Non-hypoxic Cells Research Articles

Application of a Specific and Sensitive NQO1 Turn-On Near-Infrared Fluorescence Probe for Live Cancer Cell and Xenografted Tumor Imaging in Nude Mice.

Sensitive activity stains for enzymes selectively expressed in human cancers offer valuable tools for imaging with wide applications in experimental, diagnostic, and therapeutic settings. The scant expression of the antioxidant enzyme NQO1 in normal tissues and its great abundance in malignant counterparts due to the increased redox stress and hypoxia is one such example. Previously, we described a potent nontoxic probe that remains nonfluorescent but releases an intense fluorogenic compound after intracellular cleavage by NQO1 catalysis. This infrared probe with a 644nm emission has excellent tissue penetrating ability and low background absorption. Described here are methods (fluorescence microscopy, flow cytometry, and in vivo animal imaging) to rapidly image NQO1 activity in hypoxic and non-hypoxic cancer cells and tumors developed in live mouse xenograft models. The specificity of the dye for NQO1 in all three procedures was verified, and the methods should be useful for both in vitro and in vivo studies.

MODL-25. INTERNAL CAROTID INJECTION MODEL OF BRAIN METASTASIS DESCRIBES LEPTOMENINGEAL DISEASE

Abstract Leptomeningeal disease (LMD), the most aggressive form of central nervous system (CNS) metastasis in which cancer cells infiltrate leptomeninges, is universally and rapidly fatal due to poor detection and no effective treatment. Treatment of LMD is challenged by the diffuse nature of the disease, low specificity of detection, poor drug penetration into CNS and high neurotoxicity. LMD is a heterogeneous disease: co-existence of leptomeningeal spread and parenchymal brain metastases in not uncommon, and LMD tumors vary in size and may be found at various locations throughout entire neuroaxis. Radiographic detection (i.e., MRI) works well for big “bulky” metastases but may produce normal results in patients with active disease identified by cerebrospinal fluid (CSF) tap. This spatial and morphological heterogeneity of LMD may ultimately be responsible for a varied and limited therapy response. Mechanistic and translational studies of LMD are hampered by the lack of adequate in vivo models that recapitulate the consecutive steps of LMD progression separate from systemic disease. In this study, we show that the internal carotid (ICA) injection of tumor cells, which is widely used to model brain metastasis, produces leptomeningeal spread. The ICA injection of various tumor cell lines faithfully recapitulated the stages, heterogeneity, and spatial distribution of human LMD. While some cell lines demonstrated clear preference for growth in brain parenchyma, they still had a measurable population of cells within the leptomeninges. Most bulky LMD metastases formed and grew near fenestrated endothelium, where the direct access to blood might support tumor growth and survival. A population not associated with the fenestrated endothelium consisted mostly of single non-dividing non-hypoxic cells. This diffuse cancer spread limited the efficacy of standard whole-brain radiation therapy. Thus, leptomeningeal spread should be taken into account when interpreting the data from the ICA model that is widely used to study brain metastasis.

NF-κB mediated regulation of tumor cell proliferation in hypoxic microenvironment.

Hypoxia is caused by a cancer-promoting milieu characterized by persistent inflammation. NF-κB and HIF-1α are critical participants in this transition. Tumor development and maintenance are aided by NF-κB, while cellular proliferation and adaptability to angiogenic signals are aided by HIF-1α. Prolyl hydroxylase-2 (PHD-2) has been hypothesized to be the key oxygen-dependent regulator of HIF-1α and NF-transcriptional B's activity. Without low oxygen levels, HIF-1α is degraded by the proteasome in a process dependent on oxygen and 2-oxoglutarate. As opposed to the normal NF-κB activation route, where NF-κB is deactivated by PHD-2-mediated hydroxylation of IKK, this method actually activates NF-κB. HIF-1α is protected from degradation by proteasomes in hypoxic cells, where it then activates transcription factors involved in cellular metastasis and angiogenesis. The Pasteur phenomenon causes lactate to build up inside the hypoxic cells. As part of a process known as lactate shuttle, MCT-1 and MCT-4 cells help deliver lactate from the blood to neighboring, non-hypoxic tumour cells. Non-hypoxic tumour cells use lactate, which is converted to pyruvate, as fuel for oxidative phosphorylation. OXOPHOS cancer cells are characterized by a metabolic switch from glucose-facilitated oxidative phosphorylation to lactate-facilitated oxidative phosphorylation. Although PHD-2 was found in OXOPHOS cells. There is no clear explanation for the presence of NF-kappa B activity. The accumulation of the competitive inhibitor of 2-oxo-glutarate, pyruvate, in non-hypoxic tumour cells is well established. So, we conclude that PHD-2 is inactive in non-hypoxic tumour cells due to pyruvate-mediated competitive suppression of 2-oxo-glutarate. This results in canonical activation of NF-κB. In non-hypoxic tumour cells, 2-oxoglutarate serves as a limiting factor, rendering PHD-2 inactive. However, FIH prevents HIF-1α from engaging in its transcriptional actions. Using the existing scientific literature, we conclude in this study that NF-κB is the major regulator of tumour cell growth and proliferation via pyruvate-mediated competitive inhibition of PHD-2.

