Radioresistant Cancer Stem Cells Research Articles

Abstract 5048: Combatting pancreatic cancer stem cells by novel titanium peroxides nanoparticles combined with X-ray radiation

Abstract Background: Radiotherapy is widely used as the main treatment for multiple malignancies. However, several types of cancers, including pancreatic cancer, show resistance to radiation therapy. The presence of cancer stem cells (CSCs) are the major cause of radiation resistance in pancreatic cancer. Whereas, titanium peroxides nanoparticles (TiOxNPs) which are a modified form of titanium oxide nanoparticles promote enhanced remarkable efficacy of radiation therapy. Therefore, we examined the efficacy of TiOxNPs as radiosensitizers to eradicate pancreatic cancer stem cells. Methods and materials: In vitro, Sphere-forming assay, survival assay, migration, and invasion assay were evaluated after using TiOxNPs prior to radiation exposure to pancreatic cancer cell line. In vivo, tumor-bearing nude mice were injected by TiOxNPs either intratumoral or intravenous one hour prior to radiation treatment, and the tumor volume, body weight, and mice survival was calculated. In addition, proteins-related stemness were measured in vitro and in vivo to evaluate the usage of TiOxNPs as radiosensitizers to pancreatic CSCs. Moreover, we planned to evaluate the mechanism beyond the efficacy of TiOxNPs as radiosensitizers to CSCs by detecting reactive oxygen species (ROS) production, mitochondrial function, and the phosphorylation of some signaling proteins. We found that TiOxNPs combined with ionizing radiation showed anti-cancer effects on radioresistant CSCs both in vitro and in vivo. Results: In vitro, a marked reduction in growth was detected after exposing the TiOxNPs-treated cells to radiation therapy, specifically with a 5Gy dose compared with 2Gy dose. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. In vivo, we first established an aggressive xenograft by injecting MIA PaCa-2 sphere cells into the flank region of BALB/c nude mice, and found that animals treated with combined TiOxNPs and irradiation showed a dramatic reduction in tumor volume and weight compared to the untreated group. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Radiosensitizing TiOxNPs suppressed pancreatic xenograft outgrowth after primary or dissociating sphere MIA PaCa-2 cell implantation. It is assumed that synergy is created by inactivating the AKT signaling pathway and producing unbearable amounts of ROS. Conclusion: Our findings showed that using TiOxNPs in combination with radiation might be a favorable therapeutic approach to eradicate pancreatic CSCs. Citation Format: Mohammed Salah, Hiroki Kawaguchi, Hiroaki Akasaka, Yasuyuki Shimizu, Kenta Morita, Yuya Nishimura, Hikaru Kubota, Tomomi Sogawa, Naritoshi Mukumoto, Chiaki Ogino, Ryohei Sasaki. Combatting pancreatic cancer stem cells by novel titanium peroxides nanoparticles combined with X-ray radiation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5048.

Reactive oxygen species-inducing titanium peroxide nanoparticles as promising radiosensitizers for eliminating pancreatic cancer stem cells

BackgroundDespite recent advances in radiotherapy, radioresistance in patients with pancreatic cancer remains a crucial dilemma for clinical treatment. Cancer stem cells (CSCs) represent a major factor in radioresistance. Developing a potent radiosensitizer may be a novel candidate for the eradication of pancreatic CSCs.MethodsCSCs were isolated from MIA PaCa-2 and PANC1 human pancreatic cancer cell lines. Titanium peroxide nanoparticles (TiOxNPs) were synthesized from titanium dioxide nanoparticles (TiO2NPs) and utilized as radiosensitizers when added one hour prior to radiation exposure. The antitumor activity of this novel therapeutic strategy was evaluated against well-established pancreatic CSCs model both in vitro and in vivo.ResultsIt is shown that TiOxNPs combined with ionizing radiation exhibit anti-cancer effects on radioresistant CSCs both in vitro and in vivo. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Notably, radiosensitizing TiOxNPs suppressed the growth of pancreatic xenografts following primary or dissociating sphere MIA PaCa-2 cell implantation. It is inferred that synergy is formed by generating intolerable levels of reactive oxygen species (ROS) and inactivating the AKT signaling pathway.ConclusionsOur data suggested the use of TiOxNPs in combination with radiation may be considered an attractive therapeutic strategy to eliminate pancreatic CSCs.

