extracts. Colonies formed after 15 days of culture of GIC3 under hypoxia (b) allow evaluating the self-renewal capacity by colonies counting (c). The relative expression of CD133 (d) and GFAP (e) was quantified from blots and the values were normalized by actin expression and normoxia.
rapidly numerous large spheres in basal condition contrary to GIC9 (Fig. 2a). Cetuximab was not able to prevent the formation of spheres for GICs but can decrease significantly the diameter of GIC spheres (Fig. 2b). Indeed, hypoxia increased significantly the size of spheres for GICs (30% of increase); nevertheless cetuximab is able to reduce it significantly at 12% and 20% for GIC3 and GIC9, respectively. The sensitivity of GICs to cetuximab was assessed by cell counting (Fig. 3a) and MTT assay (Fig. 3b). Cetuximab decreased significantly the viability of GIC3 cells in normoxia of approximately 37 and 31% evaluated by cell counting and MTT assay, respectively. The sensitivity of GIC9 for cetuximab was also important in normoxia, and despite a lower EGFR expression, the cell number was significantly decreased at 40%, but the decrease of MTT value was only at 19%. In hypoxia condition, cell viability was increased but cetuximab had no effect on the cell viability whatever the tumor origin.
differentiated cells) using Western blotting and their self-renewal capacity by using forming colony assay (Fig. 4). Cetuximab decreased the expression of CD133 for GIC3 and GIC9 but CD133 was detected more weakly for GIC9 whatever the culture condition (Fig. 4a). Even if hypoxia increased the expression of CD133, cetuximab was able to counteract this overexpression. Oppositely, the expression of GFAP increased after exposure to cetuximab. The number of colonies of GIC3 was significantly increased after a short period of 48 h under hypoxia of approximately 100%; nevertheless the same period of treatment with cetuximab was able to inhibit this effect (Fig. 4b and c). No colonies for GIC9 were observed whatever the conditions in the semi-solid medium.
3.4. Effect of cetuximab on the angiogenic potential of GICs 3.3. Effect of cetuximab on CD 133 and GFAP expression and self-renewal capacity of GICs The effect of cetuximab to affect the stem properties of GICs was evaluated by the expression of CD133 and GFAP (marker of
In order to evaluate the effect of cetuximab and hypoxia on the behavior of GICs on their microenvironment in particular on endothelial cells, conditioned media were harvested and put on endothelial cells. Angiogenesis was evaluated by Matrigel™ assay (Fig. 5) and endothelial cell migration assay (Fig. 6).
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The culture of endothelial cells on Matrigel™ allowed the formation of an endothelial network and the quantification of angiogenic parameters such as the number of junctions (Fig. 5a), of tubules (Fig. 5b) and the length of tubules (Fig. 5c). Nevertheless even hypoxia seemed to increase the angiogenic properties in particular for GIC3; no significant differences were observed on Matrigel whatever the cell origin or the culture conditions.
Fig. 6. Effect of cetuximab and hypoxia on the capacity of GICs to induce the migration of endothelial cells. A wound was made on endothelial cell monolayers and the distance of cell migration after 16 h of contact with the different conditioned media was measured.
Migration of endothelial cells represents another mechanism involved in angiogenesis. Hypoxia induced an increase of the migration of endothelial cells cultured with GIC conditioned media (Fig. 6). Cetuximab inhibited the migration of endothelial cells significantly of approximately 60% in hypoxia. In normoxia, the effect of cetuximab is different following cell origin. Indeed, strangely cetuximab induced an increase of endothelial cell migration for GIC9.
4. Discussion
Fig. 5. Effect of cetuximab and hypoxia on the capacity of GICs to induce the formation of endothelial network. Endothelial cells were cultured on Matrigel™ in presence of conditioned media obtained from GIC and junction number (a), tubule number (b) as well as the total tubule length (c) were quantified by AngioSys software (TCS Cellworks, UK).