P16.09 Regulation of chromatin accessibility in the hypoxic tumor microenvironment of glioblastoma

Abstract BACKGROUND Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most aggressive type of primary brain tumor. GBM has the poorest prognosis with no efficient cure to date due to diffusive growth into the brain, resistance to treatments and the immunosuppressive tumor microenvironment (TME). The growth and invasiveness of GBM is supported by the heterogeneous TME including local microglia and bone-marrow-derived macrophages (collectively known as glioma-associated microglia and macrophages, GAMs). In addition, tumor hypoxia is a key factor in the progression of GBM, as it can globally and rapidly alter gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can influence the pro-tumorigenic function of GAMs by inducing the expression of cytokines and cell surface receptors. However, little is known on the hypoxia-imposed chromatin changes of GAMs and GBM cells, which can in turn impact the interaction between these cell populations. Here we analyze these changes using a single-cell method, which preserves in situ hypoxia within the TME of GBM. MATERIAL AND METHODS Single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing) method in a GL261 murine glioma model was used to simultaneously assess genome-wide chromatin accessibility and expression of intracellular protein markers in single cells, enabling accurate selection of hypoxic and non-hypoxic tumor cells and GAMs. Pi-ATAC-seq is used on paraformaldehyde-perfused tumors and therefore allows capturing unaltered hypoxia-dependent cellular states, that often become distorted during dissociation and preparation of fresh material in most common single-cell methods. RESULTS We optimized Pi-ATAC method in a GL261 GBM mouse model, with specific sorting of GAMs using CD11b+ immunosorting followed by separation of microglia and macrophages, based on intensity of CD45 staining. HIF-1α induction and binding of pimonidazole were used to mark hypoxic populations. Currently, we are investigating the chromatin accessibility profiles of cancer cells and GAMs within the hypoxic tumor microenvironment of GBM. Exploring open chromatin profiles in GAMs and glioma-microglia co-cultures will allow to unravel the mechanisms of chromatin accessibility modulation in the oxygen-dependent manner. CONCLUSION In summary, we optimized the Pi-ATAC method in a mouse GBM model to characterize the chromatin openness changes in GAMs and cancer cells in response to hypoxic stress. Further validation of these results will provide the potential to identify novel markers for GAMs/glioma interactions in hypoxic GBMs and develop novel therapeutic targets.

OTME-2. Regulation of chromatin accessibility in the hypoxic tumor microenvironment of glioblastoma

Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most common primary brain tumor in adults. GBM has the poorest prognosis and no efficient cure to date due to diffusive growth into the surrounding brain, preventing complete surgical resection and leading to inevitable tumor relapse. Tumor microenvironment (TME) of GBM contains brain-residing microglia and bone-marrow derived macrophages (collectively known as glioma-associated microglia/macrophages, GAMs) that constitute up to 30% of the tumor mass and promote tumor invasion. Hypoxia (a shortage of oxygen) is a key factor in tumor progression of GBM as it can globally and rapidly alter the gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can enhance the pro-tumorigenic function of GAMs, e.g. by inducing expression of cytokines and cell surface receptors both in GAMs and glioma cells, but little is known about chromatin alterations of GBM under hypoxia. Since regulation of expression of such molecules could depend on the epigenetic alterations, we hypothesize that hypoxia may potently alter the chromatin accessibility and functions of GAMs and glioma cells.We determine the genome-wide changes in chromatin accessibility in GAMs and glioma cells in response to hypoxic stress using single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing), which allows simultaneous genome-wide assessment of chromatin accessibility and expression of intracellular protein markers in single cells, allowing faithful selection of hypoxic and non-hypoxic cells. Secondly, we are employing an oxygen-dependent co-culture model in vitro to study the mechanisms of chromatin alterations in GAMs and glioma cells under controlled hypoxic conditions and test how these changes depend on the glioma - GAMs cross-communication. In summary, we characterize the interactions between innate immune cells and glioma cells by looking at their chromatin alterations under hypoxia.Supported by the National Science Center grant (Poland) 2019/33/B/NZ1/01556 (KBL).

