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Supplementary Data from Plasticity within Aldehyde Dehydrogenase–Positive Cells Determines Prostate Cancer Radiosensitivity

Supplementary Figure from Plasticity within Aldehyde Dehydrogenase–Positive Cells Determines Prostate Cancer Radiosensitivity

Regulation of the genes involved in WNT signaling pathway in the ALDH+ population of DU145 radioresistant (RR) cells versus ALDH- population of DU145 RR cells.

Radiotherapy is a mainstay of curative prostate cancer treatment, but risks of recurrence after treatment remain significant in locally advanced disease. Given that tumor relapse can be attributed to a population of cancer stem cells (CSC) that survives radiotherapy, analysis of this cell population might illuminate tactics to personalize treatment. However, this direction remains challenging given the plastic nature of prostate cancers following treatment. We show here that irradiating prostate cancer cells stimulates a durable upregulation of stem cell markers that epigenetically reprogram these cells. In both tumorigenic and radioresistant cell populations, a phenotypic switch occurred during a course of radiotherapy that was associated with stable genetic and epigenetic changes. Specifically, we found that irradiation triggered histone H3 methylation at the promoter of the CSC marker aldehyde dehydrogenase 1A1 (ALDH1A1), stimulating its gene transcription. Inhibiting this methylation event triggered apoptosis, promoted radiosensitization, and hindered tumorigenicity of radioresistant prostate cancer cells. Overall, our results suggest that epigenetic therapies may restore the cytotoxic effects of irradiation in radioresistant CSC populations. Cancer Res; 76(9); 2637–51. ©2016 AACR.

Purpose:The heavy chain of the CD98 protein (CD98hc) is encoded by the SLC3A2 gene. Together with the light subunit LAT1, CD98hc constitutes a heterodimeric transmembrane amino acid transporter. High SLC3A2 mRNA expression levels are associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy. Little is known regarding the CD98hc protein–mediated molecular mechanisms of tumor radioresistance.Experimental Design:CD98hc protein expression levels were correlated with corresponding tumor control dose 50 (TCD50) in HNSCC xenograft models. Expression levels of CD98hc and LAT1 in HNSCC cells were modulated by siRNA or CRISPR/Cas9 gene editing. HNSCC cell phenotypes were characterized by transcription profiling, plasma membrane proteomics, metabolic analysis, and signaling pathway activation. Expression levels of CD98hc and LAT1 proteins were examined by IHC analysis of tumor tissues from patients with locally advanced HNSCC treated with primary radiochemotherapy (RCTx). Primary endpoint was locoregional tumor control (LRC).Results:High expression levels of CD98hc resulted in an increase in mTOR pathway activation, amino acid metabolism, and DNA repair as well as downregulation of oxidative stress and autophagy. High expression levels of CD98hc and LAT1 proteins were significantly correlated and associated with an increase in radioresistance in HNSCC in vitro and in vivo models. High expression of both proteins identified a poor prognosis subgroup in patients with locally advanced HNSCC after RCTx.Conclusions:We found that CD98hc-associated signaling mechanisms play a central role in the regulation of HNSCC radioresistance and may be a promising target for tumor radiosensitization.

Regulation of the genes that were not mapped within the deleted or amplified chromosomal regions in the entire population of DU145 parental (P) cells versus DU145 radioresistant (RR) cells.

Regulation of the genes involved in WNT signaling pathway in the ALDH+ population of DU145 parental (P) cells versus ALDH- population of DU145 P cells.

Gene set enrichment analysis (GSEA) for DU145 radioresistant (RR) cells versus DU145 parental (P) cells, NOM p value < 0.05.

Irradiation-induced changes in the expression of stem cell markers (S1); Genetic alterations in the irradiated prostate cancer cells (S2); Regulation of histone methylation in response to cell irradiation (S3); Differential expression of stem cell and EMT markers in response to irradiation in the parental and radioresistant prostate cancer cells (S4); The role of ALDH1A1 expression in regulation of spherogenicity, tumorigenicity and radioresistance of the parental and radioresistant prostate cancer cells (S5); Analysis and validation of the global gene expression data (S6); Analysis of cell viability, plating efficacy and post-irradiation clonogenic survival in response to inhibition of the histone methylation activity (S7); Effect of DZNep treatment on apoptosis induction and γH2A.X level in prostate cancer cell lines (S8); Analysis of apoptosis induction and γH2A.X increase by DZNep treatment in prostate tumor tissues and primary cell cultures (S9); Co-regulation of EZH2, ALDH1A1 and CTNNB1 in cell lines and prostate tumors (S10).

Supplementary discussion; Supplementary methods; Supplementary tables S1-S8; Supplementary figures S1-S8.