Recently, it has been demonstrated that a specific cancer cell population possesses stemness properties such as tumor initiation, selfrenewal capacity and multilineage differentiation [28]. This tumor initiating cells could induce resistance to radiotherapy or chemotherapy and could be involved in tumor recurrence [2]. In this study, we evaluated for the first time, to the best of our knowledge, the in vitro response of GICs to anti-EGFR monoclonal antibody therapy such as cetuximab depending on the oxygenation level. We envisaged also the benefit of EGFR inhibition on the relationship between this cancer specific cell population and endothelial cells. In our GIC models with different EGFR status, the capacity of GICs to form spheres in vitro was corroborated with EGFR expression as previously demonstrated by Mazzoleni et al. As expected, cetuximab decreased the activation of EGFR; however the dependence to EGFR signal seems to be less important for GICs under hypoxia. It has been recently demonstrated that GICs are preferentially localized in the tumors in two specific regions extremely different in terms of oxygen level, the hypoxic and the peri-vascular niches [5,8]. The response of GICs to cetuximab could be largely dependent of the conditions encountered in their microenvironment as oxygen tension. We showed that oxygenation tension modified the sensitivity of GICs to cetuximab. Indeed, hypoxia inhibited the sensitivity of GICs to cetuximab probably by the induction of cell survival pathways such as HIF signaling pathway [19]. Moreover, cetuximab could change the activation of NF-kappa B (nuclear factor kappa B) which is a direct modulator of HIF-1 alpha expression as demonstrated by van Uden et al. [32]. In GICs, it seems that the up-regulation of the HIF2α sub-unit is specifically involved in the response to hypoxia [20]. Mazzoleni et al. [21] hypothesized that in tumors different subtypes of GICs could express more or less EGFR and could explain the tumor relapse after EGFR therapy. Cetuximab is well known to inhibit cell survival/proliferation signaling pathways [22]. Nevertheless, it is interesting to note that cetuximab was relatively
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efficient in normoxia even on GICs expressing weakly EGFR as also shown previously by Clark et al. [10]. Our two GIC models did not express EGFR variant III, which is a mutated form of EGFR activated constitutively, and can be involved in the resistance mechanism to cetuximab [31]. 30% of glioma expresses the active mutant EGFRvIII protein [12] and the mutation promotes glioma growth and vascularization through NF-Kappa B signaling [6]. It would be interesting to evaluate the self-renewal of GICs expressing EGFRvIII to cetuximab due to the ability of cetuximab to decrease strongly the in vitro activity of EGFRvIII [17]. Hypoxia promoted the expansion of GICs probably by selecting CD133 positive cells as previously described by Soeda et al. [30]. A recent work of Eimer et al. [11] showed that EGFR inhibition using erlotinib decreased cell proliferation without altering self-renewal capacity. The decrease of CD133 expression and the increase of GFAP observed in our study under hypoxia with the cetuximab could lead to an early differentiation of GICs and enhance the effectiveness of conventional therapies used concomitantly as the radiotherapy or chemotherapy in particular in hypoxic regions. Nevertheless, Clark and colleagues show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members enabling GIC proliferation, and therefore simultaneous blockade of multiple HER family members may be required for more efficacious GBM therapy [10]. The establishment of new blood vessels in the tumors (neoangiogenesis) is due to the release of molecular signals (growth factors) by tumor cells with a strong involvement of GICs that leads to the tumor growth [4,7]. In our models, cetuximab did not prevent the endothelial network formation induced by GICs but the EGFR inhibition of GICs decreased strongly endothelial cell migration under hypoxia. Cetuximab is known to modify the release of growth factors by cancer cells [16]. Nevertheless, it will be interesting to evaluate in our models the release of growth factors such as TGFβ which is known to modulate endothelial cell migration [13] or PDGF which is strongly involved in glioma progression [24]. As endothelial cell migration is essential to neoangiogenesis [23], the use of cetuximab could enhance the effect of anti-angiogenic therapies such as bevacizumab or cilengitide. Moreover, in limiting the attractiveness of GICs for endothelial cells, cetuximab could promote GIC differentiation. Indeed, the cell–cell communication involving GICs and endothelial cells is very important to maintain the stemness properties of GICs [33].
5. Conclusion Taking into account all our results, EGFR inhibition by using cetuximab is efficient on GICs in a high level of oxygenation as those found in the peri-vascular niches but can also affect their self-renewal capacity in hypoxic regions. Moreover, cetuximab could alter the relationship between GICs and the endothelial cells and enhance the therapeutic potential of anti-angiogenic therapeutic agents. Nevertheless, further investigations using in vivo preclinical models will be necessary to elucidate the real impact of anti-EGFR therapies on the relationship between GICs and endothelial cells and their involvement on the recurrence phenomena.
Conflicts of interest The authors declare that they have no conflict of interest.
Acknowledgment This study was supported by grants from the “Université de Lorraine”, the “Région Lorraine” and the French “Ligue Nationale contre le Cancer”.
