Tumor hypoxia is a common microenvironmental factor that adversely influences tumor phenotype and treatment response. Cellular adaptation to hypoxia occurs through multiple mechanisms, including activation of the unfolded protein response (UPR). Recent reports have indicated that hypoxia activates a lysosomal degradation pathway known as autophagy, and here we show that the UPR enhances the capacity of hypoxic tumor cells to carry out autophagy, and that this promotes their survival. In several human cancer cell lines, hypoxia increased transcription of the essential autophagy genes microtubule-associated protein 1 light chain 3β (MAP1LC3B) and autophagy-related gene 5 (ATG5) through the transcription factors ATF4 and CHOP, respectively, which are regulated by PKR-like ER kinase (PERK, also known as EIF2AK3). MAP1LC3B and ATG5 are not required for initiation of autophagy but mediate phagophore expansion and autophagosome formation. We observed that transcriptional induction of MAP1LC3B replenished MAP1LC3B protein that was turned over during extensive hypoxia-induced autophagy. Correspondingly, cells deficient in PERK signaling failed to transcriptionally induce MAP1LC3B and became rapidly depleted of MAP1LC3B protein during hypoxia. Consistent with these data, autophagy and MAP1LC3B induction occurred preferentially in hypoxic regions of human tumor xenografts. Furthermore, pharmacological inhibition of autophagy sensitized human tumor cells to hypoxia, reduced the fraction of viable hypoxic tumor cells, and sensitized xenografted human tumors to irradiation. Our data therefore demonstrate that the UPR is an important mediator of the hypoxic tumor microenvironment and that it contributes to resistance to treatment through its ability to facilitate autophagy.

The epidermal growth factor receptor (EGFR) is highly expressed in a significant number of human malignancies, and its expression is associated with tumor aggressiveness and overall treatment resistance. The monoclonal antibody cetuximab is increasingly used in clinical settings as a treatment modality in combination with more conventional therapies, such as radio-and chemotherapy. Currently, little is known about tumorspecific uptake and overall pharmacokinetics. Noninvasive quantification of cetuximab uptake could provide important diagnostic information for patient selection and therapy evaluation. To this end, we have developed and validated a novel probe using cetuximab labeled with the long-lived positron emitter 89 Zr for PET imaging. Methods: Tumor cell lines with varying EGFR expression levels were used for in vivo tumor imaging experiments. PET with 89 Zr-labeled cetuximab (3.75 6 0.14 MBq) was performed on tumor-bearing NMRI-nu mice at multiple time points after injection (ranging from 1 to 120 h) and quantified by drawing regions of interest on selected tissues. Uptake was compared by biodistribution g-counting, and ex vivo EGFR expression levels were quantified using Western blot analysis. Results: Uptake of 89 Zr-labeled cetuximab was demonstrated in the EGFR-positive tumors. However, the EGFR levels measured in vivo did not correlate with the relative signal obtained by PET. Tumor-to-blood ratios were significantly higher in the cell lines with intermediate (compared with the high) EGFR expression starting from 24 h after injection. Normal tissue uptake was unaffected by the different tumor types. Ex vivo g-counting experiments confirmed the observed in vivo PET results. A similar disparity was found between 89 Zr-labeled cetuximab tumor uptake and in vivo EGFR expression levels as demonstrated by Western blotting. Conclusion: The 89 Zr-labeled cetuximab imaging probe is a promising tool for noninvasive evaluation of cetuximab uptake. Our results demonstrate a disparity between in vivo EGFR expression levels and cetuximab uptake. In a general sense, the results indicate a disparity between antibody uptake and expression levels of a biologic target in a tumor, suggesting that additional pharmacokinetic or pharmacodynamic mechanisms influence tumor delivery of this therapy. These additional mechanisms may explain why receptor expression levels alone are not sufficient to predict patient response.

Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxiainducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers.growth delay | irradiation | acute hypoxia S olid tumor microenvironments are characterized by extreme heterogeneities in oxygenation that arise as a result of poorly developed vascular networks. Gradients in oxygen are frequently found surrounding perfused vessels, ranging from normal values (∼5%) near the blood vessel to complete anoxia adjacent to necrosis. This gradient of hypoxia is generally referred to as "chronic" or "diffusion-limited" hypoxia and results from cellular oxygen consumption. Hypoxia can also arise in a temporal manner as a consequence of transient changes in oxygen delivery caused by changes in vessel perfusion (1). This "acute" or "perfusion-limited" hypoxia (2) is often cyclic in nature and can account for a large proportion of hypoxic cells at any given time (3). The steady-state proportion of hypoxic cells in tumors is influenced by the tolerance of individual tumor cells to these different types of hypoxia and varies remarkably among tumors with otherwise similar clinical features (4). For example, in head and neck cancer, oxygen electrode measurements of the hypoxic fraction range from 0% to 97% (5). These differences are important, because the fraction of viable hypoxic cells is a major determinant of outcome, as hypoxic cells are highly resistant to chemotherapy and radiation therapy (5, 6). Reducing cellular tolerance to hypoxia is therefore a strategy to reduce the proportion of hypoxic cells in tumors to improve current cancer therapy.The mechanisms influencing hypoxia tolerance and therapy resistance in solid tumors are only partially understood. Hypoxiainducible factor (HIF) ...