Description of additional methods and procedures used in the study. Also includes Supplementary Reference.

Radiotherapy is a curative treatment option in prostate cancer. Nevertheless, patients with high-risk prostate cancer are prone to relapse. Identification of the predictive biomarkers and molecular mechanisms of radioresistance bears promise to improve cancer therapies. In this study, we show that aldehyde dehydrogenase (ALDH) activity is indicative of radioresistant prostate progenitor cells with an enhanced DNA repair capacity and activation of epithelial–mesenchymal transition (EMT). Gene expression profiling of prostate cancer cells, their radioresistant derivatives, ALDH+ and ALDH− cell populations revealed the mechanisms, which link tumor progenitors to radioresistance, including activation of the WNT/β-catenin signaling pathway. We found that expression of the ALDH1A1 gene is regulated by the WNT signaling pathway and co-occurs with expression of β-catenin in prostate tumor specimens. Inhibition of the WNT pathway led to a decrease in ALDH+ tumor progenitor population and to radiosensitization of cancer cells. Taken together, our results indicate that ALDH+ cells contribute to tumor radioresistance and their molecular targeting may enhance the effectiveness of radiotherapy. Cancer Res; 75(7); 1482–94. ©2015 AACR.

Supplementary Methods, Table 1, Figure S1-S9 (i) supplementary methods: Human tumor tissue dissociation, Immunofluorescence microscopy, Clonogenic cell survival assay, Sphere formation assay, Cell proliferation and cytotoxicity assay, Wound healing assay, Transwell migration assay, Western blot analysis, Flow cytometry analysis, Chromatin immunoprecipitation assay, siRNA-mediated gene silencing, Luciferase reporter assay, Histology, Microarray analysis of the xenograft tumors, (ii) supplementary table 1: Primary tumor tissues used in the study and (iii) supplementary figures including Figure S1 (analysis of ALDH1A1 expression in normal and tumor prostate tissues), Figure S2 (prostate cancer ALDH+ cells possess CSC properties), Figure S3 (analysis of CSC marker expression after irradiation), Figure S4 (clonogenicity and tumorigenisity of ALDH+ and ALDH- cells after irradiation), Figure S5 (characterization of the radioresistant prostate cancer cell sublines), Figure S6 (the mechanisms of prostate cancer cell radioresistance), Figure S7 (comparative gene expression profiling of ALDH+ and radioresistant DU145 cells), Figure S8 (EMT properties of ALDH+ and radioresistant prostate cancer cells), Figure S9 (WNT/β-catenin pathway regulates ALDH1A1 expression)

It is elusive whether clonal selection of tumor cells in response to ionizing radiation (IR) is a deterministic or stochastic process. With high resolution clonal barcoding and tracking of over 400 000 HNSCC patient-derived tumor cells the clonal dynamics of tumor cells in response to IR was analyzed. Fractionated IR induced a strong selective pressure for clonal reduction which significantly exceeded uniform clonal survival probabilities indicative for a strong clone-to-clone difference within tumor cell lines. IR induced clonal reduction affected the majority of tumor cells ranging between 96% and 75% and correlated to the degree of radiation sensitivity. Survival to IR is driven by a deterministic clonal selection of a smaller population which commonly survives radiation, while increased clonogenic capacity is a result of clonal competition of cells which have been selected stochastically. A 2-fold increase in radiation resistance results in a 4-fold (P

Cancer stem cells (CSCs) are a major cause of tumor therapy failure. This is mainly attributed to increased DNA repair capacity and immune escape. Recent studies have shown that functional DNA repair via homologous recombination (HR) prevents radiation-induced accumulation of DNA in the cytoplasm, thereby inhibiting the intracellular immune response. However, it is unclear whether CSCs can suppress radiation-induced cytoplasmic dsDNA formation. Here, we show that the increased radioresistance of ALDH1-positive breast cancer stem cells (BCSCs) in S phase is mediated by both enhanced DNA double-strand break repair and improved replication fork protection due to HR. Both HR-mediated processes lead to suppression of radiation-induced replication stress and consequently reduction of cytoplasmic dsDNA. The amount of cytoplasmic dsDNA correlated significantly with BCSC content (p=0.0002). This clearly indicates that HR-dependent avoidance of radiation-induced replication stress mediates radioresistance and contributes to its immune evasion. Consistent with this, enhancement of replication stress by inhibition of ataxia telangiectasia and RAD3 related (ATR) resulted in significant radiosensitization (SER37 increase 1.7-2.8 Gy, p

The development of hybrid materials, which combine inorganic with organic materials, is receiving increasing attention by researchers. As a consequence of carbon nanostructures high chemical versatility, they exhibit enormous potential for new highly engineered multifunctional nanotherapeutic agents for cancer therapy. Whereas many groups are working on drug delivery systems for chemotherapy, the use of carbon nanohybrids for radiotherapy is rarely applied. Thus, nanotechnology offers a wide range of solutions to overcome the current obstacles of conventional chemo- and/or radiotherapies. Within this review, the structure and properties of carbon nanostructures (carbon nanotubes, nanographene oxide) functionalized preferentially with different types of polymers (synthetic, natural) are discussed. In short, synthesis approaches, toxicity investigations and anticancer efficacy of different carbon nanohybrids are described.