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References [1] T.R. Alves, F.R. Lima, S.A. Kahn, D. Lobo, L.G. Dubois, R. Soletti, H. Borges, V.M. Neto, Glioblastoma cells: a heterogeneous and fatal tumor interacting with the parenchyma, Life Sci. 89 (2011) 532–539. [2] S. Bao, Q. Wu, Z. Li, S. Sathornsumetee, H. Wang, R.E. McLendon, A.B. Hjelmeland, J.N. Rich, Targeting cancer stem cells through L1CAM suppresses glioma growth, Cancer Res. 68 (2008) 6043–6048. [3] S. Bao, Q. Wu, R.E. McLendon, Y. Hao, Q. Shi, A.B. Hjelmeland, M.W. Dewhirst, D.D. Bigner, J.N. Rich, Glioma stem cells promote radioresistance by preferential activation of the DNA damage response, Nature 444 (2006) 756–760. [4] S. Bao, Q. Wu, S. Sathornsumetee, Y. Hao, Z. Li, A.B. Hjelmeland, Q. Shi, R.E. McLendon, D.D. Bigner, J.N. Rich, Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor, Cancer Res. 66 (2006) 7843–7848. [5] E.E. Bar, Glioblastoma, cancer stem cells and hypoxia, Brain Pathol. 21 (2011) 119–129. [6] R. Bonavia, M.M. Inda, S. Vandenberg, S.Y. Cheng, M. Nagane, P. Hadwiger, P. Tan, D.W. Sah, W.K. Cavenee, F.B. Furnari, EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway, Oncogene 31 (2012) 4054–4066. [7] J.M. Butler, H. Kobayashi, S. Rafii, Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiocrine factors, Nat. Rev. Cancer 10 (2010) 138–146. [8] C. Calabrese, H. Poppleton, M. Kocak, T.L. Hogg, C. Fuller, B. Hamner, E.Y. Oh, M.W. Gaber, D. Finklestein, M. Allen, A. Frank, I.T. Bayazitov, S.S. Zakharenko, A. Gajjar, A. Davidoff, R.J. Gilbertson, A perivascular niche for brain tumor stem cells, Cancer Cell 11 (2007) 69–82. [9] S.A. Choi, K.C. Wang, J.H. Phi, J.Y. Lee, C.K. Park, S.H. Park, S.K. Kim, A distinct subpopulation within CD133 positive brain tumor cells shares characteristics with endothelial progenitor cells, Cancer Lett. 324 (2012) 221–230. [10] P.A. Clark, M. Iida, D.M. Treisman, H. Kalluri, S. Ezhilan, M. Zorniak, D.L. Wheeler, J.S. Kuo, Activation of multiple ERBB family receptors mediates glioblastoma cancer stem-like cell resistance to EGFR-targeted inhibition, Neoplasia 14 (2012) 420–428. [11] S. Eimer, F. Dugay, K. Airiau, T. Avril, V. Quillien, M.A. Belaud-Rotureau, F. Belloc, Cyclopamine cooperates with EGFR inhibition to deplete stem-like cancer cells in glioblastoma-derived spheroid cultures, Neuro Oncol. 14 (2012) 1441–1451. [12] Faulkner C, Palmer A, Williams H, Wragg C, Haynes HR, White P, DeSouza RM, Williams M, Hopkins K, Kurian KM, 2015. EGFR and EGFRvIII analysis in glioblastoma as therapeutic biomarkers. Br. J. Neurosurg. 29, 23–29. [13] K. Giehl, A. Menke, Moving on: molecular mechanisms in TGFβ-induced epithelial cell migration, Signal Transduct. 6 (2006) 355–364. [14] B. Hasselbalch, U. Lassen, S. Hansen, M. Holmberg, M. Sørensen, M. Kosteljanetz, H. Broholm, M.T. Stockhausen, H.S. Poulsen, Cetuximab, bevacizumab, and irinotecan for patients with primary glioblastoma and progression after radiation therapy and temozolomide: a phase II trial, Neuro Oncol. 12 (2010) 508–516. [15] Hoelzinger DB, Mariani L, Weis J, Woyke T, Berens TJ, McDonough WS, Sloan A, Coons SW, Berens ME, 2005. Gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets. Neoplasia 7, 7–16. [16] V. Jouan-Hureaux, C. Boura, J.L. Merlin, B. Faivre, Modulation of endothelial cell network formation in vitro by molecular signaling of head and neck squamous cell carcinoma (HNSCC) exposed to cetuximab, Microvasc. Res. 83 (2012) 131–137. [17] B. Jutten, L. Dubois, Y. Li, H. Aerts, B.G. Wouters, P. Lambin, J. Theys, G. Lammering, Binding of cetuximab to the EGFRvIII deletion mutant and its biological consequences in malignant glioma cells, Radiother. Oncol. 