Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

International audienceDespite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a "side population" (SP). As the SP cells represent a low percentage (<5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of β-catenin

Overcoming Resistance to Irradiation of Head and Neck Squamous Cell Carcinoma by Targeting the Cancer Stem Cell Subpopulation

International audienceTo date, for some cancer, radiotherapy leads to low benefits in terms of local control and long-term survival. One hypothesis for this failure is that these malignancies have acquired, or are inherently, radioresistant to low-LET radiation. Therefore, radiotherapy is undergoing an evolution by shifting to the use of heavier ion. Hadrontherapy has demonstrated favorable results for many of the malignancies. However, for some cancers, such as head and neck squamous cell carcinoma (HNSCC), local control remains low. In order to obtain more insights into this resistance mechanism and tumour escape, we have investigated the role of cancer stem cells (CSC) obtained from radioresistant HNSCC cell lines

Radiological studies with low energy protons at the Radiograaff Platform

International audienceA horizontal beam facility - equipped with a biology laboratory - for radiobiological experiments with medium-energy protons has been setup at the 4MV Van de Graaff accelerator of the Institut de Physique Nucléaire de Lyon. At this energy, the Relative Biological Effectiveness (RBE) of protons may amount to 2-7 and this platform constitutes a tool to study the specific effects of high Linear Energy Transfert (LET) radiations to cells. For such macroscopic-irradiation facilities, the dose distributions over the cell samples has to be uniform with accuracy better than +/- 5% with a controlled dose rate (2Gy/min) for clinical interest. In addition, the variation of the LET between the entrance and the exit of the cells does not exceed 10%. A homogenous irradiation field with a suitable proton flux is obtained by means of collimators and Au-scattering foils arrangements. The set-up was stimulated by the GATE v6.1 Monte-Carlo platform. Proton energy measurement, fluence-homogeneity evaluation and monitoring system calibration were tested with 3.5 MeV protons extracted in air. With a double scattering system, a fluence heterogeneity of -13% over a circular field of 20 nm diameter was obtained. A preliminary biological experiment was performed to test protocols with two human Head and Neck Squamous cell line carcinoma with quite different radiosensitivities. Cells were irradiated at 2Gy with a dose rate of 2Gy/min. DNA Double Strand Break induction and repair, were measured by scoring the gamma-H2AX foci

Essential role of HIF-1α in both resistance to photon and carbon ion exposures and invasiveness of Head and Neck Squamous Cell Carcinoma (HNSCC) cancer stem cells

International audiencePurpose: HNSCC have a poor prognosis, which is partly due to the presence of cancer stem cells (CSCs) resistant to chemo- and radio-therapies. CSCs are located in a hypoxic environment which leads to HIF-1a stabilization and induces epithelial mesenchymal transition. Reactive Oxygen Species (ROS), produced in response to hypoxia and/or irradiation, play a key role in this stabilization. However, mechanisms leading to HIF-1a stabilization and their consequences on the invasiveness need to be clarified in CSCs exposed to carbon or photon radiation in hypoxia.Methods: Two HNSCC cells lines and their CSCs, sorted by flow cytometry, were grown under normoxia or hypoxia (1% O2). Oxygen Enhancement Ratio (OER) was calculated at 10% survival after photon and carbon ion (75MeV/n) irradiation. The expression of HIF-1a was followed by Western-Blots. ROS were quantified with a CM-H2DCFDA dye and Migration/Invasion with Boyden chambers.Results: Both parental and CSCs sub-populations are more resistant to photons under hypoxia (OER>1.2) whereas the oxygen effect is canceled after carbon ion exposure (OER=1). Interestingly, as for the OER values, HIF-1a expression is function of the type of irradiation, stabilized with photons and fully inhibited with carbon ions. Additionally, under hypoxia, HIF-1a appears earlier in CSCs than in non-CSCs, and is correlated with ROS levels, confirming their adaptive properties to hypoxia. Finally, the invasion/migration abilities of CSCs are more important than in non-CSCs under normoxia, and that, even more significantly under hypoxia. After carbon ion exposure, in both conditions of oxygenation, invasion/migration processes are decreased compared with photons.Conclusion: HIF-1a plays a key role in the radioresistance and the invasiveness of CSCs. No expression of HIF-1a was found after carbon ion exposure suggesting that hadrontherapy could be a relevant alternative to kill CSCs.Supported by ANR-11-LABX-0063, ANR-11-INBS-0007, INCa-DGOS-466