Purpose: Despite technological improvement and advances in biology-driven patient stratification, many patients still fail radiotherapy resulting in loco-regional and distant recurrence. Tumor heterogeneity remains a key challenge to effective cancer treatment, and reliable stratification of cancer patients for prediction of outcomes is highly important. Intratumoral heterogeneity is manifested at the different levels, including different tumorigenic properties of cancer cells. Since John Dick et al. isolated leukemia initiating cells in 1990, the populations of tumor initiating or cancer stem cells (CSCs) were identified and characterized also for a broad spectrum of solid tumor types. The properties of CSCs are of considerable clinical relevance: CSCs have self-renewal and tumor initiating potential, and the metastases are initiated by the CSC clones with the ability to disseminate from the primary tumor site. Conclusion: Evidence from both, experimental and clinical studies demonstrates that the probability of achieving local tumor control by radiation therapy depends on the complete eradication of CSC populations. The number, properties and molecular signature of CSCs are highly predictive for clinical outcome of radiotherapy, whereas targeted therapies against CSCs combined with conventional treatment are expected to provide an improved clinical response and prevent tumor relapse. In this review, we discuss the modern methods to study CSCs in radiation biology, the role of CSCs in personalized cancer therapy as well as future directions for CSC research in translational radiooncology.

Within the hematopoietic system, the Notch pathway is critical for promoting thymic T cell development and suppressing the B and myeloid lineage fates; however, its impact on NK lymphopoiesis is less understood. To study the role of Notch during NK cell development in vivo, we investigated different NK cell compartments and function in Rbp-Jkfl/flVav-Cretg/+ mice, in which Rbp-Jk, the major transcriptional effector of canonical Notch signaling, was specifically deleted in all hematopoietic cells. Peripheral conventional cytotoxic NK cells in Rbp-Jk–deleted mice were significantly reduced and had an activated phenotype. Furthermore, the pool of early NK cell progenitors in the bone marrow was decreased, whereas immature NK cells were increased, leading to a block in NK cell maturation. These changes were cell intrinsic as the hematopoietic chimeras generated after transplantation of Rbp-Jk–deficient bone marrow cells had the same NK cell phenotype as the Rbp-Jk–deleted donor mice, whereas the wild-type competitors did not. The expression of several crucial NK cell regulatory pathways was significantly altered after Rbp-Jk deletion. Together, these results demonstrate the involvement of canonical Notch signaling in regulation of multiple stages of NK cell development.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Here, we found that radioresistant PCa cells and prostate cancer stem cells (CSCs) have a high glutamine demand. Glutaminase (GLS)-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as glutaminase (GLS) and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

The impact of functional heterogeneity in response to radiation therapy is poorly understood to the present. It remains elusive whether clonal selection of tumor cells in response to ionizing radiation (IR) is a deterministic or stochastic process. We applied high-resolution lentiviral cellular barcoding for quantitative clonal tracking and deconvolution of clonal dynamics in response to IR. Clonal tracking of over 400.000 HNSCC patient-derived tumor cells and the analyses of over 1500 million sequencing reads in clonogenic survival assays reveals that fractionated IR induced a strong selective pressure for clonal reduction. This significantly exceeded uniform clonal survival probabilities indicative for a strong clone-to clone difference within tumor cell lines. IR induced clonal reduction affected the majority of tumor cells ranging between 96-75% and correlated to the degree of radiation sensitivity. Survival and clonogenicity is characterized by an intensive clonal distortion and dominance of individual tumor cells. Survival to IR is driven by a deterministic clonal selection of a smaller population which commonly survives radiation, while increased clonogenic capacity is a result of clonal competition of cells which have been selected stochastically. A 2-fold increase in radiation resistance results in a 4-fold (p