Phenotypic variation modulates the growth dynamics and response to radiotherapy of solid tumours under normoxia and hypoxia

In cancer, treatment failure and disease recurrence have been associated with small subpopulations of cancer cells with a stem-like phenotype. In this paper, we develop and investigate a phenotype-structured model of solid tumour growth in which cells are structured by a stemness level, which varies continuously between stem-like and terminally differentiated behaviours. Cell evolution is driven by proliferation and death, as well as advection and diffusion with respect to the stemness structure variable. Here, the magnitude and sign of the advective flux are allowed to vary with the oxygen level. We use the model to investigate how the environment, in particular oxygen levels, affects the tumour’s population dynamics and composition, and its response to radiotherapy. We use a combination of numerical and analytical techniques to quantify how under physiological oxygen levels the cells evolve to a differentiated phenotype and under low oxygen level (i.e., hypoxia) they de-differentiate. Under normoxia, the proportion of cancer stem cells is typically negligible and the tumour may ultimately become extinct whereas under hypoxia cancer stem cells comprise a dominant proportion of the tumour volume, enhancing radio-resistance and favouring the tumour’s long-term survival. We then investigate how such phenotypic heterogeneity impacts the tumour’s response to treatment with radiotherapy under normoxia and hypoxia. Of particular interest is establishing how the presence of radio-resistant cancer stem cells can facilitate a tumour’s regrowth following radiotherapy. We also use the model to show how radiation-induced changes in tumour oxygen levels can give rise to complex re-growth dynamics. For example, transient periods of hypoxia induced by damage to tumour blood vessels may rescue the cancer cell population from extinction and drive secondary regrowth.

Microsecond Pulsed Electric Fields: An Effective Way to Selectively Target and Radiosensitize Medulloblastoma Cancer Stem Cells

Cancer stem cells constitute an endless reserve for the maintenance and progression of tumors, and they could be the reason for conventional therapy failure. New therapeutic strategies are necessary to specifically target them. In this context, microsecond pulsed electric fields have been selected to expose D283Med cells, a human medulloblastoma cell line resulted to be rich in cancer stem cells, and normal human astrocytes. We analyzed invitro different endpoints at different times after microsecond pulsed electric field exposure, such as permeabilization, reactive oxygen species generation, cell viability/proliferation, cell cycle, and clonogenicity, as well as the expression of different genes involved in cell cycle, apoptosis, and senescence. Furthermore, the response of D283Med cells exposed to microsecond pulsed electric fields was validated invivo in a heterotopic mouse xenograft model. Our invitro results showed that a specific pulse protocol (ie, 0.3 MV/m, 40 μs, 5 pulses) was able to induce irreversible membrane permeabilization and apoptosis exclusively in medulloblastoma cancer stem cells. In the surviving cells, reactive oxygen species generation was observed, together with a transitory G2/M cell-cycle arrest with a senescence-associated phenotype via the upregulation of GADD45A. Invivo results, after pulsed electric field exposure, demonstrated a significant tumor volume reduction with no eradication of tumor mass. In conjunction, we verified the efficacy of electric pulse pre-exposure followed by ionizing irradiation invivo to enable complete inhibition of tumor growth. Our data reveal novel therapeutic options for the targeting of medulloblastoma cancer stem cells, indicating nonionizing pulsed electric field pre-exposure as an effective means to overcome the radioresistance of cancer stem cells.

A multimodal treatment of carbon ions irradiation, miRNA-34 and mTOR inhibitor specifically control high-grade chondrosarcoma cancer stem cells

Background and purposeHigh-grade chondrosarcomas are chemo- and radio-resistant cartilage-forming tumors of bone that often relapse and metastase. Thus, new therapeutic strategies are urgently needed. Material and methodsChondrosarcoma cells (CH-2879) were exposed to carbon-ion irradiation, combined with miR-34 mimic and/or rapamycin administration. The effects of treatment on cancer stem cells, stemness-associated phenotype, radioresistance and tumor-initiating properties were evaluated. ResultsWe show that high-grade chondrosarcoma cells contain a population of radioresistant cancer stem cells that can be targeted by a combination of carbon-ion therapy, miR-34 mimic administration and/or rapamycin treatment that triggers FOXO3 and miR-34 over-expression. mTOR inhibition by rapamycin triggered FOXO3 and miR-34, leading to KLF4 repression. ConclusionOur results show that particle therapy combined with molecular treatments effectively controls cancer stem cells and may overcome treatment resistance of high-grade chondrosarcoma.

CSC Radioresistance: A Therapeutic Challenge to Improve Radiotherapy Effectiveness in Cancer.

