Hypoxia and its therapeutic possibilities in paediatric cancers

In tumours, hypoxia—a condition in which the demand for oxygen is higher than its availability—is well known to be associated with reduced sensitivity to radiotherapy and chemotherapy, and with immunosuppression. The consequences of hypoxia on tumour biology and patient outcomes have therefore led to the investigation of strategies that can alleviate hypoxia in cancer cells, with the aim of sensitising cells to treatments. An alternative therapeutic approach involves the design of prodrugs that are activated by hypoxic cells. Increasing evidence indicates that hypoxia is not just clinically significant in adult cancers but also in paediatric cancers. We evaluate relevant methods to assess the levels and extent of hypoxia in childhood cancers, including novel imaging strategies such as oxygen-enhanced magnetic resonance imaging (MRI). Preclinical and clinical evidence largely supports the use of hypoxia-targeting drugs in children, and we describe the critical need to identify robust predictive biomarkers for the use of such drugs in future paediatric clinical trials. Ultimately, a more personalised approach to treatment that includes targeting hypoxic tumour cells might improve outcomes in subgroups of paediatric cancer patients.Subject terms: Tumour biomarkers, Paediatric cancer, Cancer microenvironment, Cancer therapy     

Tumour Oxygenation: The Importance of Hypoxia,Anemia, and Angiogenesis in Radiation Therapy

It is known that tumour oxygenation levels play a role in the success of cancer treatment, especially radiation therapy. Current research continues to show that tumour hypoxia adversely affects patient outcome and is focused on finding methods of improving tumour oxygen levels or using poor oxygenation as a tumour-specific characteristic in order to develop new therapeutic methods.The radiobiology of oxygen in radiation-induced cell eradication will be discussed, as well as the effects of tumour hypoxia on malignant progression and metastases. There are currently methods to improve poor oxygenation, or drugs can be used that mimic the effect of oxygen in the cell, or target other therapies to the hypoxic cells while sparing normal tissues.Anemia and angiogenesis are related topics that are important to tumour oxygenation and patient outcome. Novel ideas on improving anemia and influencing tumour angiogenesis to increase treatment success have also been included.     

Relationship Between Tumour Oxygenation, Bioenergetic Status and Radiobiological Hypoxia in an Experimental Model

Tumour oxygenation and bioenergetic status were measured in the same tumour and these results related to radiobiological hypoxia. A C3H mouse mammary carcinoma grown in the feet of CDF1 mice was used. Bioenergetic status was assessed by ³¹P MRS using a SISCO 7 Tesla magnet, oxygen measurements were done by a polarographic electrode and the hypoxic fraction was determined from direct analysis of the radiation dose-response data. During all examinations restrained, non-anaesthetized mice were allowed to breathe either 100% oxygen, carbogen, normal air, carbon monoxide (CO) at 75, 220, or 660 ppm or had blood flow occluded by clamping. Results showed a significant correlation between the radiobiological hypoxic fraction and % pO₂ ≤5 mmHg under the different treatment conditions, whereas no correlation was found between beta nucleosidetriphosphate/inorganic phosphate (β-NTP/Pi) ratio and either the hypoxic fraction or the % of pO₂ values ≤5 mmHg under the different treatment conditions. In conclusion, oxygen electrode measurements were sensitive to changes in tumour hypoxia whereas the bioenergetic status alone seemed to be a less precise measure of hypoxia in this tumour model. Furthermore, the present study demonstrated that tumour cells in vivo can actually maintain the bioenergetic status during a period of severe hypoxia.     

Hypoxia and its impact on the tumour microenvironment of gastroesophageal cancers

The malfeasant role of the hypoxic tumour microenvironment (TME) in cancer progression was recognized decades ago but the exact mechanisms that augment the hallmarks of cancer and promote treatment resistance continue to be elucidated. Gastroesophageal cancers (GOCs) represent a major burden of worldwide disease, responsible for the deaths of over 1 million people annually. Disentangling the impact of hypoxia in GOCs enables a better overall understanding of the disease pathogenesis while shining a light on novel therapeutic strategies and facilitating precision treatment approaches with the ultimate goal of improving outcomes for patients with these diseases. This review discusses the underlying principles and processes of the hypoxic response and the effect of hypoxia in promoting the hallmarks of cancer in the context of GOCs. We focus on its bidirectional influence on inflammation and how it drives angiogenesis, innate and adaptive immune evasion, metastasis, and the reprogramming of cellular bioenergetics. The contribution of the hypoxic GOC TME to treatment resistance is examined and a brief overview of the pharmacodynamics of hypoxia-targeted therapeutics is given. The principal methods that are used in measuring hypoxia and how they may enhance prognostication or provide rationale for individually tailored management in the case of tumours with significant hypoxic regions are also discussed.     