Simple Summary The overall five-year survival rate of patients with loco-regional advanced head and neck squamous cell carcinomas (HNSCC) is only around 40%. The curability of HNSCC with radiochemotherapy was previously found to be associated with clinical and biological parameters including tumor volume, hypoxia, epidermal growth factor receptor expression, and human papillomavirus infection status. Different signaling pathways, e.g., constitutively activated receptor tyrosine kinase signaling, increased DNA damage repair and intracellular defense against reactive oxygen species were identified as factors driving HNSCC progression and its resistance to therapy. c-MET was found to be hyperactivated in HNSCC and has been reported to drive tumor progression, therapy resistance, and metastatic spread. Here, we investigated the therapeutic potential of c-MET targeting strategies for HNSCC radiosensitization and discovered putative resistance mechanisms impeeding success of therapeutic intervention. This study highlights the importance of detailed knowledge about biological mechanisms and regulatory networks for future patient stratification and individualized treatment concepts. Abstract The receptor tyrosine kinase c-MET activates intracellular signaling and induces cell proliferation, epithelial-to-mesenchymal-transition and migration. Within the present study, we validated the prognostic value of c-MET in patients with head and neck squamous cell carcinoma (HNSCC) treated with radio(chemo)therapy using the Cancer Genome Atlas database and found an association of increased MET gene expression and protein phosphorylation with reduced disease-specific and progression-free survival. To investigate the role of c-MET-dependent radioresistance, c-MET-positive cells were purified from established HNSCC cell lines and a reduced radiosensitivity and enhanced sphere-forming potential, compared to the c-MET-depleted cell population, was found in two out of four analyzed cell lines pointing to regulatory heterogeneity. We showed that c-MET is dynamically regulated after irradiation in vitro and in vivo. Interestingly, no direct impact of c-MET on DNA damage repair was found. The therapeutic potential of eight c-MET targeting agents in combination with irradiation demonstrated variable response rates in six HNSCC cell lines. Amongst them, crizotinib, foretinib, and Pha665752 exhibited the strongest radiosensitizing effect. Kinase activity profiling showed an association of crizotinib resistance with compensatory PI3K/AKT and MAP kinase signaling. Overall, our results indicate that c-MET is conferring radioresistance in HNSCC through modulation of intracellular kinase signaling and stem-like features.

Despite the progress in the prevention, diagnostics and treatment, cancer remains a major cause of morbidity and mortality around the world. According to the world health organization (WHO), more than 14 million newly diagnosed cases and more than 8 million deaths were reported in 2012, and increase in new cases of cancer by 70% and cancer related deaths by 45% is expected over the next 2 decades. Metastatic tumors are the cause of more than 90% of cancer related deaths due to the fact that current therapies frequently fail to provide durable curative response if tumor is spread. In this review, we summarize the main common hallmarks and evolving concept of cancer stem cells and metastasis initiating cells, their dynamic interaction with microenvironmental factors, discuss perspectives of using cancer stem cells and circulating tumor cells as prognostic and predictive tumor biomarkers as well as possible strategies for their targeting in the clinical setting.

Radiotherapy is a mainstay of curative prostate cancer treatment, but risks of recurrence after treatment remain significant in locally advanced disease. Given that tumor relapse can be attributed to a population of cancer stem cells (CSC) that survives radiotherapy, analysis of this cell population might illuminate tactics to personalize treatment. However, this direction remains challenging given the plastic nature of prostate cancers following treatment. We show here that irradiating prostate cancer cells stimulates a durable upregulation of stem cell markers that epigenetically reprogram these cells. In both tumorigenic and radioresistant cell populations, a phenotypic switch occurred during a course of radiotherapy that was associated with stable genetic and epigenetic changes. Specifically, we found that irradiation triggered histone H3 methylation at the promoter of the CSC marker aldehyde dehydrogenase 1A1 (ALDH1A1), stimulating its gene transcription. Inhibiting this methylation event triggered apoptosis, promoted radiosensitization, and hindered tumorigenicity of radioresistant prostate cancer cells. Overall, our results suggest that epigenetic therapies may restore the cytotoxic effects of irradiation in radioresistant CSC populations. Cancer Res; 76(9); 2637–51. ©2016 AACR.

Purpose: The heavy chain of the CD98 protein (CD98hc) is encoded by the SLC3A2 gene. Together with the light subunit LAT1, CD98hc constitutes a heterodimeric transmembrane amino acid transporter. High SLC3A2 mRNA expression levels are associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy. Little is known regarding the CD98hc protein–mediated molecular mechanisms of tumor radioresistance. Experimental Design: CD98hc protein expression levels were correlated with corresponding tumor control dose 50 (TCD50) in HNSCC xenograft models. Expression levels of CD98hc and LAT1 in HNSCC cells were modulated by siRNA or CRISPR/Cas9 gene editing. HNSCC cell phenotypes were characterized by transcription profiling, plasma membrane proteomics, metabolic analysis, and signaling pathway activation. Expression levels of CD98hc and LAT1 proteins were examined by IHC analysis of tumor tissues from patients with locally advanced HNSCC treated with primary radiochemotherapy (RCTx). Primary endpoint was locoregional tumor control (LRC). Results: High expression levels of CD98hc resulted in an increase in mTOR pathway activation, amino acid metabolism, and DNA repair as well as downregulation of oxidative stress and autophagy. High expression levels of CD98hc and LAT1 proteins were significantly correlated and associated with an increase in radioresistance in HNSCC in vitro and in vivo models. High expression of both proteins identified a poor prognosis subgroup in patients with locally advanced HNSCC after RCTx. Conclusions: We found that CD98hc-associated signaling mechanisms play a central role in the regulation of HNSCC radioresistance and may be a promising target for tumor radiosensitization.

