CD44, γ-H2AX, and p-ATM Expressions in Short-Term Ex Vivo Culture of Tumour Slices Predict the Treatment Response in Patients with Oral Squamous Cell Carcinoma.

Squamous cell carcinoma is the most common type of head and neck cancer (HNSCC) with a disease-free survival at 3 years that does not exceed 30%. Biomarkers able to predict clinical outcomes are clearly needed. The purpose of this study was to investigate whether a short-term culture of tumour fragments irradiated ex vivo could anticipate patient responses to chemo- and/or radiotherapies. Biopsies were collected prior to treatment from a cohort of 28 patients with non-operable tumours of the oral cavity or oropharynx, and then cultured ex vivo. Short-term biopsy slice culture is a robust method that keeps cells viable for 7 days. Different biomarkers involved in the stemness status (CD44) or the DNA damage response (pATM and γ-H2AX) were investigated for their potential to predict the treatment response. A higher expression of all these markers was predictive of a poor response to treatment. This allowed the stratification of responder or non-responder patients to treatment. Moreover, the ratio for the expression of the three markers 24 h after 4 Gy irradiation versus 0 Gy was higher in responder than in non-responder patients. Finally, combining these biomarkers greatly improved their predictive potential, especially when the γ-H2AX ratio was associated with the CD44 ratio or the pATM ratio. These results encourage further evaluation of these biomarkers in a larger cohort of patients.

Ceramide-Enriched Membrane Domains Contribute to Targeted and Nontargeted Effects of Radiation through Modulation of PI3K/AKT Signaling in HNSCC Cells.

We investigated the potential involvement of ceramide-enriched membrane domains in radiation-induced targeted and nontargeted effects using head and neck squamous cell carcinoma with opposite radiosensitivities. In radiosensitive SCC61 cells, the proportion of targeted effects was 34% and nontargeted effects killed 32% of cells. In contrast, only targeted effects (30%) are involved in the overall death of radioresistant SQ20B cells. We then demonstrated in SCC61 cells that nontargeted cell response was driven by the formation of the radiation-induced ceramide-enriched domain. By contrast, the existence of these platforms in SQ20B cells confers a permissive region for phosphatidylinositol-3-kinase (PI3K)/AKT activation. The disruption of lipid raft results in strong inhibition of PI3K/AKT signaling, leading to radiosensitization and apparition of nontargeted effects. These results suggest that ceramide-enriched platforms play a significant role in targeted and nontargeted effects during radiotherapy and that drugs modulating cholesterol levels may be a good alternative for improving radiotherapy effectiveness.

Differential pattern of HIF-1α expression in HNSCC cancer stem cells after carbon ion or photon irradiation: one molecular explanation of the oxygen effect.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) are resistant to standard treatments, partly due to cancer stem cells (CSCs) localised in hypoxic niches. Compared to X-rays, carbon ion irradiation relies on better ballistic properties, higher relative biological effectiveness and the absence of oxygen effect. Hypoxia-inducible factor-1α (HIF-1α) is involved in the resistance to photons, whereas its role in response to carbon ions remains unclear. METHODS: Two HNSCC cell lines and their CSC sub-population were studied in response to photons or carbon ion irradiation, in normoxia or hypoxia, after inhibition or not of HIF-1α. RESULTS: Under hypoxia, compared to non-CSCs, HIF-1α is expressed earlier in CSCs. A combined effect photons/hypoxia, less observed with carbon ions, results in a synergic and earlier HIF-1α expression in both subpopulations. The diffuse ROS production by photons is concomitant with HIF-1α expression and essential to its activation. There is no oxygen effect in response to carbon ions and the ROS localised in the track might be insufficient to stabilise HIF-1α. Finally, in hypoxia, cells were sensitised to both types of radiations after HIF-1α inhibition. CONCLUSIONS: Hypoxia-inducible factor-1α plays a main role in the response of CSCs and non-CSCs to carbon ion and photon irradiations, which makes the HIF-1α targeting an attractive therapeutic challenge.

Gadolinium-based nanoparticles as sensitizing agents to carbon ions in head and neck tumor cells.