Several mutations in nuclear genes encoding for mitochondrial components have been associated with an increased cancer risk or are even causative, e.g. succinate dehydrogenase (SDHB, SDHC and SDHD genes) and iso-citrate dehydrogenase (IDH1 and IDH2 genes). Recently, studies have suggested an eminent role for mitochondrial DNA (mtDNA) mutations in the development of a wide variety of cancers. Various studies associated mtDNA abnormalities, including mutations, deletions, inversions and copy number alterations, with mitochondrial dysfunction. This might, explain the hampered cellular bioenergetics in many cancer cell types. Germline (e.g. m.10398A>G; m.6253T>C) and somatic mtDNA mutations as well as differences in mtDNA copy number seem to be associated with cancer risk. It seems that mtDNA can contribute as driver or as complementary gene mutation according to the multiple-hit model. This can enhance the mutagenic/clonogenic potential of the cell as observed for m.8993T>G or influences the metastatic potential in later stages of cancer progression. Alternatively, other mtDNA variations will be innocent passenger mutations in a tumor and therefore do not contribute to the tumorigenic or metastatic potential. In this review, we discuss how reported mtDNA variations interfere with cancer treatment and what implications this has on current successful pharmaceutical interventions. Mutations in MT-ND4 and mtDNA depletion have been reported to be involved in cisplatin resistance. Pharmaceutical impairment of OXPHOS by metformin can increase the efficiency of radiotherapy. To study mitochondrial dysfunction in cancer, different cellular models (like ρ(0) cells or cybrids), in vivo murine models (xenografts and specific mtDNA mouse models in combination with a spontaneous cancer mouse model) and small animal models (e.g. Danio rerio) could be potentially interesting to use. For future research, we foresee that unraveling mtDNA variations can contribute to personalized therapy for specific cancer types and improve the outcome of the disease.

In this study, we performed a systematic literature search linking radiomics to tumor biology. All but two studies (n = 39) revealed that radiomic features derived from ultrasound, CT, PET and/or MR are significantly associated with one or several specific tumor biologic substrates, from somatic mutation status to tumor histopathologic grading and metabolism. Considerable inter-observer differences were found with regard to RQS scoring, while important questions were raised concerning the interpretability of the outcome of such scores.

Specific inhibition of CAIX activity enhanced the effect of tumor irradiation and might, therefore, be an attractive strategy to improve overall cancer treatment.

HighlightsHypoxia-activated prodrugs have yielded promising results up to phase II trials.Implementation of hypoxia-activated prodrugs in the clinic has not been successful.Phase III clinical trials lack patient stratification based on tumor hypoxia status.Stratification will decrease the number of patients needed and increase success.Improvements in hypoxia-activated prodrug design can also increase success rates.

Hypoxia has been shown to be an important microenvironmental parameter influencing tumor progression and treatment efficacy. Patient guidance for hypoxia-targeted therapy requires evaluation of tumor oxygenation, preferably in a noninvasive manner. The aim of this study was to evaluate and validate the uptake of [ 18 F] HX4, a novel developed hypoxia marker for PET imaging. A heterogeneous accumulation of [ 18 F]HX4 was found within rat rhabdomyosarcoma tumors that was significantly (P < 0.0001) higher compared with the surrounding tissues, with temporal increasing tumor-to-blood ratios reaching a plateau of 7.638 ± 0.926 and optimal imaging properties 4 h after injection. [ 18 F]HX4 retention in normal tissues was found to be short-lived, homogeneous and characterized by a fast progressive temporal clearance. Heterogeneity in [ 18 F]HX4 tumor uptake was analyzed based on 16 regions within the tumor according to the different orthogonal planes at the largest diameter. Validation of heterogeneous [ 18 F]HX4 tumor uptake was shown by a strong and significant relationship (r = 0.722; P < 0.0001) with the hypoxic fraction as calculated by the percentage pimonidazole-positive pixels. Furthermore, a causal relationship with tumor oxygenation was established, because combination treatment of nicotinamide and carbogen resulted in a 40% reduction (P < 0.001) in [ 18 F]HX4 tumor accumulation whereas treatment with 7% oxygen breathing resulted in a 30% increased uptake (P < 0.05). [ 18 F]HX4 is therefore a promising candidate for noninvasive detection and evaluation of tumor hypoxia at a macroscopic level.cancer | nuclear medicine | experimental research T he presence of hypoxic regions due to abnormalities in tumor vasculature, heterogeneously spread within solid tumors influences clinical outcome; as it is an independent predictor of poor prognosis-free survival in several types of cancer (1). In contrast, this unique tumor characteristic makes it an attractive target for novel drugs to increase the therapeutic effect of conventional cancer treatment modalities. Another approach is the use of intensity-modulated radiotherapy to give a higher dose to hypoxic areas while sparing the surrounding normal tissue (2, 3). Although treatments to counteract the negative effect of intratumoral hypoxia are under investigation, not all patients will benefit from such selective treatments. Therefore, to guide hypoxiadirected therapies in individual patients, it is important to evaluate tumor oxygenation using a reliable noninvasive method.To date, a variety of methods are available for assessment of tumor oxygenation in solid tumors, of which polarographic oxygen electrodes and immunohistological assays remain the gold standard (4). These standard invasive modalities have not yielded reliable 3D images of the whole tumor for clinical use, and therefore research has been focused on noninvasive imaging techniques, such as positron-emission tomography (PET) using nitroimidazoles. The 2-nitroimidazole derivative fluoromisonidazole (FMISO)...