Radiotherapy (RT) is a modality of oncologic treatment that can be used to treat approximately 50% of all cancer patients either alone or in combination with other treatment modalities such as surgery, chemotherapy, immunotherapy, and therapeutic targeting. Despite the technological advances in RT, which allow a more precise delivery of radiation while progressively minimizing the impact on normal tissues, issues like radioresistance and tumor recurrence remain important challenges. Tumor heterogeneity is responsible for the variation in the radiation response of the different tumor subpopulations. A main factor related to radioresistance is the presence of cancer stem cells (CSC) inside tumors, which are responsible for metastases, relapses, RT failure, and a poor prognosis in cancer patients. The plasticity of CSCs, a process highly dependent on the epithelial–mesenchymal transition (EMT) and associated to cell dedifferentiation, complicates the identification and eradication of CSCs and it might be involved in disease relapse and progression after irradiation. The tumor microenvironment and the interactions of CSCs with their niches also play an important role in the response to RT. This review provides a deep insight into the characteristics and radioresistance mechanisms of CSCs and into the role of CSCs and tumor microenvironment in both the primary tumor and metastasis in response to radiation, and the radiobiological principles related to the CSC response to RT. Finally, we summarize the major advances and clinical trials on the development of CSC-based therapies combined with RT to overcome radioresistance. A better understanding of the potential therapeutic targets for CSC radiosensitization will provide safer and more efficient combination strategies, which in turn will improve the live expectancy and curability of cancer patients.

Nuclear Accumulation of β-Catenin in Cancer Stem Cell Radioresistance and Stemness in Human Colon Cancer.

The aim of this study was to examine whether the Wnt/β-catenin signal activation is a cause of radioresistance in colon cancer by assessing the β-catenin localization and its correlation with cancer stem cells (CSCs). The nuclear levels of β-catenin, the hallmark of Wnt activation, were analyzed in HCT116 and SW480 cells by immunohistochemistry, before and after irradiation. Further, we assessed CSC populations by staining for aldehyde dehydrogenase-1 (ALDH1) and CD44. β-catenin was localized predominantly in the nucleus and plasma membrane in SW480 and HCT116 cells, respectively. Compared to HCT116 cells, SW480 cells displayed higher Wnt activation. At 24 h after irradiation, most of the DSBs in SW480 cells were repaired, but were still present in HCT116 cells. Additionally, compared to HCT116 cells, a significantly higher proportion of SW480 cells were ALDH1- and CD44-positive. Colon cancers with nuclear β-catenin accumulation demonstrated greater radio-resistance with a higher number of CSCs.

Cancer Stem Cells and Radioresistance: DNA Repair and Beyond.

The current preclinical and clinical findings demonstrate that, in addition to the conventional clinical and pathological indicators that have a prognostic value in radiation oncology, the number of cancer stem cells (CSCs) and their inherent radioresistance are important parameters for local control after radiotherapy. In this review, we discuss the molecular mechanisms of CSC radioresistance attributable to DNA repair mechanisms and the development of CSC-targeted therapies for tumor radiosensitization. We also discuss the current challenges in preclinical and translational CSC research including the high inter- and intratumoral heterogeneity, plasticity of CSCs, and microenvironment-stimulated tumor cell reprogramming.

Effect of Radiation on self-renewality of prostate cancer stem cells.

Introduction: CSCs have been identified in prostate cancer (PCa), one of the most diagnosed malignancies in men over the world, for which radiation resistance is a major problem in the treatment of advanced stages. Cancer stem cells (CSCs) have the ability to self-renew and differentiate to give rise to heterogeneous phenotype of the tumor cells. It is believed that CSCs are involved in metastasis, recurrence and therapy resistance in various cancers. Recently CSCs have been identified in prostate cancer. In vitro one of the main properties used to enriched cancer stem cells is their capacity to form spheres in non-adherent culture conditions. Materials and Methods: Human prostate carcinoma cell line (DU145) were plated in serum-free suspension culture system allowed for tumorsphere forming that enriched cancer stem cells. To identify prostate CSCs in adherent cells and nonadherent sphere cells of prostate cancer cell lines, the expression of the CD133 and CD44 cell-surface markers were assessed by flowcytometry. To evaluate of different doses of radiation (electron 6 MeV) on the self-renewal and proliferation of the Du145, sphere-formation assay (SFA) and plating efficiency (colony formation assay (CFA)) were assessed. The results were comparing with the parental cells. SFA is based on the self-renewing properties unique to CSCs. CFA is used as a measure of the proliferative potential of cells. Results: The results show that both SF (CFA) and SF (SFA) declined with the increase of radiation dose in cells treated as monolayer or sphere culture. However, the extent of reduction in cells treated as monolayer culture was significantly higher compared to cells treated as spheroids, suggesting higher resistance of spheroids to radiation. According to these results, tumorspheres from PCa showed more radioresistant than bulk of tumor. Conclusion: Due to the relative resistance of CSCs to radiotherapy, determination and characterization of radioresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.