Abstract 1078: Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features

Abstract Trastuzumab and trastuzumab emtansine (T-DM1) targeted therapies are the main choice for the treatment of HER2-overexpressing breast cancer patients. However, de novo or acquired resistance is still the major obstacle in clinical practice. It has been shown that several pathways, including HER2, can lead to HIF-α stabilization in breast cancer cells. Thus, the purpose of our study was to analyse the effect of hypoxia in acquired resistance to anti-HER2 therapies. We used HER2-overexpressing BT-474 and non-overexpressing MCF-7 human breast cancer cell lines. As an acute hypoxia model, we added CoCl2 (100 µM) to cell culture medium. The hypoxic status of the cells was evaluated by a Western blot analysis, showing a peak of HIF-1α expression after 6 hours of CoCl2 exposure. This result correlated with VEGF induction, as measured by RT-qPCR (p<0.05). Interestingly, under hypoxia, BT-474 cells treated with increasing concentrations of trastuzumab and T-DM1 for 72 hours presented a significantly higher viability compared with non-hypoxic cells (p<0.05). However, although the drugs decreased membrane HER2 protein expression (p<0.01), HER2 levels did not change under hypoxic conditions, as shown by flow cytometry and Western blot assays. Moreover, drug effects on MCF-7 cells did not change under hypoxic conditions. To further analyse this cytoprotective effect of hypoxia, proliferation-associated markers such as PCNA expression and AKT activation were evaluated by Western blot, while apoptosis frequency was measured by flow cytometry in BT-474 cells. PCNA and pAKT expression remained unaltered, while T-DM1-mediated apoptosis was significantly reduced under hypoxia (p<0.05). Then, using different open gene expression datasets, we identified a group of four genes (COL12A1, DKK1, CEACAM5 and ANTXR1) differentially expressed in T-DM1 resistant HER2-overexpressing breast cancer BT-474 and KPL-4 cell lines (p<0.05), which were coexpressed with hypoxia-related genes (HIF1A, EPAS1 and VEGFA) in HER2-overexpressing breast cancer tumours and cell lines from TCGA (p<0.01). Further in silico analyses revealed a direct association between those genes and the fibronectin gene (FN1, p<0.05). Remarkably, they were involved in TGF-β and angiogenesis pathways, as shown by a functional enrichment analysis (p<0.01). In addition, we found that BT-474 cells under hypoxia presented a higher mammosphere formation efficiency than non-hypoxic cells (p<0.05), showing a tendency towards a cancer stem cell phenotype. It is known that hypoxia represents a common feature of metastatic tumours and has a role in regulating stem cell behaviour. According to this, our results highlight a link between anti-HER2 therapy resistance and epithelial-to-mesenchymal transition characteristics, where hypoxia plays a key role which deserves further study. Citation Format: Virginia Judith Wolos, Ivana Jaqueline Tapia, Marianela Abrigo, Ezequiel Lacunza, Elisa Dora Bal de Kier Joffé, Gabriel León Fiszman. Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1078.

DNAPK Inhibition Preferentially Compromises the Repair of Radiation-induced DNA Double-strand Breaks in Chronically Hypoxic Tumor Cells in Xenograft Models.

Radiation-induced DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). Recently, it has been found that chronic tumor hypoxia compromises HR repair of DNA DSBs but activates the NHEJ protein DNAPK. We therefore hypothesized that inhibition of DNAPK can preferentially potentiate the sensitivity of chronically hypoxic cancer cells to radiation through contextual synthetic lethality in vivo In this study, we investigated the impact of DNAPK inhibition by a novel selective DNAPK inhibitor, NU5455, on the repair of radiation-induced DNA DSBs in chronically hypoxic and nonhypoxic cells across a range of xenograft models. We found that NU5455 inhibited DSB repair following radiation in both chronically hypoxic and nonhypoxic tumor cells. Most importantly, the inhibitory effect was more pronounced in chronically hypoxic tumor cells than in nonhypoxic tumor cells. This is the first in vivo study to indicate that DNAPK inhibition may preferentially sensitize chronically hypoxic tumor cells to radiotherapy, suggesting a broader therapeutic window for transient DNAPK inhibition combined with radiotherapy.