Many tumors including prostate cancer are maintained by cancer stem cells (CSCs), which might cause tumor relapse if not eradicated during the course of treatment. Specific targeting or radiosensitization of CSCs bear promise to improve tumor curability by synergistic effects in combination with radiotherapy. Carbon nanotubes (CNTs) can be used as promising drug delivery systems for anticancer drugs such as the flavonoid catechin. Catechin is an extensively studied active ingredient of the different plants, including green tea, and it is widely recognized as co-adjuvant in cancer therapy. Here we describe the synthesis of biocompatible, catechin-loaded and gelatin-conjugated CNTs (Gel_CT_CNTs) with anticancer properties and demonstrate their potential for the eradication of prostate CSCs in combination with X-ray irradiation. Gel_CT_CNTs showed a significant enhancement of in vitro anticancer activity as compared to catechin alone. Moreover, treatment of prostate cancer cells with Gel_CT_CNT nanohybrids inhibited the tumorigenic cell population defined by a high aldehyde dehydrogenase (ALDH) activity. A combination of X-ray irradiation and treatment with Gel_CT_CNTs caused a decrease in the protein level of stem cell-related transcription factors and regulators including Nanog, Oct4 and β-catenin and led to an increase of cancer cell radiosensitivity as demonstrated by clonogenic and spherogenic cell survival assays. Taken together, our results suggest that a combination of irradiation and Gel_CT_CNTs can be potentially used for the radiosensitization and eradication of prostate CSC populations.

Purpose : Radioresistance of cancer cells remains a fundamental barrier for maximum effi cient radiotherapy. Tumor heterogeneity and the existence of distinct cell subpopulations exhibiting diff erent genotypes and biological behaviors raise diffi culties to eradicate all tumorigenic cells. Recent evidence indicates that a distinct population of tumor cells, called cancer stem cells (CSC), is involved in tumor initiation and recurrence and is a putative cause of tumor radioresistance. There is an urgent need to identify the intrinsic molecular mechanisms regulating the generation and maintenance of resistance to radiotherapy, especially within the CSC subset. The chemokine C-X-C motif receptor 4 (CXCR4) has been found to be a prognostic marker in various types of cancer, being involved in chemotaxis, stemness and drug resistance. The interaction of CXCR4 with its ligand, the chemokine C-X-C motif ligand 12 (CXCL12), plays an important role in modulating the tumor microenvironment, angiogenesis and CSC niche. Moreover, the therapeutic inhibition of the CXCR4/CXCL12 signaling pathway is sensitizing the malignant cells to conventional anti-cancer therapy. Content : Within this review we are summarizing the role of the CXCR4/CXCL12 axis in the modulation of CSC properties, the regulation of the tumor microenvironment in response to irradiation, therapy resistance and tumor relapse. Conclusion : In light of recent fi ndings, the inhibition of the CXCR4/CXCL12 signaling pathway is a promising therapeutic option to refi ne radiotherapy.

Introduction Pathophysiological conditions within the tumour microenvironment induce metabolic adaptations of cancer cells. These metabolic features correlating with aggressive tumour growth pattern and the relapse risk after radiotherapy. Beside glucose, fast-growing cancer cells consume glutamine for energy production. Within the presented project we investigated the potential of the glutamine metabolism as putative therapeutic target and predictive biomarker for an individualised radiotherapy of prostate cancer (PC) patients. Material and methods Genome and metabolome analysis of radioresistant PC cells identified the glutamine metabolism as regulator for intrinsic sensitivity to irradiation. Radiosensitizing effects of glutamine deprivation, molecular targeting and genetic suppression of key enzymes were determined for clonogenic survival, DNA repair and sphere-formation ability to determine the cancer stem cell (CSC) potential in PC cell lines, primary culture models and ex vivo treated primary biopsies. Analysis of tumour growth after glutamine deprivation were performed in xenograft models. The intracellular level of the glutamine metabolism and tricarboxylic acid cycle metabolites, reactive oxygen species (ROS) and glutathione (GSH) were determined. The clinical validation of the identified metabolic biomarkers was carried out with the NanoString technology. Results and discussions Radioresistant PC cells exhibit an accelerated glutaminolysis with enhanced α-ketoglutarate to succinate ratio. The elevated α-ketoglutarate is leading to GSH consumption, increased intracellular ROS level, modulated epigenetic regulators and induction of a CSC phenotype. Metabolic, chemical and genetic targeting of glutaminolysis results in the inhibition of mTOR signalling, enhanced endoplasmic reticulum stress and reduction of DNA repair. In combination with irradiation this targeting therapy is effectively radiosensitizing PC cells in vitro and in vivo . Moreover, the c-MYC gene expression, a key regulator of glutaminolysis, significantly correlates with the PSA-free survival after radiotherapy. Conclusion This study shows that the enhanced glutaminolysis of PC cells is conferring resistance to radiotherapy. The therapeutic targeting of the glutamine metabolism is elevating the cytotoxic effects of irradiation. In addition, metabolic enzymes involved in the glutamine metabolism can be potentially used to predict clinical outcome of PC patients after radiotherapy.