Calcium channel α2δ1 subunit (CACNA2D1) enhances radioresistance in cancer stem-like cells in non-small cell lung cancer cell lines.

PurposeRadiotherapy is a major treatment method for patients with non-small cell lung cancer (NSCLC). However, the presence of radioresistant cancer stem cells (CSCs) may be associated with disease relapse or a poor outcome after radiotherapy. Voltage-gated calcium channel α2δ1 subunit (encoded by the gene CACNA2D1) isoform 5 is a marker of CSCs in hepatocellular carcinoma. This study aimed to investigate the radiosensitivity of α2δ1-high cells in NSCLC cell lines.Materials and methodsNSCLC cell lines A549, H1975, H1299, and PC9 were used. CACNA2D1-knockdown and CACNA2D1-overexpressing cell lines were established by lentiviral infection. Colony formation assay was performed to determine radiosensitivity. Sphere formation assay in serum-free medium was performed to evaluate self-renewal capacity. Proteins associated with DNA damage repair were analyzed by immunofluorescence or Western blot. The monoclonal antibody of α2δ1 was applied alone or in combination with radiation either in vitro or in vivo to determine the anti-tumor effect of the antibody.Resultsα2δ1-high cells showed greater sphere-forming efficiency than α2δ1-low cells and were relatively resistant to radiation. CACNA2D1 knockdown in A549 cells enhanced radiosensitivity, whereas CACNA2D1 overexpression in PC9 and H1975 cells reduced radiosensitivity, suggesting that α2δ1 imparted radioresistance to NSCLC cells. Analysis of proteins involved in DNA damage repair suggested that α2δ1 enhanced the efficiency of DNA damage repair. The monoclonal antibody of α2δ1 had a synergistic effect with that of radiation to block the self-renewal of α2δ1-high cells and enhanced the radiosensitivity of α2δ1-positive cells in colony formation assays. The combination of the α2δ1 antibody with radiation repressed A549 xenograft growth in vivo.Conclusionα2δ1 enhances radioresistance in cancer stem-like cells in NSCLC. The α2δ1 monoclonal antibody sensitizes α2δ1-high cells to radiation, suggesting that the antibody may be used to improve the treatment outcome when combined with radiation in NSCLC.

Breast cancer stem cell-like cells generated during TGFβ-induced EMT are radioresistant.

Failure of conventional antitumor therapy is commonly associated with cancer stem cells (CSCs), which are often defined as inherently resistant to radiation and chemotherapeutic agents. However, controversy about the mechanisms involved in the radiation response remains and the inherent intrinsic radioresistance of CSCs has also been questioned. These discrepancies observed in the literature are strongly associated with the cell models used. In order to clarify these contradictory observations, we studied the radiosensitivity of breast CSCs using purified CD24−/low/CD44+ CSCs and their corresponding CD24+/CD44low non-stem cells. These cells were generated after induction of the epithelial-mesenchymal transition (EMT) by transforming growth factor β (TGFβ) in immortalized human mammary epithelial cells (HMLE). Consequently, these 2 cellular subpopulations have an identical genetic background, their differences being related exclusively to TGFβ-induced cell reprogramming. We showed that mesenchymal CD24−/low/CD44+ CSCs are more resistant to radiation compared with CD24+/CD44low parental cells. Cell cycle distribution and free radical scavengers, but not DNA repair efficiency, appeared to be intrinsic determinants of cellular radiosensitivity. Finally, for the first time, we showed that reduced radiation-induced activation of the death receptor pathways (FasL, TRAIL and TNF-α) at the transcriptional level was a key causal event in the radioresistance of CD24−/low/CD44+ cells acquired during EMT.