HnRNPA1 ISOFORM BRING DIVERSITY IN GLIOMA CELL SURVIVAL

Hypoxia response plays a critical impact on tumor growth and drug resistance development. Under hypoxic and non-hypoxic growth environment U87MG and A172 glioma cells behaved differently. The presence of hnRNPA1 isoforms variant showed critical regulatory step in directing glioma cell adaptability. Knocking hnRNPA1, distinctly affecting the hnRNPA1 isoforms in two glioma cell lines. U87 MG cells, in response two different siRNA against hnRNPA1, suppresses higher isoform of hnRNPA1 compare to lower isoforms, on contrary A172 responses predominantly with up regulation of hnRNPA1vII (higher isoform) only. Leading expression of HnRNPA1vII isoform down regulates phospho-ERK½ activation. Additionally, knock down of GSK3 alpha also enhances the generation of higher isoform of hnRNPA1 in non-hypoxic cells. Remarkably, cells grown under hypoxic condition, phospho-ERK½ activation was observed only in presence of lower abundant isoform of hnRNPA1. Furthermore, immunohistochemistry was conducted to recapitulate the moderate to weak expression of GSK3 alpha correlated with strong expression of hnRNPA1 in glioma hypoxic tumor. Nonetheless, selective knock down of hnRNPA1vII (higher form) and vI (lower isoform) in U87MG were examined, evidently the phospho-ERK½ expression was downregulated in hnRNPA1vI knock down, compared with higher phospho-ERK½ in hnRNPA1 higher isoform (vII) knock down was observed. Interestingly, knock down of hnRNPA1 vII (higher isoform) also reduces the expression of hnRNPA2/B1 and SF2/ASF1 (splice factor RNA binding protein), in U87 MG cells. In conclusion, selective targeting hnRNPA1 isoforms attenuate glioma cell metabolism, and promote therapeutic sensitization.

LOXL2-enriched small extracellular vesicles mediate hypoxia-induced premetastatic niche and indicates poor outcome of head and neck cancer.

Small extracellular vesicles (sEVs) operate as a signaling platform due to their ability to carry functional molecular cargos. However, the role of sEVs in hypoxic tumor microenvironment-mediated premetastatic niche formation remains poorly understood.Methods: Protein expression profile of sEVs derived from normoxic and hypoxic head and neck squamous cell carcinoma (HNSCC) cells were determined by Isobaric Tagging Technology for Relative Quantitation. In vitro invasion assay and in vivo colonization were performed to evaluate the role of sEV-delivering proteins.Results: We identified lysyl oxidase like 2 (LOXL2) which had the highest fold increase in hypoxic sEVs compared with normoxic sEVs. Hypoxic cell-derived sEVs delivered high amounts of LOXL2 to non-hypoxic HNSCC cells to elicit epithelial-to-mesenchymal transition (EMT) and induce the invasion of the recipient cancer cells. Moreover, LOXL2-enriched sEVs were incorporated by distant fibroblasts and activate FAK/Src signaling in recipient fibroblasts. Increased production of fibronectin mediated by FAK/Src signaling recruited myeloid-derived suppressor cells to form a premetastatic niche. Serum sEV LOXL2 can reflect a hypoxic and aggressive tumor type and can serve as an alternative to tissue LOXL2 as an independent prognostic factor of overall survival for patients with HNSCC.Conclusion: sEVs derived from the hypoxic tumor microenvironment of HNSCC can drive local invasion of non-hypoxic HNSCC cells and stimulate premetastatic niche formation by delivering LOXL2 to non-hypoxic HNSCC cells and fibroblasts to induce EMT and fibronectin production, respectively.

PH‐Responsive Polymers for Improving the Signal‐to‐Noise Ratio of Hypoxia PET Imaging with [18F]Fluoromisonidazole

Front Cover: Positron emission tomography (PET) imaging of hypoxia using the radiotracer [18F]FMISO suffers from a poor signal-to-noise ratio due to the unwanted uptake and slow clearance of the radiotracer in surrounding non-hypoxic cells. In article 2000061, Jason S. Lewis, Thomas P. Davis, and co-workers decrease the uptake in non-hypoxic cells by delivering the radiotracer using pH-responsive polymers, which only release the radiotracer in the acidic microenvironment of hypoxic cells. Cover design credit: Memorial Sloan Kettering Cancer Center.

Abstract 2433: The hypoxic tumor microenvironment in vivo selects the tumor cells with cancer stem cell characteristics and increased resistance against genotoxic stresses