Hypoxia and oxidative stress. Tumour hypoxia--therapeutic considerations

Conclusive research has shown that regions of acute/chronic hypoxia, which exist within the majority of solid tumours, have a profound influence on the therapeutic outcome of cancer chemotherapy and radiotherapy and are a strong prognostic factor of disease progression and survival. A strong argument therefore exists for assessing the hypoxic fraction of tumours, prior to patient treatment, and to tailor this treatment accordingly. Tumour hypoxia also provides a powerful physiological stimulus that can be exploited as a tumour-specific condition, allowing for the rationale design of hypoxia-activated anticancer drugs or novel hypoxia-regulated gene therapy strategies.     

Hypoxia imaging using Positron Emission Tomography in non-small cell lung cancer: Implications for radiotherapy

Tumour hypoxia is an important contributor to radioresistance. Thus, increasing the radiation dose to hypoxic areas may result in improved locoregional tumour control. However, this strategy requires accurate detection of the hypoxic sub-volume using PET imaging. Secondly, hypoxia imaging may also provide prognostic information and may be of help to monitor treatment response. Therefore, a systematic review of the scientific literature was carried out on the use of Positron Emission Tomography (PET) to image Tumour hypoxia in non-small cell lung cancer (NSCLC). More specifically, the purpose of this review was (1) to summarize the different hypoxia tracers used, (2) to investigate whether Tumour hypoxia can be detected in NSCLC and finally (3) whether the presence of hypoxia can be used to predict outcome.     

Hypoxia in human soft tissue sarcomas: adverse impact on survival and no association with p53 mutations

Clinical and experimental studies have suggested that tumour hypoxia is associated with poor treatment outcome and that loss of apoptotic potential may play a role in malignant progression of neoplastic cells. The tumour suppressor gene p53 induces apoptosis under certain conditions and microenvironmental tumour hypoxia may select for mutant tumour cells with diminished apoptotic potential due to lack of p53 function. The aim of this study was to evaluate the prognostic relevance of oxygenation status for treatment outcome and to compare pre-treatment tumour oxygenation measurements were done in 31 of those by PCR using DNA extracted from paraffin-embaedded sections (n = 2) or frozen biopsies (n = 29). The overall median of the tumour median pO(2)was 19 mmHg (range 1-58 mmHg). Only 6 tumours had functional p53 mutations and no association was found between mutant p53 and tumour hypoxia. Five out of 6 STS with lower histopathological grade were well-oxygenated whereas high-grade STS were both hypoxic and well-oxygenated. At a median follow-up of 74 months, 16 patients were still alive among 28 available for survival analysis. When stratifying into hypoxic and well-oxygenated tumours patients with the most hypoxic tumours has a statistically poorer disease-specific and overall survival at 5 years. In conclusion hypoxia was an indicator for both a poorer disease specific and overall survival in human STS but hypoxic tumours were not characterized by mutations in the p53 gene.     

Hypoxia in larynx carcinomas assessed by pimonidazole binding and the value of CA-IX and vascularity as surrogate markers of hypoxia