ROLE DU MICROENVIRONNEMENT HYPOXIQUE DANS L’ECHAPPEMENT TUMORAL DES CANCERS EPIDERMOÏDES DE LA TETE ET DU COU EN REPONSE A L’IRRADIATION PHOTONIQUE ET PAR IONS CARBONE

International audienceObjectifs: Les carcinomes épidermoïdes de la tête et du cou (HNSCC) possèdent un taux de récidive élevé du fait d’une résistance aux thérapies anti-cancéreuses. L’hadronthérapie est une alternative à la radiothérapie photonique du fait de ses propriétés balistiques et d’une Efficacité Biologique Relative élevée. Cependant, le contrôle local des HNSCC traités par ions carbone reste inférieur à celui des autres cancers des VADS. La présence d’une sous-population de cellules souches cancéreuses (CSCs) résistante aux traitements par chimio- et radio-thérapies et localisée dans des niches hypoxiques participerait à l’échappement tumoral. En effet, l’environnement créé active les voies de signalisation de l’hypoxie dont le principal marqueur est la protéine HIF-1α. Stabilisée et donc activée par l’hypoxie, HIF-1α permet de moduler la transcription de gènes cibles impliqués notamment dans les phénomènes de migration et d’invasion. Nous nous sommes donc intéressés aux mécanismes impliqués dans la radiorésistance et l’invasion-migration de lignées HNSCC et leur sous-population souche en conditions hypoxique et normoxique

Carbon ions Versus γ-Irradiation: The Telomeric Effect in Cancer Cells

International audienceThe higher biological effect of Carbon ions hadrontherapy (C+) is explained by the nature of the DNA damages. It is known that cell response to γ-irradiation (γ-IR), but not to C+, is correlated with telomere length in different type of cancer cells. Here, we propose that this " telomeric effect " must result from an effect of ROS in γ-IR compared to C+

A key role of HIF-1α in the radioresistance of Cancer Stem Cells in their hypoxic microenvironment

International audienceThe resistance of Head and Neck Squamous Cell Carcinoma (HNSCC) to chemo- and radio-therapies is partly explained by the presence of Cancer Stem Cells (CSCs) located in a hypoxic microenvironment. In these niches, poor in oxygen, the HIF-1α protein is expressed and Reactive Oxygen Species (ROS), produced in response to hypoxia and/or irradiation, play a key role in HIF-1α stabilization. However, mechanisms leading to HIF-1 expression and their consequences on the CSCs survival need to be clarified in response to carbon or photon radiations under hypoxia. For two HNSCC cell lines and their CSCs subpopulation, we observed a radioresistance in response to photons under hypoxia (OER>1.2). This result is associated with a stabilization of HIF-1α. Additionally, under hypoxia, HIF-1 appears earlier in CSCs than in non-CSCs, and its expression is correlated with ROS levels, confirming CSCs adaptive properties to hypoxia. To go further in the understanding of HIF-1α activation’s mechanism, we used DMSO as a ROS scavenger and observed the consequences on HIF-1α, whose expression was significantly decreased. Finally, inhibition of HIF-1α with a siRNA leads to the radiosensitization of the CSCs in their hypoxic microenvironment and even more after exposure to photons. Compared with photons, the oxygen effect is canceled after carbon ion exposure (OER=1) and interestingly, HIF-1 expression is fully inhibited following carbon ion irradiation. These results taken together demonstrate that HIF-1 plays a key role in the radioresistance of HNSCC CSCs and suggest that a therapeutic which silence HIF-1α could be a relevant alternative to kill or sensitize CSCs as observed with carbon ion irradiation.This work was supported by CPER ETOILE/UCBL, Labex Primes (ANR-11-LABX-0063), France Hadron (ANR-11-INBS-0007) and Lyric ( INCa-DGOS-4664

Cellular and molecular portrait of eleven human glioblastoma cell lines irradiated with photons or carbon ions