Tumors are known to be heterogeneous containing a dynamic mixture of phenotypically and functionally different tumor cells. The two concepts attempting to explain the origin of intratumor heterogeneity are the cancer stem cell hypothesis and the clonal evolution model. The stochastic model argues that tumors are biologically homogenous and all cancer cells within the tumor have equal ability to propagate the tumor growth depending on continuing mutations and selective pressure. By contrast, the stem cells model suggests that cancer heterogeneity is due to the hierarchy that originates from a small population of cancer stem cells (CSCs) which are biologically distinct from the bulk tumor and possesses self-renewal, tumorigenic and multilineage potential. Although these two hypotheses have been discussed for a long time as mutually exclusive explanations of tumor heterogeneity, they are easily reconciled serving as a driving force of cancer evolution and diversity. Recent discovery of the cancer cell plasticity and heterogeneity makes the CSC population a moving target that could be hard to track and eradicate. Understanding the signaling mechanisms regulating CSCs during the course of cancer treatment can be indispensable for the optimization of current treatment strategies.

The chemokine receptor type 4 (CXCR4) is known to be involved in immunodeficiency disorders and contributes to different stages of cancer development. The CXCR4 expression level in cancer cells is an adverse prognostic indicator independent from other prognostic factors. Novel findings are pointing out the expression of CXCR4 in the tumor-initiating cancer stem cells (CSCs), which are involved in therapy resistance, relapse, metastasis and poor clinical outcome. CSCs are regulated by signals generated by the tumor microenvironment, but the exact mechanisms are not fully understood. Recent studies provide evidence for an important role of the CXCR4/CXCL12 axis for CSC maintenance, dissemination and metastatic colonization. In addition, this signaling pathway has a crucial contribution in modulation of the tumor microenvironment by inducing neo-angiogenesis and the recruitment of pro-tumorigenic myeloid cells to impede innate and adaptive immune mechanisms of tumor destruction. Moreover, binding of the chemokine ligand CXCL12 to its receptor CXCR4 has a direct effect on cell survival and growth of malignant cells. The correlation of CXCR4 expression with cancer stage and patient outcome makes CXCR4 an important prognostic marker as well as a druggable target with great potential for tumor sensitization to anti-cancer therapies.

Many tumors including prostate cancer are maintained by cancer stem cells (CSCs), which might cause tumor relapse if not eradicated during the course of treatment. Specific targeting or radiosensitization of CSCs bear promise to improve tumor curability by synergistic effects in combination with radiotherapy. Carbon nanotubes (CNTs) can be used as promising drug delivery systems for anticancer drugs such as the flavonoid catechin. Catechin is an extensively studied active ingredient of the different plants, including green tea, and it is widely recognized as co-adjuvant in cancer therapy. Here we describe the synthesis of biocompatible, catechin-loaded and gelatin-conjugated CNTs (Gel_CT_CNTs) with anticancer properties and demonstrate their potential for the eradication of prostate CSCs in combination with X-ray irradiation. Gel_CT_CNTs showed a significant enhancement of in vitro anticancer activity as compared to catechin alone. Moreover, treatment of prostate cancer cells with Gel_CT_CNT nanohybrids inhibited the tumorigenic cell population defined by a high aldehyde dehydrogenase (ALDH) activity. A combination of X-ray irradiation and treatment with Gel_CT_CNTs caused a decrease in the protein level of stem cell-related transcription factors and regulators including Nanog, Oct4 and β-catenin and led to an increase of cancer cell radiosensitivity as demonstrated by clonogenic and spherogenic cell survival assays. Taken together, our results suggest that a combination of irradiation and Gel_CT_CNTs can be potentially used for the radiosensitization and eradication of prostate CSC populations.