Hadrontherapy presents the major advantage of improving tumor sterilization while sparing surrounding healthy tissues because of the particular ballistic (Bragg peak) of carbon ions. However, its efficacy is still limited in the most resistant cancers, such as grade III-IV head and neck squamous cell carcinoma (HNSCC), in which the association of carbon ions with gadolinium-based nanoparticles (AGuIX®) could be used as a Trojan horse. We report for the first time the radioenhancing effect of AGuIX® when combined with carbon ion irradiation in human tumor cells. An increase in relative biological effectiveness (1.7) in three HNSCC cell lines (SQ20B, FaDu, and Cal33) was associated with a significant reduction in the radiation dose needed for killing cells. Radiosensitization goes through a higher number of unrepaired DNA double-strand breaks. These results underline the strong potential of AGuIX® in sensitizing aggressive tumors to hadrontherapy and, therefore, improving local control while lowering acute/late toxicity.

Telomerase inhibition improves tumor response to radiotherapy in a murine orthotopic model of human glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most frequent and aggressive type of adult brain tumor. Most GBMs express telomerase; a high level of intra-tumoral telomerase activity (TA) is predictive of poor prognosis. Thus, telomerase inhibitors are promising options to treat GBM. These inhibitors increase the response to radiotherapy (RT), in vitro as well as in vivo. Since typical treatments for GBM include RT, our objective was to evaluate the efficiency of Imetelstat (TA inhibitor) combined with RT. FINDINGS: We used a murine orthotopic model of human GBM (N = 8 to11 mice per group) and µMRI imaging to evaluate the efficacy of Imetelstat (delivered by intra-peritoneal injection) alone and combined with RT. Using a clinically established protocol, we demonstrated that Imetelstat significantly: (i) inhibited the TA in the very center of the tumor, (ii) reduced tumor volume as a proportion of TA inhibition, and (iii) increased the response to RT, in terms of tumor volume regression and survival increase. CONCLUSIONS: Imetelstat is currently evaluated in refractory brain tumors in young patients (without RT). Our results support its clinical evaluation combined with RT to treat GBM.

Differential Formation of Stress Granules in Radiosensitive and Radioresistant Head and Neck Squamous Cell Carcinoma Cells.

PURPOSE: Stress granules (SGs) are cytoplasmic aggregates in which mRNAs and specific proteins are trapped in response to a variety of damaging agents. They participate in the cellular defense mechanisms. Currently, their mechanism of formation in response to ionizing radiation and their role in tumor-cell radiosensitivity remain elusive. METHODS AND MATERIALS: The kinetics of SG formation was investigated after the delivery of photon irradiation at different doses to head and neck squamous cell carcinoma cell lines with different radiosensitivities and the HeLa cervical cancer cell line (used as reference). In parallel, the response to a canonical inducer of SGs, sodium arsenite, was also studied. Immunolabeling of SG-specific proteins and mRNA fluorescence in situ hybridization enabled SG detection and quantification. Furthermore, a ribopuromycylation assay was used to assess the cell translational status. To determine whether reactive oxygen species were involved in SG formation, their scavenging or production was induced by pharmacologic pretreatment in both SCC61 and SQ20B cells. RESULTS: Photon irradiation at different doses led to the formation of cytoplasmic foci that were positive for different SG markers. The presence of SGs gradually increased from 30 minutes to 2 hours postexposure in HeLa, SCC61, and Cal60 radiosensitive cells. In turn, the SQ20B and FaDu radioresistant cells did not form SGs. These results indicated a correlation between sensitivity to photon irradiation and SG formation. Moreover, SG formation was significantly reduced by reactive oxygen species scavenging using dimethyl sulfoxide in SCC61 cells, which supported their role in SG formation. However, a reciprocal experiment in SQ20B cells that depleted glutathione using buthionine sulfoximide did not restore SG formation in these cells. CONCLUSIONS: SGs are formed in response to irradiation in radiosensitive, but not in radioresistant, head and neck squamous cell carcinoma cells. Interestingly, compared with sodium arsenite-induced SGs, photon-induced SGs exhibited a different morphology and cellular localization. Moreover, photon-induced SGs were not associated with the inhibition of translation; rather, they depended on oxidative stress.

p53-independent early and late apoptosis is mediated by ceramide after exposure of tumor cells to photon or carbon ion irradiation.