International audienceThe multiform glioblastoma (GBM) is a heterogeneous and highly invasive entity, making it the most aggressive brain tumor. The standard-of-care for glioblastoma consists of surgical resection, radiotherapy and chemotherapy, but despite the recent improvement of therapeutic protocols, the recurrence seems inevitable. Due to a better dose localization in the tumor volume and a greater Relative Biological Efficiency (RBE), carbon ion therapy seems to be a promising alternative to conventional radiotherapy. However, to optimize individual treatment by hadrontherapy the exact carbon equivalent dose needs to be determined from data obtained after photon irradiation. Therefore, molecular and cellular investigations of GBM are required in order to improve the prediction and treatment of brain tumors. In this study, we have examined the radiobiological features of 11 human glioma cell lines displaying gradual radiosensitivity, following photon- or carbon-therapy in order to optimize and secure antiglioma strategies. Independent of p53 or O6-methylguanine-DNA methyltransferase (MGMT) status, all cell lines respond to both types of radiation by a G2/M phase arrest followed by the appearance of mitotic catastrophe, which is concluded by a ceramide-dependent-apoptotic cell death. Statistical analyses demonstrate that: (i) the surviving fraction at 2Gy (SF2) and the dose for 10% survival (D10) photon values are correlated with that obtained in response to carbon ions; (ii) regardless of p53, MGMT status, and radiosensitivity, the release of ceramide is associated with the induction of late apoptosis; (iii) the appearance of polyploid cells after photon irradiation could predict the RBE to carbon ions.The present study clearly provides a consistent database of the cellular and molecular response of glioblastoma cell lines to photon irradiation, and to the best of our knowledge represents the largest archive for carbon ion hadrontherapy response of glioblastoma cells. This type of archive is intended to customize the treatment of patients by allowing the development of new predictive mathematical models for the response of tumors to radiation, and ultimately could improve hadrontherapy treatment plans

Ciblage de la famille HER dans les cancers ORL : inhibition de la prolifération cellulaire et de l’invasion-migration cellulaire en réponse à l’association cetuximab-pertuzumab combinée à l’irradiation photonique

National audienceObjet : Les cancers épidermoïdes ORL sont associés à un fort taux de récidive loco-régionale et métastatique. Les cellules souches cancéreuses (CSCs), sous-population hautement migratoire, semblent être une hypothèse majeure à l’origine de la résistance aux traitements. L’objectif du travail était de comparer l’efficacité du blocage pan-HER par une association cetuximab-pertuzumab associé à l’irradiation photonique dans les processus d’invasion et migration de la lignée SQ20B et sa sous-population souche.Méthodes : La sous-population de CSCs de la lignée SQ20B a été isolée par double tri. La prolifération des cellules SQ20B et SQ20B/CSCs a été étudiée après traitement au cetuximab 5nM et/ou pertuzumab 20microg/mL +/- irradiation photonique à 10 Gy. L’analyse de la migration et de l’invasion a été réalisée par test de blessure avec et sans matrigel (IncuCyte(R)). L’activation de l’EGFR (Tyr1068) et des voies de signalisation intracellulaires (Phospho-AKT et Phospho-MEK1/2) a été étudiée en réponse aux traitements par western-blot (WES(R)).Résultat : Le cetuximab inhibe la prolifération cellulaire des cellules SQ20B et non celle de la sous-population souche. L’association cetuximab-pertuzumab inhibe significativement la prolifération des cellules SQ20B et SQ20B/CSCs. La double association cetuximab-pertuzumab associée à l’irradiation 10Gy inhibe significativement la migration et l’invasion des deux populations cellulaires. Le double traitement inhibe la phosphorylation d’EGFR dans les deux populations. Les cellules SQ20B expriment fortement phospho-AKT à l’inverse des SQ20B/CSCs qui expriment phospho-MEK1/2. Enfin, l’association cetuximab-pertuzumab-10Gy inhibe fortement l’expression de phospho-AKT et phospho-MEK1/2.Conclusion : Le double traitement cetuximab-pertuzumab combiné à l’irradiation photonique inhibe significativement la prolifération, la migration et l’invasion de la lignée SQ20B et sa sous-population souche.This work was supported by LabEx PRIMES, France Hadro