Abstract Tumor hypoxia has been established as an independent prognostic factor associated with poor patient survival and resistance to conventional therapies. Emerging evidence suggests that hypoxia has the potential to maintain or enhance the stem cell phenotype and confers chemo- or radio-resistance to tumor cells. However, it remains to be determined whether cell fate and acquisition of therapy resistance is regulated in vivo by the hypoxic tumor microenvironment (TME). In this study, we have developed a hypoxia-sensing human breast cancer cell xenograft model using the hypoxia-response element (HRE)-driven enhanced green fluorescence protein (EGFP) as a hypoxia reporter. We have found that the hypoxic tumor cells isolated from xenografts contain increased subpopulations of tumor cells showing the cancer stem cell (CSC) characteristics. Upon re-implantation, the CSC characteristics of the hypoxic tumor cells continue to be further enriched during in vivo growth, whereas secondary xenografts derived from the non-hypoxic tumor cells remain similar to the primary xenografts. Consistent with this finding, the phenotypes exhibited by the hypoxic tumor cells are stably different from those of the non-hypoxic tumor cells isolated from the same tumor mass even when they are maintained under the same ambient culture conditions. Mechanistically, we find that the PI3K/AKT pathway is strongly potentiated in the hypoxic tumor cells and is required to maintain the CSC phenotype. Cell fate differences between hypoxic and non-hypoxic tumor cells are found only in tumor cells isolated from the hypoxic TME in vivo, and are not seen in tumor cells treated by hypoxia in vitro alone. In addition, we have found that naturally hypoxic tumor cells in a breast cancer xenograft model are moderately radio- and chemo-resistant compared to their neighboring non-hypoxic tumor cells in the same tumor. These naturally hypoxic tumor cells are proficient at repairing DNA damages and resist apoptosis induced by genotoxic stresses. The ex vivo hypoxic tumor cells are capable of proficient activation of the DNA damage-sensing pathways, especially the ATM pathway. These previously unrecognized observations suggest that the hypoxic TME may promote malignant progression by coordinating induction, selection and preferential maintenance of the CSC phenotype in tumor cells. This presentation will review our results which strongly suggest that the hypoxic TME may induce adaptive responses, either by clonal evolution or selection, such that hypoxic tumor cells in vivo acquire new functional traits that confer resistance to genotoxic stresses and enhance survival. It can be postulated that previously hypoxic tumor cells may continue to exhibit their aggressive phenotypes long after they have migrated into non-hypoxic regions or after their native niche become normoxic. Citation Format: Hoon Kim, Qun Lin, Zhong Yun. The hypoxic tumor microenvironment in vivo selects the tumor cells with cancer stem cell characteristics and increased resistance against genotoxic stresses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2433.

The hypoxic tumor microenvironment in vivo selects tumor cells with increased survival against genotoxic stresses

Tumor sensitivity to radiation therapy has been known to be dependent on O2 concentrations. However, radiosensitivity of naturally occurring hypoxic tumor cells remains to be well fully investigated in direct comparison to that of their adjacent non-hypoxic tumor cells within the same tumor. We developed a hypoxia-sensing xenograft model using the hypoxia-response element (HRE)-driven enhanced green fluorescence protein (EGFP) as a hypoxia reporter to identify hypoxic tumor cells in situ. Here, we have found that naturally hypoxic tumor cells are moderately radioresistant compared to their neighboring non-hypoxic tumor cells in the same tumor. These naturally hypoxic tumor cells are proficient at repairing DNA damages and resist apoptosis induced by genotoxic stresses, which involves activation of the ATM/CHK1/CHK2 DNA damage-sensing pathway. Inhibition of the checkpoint kinases sensitizes the ex vivo hypoxic tumor cells to ionizing irradiation. Second, the new functional phenotypes acquired by the hypoxic tumor cells in vivo are stable even after they are maintained under non-hypoxic conditions. These new results strongly suggest that the hypoxic tumor microenvironment is capable of selecting stable tumor cell populations with increased resistance to genotoxic stresses and enhanced survival.

The hypoxic tumor microenvironment in vivo selects the cancer stem cell fate of breast cancer cells

BackgroundTumor hypoxia is an independent prognostic factor associated with poor patient survival. Emerging evidence suggests that hypoxia can potentially maintain or enhance the stem cell phenotype of both normal stem cells and cancer cells. However, it remains to be determined whether cell fate is regulated in vivo by the hypoxic tumor microenvironment (TME).MethodsWe established a hypoxia-sensing xenograft model to identify hypoxic tumor cell in vivo primarily using human breast cancer cell lines MDA-MB-231 and MCF7. Hypoxic tumor cells were identified in situ by fluorescence of green fluorescence protein. They were further isolated from xenografts, purified and sorted by flow cytometry for detailed analysis of their stem cell characteristics.ResultsWe have found that hypoxic tumor cells freshly isolated from xenografts contain increased subpopulations of tumor cells with cancer stem cell (CSC)-like characteristics. The CSC characteristics of the hypoxic tumor cells are further enhanced upon re-implantation in vivo, whereas secondary xenografts derived from the non-hypoxic tumor cells remain similar to the primary xenografts. Interestingly, the phenotypes exhibited by the hypoxic tumor cells are stable and remain distinctively different from those of the non-hypoxic tumor cells isolated from the same tumor mass even when they are maintained under the same ambient culture conditions. Mechanistically, the PI3K/AKT pathway is strongly potentiated in the hypoxic tumor cells and is required to maintain the CSC-like phenotype. Importantly, the differential cell fates between hypoxic and non-hypoxic tumor cells are only found in tumor cells isolated from the hypoxic TME in vivo and are not seen in tumor cells treated by hypoxia in vitro alone.ConclusionsThese previously unknown observations suggest that the hypoxic TME may promote malignant progression and therapy resistance by coordinating induction, selection and/or preferential maintenance of the CSC-like phenotype in tumor cells.