BACKGROUND: To determine whether ceramide is responsible for the induction of p53-independent early or late apoptosis in response to high- and low-Linear-Energy-Transfer (LET) irradiation. METHODS: Four cell lines displaying different radiosensitivities and p53-protein status were irradiated with photons or 33.4 or 184 keV/µm carbon ions. The kinetics of ceramide production was quantified by fluorescent microscopy or High-Performance-Liquid-Chromatogaphy and the sequence of events leading to apoptosis by flow cytometry. RESULTS: Regardless of the p53-status, both low and high-LET irradiation induced an early ceramide production in radiosensitive cells and late in the radioresistant. This production strongly correlated with the level of early apoptosis in radiosensitive cells and delayed apoptosis in the radioresistant ones, regardless of radiation quality, tumor type, radiosensitivity, or p53-status. Inhibition of caspase activity or ceramide production showed that, for both types of radiation, ceramide is essential for the initiation of early apoptosis in radiosensitive cells and late apoptosis following mitotic catastrophe in radioresistant cells. CONCLUSIONS: Ceramide is a determining factor in the onset of early and late apoptosis after low and high-LET irradiation and is the mediator of the p53-independent-apoptotic pathway. We propose that ceramide is the molecular bridge between mitotic catastrophe and the commitment phase of delayed apoptosis in response to irradiation.

Circulating Tumor Cell Detection during Neoadjuvant Chemotherapy to Predict Early Response in Locally Advanced Oropharyngeal Cancers: A Prospective Pilot Study.

Patients with locally advanced oropharyngeal carcinoma treated with neoadjuvant chemotherapy are reassessed both radiologically and clinically to adapt their treatment after the first cycle. However, some responders show early tumor progression after adjuvant radiotherapy. This cohort study evaluated circulating tumor cells (CTCs) from a population of locally advanced oropharyngeal carcinoma patients treated with docetaxel, cisplatin, and 5-fluorouracil (DCF) induction chemotherapy or DCF with a modified dose and fractioned administration. The counts and phenotypes of CTCs were assessed at baseline and at day 21 of treatment, after isolation using the RosetteSepTM technique based on negative enrichment. At baseline, 6 out of 21 patients had CTCs (28.6%). On day 21, 5 out of 11 patients had CTCs (41.6%). There was no significant difference in the overall and progression-free survival between patients with or without CTCs at baseline (p = 0.44 and 0.78) or day 21 (p = 0.88 and 0.5). Out of the 11 patients tested at day 21, 4 had a positive variation of CTCs (33%). Patients with a positive variation of CTCs display a lower overall survival. Our findings suggest that the variation in the number of CTCs would be a better guide to the management of treatment, with possible early changes in treatment strategy.

Combining radiation to EGFR and Bcl-2 blockade: a new approach to target cancer stem cells in head and neck squamous cell carcinoma.

PURPOSE: The clinical outcome of head and neck squamous cell carcinoma (HNSCC) remains poor, partly due to the presence of resistant cancer stem cells (CSCs) which are responsible of recurrences. CSCs have low EGFR expression and, conversely, overexpress the anti-apoptotic Bcl-2 protein, which is involved in resistance to apoptosis and the invasion/migration capacities of tumour cells. METHODS: The combination therapy of ABT-199, a Bcl-2 inhibitor, cetuximab an EGFR inhibitor, and radiation using an HNSCC model (SQ20B cell line) and its corresponding CSC subpopulation were evaluated in vitro (2D/3D cell proliferation; invasion/migration and apoptosis using videomicroscopy) and in vivo. RESULTS: Cetuximab strongly inhibited 2D and 3D cell proliferation, as well as invasion/migration, only in non-CSC-SQ20B cells, whereas ABT-199 selectively inhibited these mechanisms in SQ20B/CSCs. The combination of irradiation + cetuximab + ABT-199 increased the inhibition of the 2D and 3D cell proliferation, invasion/migration, and resistance to apoptosis in both cell sub-populations. In addition, in a nude mouse model with heterotopic tumour xenograft, a treatment combining cetuximab + ABT-199 with fractional irradiation strongly delayed the tumour growth and increased in vivo lifespan without side effects. CONCLUSION: Based on the present results, this triple combination therapy may represent a new opportunity for testing in clinical trials, particularly in locally advanced HNSCC.