Tumour hypoxia as driving force in tumour progression and treatment resistance has been well established. Assessment of oxygenation status of tumours may provide important prognostic information and improve selection of patients for treatment. In this study, a large homogenous group of 103 laryngeal carcinomas has been investigated in the presence of hypoxia by pimonidazole binding and the usefulness of Carbonic anhydrase IX (CA-IX) and vascular parameters as surrogate markers of hypoxia. These parameters are further related to clinical and biological characteristics.One hundred and three patients with T2–T4 larynx carcinoma were included. They were given the hypoxia marker pimonidazole intravenously (i.v.) 2 h prior to taking a biopsy. Expression of all the parameters was examined by immunohistochemistry, excluding large necrotic areas. Among tumours a large variation in pimonidazole positivity (hypoxic fraction based on pimonidazole, HFpimo) (range 0–19%) and CA-IX expression (hypoxic fraction based on CA-IX staining, HFCA-IX) (range 0–34%) was observed. In 67% of the tumours, hypoxia involved 1% of the viable tumour area. HFpimo and HFCA-IX correlated significantly albeit weak (p = 0.04). Both parameters showed weak inverse correlations with the relative vascular area (RVA) (p = 0.01). HFpimo was further associated with histopathological grade, with poorly differentiated tumours being more hypoxic. The fraction of the tumour area positive for both pimonidazole and CA-IX correlated significantly with N stage.From these results, it was concluded that CA-IX and RVA have only limited value for measuring hypoxia and are not as robust as pimonidazole, probably due to the influence of other factors in the microenvironment. A combination of staining patterns of exogenous and endogenous markers might give important additive information about tumour biology and behaviour.     

Therapeutic Modification of Hypoxia

Regions of reduced oxygenation (hypoxia) are a characteristic feature of virtually all animal and human solid tumours. Numerous preclinical studies, both in vitro and in vivo, have shown that decreasing oxygen concentration induces resistance to radiation. Importantly, hypoxia in human tumours is a negative indicator of radiotherapy outcome. Hypoxia also contributes to resistance to other cancer therapeutics, including immunotherapy, and increases malignant progression as well as cancer cell dissemination. Consequently, substantial effort has been made to detect hypoxia in human tumours and identify realistic approaches to overcome hypoxia and improve cancer therapy outcomes. Hypoxia-targeting strategies include improving oxygen availability, sensitising hypoxic cells to radiation, preferentially killing these cells, locating the hypoxic regions in tumours and increasing the radiation dose to those areas, or applying high energy transfer radiation, which is less affected by hypoxia. Despite numerous clinical studies with each of these hypoxia-modifying approaches, many of which improved both local tumour control and overall survival, hypoxic modification has not been established in routine clinical practice. Here we review the background and significance of hypoxia, how it can be imaged clinically and focus on the various hypoxia-modifying techniques that have undergone, or are currently in, clinical evaluation.     

Hypoxia and oxidative stress in breast cancer. Hypoxia and tumourigenesis

The microenvironmental hypoxia that arises as a consequence of the development of a solid tumour also acts to promote tumour growth. Hypoxia induces the expression of key components of the angiogenic and apoptotic signalling cascades, the glycolytic pathway and various cell-cycle control proteins. At the cellular level it mediates the infiltration and accumulation of tumour-associated macrophages within avascular tumour regions. Complex interactions between tumour cell and macrophage hypoxia-regulated gene products and their associated pathways form the basis for the hypoxic promotion of tumourigenesis and malignant progression.     

Contribution of Transient Blood Flow to Tumour Hypoxia in Mice

Tumours grown in mice typically exhibit regions of hypoxia believed to result from two different processes: chronic oxygen deprivation due to consumption/diffusion limitations, and periodic deprivation resulting from transient reductions in tumour blood flow. The relative contribution of each is, however, not generally known. We have addressed this issue in transplanted SCCVII squamous cell carcinomas in C3H mice, using a quantitative extension of the fluorescence 'mismatch' technique coupled with cell sorting from irradiated tumours. At least half of the vessels in these tumours exhibit transient perfusion changes. Additionally, a majority of the 15-20% of cells that are sufficiently hypoxic to be resistant to radiation in the SCCVII tumours appear to result from cyclic, not continuous (diffusion-limited) hypoxia. Since different strategies may be necessary to counteract cyclic hypoxia in tumours, the possibility of transient blood flow changes should not be ignored when planning cancer therapy for humans.     