Natural killer (NK) cells are cytotoxic lymphocytes and play a vital role in controlling viral infections and cancer. In contrast to B and T lymphopoiesis where cellular and regulatory pathways have been extensively characterized, the cellular stages of early human NK cell commitment remain poorly understood. Here we demonstrate that a Lin(-)CD34(+)CD38(+)CD123(-)CD45RA(+)CD7(+)CD10(+)CD127(-) population represents a NK lineage-restricted progenitor (NKP) in fetal development, umbilical cord blood, and adult tissues. The newly identified NKP has robust NK cell potential both in vitro and in vivo, generates functionally cytotoxic NK cells, and lacks the ability to produce T cells, B cells, myeloid cells, and innate lymphoid-like cells (ILCs). Our findings identify an early step to human NK cell commitment and provide new insights into the human hematopoietic hierarchy.

Resistance to current chemotherapeutic or radiation-based cancer treatment strategies is a serious concern. Cancer stem cells (CSCs) are typically able to evade treatment and establish a recurrent tumor or metastasis, and it is these that lead to the majority of cancer deaths. Therefore, a major current goal is to develop treatment strategies that eliminate the resistant CSCs as well as the bulk tumor cells in order to achieve complete disease clearance. Aldehyde dehydrogenases (ALDHs) are important for maintenance and differentiation of stem cells as well as normal development. There is expanding evidence that ALDH expression increases in response to therapy and promotes chemoresistance and survival mechanisms in CSCs. This perspective will discuss a paper by Cojoc and colleagues recently published in Cancer Research, that indicates ALDHs play a key role in resistance to radiation therapy and tumor recurrence in prostate cancer. The authors suggest that ALDHs are a potential therapeutic target for treatment prostate cancer patients to limit radiation resistance and disease recurrence. The findings are consistent with work from other cancers showing ALDHs are major contributors of CSC signaling and resistance to anti-cancer treatments. This perspective will address representative work concerning the validity of ALDH and the associated retinoic acid signaling pathway as chemotherapeutic targets for prostate as well as other cancers.

Background : Tumor initiation, growth and relapse after therapy are thought to be driven by a population of cells with stem cell characteristics, named cancer stem cells (CSC). The regulation of their radiation resistance and their maintenance is poorly understood. CSC are believed to reside preferentially in special microenvironmental niches located within tumor tissues. The features of these niches are of crucial importance for CSC self-renewal, metastatic potential and therapy resistance. One of the characteristics of solid tumors is occurrence of less oxygenated (hypoxic regions), which are believed to serve as so-called hypoxic niches for CSC. Purpose : The purpose of this review was the critical discussion of the supportive role of hypoxia and hypoxia-related pathways during cancer progression and radiotherapy resistance and the relevance for therapeutic implications in the clinic. Conclusion : It is generally known since decades that hypoxia inside solid tumors impedes chemo- and radiotherapy. However, there is limited evidence to date that targeting hypoxic regions during conventional therapy is eff ective. Nonetheless improved hypoxia-imaging technologies and image guided individualized hypoxia targeted therapy in conjunction with the development of novel molecular targets may be able to challenge the protective eff ect on the tumor provided by hypoxia.

Cancer is a multigene disease which arises as a result of mutational and epigenetic changes coupled with activation of complex signaling networks. The use of biomarkers for early cancer detection, staging and individualization of therapy might improve patient care. A few fundamental issues such as tumor heterogeneity, a highly dynamic nature of the intrinsic and extrinsic determinants of radio- and chemoresistance, along with the plasticity and diversity of cancer stem cells (CSC) make biomarker development a challenging task. In this review we outline the preclinical strategies of cancer biomarker discovery including genomic, proteomic, metabolomic and microRNomic profiling, comparative genome hybridization (CGH), single nucleotide polymorphism (SNP) analysis, high throughput screening (HTS) and next generation sequencing (NGS). Other promising approaches such as assessment of circulating tumor cells (CTC), analysis of CSC-specific markers and cell-free circulating tumor DNA (ctDNA) are also discussed.The emergence of powerful proteomic and genomic technologies in conjunction with advanced bioinformatic tools allows the simultaneous analysis of thousands of biological molecules. These techniques yield the discovery of new tumor signatures, which are sensitive and specific enough for early cancer detection, for monitoring disease progression and for proper treatment selection, paving the way to individualized cancer treatment.