Nuclear EGFR-PKM2 axis induces cancer stem cell-like characteristics in irradiation-resistant cells

Radiation therapy has become an important tool in the treatment of cancer patients, but most patients relapse within 5 years. Relapse is due to the presence of cancer stem cells (CSCs), but the molecular mechanism of radioresistance in CSCs remains largely elusive. Here, we found that irradiation-resistant (IR) cells exhibited increased stem cell-like properties together with elevated anchorage-independent growth and metastasis ability. EGFR not only leads to increased acquisition of endometrial cancer stem cell markers in radioresistant sublines but is critical for the cancer stem-cell phenotype and tumorigenicity. Moreover, PKM2 functions as an interacting partner of EGFR, which induces the EMT phenotype and stem cell-like properties in IR cells. Finally, we found that the regulatory function of the EGFR-PKM2 axis is dependent on nuclear EGFR. In sum, our study indicated that EGFR and PKM2 directly interact and bind with each other to regulate the transcription of stemness-related genes and promote the stem-like phenotype, thus promoting invasion and metastasis.

Tumor Tissue Analogs for the Assessment of Radioresistance in Cancer Stem Cells.

Over the years, radiotherapy-related research has been based on local tumor control as an experimental endpoint, yielding a wealth of data demonstrating the importance of cancer stem cells in tumor reoccurrence after radiotherapy. Literature is replete with experimental and clinical evidence that the cancer stem cell population in a tumor affects its radiocurability. An important consideration for radiotherapy is the microenvironmental stimuli in the CSC niche that results from factors such as hypoxia, extracellular matrix (ECM) elements and their intercellular interaction with non-stem cells and other cell types that prevail in the tumor milieu. In this chapter, we have described the methodology to develop in vitro 3D tumor models that incorporate these microenvironmental characteristics and design experiments that generate endpoints for understanding radioresistance in cancer stem cells.

Abstract 3898: Emerging role of Fra-1 as a novel target for radiosensitization in HPV-associated cancers

Abstract Background: Activator protein-1 (AP-1) plays a central role in HPV-mediated cervical and tongue cancer (subset of HNSCC). AP-1, a homo/heterodimeric protein composed of Jun and Fos family proteins, has been implicated in chemoradioresistance but the mechanism(s) remained unexplored. In the present study, AP-1 family protein Fra-1 has been shown to play a critical role in promoting radiosensitization in enriched cervical cancer stem-like cells (CaCxSLCs) and can serve as a target for drug intervention in cervical and other HPV associated cancers. Experimental Procedure: Cervical cancer stem-like cells (CaCxSLCs) were isolated and enriched by sequential gating HPV+ve and HPV-ve human cervical cancer cell lines, SiHa, and C33a using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media (DCM) with intermittent culturing. CaCxSLCs were also assessed for their cervicosphere forming ability, exclusion of DCV dye for generating SP cells, self renewability and quiescenceness. Both CaCxSLCs and Non CaCxSLCs were exposed to different dose of UV irradiation and a herbal compound curcumin, a potent inhibitor of AP-1 was used to examine the role of AP-1 and its family proteins in the radiosensitization of cancer stem cells. Results: Upon UV irradiation, CaCxSLCs showed a dose-dependent higher proliferation and highly increased AP-1 activity whereas it was completely abolished in non-stem cancer cells. CaCxSLCs also showed differential overexpression of c-Fos and c-Jun at transcript as well as in protein level. The loss of AP-1 activity and expression was accompanied by decrease in cell viability and proliferation in UV irradiated non-stem cancer cells. Interestingly, CaCxSLCs treated with curcumin prior to UV irradiation abolished AP-1 activity and a concomitant reduction in SP cells leading to abrogation of sphere forming ability, loss of proliferation, induction of apoptosis and the cells were poorly tumorigenic. The curcumin pre-treatment abolished the expression of c-Fos and c-Jun but upregulated Fra-1 expression in irradiated CaCxSLCs. Conclusion: The study suggests a critical role of AP-1 protein in the manifestation of radioresistance but curcumin pretreatment helps in radiosensitizing CaCxSLCs through upregulation of Fra-1 which may serve as a potential target for elimination of radioresistant cancer stem cells in HPV-associated malignancies. Note: This abstract was not presented at the meeting. Citation Format: Abhishek Tyagi, Shilpi Gupta, Prabhat Kuamr, Alok C. Bharti, Bhudev C. Das. Emerging role of Fra-1 as a novel target for radiosensitization in HPV-associated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3898. doi:10.1158/1538-7445.AM2017-3898

Abstract 937: Plasticity in heterogenous cancer stem cells promotes glioblastoma radioresistance