Involvement of HIF-1α in the Detection, Signaling, and Repair of DNA Double-Strand Breaks after Photon and Carbon-Ion Irradiation.

Hypoxia-Inducible Factor 1α (HIF-1α), which promotes cancer cell survival, is the main regulator of oxygen homeostasis. Hypoxia combined with photon and carbon ion irradiation (C-ions) stabilizes HIF-1α. Silencing HIF-1α under hypoxia leads to substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and C-ions. Thus, this study aimed to clarify a potential involvement of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). After confirming the nucleoshuttling of HIF-1α in response to both exposure under hypoxia, we showed that silencing HIF-1α in non-CSCs and CSCs decreased the initiation of the DSB detection (P-ATM), and increased the residual phosphorylated H2AX (γH2AX) foci. While HIF-1α silencing did not modulate 53BP1 expression, P-DNA-PKcs (NHEJ-c) and RAD51 (HR) signals decreased. Altogether, our experiments demonstrate the involvement of HIF-1α in the detection and signaling of DSBs, but also in the main repair pathways (NHEJ-c and HR), without favoring one of them. Combining HIF-1α silencing with both types of radiation could therefore present a potential therapeutic benefit of targeting CSCs mostly present in tumor hypoxic niches.

Impact of hypoxia on the double-strand break repair after photon and carbon ion irradiation of radioresistant HNSCC cells.

DNA double-strand breaks (DSBs) induced by photon irradiation are the most deleterious damage for cancer cells and their efficient repair may contribute to radioresistance, particularly in hypoxic conditions. Carbon ions (C-ions) act independently of the oxygen concentration and trigger complex- and clustered-DSBs difficult to repair. Understanding the interrelation between hypoxia, radiation-type, and DNA-repair is therefore essential for overcoming radioresistance. The DSBs signaling and the contribution of the canonical non-homologous end-joining (NHEJ-c) and homologous-recombination (HR) repair pathways were assessed by immunostaining in two cancer-stem-cell (CSCs) and non-CSCs HNSCC cell lines. Detection and signaling of DSBs were lower in response to C-ions than photons. Hypoxia increased the decay-rate of the detected DSBs (γH2AX) in CSCs after photons and the initiation of DSB repair signaling (P-ATM) in CSCs and non-CSCs after both radiations, but not the choice of DSB repair pathway (53BP1). Additionally, hypoxia increased the NHEJ-c (DNA-PK) and the HR pathway (RAD51) activation only after photons. Furthermore, the involvement of the HR seemed to be higher in CSCs after photons and in non-CSCs after C-ions. Taken together, our results show that C-ions may overcome the radioresistance of HNSCC associated with DNA repair, particularly in CSCs, and independently of a hypoxic microenvironment.

Comparison of biophysical models with experimental data for three cell lines in response to irradiation with monoenergetic ions.

The relative biological effectiveness (RBE) in particle therapy is currently estimated using biophysical models. We compared experimental measurements to the α curves as function of linear energy transfer computed by the Local Effect Model (LEM I-IV), the Microdosimetric Kinetic Model (MKM) and the NanOx model for HSG, V79 and CHO-K1 cells in response to monoenergetic irradiations. Although the LEM IV and the MKM predictions accurately reproduced the trend observed in the data, NanOx yielded a better agreement than the other models for more irradiation configurations. Its χ 2 estimator was indeed the lowest for three over seven considered cases.

ROS Production and Distribution: A New Paradigm to Explain the Differential Effects of X-ray and Carbon Ion Irradiation on Cancer Stem Cell Migration and Invasion.