92 (2009) 393–398. [18] J. Lee, S. Kotliarova, Y. Kotliarov, A. Li, Q. Su, N.M. Donin, S. Pastorino, B.W. Purow, N. Christopher, W. Zhang, J.K. Park, H.A. Fine, Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines, Cancer Cell 9 (2006) 391–403. [19] X. Li, Y. Lu, K. Liang, T. Pan, J. Mendelsohn, Z. Fan, Requirement of hypoxia-inducible factor-1alpha down-regulation in mediating the antitumor activity of the antiepidermal growth factor receptor monoclonal antibody cetuximab, Mol. Cancer Ther. 7 (2008) 1207–1217. [20] Z. Li, S. Bao, Q. Wu, H. Wang, C. Eyler, S. Sathornsumetee, Q. Shi, Y. Cao, J. Lathia, R.E. McLendon, A.B. Hjelmeland, J.N. Rich, Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells, Cancer Cell 15 (2009) 501–513. [21] S. Mazzoleni, L.S. Politi, M. Pala, M. Cominelli, A. Franzin, L. Sergi Sergi, A. Falini, M. De Palma, A. Bulfone, P.L. Poliani, R. Galli, Epidermal growth factor receptor expression identifies functionally and molecularly distinct tumor-initiating cells in human glioblastoma multiforme and is required for gliomagenesis, Cancer Res. 70 (2010) 7500–7513. [22] J. Mriouah, C. Boura, S. Pinel, A.S. Chretien, A. Fifre, J.L. Merlin, B. Faivre, Cellular response to cetuximab in PTEN-silenced head and neck squamous cell carcinoma cell line, Int. J. Oncol. 37 (2010) 1555–1563. [23] J. Mriouah, C. Boura, M. Thomassin, T. Bastogne, D. Dumas, B. Faivre, M. BarberiHeyob, Tumor vascular responses to antivascular and antiangiogenic strategies: looking for suitable models, Trends Biotechnol. 30 (2012) 649–658. [24] I. Nazarenko, S.M. Hede, X. He, A. Hedrén, J. Thompson, M.S. Lindström, M. Nistér, PDGF and PDGF receptors in glioma, Ups. J. Med. Sci. 117 (2012) 99–112. [25] S. Pinel, J. Mriouah, M. Vandamme, A. Chateau, F. Plénat, E. Guérin, L. Taillandier, V. Bernier-Chastagner, J.L. Merlin, P. Chastagner, Synergistic antitumor effect between gefitinib and fractionated irradiation in anaplastic oligodendrogliomas cannot be predicted by the Egfr signaling activity, PLoS One 8 (2013) e68333. [26] F. Pistollato, S. Abbadi, E. Rampazzo, L. Persano, A. Della Puppa, C. Frasson, E. Sarto, R. Scienza, D. D'avella, G. Basso, Intratumoral hypoxic gradient drives stem cells distribution and MGMT expression in glioblastoma, Stem Cells 28 (2010) 851–862.
80
T. Randriarimanana et al. / Life Sciences 137 (2015) 74–80
[27] B.J. Shin, J.K. Burkhardt, H.A. Riina, J.A. Boockvar, Superselective intra-arterial cerebral infusion of novel agents after blood–brain disruption for the treatment of recurrent glioblastoma multiforme: a technical case series, Neurosurg. Clin. N. Am. 23 (2012) 323–329. [28] S.K. Singh, I.D. Clarke, M. Terasaki, V.E. Bonn, C. Hawkins, J. Squire, P.B. Dirks, Identification of a cancer stem cell in human brain tumors, Cancer Res. 63 (2003) 5821–5828. [29] S.K. Singh, C. Hawkins, I.D. Clarke, J.A. Squire, J. Bayani, T. Hide, R.M. Henkelman, M.D. Cusimano, P.B. Dirks, Identification of human brain tumour initiating cells, Nature 432 (2004) 396–401. [30] A. Soeda, M. Park, D. Lee, A. Mintz, A. Androutsellis-Theotokis, R.D. McKay, J. Engh, T. Iwama, T. Kunisada, A.B. Kassam, I.F. Pollack, D.M. Park, Hypoxia promotes
expansion of the CD133-positive glioma stem cells through activation of HIF1alpha, Oncogene 28 (2009) 3949–3959. [31] J.C. Sok, F.M. Coppelli, S.M. Thomas, M.N. Lango, S. Xi, J.L. Hunt, M.L. Freilino, M.W. Graner, C.J. Wikstrand, D.D. Bigner, W.E. Gooding, F.B. Furnari, J.R. Grandis, Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targeting, Clin. Cancer Res. 12 (2006) 5064–5073. [32] P. van Uden, N.S. Kenneth, S. Rocha, Regulation of hypoxia-inducible factor-1alpha by NF-kappaB, Biochem. J. 412 (2008) 477–484. [33] T.S. Zhu, M.A. Costello, C.E. Talsma, C.G. Flack, J.G. Crowley, L.L. Hamm, X. He, S.L. Hervey-Jumper, J.A. Heth, K.M. Muraszko, F. DiMeco, A.L. Vescovi, X. Fan, Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells, Cancer Res. 71 (2011) 6061–6072.