Abstract Some cancers including Glioblastoma (GBM) have hierarchical cellular architecture with cancer stem cells (CSCs) as a minor tumorigenic subpopulation. While CSCs have been touted to contribute to initiate recurrence, post-surgical therapy-induced phenotypic changes in CSCs remain elusive. Here we report that treatment of GBM cells with ionizing radiation (IR) promotes a wound-healing mesenchymal signature, accompanied by conversion of naïve CSCs expressing CD133 to post-treatment more radioresistant CSCs expressing the GPI-anchored, TGFb-inhibitory protein CD109 in an ATM, NF-kB, CEBP-b dependent manner. In post-IR GBM, these therapy-escaped CD133-/CD109+ GBM cells, but not the rest of the tumor, uniquely retain clonogenic, tumorigenic, and multi-potent capacities both in vitro and in vivo. CD109 silencing unexpectedly eliminates cancer stemness via controlling YAP/TAZ as the downstream target and this CD109-YAP/TAZ signaling axis is evolutionally conserved between drosophila and human brain tumors. Clinically, both CD133 and CD109 are independently associated with poorer prognosis of GBM patients, while the poorest prognosis group of GBM expressed both CD133 and CD109 at higher rates. These data indicate a possibility of the post-IR CSC plasticity and subsequent heterogeneity, and the activation of the CD109-YAP/TAZ signaling axis as previously unidentified therapeutic targets for post-IR recurrent GBM. Citation Format: Mutsuko Minata, Alessandra Audia, Junfeng Shi, Songjian Lu, Marat Pavlyukov, Yancey Gillespie Gillespie, Krishna Bhat, Ichiro Nakano. Plasticity in heterogenous cancer stem cells promotes glioblastoma radioresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 937. doi:10.1158/1538-7445.AM2017-937

Abstract 3907: Targeting cancer stem cells with microRNA therapeutics and particle radiation in challenging cancer models

Abstract Remarkable progress has been made in the understanding and treatment of human cancer, resulting in greatly improved survival for many patients. However, such achievements remain incomplete or out of reach for some hard-to-treat cancers, such as pancreatic cancer or triple-negative breast cancer. Chondrosarcoma is resistant to conventional radiation therapy and to chemotherapy. Innovative approaches focusing on those challenging cancer models are therefore highly expected. There is now wide acknowledgment that tumors are generally heterogeneous and that cancer treatment failure, relapse or metastasis may be related a small population of stem-like cells that are capable of self-renewing and of causing the different lineage of cancer cells comprising a tumor. These cancer stem cells (CSCs) are more radio-resistant than their non-CSC counterparts and have a distinct molecular signature. CSCs and differentiated cancer cells seem to coexist in a dynamic equilibrium influenced by the micro-environment and by specific micro-RNA expression patterns. The development of new therapeutic strategies (such as micro-RNA modulating strategies) that selectively target CSCs is currently receiving increasing attention. Particle radiation therapy (such as carbon-ion therapy) is effective at treating various radioresistant tumors and has already achieved promising results. Approaches combining high-LET radiation therapy and targeted-therapy have been suggested for tumors with bad prognosis.We have described a new molecular pathway in basal-like breast cells, resulting in the generation of breast CSCs. Maintenance of the CSC phenotype required the downregulation of miR-29 expression and the upregulation of Klf4 expression. Restoring miR-29 with mimic administration might therefore improve breast cancer therapy efficiency by targeting CSCs. For those reasons, we have evaluated the ability of several microRNA-modulating treatments to reverse CSC phenotype in three experimental in vitro models (pancreatic cancer, chondrosarcoma, triple-negative breast cancer), in combination with exposure spread-out Bragg peak (SOBP) carbon-ion beam (at the HIMAC medical accelerator). In triple-negative breast cells, miR-29 mimic administration increased radiation-induced cell death and decreased the proportion of CSCs. We identified several other microRNA mimic treatments capable of improving carbon-ion therapy efficiency by targeting radioresistant CSCs in those experimental models. Citation Format: Guillaume Vares, Yannick Saintigny, Sei Sai, Hirotaka Sugawara, Tetsuo Nakajima. Targeting cancer stem cells with microRNA therapeutics and particle radiation in challenging cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3907. doi:10.1158/1538-7445.AM2017-3907

Abstract 2536: AP-1/Fra-1 as a target for therapy promotes chemoradiosensitivity of cancer and cancer stem cells