Although conventional radiotherapy promotes the migration/invasion of cancer stem cells (CSCs) under normoxia, carbon ion (C-ion) irradiation actually decreases these processes. Unraveling the mechanisms of this discrepancy, particularly under the hypoxic conditions that pertain in niches where CSCs are preferentially localized, would provide a better understanding of the origins of metastases. Invasion/migration, proteins involved in epithelial-to-mesenchymal transition (EMT), and expression of MMP-2 and HIF-1α were quantified in the CSC subpopulations of two head-and-neck squamous cell carcinoma (HNSCC) cell lines irradiated with X-rays or C-ions. X-rays triggered HNSCC-CSC migration/invasion under normoxia, however this effect was significantly attenuated under hypoxia. C-ions induced fewer of these processes in both oxygenation conditions. The differential response to C-ions was associated with a lack of HIF-1α stabilization, MMP-2 expression, or activation of kinases of the main EMT signaling pathways. Furthermore,we demonstrated a major role of reactive oxygen species (ROS) in the triggering of invasion/migration in response to X-rays. Monte-Carlo simulations demonstrated that HO● radicals are quantitatively higher after C-ions than after X-rays, however they are very differently distributed within cells. We postulate that the uniform distribution of ROS after X-rays induces the mechanisms leading to invasion/migration, which ROS concentrated in C-ion tracks are unable to trigger.

Radiobiologic parameters and local effect model predictions for head-and-neck squamous cell carcinomas exposed to high linear energy transfer ions.

PURPOSE: To establish the radiobiologic parameters of head-and-neck squamous cell carcinomas (HNSCC) in response to ion irradiation with various linear energy transfer (LET) values and to evaluate the relevance of the local effect model (LEM) in HNSCC. METHODS AND MATERIALS: Cell survival curves were established in radiosensitive SCC61 and radioresistant SQ20B cell lines irradiated with [33.6 and 184 keV/n] carbon, [302 keV/n] argon, and X-rays. The results of ion experiments were confronted to LEM predictions. RESULTS: The relative biologic efficiency ranged from 1.5 to 4.2 for SCC61 and 2.1 to 2.8 for SQ20B cells. Fixing an arbitrary D(0) parameter, which characterized survival to X-ray at high doses (>10 Gy), gave unsatisfying LEM predictions for both cell lines. For D(0) = 10 Gy, the error on survival fraction at 2 Gy amounted to a factor of 10 for [184 keV/n] carbon in SCC61 cells. We showed that the slope (s(max)) of the survival curve at high doses was much more reliable than D(0). Fitting s(max) to 2.5 Gy(-1) gave better predictions for both cell lines. Nevertheless, LEM could not predict the responses to fast and slow ions with the same accuracy. CONCLUSIONS: The LEM could predict the main trends of these experimental data with correct orders of magnitude while s(max) was optimized. Thus the efficiency of carbon ions cannot be simply extracted from the clinical response of a patient to X-rays. LEM should help to optimize planning for hadrontherapy if a set of experimental data is available for high-LET radiations in various types of tumors.

Differential regulation of cell death in head and neck cell carcinoma through alteration of cholesterol levels in lipid rafts microdomains.

Lipid rafts are cholesterol-enriched microdomains in the plasma membrane. They act as molecular platforms that spatially organize membrane receptor molecules and are involved in the transduction of various signaling pathways. We recently reported that in the radiosensitive squamous cell carcinoma SCC61 line, gamma-irradiation results in a rearrangement of the plasma membrane rafts and signaling platforms leading to radiation-induced apoptosis in a ceramide-dependent pathway. By contrast, this reorganization was found to be defective in the radioresistant counterpart cell line, SQ20B. As the cholesterol content of lipid rafts is two times higher in SQ20B compared with SCC61 cells, we investigated the modulation of these microdomains using methyl-beta-cyclodextrin (MbetaCDX), a widely used cholesterol-depleting agent, in order to disrupt raft organization in both cells. Here, we report that MbetaCDX treatment resulted in the triggering of apoptosis in SCC61 cells involving mitochondrial events and associated with the clustering of Fas, the formation of Fas-FADD complexes and the cleavage of procaspase 8. The ligand-independent activation of this death receptor was totally absent in SQ20B cells, which remained resistant to MbetaCDX-triggered apoptosis. However, treatment of SQ20B with MbetaCDX resulted in a ligand-independent activation of the epidermal growth factor receptor (EGFR) survival pathway, as evidenced by an increased tyrosine phosphorylation of EGFR. Taken altogether, our results indicate that lipid raft integrity is intimately involved in the triggering of apoptotic cell death and/or survival pathways in head and neck carcinoma cells.