Oxygen Enhancement Ratio in Radiation-Induced Initial DSBs by an Optimized Flow Cytometry-based Gamma-H2AX Analysis in A549 Human Cancer Cells.

High-linear energy transfer (LET) heavy ions cause higher therapeutic effects than low-LET radiation due to lower dependency on oxygen concentration in tumor cell killing. The lethality after irradiation largely depends on DNA double-strand breaks (DSBs), however the detailed LET dependency for DSB induction under oxic and hypoxic conditions has not been reported. Therefore, we evaluated the oxygen enhancement ratio (OER) of heavy ion-induced DSB induction using a highly-optimized flow cytometry-based method of γ-H2AX detection. Non-small cell lung cancer (NSCLC) A549 cells were exposed to X-ray, carbon-ion and iron-ion radiations under oxic or hypoxic condition. As a DSB marker, the γ-H2AX signal was measured 1 h postirradiation and analyzed by flow cytometry. DSB slope values were calculated as DSB induction per Gy. Our method was able to detect high-LET radiation-induced DSBs even from clustered DNA damage sites. We also showed a decrease in OER value in an LET-dependent manner regardless of radiation type. In summary, we demonstrated a simple, quick and highly-optimized flow cytometry-based method of DSB analysis that detects DSBs induced by heavy-ion radiation for hypoxic and nonhypoxic cancer cells. Our study may provide a useful biological basis for heavy-ion radiotherapy.

Transcriptional reprogramming of metabolic pathways in critically ill patients.

BackgroundCritical illness causes a shift away from mitochondrial metabolism towards a greater dependence on glycolysis. This metabolic shift is thought to be associated with lactic acidosis, organ dysfunction and poor clinical outcomes. The current paradigm is that low oxygen supply causes regional hypoxia, which in turn drives such a metabolic shift. In this study, we evaluated whether the shift towards glycolysis can also occur in cells where oxygen supply is plentiful.MethodsWe used circulating blood cells from non-hypoxic critically ill patients (n = 47) as a model to study cellular metabolism in a normal oxygen milieu. We measured the transcriptomic profiles of canonical metabolic pathways in these cells and compared them to cells obtained from healthy controls (n = 18).ResultsTranscriptomic profiling revealed a significant reprogramming of metabolic pathways during critical illness. In well-oxygenated cells, there was a reduced expression of tricarboxylic acid cycle genes and genes associated with pyruvate entry into the mitochondria suggesting decreased mitochondrial oxidation. In contrast, glycolysis was accelerated, as reflected by an up-regulation of genes coding for enzymes of early and late glycolytic pathway that were associated with increased lactate production. The pentose phosphate pathway genes for NADPH production were also up-regulated suggesting enhanced antioxidant production during increased oxidative stress.ConclusionsContrary to the established paradigm, aerobic glycolysis does occur in non-hypoxic cells during critical illness and its occurrence may represent an adaptive strategy common to cells under increased oxidative stress. Further study of this previously under-recognized metabolic phenomenon might identify novel drug target for antioxidant therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s40635-016-0094-1) contains supplementary material, which is available to authorized users.

Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality

PurposePoly(ADP-ribose) polymerase (PARP) inhibitors potentiate radiation therapy in preclinical models of human non-small cell lung cancer (NSCLC) and other types of cancer. However, the mechanisms underlying radiosensitization in vivo are incompletely understood. Herein, we investigated the impact of hypoxia on radiosensitization by the PARP inhibitor olaparib in human NSCLC xenograft models.Methods and MaterialsNSCLC Calu-6 and Calu-3 cells were irradiated in the presence of olaparib or vehicle under normoxic (21% O2) or hypoxic (1% O2) conditions. In vitro radiosensitivity was assessed by clonogenic survival assay and γH2AX foci assay. Established Calu-6 and Calu-3 subcutaneous xenografts were treated with olaparib (50 mg/kg, daily for 3 days), radiation (10 Gy), or both. Tumors (n=3/group) were collected 24 or 72 hours after the first treatment. Immunohistochemistry was performed to assess hypoxia (carbonic anhydrase IX [CA9]), vessels (CD31), DNA double strand breaks (DSB) (γH2AX), and apoptosis (cleaved caspase 3 [CC3]). The remaining xenografts (n=6/group) were monitored for tumor growth.ResultsIn vitro, olaparib showed a greater radiation-sensitizing effect in Calu-3 and Calu-6 cells in hypoxic conditions (1% O2). In vivo, Calu-3 tumors were well-oxygenated, whereas Calu-6 tumors had extensive regions of hypoxia associated with down-regulation of the homologous recombination protein RAD51. Olaparib treatment increased unrepaired DNA DSB (P<.001) and apoptosis (P<.001) in hypoxic cells of Calu-6 tumors following radiation, whereas it had no significant effect on radiation-induced DNA damage response in nonhypoxic cells of Calu-6 tumors or in the tumor cells of well-oxygenated Calu-3 tumors. Consequently, olaparib significantly increased radiation-induced growth inhibition in Calu-6 tumors (P<.001) but not in Calu-3 tumors.ConclusionsOur data suggest that hypoxia potentiates the radiation-sensitizing effects of olaparib by contextual synthetic killing, and that tumor hypoxia may be a potential biomarker for selecting patients who may get the greatest benefit from the addition of olaparib to radiation therapy.

Hypoxia Activated Prodrugs: Factors Influencing Design and Development.

Hypoxia in tumor cells is characterized by a lack of oxygen resulting from reduced blood supply to the surrounding tissue, and is a common characteristic of solid tumors as a consequence of rapid cell growth. Hypoxia in tumors is a predictor of both resistance to chemotherapy and of a metastatic/aggressive form of cancer, and as a result, development of cancer therapies which target hypoxia is of vital importance. One such targeting strategy is the development of hypoxia-activated prodrugs (HAP) which can preferentially release chemotherapeutic agents within hypoxic tumor regions. This targeting strategy is accomplished by attaching a hypoxia activated trigger to a chemotherapeutic agent and under oxygen-poor conditions, the agent (effector) is released into the tumor, while remaining intact in normal tissue, and leaving non-hypoxic cells undamaged. Overall, this strategy can achieve an increased therapeutic benefit over conventional small molecule chemotherapeutic treatments by concentrating the drugs within hypoxic tumor environments, while simultaneously reducing the side-effects and toxicity issues that surround the systemic distribution of traditional drugs on normoxic cells. In this review, we will describe the factors that should be considered when designing an effective HAP, such as the mechanism of prodrug action, the elements that influence the rational design of HAP (i.e. reduction potential), and the activating enzymes of HAP. As part of this description, we will utilize select examples from the literature to reinforce these factors, and make a case for the intelligent design of new HAPs, leading to the development of novel hypoxia targeting chemotherapeutic agents.

Cellular memory of hypoxia elicits neuroblastoma metastasis and enables invasion by non-aggressive neighbouring cells.

Therapies targeting cancer metastasis are challenging owing to the complexity of the metastatic process and the high number of effectors involved. Although tumour hypoxia has previously been associated with increased aggressiveness as well as resistance to radio- and chemotherapy, the understanding of a direct link between the level and duration of hypoxia and the individual steps involved in metastasis is still missing. Using live imaging in a chick embryo model, we have demonstrated that the exposure of neuroblastoma cells to 1% oxygen for 3 days was capable of (1) enabling cell migration towards blood vessels, (2) slowing down their velocity within blood vessels to facilitate extravasation and (3) promoting cell proliferation in primary and secondary sites. We have shown that cells do not have to be hypoxic anymore to exhibit these acquired capabilities as a long-term memory of prior hypoxic exposure is kept. Furthermore, non-hypoxic cells can be influenced by neighbouring hypoxic preconditioned cells and be entrained in the metastatic progression. The acquired aggressive phenotype relies on hypoxia-inducible factor (HIF)-dependent transcription of a number of genes involved in metastasis and can be impaired by HIF inhibition. Altogether, our results demonstrate the need to consider both temporal and spatial tumour heterogeneity because cells can 'remember' an earlier environment and share their acquired phenotype with their close neighbours. As a consequence, it is necessary to monitor the correct hypoxic markers to be able to predict the consequences of the cells' history on their behaviour and their potential response to therapies.