Abstract Transcription factor activator protein-1 (AP-1) super-family is known to modulate expression of array of genes during development of many cancers. It is also an indispensable factor for efficient expression of high risk HPV oncogenes E6/E7. Recent studies demonstrated the role of AP-1 in promoting stemness/quiescence of cancer stem cells. We have recently reported that HPVE6 oncogene controls stemness and self-renewal of cervical cancer stem cells through Hes1/NOTCH-1expression but little is known about factor(s) responsible for chemo- radioresistance of cancer stem cells. To understand this we have dissected the role of AP-1and its family members by using a herbal compound, curcumin and UV irradiation in cervical cancer. We isolated cervical cancer stem cells and enriched them by sequential gating from HPV+ve and HPV-ve human cervical cancer cell lines (SiHa and C33a) using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133). The isolated cervical cancer stem cells (SP+ve→CD49+veCD71−ve→CD133+ve) designated as CaCxSLCs displayed spheroid forming and self-renewal property with increased expression of HPVE6, AP-1 transcripts and a higher AP-1 DNA-binding activity compared to non-stem cancer cells (NSP−ve→CD49−veCD71+ve→ CD133−ve) or unsorted parental cells. Treatment with UV-radiation alone resulted in clonogenic survival of CaCxSLCs but not non-stem cancer cells with enhanced AP-1 expression and activation. In contrast, CaCxSLCs treated with UV in combination with curcumin, resulted in complete loss of AP-1 DNA-binding activity followed by decrease in sphere formation and percentage of SP cells but an increased expression of the fos-related antigen-1 (Fra-1) which has been earlier shown to us have tumor suppressor activity in cervical cancer. Our study demonstrates a critical role of AP-1/Fra-1 signaling in imparting radiosensitization of cervical cancer stem cells. Curcumin, which down regulates almost all signalling pathways, including all members of AP-1 except Fra-1 indicating its role in chemo-radiosensitization of cancer stem cells. Thus combining curcumin with conventional cancer drugs may make cancer treatment most effective. Citation Format: Bhudev C. Das, Abhishek Tyagi, Bal Gangadhar Roy, Alok C. Bharti. AP-1/Fra-1 as a target for therapy promotes chemoradiosensitivity of cancer and cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2536.

Abstract 3346: Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates

Abstract We tested the Cancer Stem Cell (CSC) hypothesis using the patented Hybrid Spheroid (HS) Assay (HSA) and by applying Koch's postulates to test its validity. The HSA is an in vitro assay that enables one to take a viable sample of an individual patient's tumor, make a single cell suspension, mix it in known proportions with human fibroblasts (AG1522) and dispense a known number of cells of the mixture into each well of Ultra Low Attachment (ULA) 96-well plates to agglomerate into 1 HS/well, each containing an average of <1 CSC. The HS provides an analog of the CSC niche, enabling the CSC to proliferate (with some daughters differentiating into Amplifying Transit Cells (ATCs)) and undergo 10-15 symmetric divisions before exhausting the nutrients. This satisfies the McCulloch and Till (spleen colony assay) requirements for a stem cell. Applying Koch's postulates: (1) Does the patient's tumor contain cells with CSC properties? Yes: (a) The HSs grow to a size consistent with containing a CSC, (b) those that grew contain cells expressing ≥ 1 OKSM factor(s), and (c) such HSs contain differentiated progeny of the initial CSC. (2) Can we isolate and propagate these cells? In progress: testing HS serial growth, dissociation and reculture. (3) Can these cells induce the tumor in vivo? In progress: testing by xenografting individual CSC-containing HSs in NSG mice. (4) Do these cells contain and express specific gene products that give them CSC properties? Yes: Demonstrated for Oct4 and Nanog. (5) If we disrupt these genes, do the cells lose their CSC properties? In progress: (shRNA). (6) If we eliminate the CSCs do we eliminate the cancer? Yes: tested by (a) mathematical modeling of single CSC growth, differentiation of some progeny into ATCs capable of a limited number of divisions, that then become terminal non-proliferative tumor cells in the HSA; if a single CSC survives/remains, the HS will grow, i.e., the cancer will recur (63% for an average of 1 CSC in a HS, with a Poisson distribution, assuming that niche sites remain available), and (b) by measuring surviving fractions after exposure to radiation and/or chemotherapy agents (where sterilization of the CSC prevents the HS from growing ≥ 10 divisions. The HSA was applied to tumor samples taken from individual endometrial adenocarcinoma patients and correctly predicted, based on CSC radioresistance, which patients would fail their standard-of-care treatments. Conclusion: The CSC hypothesis is validated in the HSA. Citation Format: Christopher S. Lange, Talal Syed, Mike Zhang, Christian Sabalvaro, Hana I. Lim, Ghadir Salame, Bhargava Chitti, Ifeanyi Ilonzo, Arsalaan Ansari, Michelle Chan, Catherine Li, Jing Xu, Xiaotong Chen. Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3346.

