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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Carbon ion irradiation withstands cancer stem cells' migration/invasion process in Head and Neck Squamous Cell Carcinoma (HNSCC).

Cancer Stem Cells (CSCs) in Head and Neck Squamous Cell Carcinoma (HNSCC) have extremely aggressive profile (high migratory and invasive potential). These characteristics can explain their resistance to conventional treatment. Efficacy of photon and carbon ion irradiation with addition of cetuximab (5 nM) is studied on clonogenic death, migration and invasion of two HNSCC populations: SQ20B and SQ20B/CSCs. SQ20B express E-cadherin and overexpress EGFR while SQ20B/CSCs express N-cadherin and low EGFR. Cetuximab strongly inhibits SQ20B proliferation but has no effect on SQ20B/CSCs. 2 Gy photon irradiation enhances migration and invasiveness in both populations (p < 0.05), while cetuximab only stops SQ20B migration (p < 0.005). Carbon irradiation significantly inhibits invasion in both populations (p < 0.05), and the association with cetuximab significantly inhibits invasion in both populations (p < 0.005). These results highlight CSCs characteristics: EGFRLow, cetuximab-resistant, and highly migratory. Carbon ion irradiation appears to be a very promising therapeutic modality counteracting migration/invasion process in both parental cells and CSCs in contrast to photon irradiation.

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.

Influence of Dose Rate on the Cellular Response to Low- and High-LET Radiations.

Nowadays, head and neck squamous cell carcinoma (HNSCC) treatment failure is mostly explained by locoregional progression or intrinsic radioresistance. Radiotherapy (RT) has recently evolved with the emergence of heavy ion radiations or new fractionation schemes of photon therapy, which modify the dose rate of treatment delivery. The aim of the present study was then to evaluate the in vitro influence of a dose rate variation during conventional RT or carbon ion hadrontherapy treatment in order to improve the therapeutic care of patient. In this regard, two HNSCC cell lines were irradiated with photons or 72 MeV/n carbon ions at a dose rate of 0.5, 2, or 10 Gy/min. For both radiosensitive and radioresistant cells, the change in dose rate significantly affected cell survival in response to photon exposure. This variation of radiosensitivity was associated with the number of initial and residual DNA double-strand breaks (DSBs). By contrast, the dose rate change did not affect neither cell survival nor the residual DNA DSBs after carbon ion irradiation. As a result, the relative biological efficiency at 10% survival increased when the dose rate decreased. In conclusion, in the RT treatment of HNSCC, it is advised to remain very careful when modifying the classical schemes toward altered fractionation. At the opposite, as the dose rate does not seem to have any effects after carbon ion exposure, there is less need to adapt hadrontherapy treatment planning during active system irradiation.

Targeting head and neck tumoral stem cells: From biological aspects to therapeutic perspectives.

Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer in the world. Effective therapeutic modalities such as surgery, radiation, chemotherapy and combinations of each are used in the management of the disease. In most cases, treatment fails to obtain total cancer cure. In recent years, it appears that one of the key determinants of treatment failure may be the presence of cancer stem cells (CSCs) that escape currently available therapies. CSCs form a small portion of the total tumor burden but may play a disproportionately important role in determining outcomes. CSCs have stem features such as self-renewal, high migration capacity, drug resistance, high proliferation abilities. A large body of evidence points to the fact that CSCs are particularly resistant to radiotherapy and chemotherapy. In HNSCC, CSCs have been increasingly shown to have an integral role in tumor initiation, disease progression, metastasis and treatment resistance. In the light of such observations, the present review summarizes biological characteristics of CSCs in HNSCC, outlines targeted strategies for the successful eradication of CSCs in HNSCC including targeting the self-renewal controlling pathways, blocking epithelial mesenchymal transition, niche targeting, immunotherapy approaches and highlights the need to better understand CSCs biology for new treatments modalities.

[Radiosensitivity and/or radioresistance of head and neck cancers: Biological angle]. / Radiosensibilité et/ou résistance des cancers ORL: aspects biologiques.

Radiation therapy is a cornerstone of head and neck cancer management. Technological improvements in recent years in radiation therapy, with intensity-modulated techniques, reinforce even more its role. However, both local and locoregional relapses are still observed. Understanding biological mechanisms of treatment resistance is a topic of major interest. From the cancer cell itself, its ability to repair and proliferate, its microenvironment and oxygenation conditions, migratory and invasive capacity, to biological parameters related to the patient, there are many mechanisms involving radiosensitivity and/or radioresistance of head and neck cancer. The present study explores the main biological mechanisms involved in radiation resistance of head and neck cancer, and describes promising therapeutic approaches.

[Cancer stem cells: Radiotherapeutic features and therapeutic targets]. / Cellules souches tumorales: aspects radiothérapeutiques et ciblage thérapeutique.

Recent evidences suggest that many types of cancers contain a cell population presenting stem cell properties. While the great majority of tumor cells are destined to differentiate, and eventually stop dividing, only a minority population of cells, termed cancer stem cells (CSCs), possesses extensive self-renewal capability and can recapitulate tumor pathophysiology in an immune-compromised animal model. Tumor initiating cells have been identified and isolated in many tumor types including brain, colon and prostate. They are virtually resistant to radiation and may contribute to treatment resistance and recurrence. Therefore, therapies specifically targeting CSCs will likely be needed for complete tumor eradication. The present study reviews published reports identifying the mechanisms of radioresistance of CSCs and potential targets based on the pathways of self-renewal. Further elucidation of pathways that regulate CSCs may provide insights into the development of novel innovative therapies.