Abstract B56: Transfer of senescent signals in a hypoxic tumor microenvironment: A mechanism of tumor cell survival mediated by exosomes

Abstract Triple Negative Breast Cancers (TNBCs) are a histological subtype of breast tumors which are innately hypoxic; a tumor microenvironment integral to chemoresistance and aggressive tumor behaviour. Importantly, hypoxia can drive viable cellular fate mechanisms rendering cancer cells capable of by-passing apoptotic cell death when chemotherapeutically challenged. Moreover, exosomes (nanovesicles 40-150nm of endocytic origin) preferentially released in the hypoxic environment can potentially be trafficked into neighbouring cells thereby mediating communication and propagating chemoresistance. The objective of this study was to establish if exosomes released from the TNBC cell line MDA-MB-231 (MSL) grown in a hypoxic environment, can mediate the transfer of viable cellular fate signatures (senescent and autophagic) to non-hypoxic cells rendering them non-apoptotic and chemoresistant. Experimentally, MDA-MB-231 (MSL) TNBC cells were cultured in hypoxia (1%) for 0-96 hours. Senescent, autophagic and cell cycle markers (LC3, p21, HDAC6, TSG101 and MAD2) were analysed by Western Blot. Nanoparticle Tracking Analysis (NTA), performed on the Nanosight LM10 microscope, allowed the enumeration and characterisation of harvested exosomes, while Dil labelled fluorescent imaging confirmed exosomal uptake in vitro. Hypoxic exposure (1%) of the MDA-MB-231 (MSL) confirmed by an increase in HIF1a, demonstrated that cellular senescence (p21), autophagy (LC3II), HDAC6 and the tumor susceptibility gene 101 (TSG101) markers increased in prolonged hypoxic exposure. Prolonged hypoxic exposure was also shown to result in an increase in the number of exosomes released from MDA-MB-231 (MSL) cells. siRNA knockdown of TSG101, a component of the ESCRT pathway involved in exosome release, resulted in a decrease in the amount of exosomes released from MDA-MB-231 (MSL) cells. In summary, it is well established in the literature that TNBCs are more hypoxic and chemoresistant than non-TNBC histological subtypes. The fact that both senescence and autophagy are induced in this environment underlies the ability of cancer cells to maintain viability. Moreover, exosomal release is enhanced in hypoxia thereby facilitating signalling between cancer cells with the potential to propagate chemoresistance. We suggest that targeting TSG101 thereby impeding exosomal release has the potential to cessate communication between cells in hypoxia thereby promoting apoptotic cell death in cells that are chemotherapeutically challenged. Funding: The Mater Surgical Oncology Research Appeal, and Mater Breast Health Research Citation Format: Luke Gubbins, Karolina Weiner-Gorzel, Shiva Sharma, Laura Cooke, Anna Salvati, Jeremy Simpson, Kenneth Dawson, Malcolm Kell, Amanda McCann. Transfer of senescent signals in a hypoxic tumor microenvironment: A mechanism of tumor cell survival mediated by exosomes. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B56. doi:10.1158/1538-7445.CHTME14-B56

Fetal Reprogramming and Senescence in Hypoplastic Left Heart Syndrome and in Human Pluripotent Stem Cells during Cardiac Differentiation

Hypoplastic left heart syndrome (HLHS) is a severe cardiac malformation characterized by left ventricle (LV) hypoplasia and abnormal LV perfusion and oxygenation. We studied hypoxia-associated injury in fetal HLHS and human pluripotent stem cells during cardiac differentiation to assess the effect of microenvironmental perturbations on fetal cardiac reprogramming. We studied LV myocardial samples from 32 HLHS and 17 structurally normal midgestation fetuses. Compared with controls, the LV in fetal HLHS samples had higher nuclear expression of hypoxia-inducible factor-1α but lower angiogenic growth factor expression, higher expression of oncogenes and transforming growth factor (TGF)-β1, more DNA damage and senescence with cell cycle arrest, fewer cardiac progenitors, myocytes and endothelial lineages, and increased myofibroblast population (P < 0.05 versus controls). Smooth muscle cells (SMCs) had less DNA damage compared with endothelial cells and myocytes. We recapitulated the fetal phenotype by subjecting human pluripotent stem cells to hypoxia during cardiac differentiation. DNA damage was prevented by treatment with a TGF-β1 inhibitor (P < 0.05 versus nonhypoxic cells). The hypoplastic LV in fetal HLHS samples demonstrates hypoxia-inducible factor-1α up-regulation, oncogene-associated cellular senescence, TGF-β1-associated fibrosis and impaired vasculogenesis. The phenotype is recapitulated by subjecting human pluripotent stem cells to hypoxia during cardiac differentiation and rescued by inhibition of TGF-β1. This finding suggests that hypoxia may reprogram the immature heart and affect differentiation and development.