237 - HIF-1α plays a key role in the response of HNSCC cancer stem cells to photon and carbon ion exposures

Purpose: Head and neck squamous cells carcinomas (HNSCC) have a poor prognosis due to escapement to anti-cancer therapies which leads to locoregional recurrences. The presence of cancer stem cells (CSCs), resistant to chemo- and radio-therapies, could explain these resistances and recurrences. Hadrontherapy has demonstrated favorable results for many cancers, since carbon ions induced more CSC’s cell death than photons. However, for the HNSCC tumor, local control remains low. CSCs are located in a hypoxic microenvironment and hypoxia is well-known for being able to induce the epithelial mesenchymal transition (EMT). The aim of this study is to investigate the response of CSCs to radiotherapy, compared with carbon ions under hypoxic conditions. Many signaling pathways involved in the resistance of CSCs and the invasion/migration process depend on the stabilization of HIF-1α. Reactive Oxygen Species (ROS), which are produced under hypoxia and in response to irradiation, play a key role in this stabilization. However, since few data are available, the mechanisms involving ROS production, HIF-1α stabilization as well as the invasion/migration process that follows, need to be clarified in HNSCC CSCs exposed to carbon or photon radiations in a hypoxic environment. Material and methods: Two HNSCC cells lines, SQ20B and FaDu, and their CSCs were grown in normoxic and hypoxic (1% O2) conditions. CSCs were isolated by flow cytometry cell sorting. Cell survival curves were performed in response to photon (250kV) and carbon ion (75MeV/n, GANIL, France) irradiations in order to define the Oxygen Enhancement Ratio (OER). The expression of HIF-1α was followed by Western-Blots. ROS were quantified with a CM-H2DCFDA dye and Migration/Invasion with Boyden chambers. Results: After photons, HNSCC cells and their CSCs appeared more resistant under hypoxia (OER>1.2) whereas the oxygen effects were cancelled after carbon ions (OER=1). Interestingly, the OER values and the expression of HIF-1α seemed to be linked. HIF-1α was weakly expressed after photon irradiation and fully inhibited after carbon ions. Additionally, under hypoxia, HIF-1α expression appeared earlier in CSCs than in the parental cell lines, confirming their adaptive properties to hypoxia. In CSCs, this expression was correlated with ROS levels. As a consequence, since HIF- 1α is known to promote EMT, under normoxia as well as hypoxia, the invasion/migration abilities of CSCs were more important than in non-CSCs. Finally, after carbon ions, either in normoxic or hypoxic conditions, invasion/migration processes were decreased compared with photons. Conclusion: HIF-1α plays a key role in the radioresistance of CSCs and in their invasiveness abilities. No expression of HIF-1α was found after carbon ion exposure suggesting that carbon ions could be a relevant therapeutic alternative to kill CSCs in their microenvironment.

Magnetic hyperthermia therapy: An emerging modality of cancer treatment in combination with radiotherapy

Magnetic hyperthermia therapy (MHT) involves heat generation using magnetic nanoparticles (MNPs) in response to an externally applied alternating current magnetic field. These MNPs can be specifically targeted to the tumor site for homogenous heating. Compared to MHT, conventional methods of HT cause heterogeneous heating of tumor and thus poor efficacy of cancer treatment. MHT has also been shown to effectively eliminate the highly chemo- and radio-resistant cancer stem cells in the tumor mass. Due to their diagnostic capability as well as heat-induced cancer cell killing ability, extensive research has been carried out to develop MNPs as potential cancer theranostic agent. The major focus of MNP research has been to design MNPs formulations for efficient targeting, increased colloidal stability, effective heat generation, and minimal inherent toxicity. A few recent MNPs formulations meet some of the required features and showed promising results in preclinical and clinical studies. HT applied through conventional modes has been combined with chemo- and radiotherapy, owing to its ability to increase oxygenation and drug supply due to vasodilation but has shown a limited success in clinic. However, a great hope has arisen from the MNPs to make combinatorial therapies more successful, not only because of the many advantages of MNPs mentioned but also due to their potential for targeted delivery of a range of anti-cancer drugs and radiosensitizing agents.