Alveolar Hypoxia Promotes Murine Lung Tumor Growth through a VEGFR-2/EGFR-Dependent Mechanism

Patients with chronic obstructive pulmonary disease (COPD) are at an increased risk for the development of lung cancer, the mechanisms for which are incompletely understood. We hypothesized that the hypoxic pulmonary microenvironment present in COPD would augment lung carcinogenesis. Mice were subjected to chemical carcinogenesis protocols and placed in either hypoxia or normoxia. Mice exposed to chronic hypoxia developed tumors with increased volume compared with normoxic controls. Both lungs and tumors from hypoxic mice showed a preferential stabilization of HIF-2α and increased expression of VEGF-A, FGF2, and their receptors as well as other survival, proliferation, and angiogenic signaling pathways regulated by HIF-2α. We showed that tumors arising in hypoxic animals have increased sensitivity to VEGFR-2/EGFR inhibition, as chemoprevention with vandetanib showed markedly increased activity in hypoxic mice. These studies showed that lung tumors arising in a hypoxic microenvironment express increased growth, angiogenic, and survival signaling that could contribute to the increased lung cancer risk in COPD. Furthermore, the differential sensitivity of tumors arising in hypoxia to VEGFR-2/EGFR inhibition suggests that the altered signaling present in tumors arising in hypoxic lung might be therapeutically exploited in patients with underlying COPD. Cancer Prev Res; 5(8); 1061-71. ?2012 AACR.     

Hypoxia and tumor response to irradiation

In radiobiology, the hypoxic cells have been shown to be more resistant to irradiation as compared to cells irradiated in conditions of normal oxygenation. Moreover, most solid tumors contain foci of clonogenic cells able to cause the failure of radiotherapy and the development of more aggressive phenotypes. To-date, numerous techniques allow the detection and quantitation of tumor hypoxia in many tumors and to correlate it with the clinical course. The impact of tumor hypoxia is evident from a careful analysis of data of several trials where the solution of the problem was attempted. In recent years, new functional imaging procedures, assays of molecular biology and new drugs that act directly on hypoxic cells, have been introduced. In the near future, this might lead to the control of this negative clinical impact of hypoxia.     

Progression and metastasis in a transgenic mouse breast cancer model: Effects of exposure to in vivo hypoxia

Hypoxia is a predictor of poor patient survival in several cancers, including breast carcinomas. One possible mechanism is genomic instability induced by oxic stress. In this study we examined this possible mechanism by exposing an in vivo breast cancer model to hypoxia/reoxygenation. MMTV-Neu transgenic mice were exposed to cycling acute (AH) or chronic hypoxia (CH) before (early) or after (late) tumour detection to study effects of hypoxia on tumour initiation and progression, respectively. We observed no effect of the hypoxic exposures on times to first tumour detection, but we saw a trend of early AH-exposed mice to develop more tumours and macrometastases than CH-exposed mice. Unexpectedly, but consistent with these findings, we observed significantly reduced 8-oxo-dG lesions levels in the mammary tissue with the greatest difference observed between the air control (AC) and AH-exposed groups. In the late gassing group, there was a similar trend for reduced 8-oxo-dG lesion levels, but interestingly mice that developed macroscopic lung metastases demonstrated significantly increased levels of 8-oxo-dG lesions in their tumours relative to those that did not, irrespective of the gassing exposure. A trend for increased macrophage content was observed in tumours from mice exposed to acute hypoxia. Our results indicate that oxic stress induced by hypoxia/reoxygenation is unlikely to be a major factor driving tumour progression of established MMTV-Neu tumours but suggest that acute and chronic hypoxia may affect tumour incidence and metastasis when applied prior to tumour development.     

Hypoxia gene expression signatures as prognostic and predictive markers in head and neck radiotherapy

Reliable methods for identification of hypoxia in radiotherapy-treated tumors have been a desirable aim in radiation oncology for decades. Hypoxia is a common feature of the microenvironment in solid tumors, and it is associated with increased aggressiveness, reduced therapeutic response, and a poorer clinical outcome. In head and neck squamous cell carcinomas, the negative effect of hypoxia on radiotherapeutic response can be counteracted and minimized by applying hypoxic modification to radiotherapy, which favors the clinical outcome after treatment. However, not all tumors are hypoxic, hence not all patients benefit from the addition of hypoxic modification. Therefore, predictive and clinically applicable methods for pretherapeutic hypoxic evaluation and categorization are needed. Hypoxia gene expression signatures are a developing strategy to approach this obstacle. This method has evolved along with the development of complementary DNA microarray analysis and classifies tumors in accordance to the expression of specific hypoxia-responsive genes in the tumor biopsy. Thus, tumors are classified and categorized in terms of the biological behavior to hypoxic conditions in the microenvironment. Until now, most of the developed hypoxia signatures have only been evaluated in terms of their prognostic impact; however, recently, a predictive impact for hypoxic modification of radiotherapy was verified. Here, we provide an overview of the hypoxic issue in radiotherapy and present the most promising hypoxia gene expression signatures developed to date.     