Radioresistance of human carcinoma cells is correlated to a defect in raft membrane clustering.

In addition to DNA damage, exposure to irradiation involves the plasma membrane in the early phases of gamma-ray-induced cell death. The involvement of raft microdomains following gamma-radiation derives essentially from the role of ceramide as a critical component leading to apoptosis. It is demonstrated here that gamma-irradiation of a radiosensitive human head and neck squamous carcinoma cell line (SCC61) results in the triggering of raft coalescence to larger membrane platforms associated with the externalization of an acid sphingomyelinase (A-SMase), leading to ceramide release in raft, 30 min postirradiation. For the first time, we show that this structural rearrangement is defective in the radioresistant SQ20B cells and associated with the lack of A-SMase activation and translocation, a result which could explain in part their resistance to apoptosis following ionizing radiation. Moreover, we show that SQ20B are protected against radiation injury through a fivefold upper level of endogenous glutathione compared to SCC61. Overcoming the endogenous antioxidant defenses of SQ20B through either H(2)O(2) treatment or GSH depletion triggers A-SMase activation and translocation, raft coalescence, and apoptosis. On the contrary, ROS scavengers abolished these events in radiosensitive SCC61 cells. Translation of this concept to tumor biology suggests that manipulation of rafts through redox equilibrium may provide opportunities for radiosensitization of tumor cells.

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an "in-incubator" video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

Preclinical models in HNSCC: A comprehensive review.

Head and neck cancer remains a significant public health concern. About 60% of patients die within 5years due to local recurrence. Head and neck squamous cell carcinoma (HNSCC) cell lines are important preclinical models in the search for new therapies against this disease. Furthermore, there is a need to test novel drugs before introduction into clinical practice. A preclinical model that closely resembles the in vivo situation would be highly valuable. In the last few decades, a multicellular spheroid model has gained attention as its behavior was comparable to in vivo tumors. Basic research is necessary to achieve an understanding of the normal and pathological state but cannot, in itself, provide sufficient information for clinical applications. Indeed, animal models are an inevitable prelude to assess the efficacy of new therapeutic approaches in HNSCC. The present review proposes an overview of HNSCC pre-clinical models in order to further understand the oncogenic properties for HNSCC and translate these findings into clinic for patients.

Dual "mAb" HER family blockade in head and neck cancer human cell lines combined with photon therapy.

Head and neck cancer stem cells (CSCs) are highly resistant to treatment. When EGFR is overexpressed in head and neck squamous cell carcinoma (HNSCC), HER2 and HER3 are also expressed. The aim of the present study was to investigate the effect of HER1/2/3 blockade through a combination of cetuximab and pertuzumab, with or without photon irradiation, on the proliferation and migration/invasion capabilities of an HNSCC chemo- and radioresistant human cell line (SQ20B) and its corresponding stem cell subpopulation. Cell proliferation, migration and invasion were studied after treatment with cetuximab +/- pertuzumab +/- 10 Gy photon irradiation. EGFR, phospho-EGFR, HER2 and HER3 protein expression levels were studied. Activation or inhibition of the RAS/MAPK and AKT-mTOR downstream signalling cascades was investigated through phospho-AKT and phospho-MEK1/2 expression. Cetuximab strongly inhibited SQ20B and FaDu cell proliferation, migration and invasion, whereas it had little effect on SQ20B-CSCs. Cetuximab-pertuzumab combined with radiation significantly inhibited SQ20B and FaDu cell and SQ20B-CSC proliferation, migration and invasion. Cetuximab-pertuzumab with 10 Gy photon irradiation switched off both phospho-AKT and phospho-MEK1/2 expression in the three populations. The triple therapy is therefore thought to inhibit SQ20B cells, SQ20B-CSCs and FaDu cells through an AKT-mTOR and Ras-MAPK downstream signalling blockade.

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

Despite 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 (CD44(high)) and the over-expression of aldehyde dehydrogenase (ALDH(high)). 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 ß-catenin.