The earliest stages of natural killer (NK)–cell development are not well characterized. In this study, we investigated in different fetal hematopoietic tissues how NK-cell progenitors and their mature NK-cell progeny emerge and expand during fetal development. Here we demonstrate, for the first time, that the counterpart of adult BM Lin−CD122+NK1.1−DX5− NK-cell progenitor (NKP) emerges in the fetal liver at E13.5. After NKP expansion, immature NK cells emerge at E14.5 in the liver and E15.5 in the spleen. Thymic NK cells arise at E15.5, whereas functionally competent cytotoxic NK cells were present in the liver and spleen at E16.5 and E17.5, respectively. Fetal NKPs failed to produce B and myeloid cells but sustained combined NK- and T-lineage potential at the single-cell level. NKPs were also found in the fetal blood, spleen, and thymus. These findings show the emergence and expansion of bipotent NK/T-cell progenitor during fetal and adult lymphopoiesis, further supporting that NK/T-lineage restriction is taking place prethymically. Uncovering the earliest NK-cell developmental stages will provide important clues, helping to understand the origin of diverse NK-cell subsets, their progenitors, and key regulators.

Natural Killer (NK) cells, the third lymphoid lineage after T and B cells, are large granular lymphocytes belonging to innate immune system, classically defined as CD3-CD56+CD16+ in human. NK cells can kill target cells without prior activation, based on the missing self-hypothesis, either due to the lack of MHC-class I molecule or up-regulation of NKG2D ligands, two properties commonly associated with transformed cells (D.H. Raulet, N. Guerra, 2009). Moreover, by secreting cytokines (such as TNF-α and IFN-γ), NK cells can also activate the response of the adaptive immune system. Experiments in mice have shown that NK cells are primarily responsible for the in vivo elimination of transplanted tumor cells (J. Wu, L. L. Lanier, 2003) and, in human, a direct correlation between low NK cytotoxicity and elevated cancer incidence was reported in a large cohort of Japanese aged over 40 (K. Imai et al., 2000). The important anti-tumor role for alloreactive NK cells has been shown in patients with acute myeloid leukemia where transplanted donor NK cells, expressing mismatch inhibitory receptors, provided graft versus leukemia effect in the absence of graft versus host disease (L. Ruggeri et al., 2006). These and many other findings are the basis of a large number of ongoing studies and clinical trials for NK cell immunotherapy against cancer (M. Terme et al., 2008). However, in contrast to B and T cell development where both cellular and regulatory pathways are well studied, the development of NK cells from hematopoietic stem cells (HSCs) is not well understood and the identity of restricted NK cell progenitor (NKP) is unknown (S. Doulatov et al., 2012). To identify human NKP, we applied the expression of known early lymphoid markers (CD45RA, CD10, CD7) and cytokine receptors (IL-7R: CD127) important for lymphoid development. Using multicolor flow cytometry, Lin-CD34+CD38+CD123-CD45RA+CD7+CD10+/-CD127+/- populations were sorted from human bone marrow (hBM) and umbilical cord blood (hUCB). Purified candidate NKPs were cultured on OP9, OP9DL1 stroma and in Terasaki cultures in the presence of specific cytokines and generation of CD3-CD56+CD16+NKp46+ NK cells, CD19+ B, CD3+ CD4+ CD8+ T and CD33+CD14+ myeloid cells was investigated. To study the ability of NKP candidate to generate NK cells after transplantation, purified NKPs were injected into new-born NOD/SCID γcnull (NSG) mice and at 11 weeks after transplantation the phenotype of generated progeny was evaluated. In addition, NK cells generated in stroma cultures and after transplantation were tested for their cytotoxic activity against K562 leukemic cell line in degranulation assay, where the surface expression of membrane glycoprotein LAMP-1 (CD107a) is measured after activation of specific killing receptors. The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate population sorted from hUCB has a robust NK cell potential in vitro at the single cell level and lacked the ability to generate T, B and myeloid cells. Furthermore, NK cells generated from this candidate NKP were functionally mature: show cytotoxic activity against K562 cells and produce cytokines: IFNγ and TNFα after activation in vitro. Further phenotypic characterization showed that the candidate NKP is highly positive for lymphoid markers CD244 and CD62L, lacks the expression of mature NK cell markers NKp46 and NKG2D, and 50% of NKPs express c-kit and Flt3 receptors. At 11 weeks after transplantation, 9 out of 17 NSG mice injected with 600 Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKPs had a significant human cells engraftment and only CD3-CD56+NKp46+CD16+/- NK cells were found in the peripheral blood, bone marrow and spleen; whereas 15 out 17 NSG mice injected with CD34+ cells were positive for the presence of human B, T, NK and myeloid cells in these tissues. Human NK cells generated after transplantation into NSG mice were functionally active and able to kill K562 leukemic cells. The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate was also found in adult hBM and showed NK-lineage restriction. Our results indicate that the Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- cells, found in human BM and UCB, represent NK-lineage restricted progenitors that generate fully mature functional NK cells. Disclosures: No relevant conflicts of interest to declare.