Hypoxia Generates a More Invasive Phenotype of Tumour Cells: An In Vivo Experimental Setup Based on the Chorioallantoic Membrane

Of all processes involved in carcinogenesis, local invasion and the formation of metastases are the clinically most relevant but the scientifically least well understood at their molecular level. Recent experimental progress has identified that tumour hypoxia not only induces tumour angiogenesis, but also modulates the expression of several genes that have been implicated in tumour invasion and metastasis. Here we developed an in vivo model to understand a number of molecular pathways and cellular mechanisms for tumour invasion in hypoxia. For this purpose fertilized chicken eggs were incubated for 10 days in normoxic conditions. Subsequently colon carcinoma cells (SW-480) were placed on the chorioallantoic membrane. During the following 6 days the eggs were incubated either in normoxic conditions or in stepwise decreasing hypoxic conditions. SW-480 colon carcinoma cells did not invade the epithelial layer in normoxic conditions. In contrast an invasion through the epithelial layer in to the mesoderm was already seen after 3 days when incubated in hypoxic conditions. The chorioallantoic membrane assay described in this paper allows investigating tumour invasion and its cellular mechanisms under defined hypoxic conditions.     

Reducing Acute and Chronic Hypoxia in Tumours by Combining Nicotinamide with Carbogen Breathing

The ability of nicotinamide and carbogen breathing to improve the radiation response of a C3H mammary carcinoma by reducing both acute and chronic hypoxia was investigated. Using a tumour growth delay assay the response of 200 mm³ foot tumours to local irradiation was found to be increased by either injecting nicotinamide (100-1 000mg/kg) 20 min prior to irradiation, or by allowing mice to breathe carbogen for 10 min before and during the radiation treatment. The greatest radiosensitization occurred when nicotinamide and carbogen were combined. With a histological fluorescent staining technique nicotinamide was shown to prevent transient stoppages in microregional blood flow, and also appeared to improve tumour oxygenation as measured with an Eppendorf oxygen electrode, both effects being consistent with its ability to decrease perfusion limited acute hypoxia. Carbogen had no effect on vessel closure, but it significantly improved tumour oxygenation, which was indicative of it reducing diffusion limited chronic hypoxia.     

Microenvironmental adaptation of experimental tumours to chronic vs acute hypoxia

This study investigated long-term microenvironmental responses (oxygenation, perfusion, metabolic status, proliferation, vascular endothelial growth factor (VEGF) expression and vascularisation) to chronic hypoxia in experimental tumours. Experiments were performed using s.c.-implanted DS-sarcomas in rats. In order to induce more pronounced tumour hypoxia, one group of animals was housed in a hypoxic atmosphere (8% O(2)) for the whole period of tumour growth (chronic hypoxia). A second group was acutely exposed to inspiratory hypoxia for only 20 min prior to the measurements (acute hypoxia), whereas animals housed under normal atmospheric conditions served as controls. Acute hypoxia reduced the median oxygen partial pressure (pO(2)) dramatically (1 vs 10 mmHg in controls), whereas in chronically hypoxic tumours the pO(2) was significantly improved (median pO(2)=4 mmHg), however not reaching the control level. These findings reflect the changes in tumour perfusion where acutely hypoxic tumours show a dramatic reduction of perfused tumour vessels (maybe the result of a simultaneous reduction in arterial blood pressure). In animals under chronic inspiratory hypoxia, the number of perfused vessels increased (compared to acute hypoxia), although the perfusion pattern found in control tumours was not reached. In the chronically hypoxic animals, tumour cell proliferation and tumour growth were significantly reduced, whereas no differences in VEGF expression and vascular density between these groups were observed. These results suggest that long-term adaptation of tumours to chronic hypoxia in vivo, while not affecting vascularity, does influence the functional status of the microvessels in favour of a more homogeneous perfusion.