Purpose Tumor hypoxia imposes a main obstacle to the efficacy of anti-cancer therapy. Understanding the cellular dynamics of individual hypoxic cells before, during and post-treatment has been hampered by the technical inability to identify and trace these cells over time. Methods and materials Here, we present a novel lineage-tracing reporter for hypoxic cells based on the conditional expression of a HIF1a-CreERT2-UnaG biosensor that can visualize hypoxic cells in a time-dependent manner and trace the fate of hypoxic cells over time. We combine this system with multiphoton microscopy, flow cytometry, and immunofluorescence to characterize the role of hypoxic cells in tumor relapse after irradiation in H1299 tumor spheroids and in vivo xenografts. Results We validate the reporter in monolayer cultures and we show that tagged cells colocalize in spheroids and human tumor xenografts with the hypoxic marker pimonidazole. We found that irradiation of H1299-HIFcreUnaG spheroids leads to preferential outgrowth of cells from the hypoxic core. Similarly, in xenografts tumors, although initially UnaG-positive-cells coincide with pimonidazole-positive tumor areas and they are merely quiescent, upon irradiation UnaG-positive cells enrich in regrowing tumors and are mainly proliferative. Conclusions Collectively, our data provide clear evidence that the hypoxic cells drive tumor relapse after irradiation.

Intratumoural hypoxia is a common characteristic of malignant treatment-resistant cancers. However, hypoxia-modification strategies for the clinic remain elusive. To date, little is known on the behaviour of individual hypoxic tumour cells in their microenvironment. To explore this issue in a spatial and temporally controlled manner, we developed a genetically encoded sensor by fusing the O-2-labile hypoxia-inducible factor 1 alpha (HIF-1 alpha) protein to eGFP and a tamoxifen-regulated Cre recombinase. Under normoxic conditions, HIF-1 alpha is degraded but, under hypoxia, the HIF-1 alpha-GFP-Cre-ERT2 fusion protein is stabilised and in the presence of tamoxifen activates a tdTomato reporter gene that is constitutively expressed in hypoxic progeny. We visualise the random distribution of hypoxic tumour cells from hypoxic or necrotic regions and vascularised areas using immunofluorescence and intravital microscopy. Once tdTomato expression is induced, it is stable for at least 4 weeks. Using this system, we could show in vivo that the post-hypoxic cells were more proliferative than non-labelled cells. Our results demonstrate that single-cell lineage tracing of hypoxic tumour cells can allow visualisation of their behaviour in living tumours using intravital microscopy. This tool should prove valuable for the study of dissemination and treatment response of post-hypoxic tumour cells in vivo at single-cell resolution. This article has an associated First Person interview with the joint first authors of the paper.

Radiation pneumonitis (RP) and radiation fibrosis (RF) are two dose-limiting toxicities of radiotherapy (RT), especially for lung, and esophageal cancer. It occurs in 5-20% of patients and limits the maximum dose that can be delivered, reducing tumor control probability (TCP) and may lead to dyspnea, lung fibrosis, and impaired quality of life. Both physical and biological factors determine the normal tissue complication probability (NTCP) by Radiotherapy. A better understanding of the pathophysiological sequence of radiation-induced lung injury (RILI) and the intrinsic, environmental and treatment-related factors may aid in the prevention, and better management of radiation-induced lung damage. In this review, we summarize our current understanding of the pathological and molecular consequences of lung exposure to ionizing radiation, and pharmaceutical interventions that may be beneficial in the prevention or curtailment of RILI, and therefore enable a more durable therapeutic tumor response.

The hypoxia-inducible transcription factors (HIF)-1/2α are the main oxygen sensors which regulate the adaptation to intratumoral hypoxia. The aim of this study was to assess the role of the HIF proteins in regulating the radiation response of a non-small cell lung cancer (NSCLC) in vitro model. To directly assess the unique and overlapping functions of HIF-1α and HIF-2α, we use CRISPR gene-editing to generate isogenic H1299 non-small cell lung carcinoma cells lacking HIF-1α, HIF-2α or both. We found that in HIF1 knockout cells, HIF-2α was strongly induced by hypoxia compared to wild type but the reverse was not seen in HIF2 knockout cells. Cells lacking HIF-1α were more radiation resistant than HIF2 knockout and wildtype cells upon hypoxia, which was associated with a reduced recruitment of γH2AX foci directly after irradiation and not due to differences in proliferation. Conversely, double-HIF1/2 knockout cells were most radiation sensitive and had increased γH2AX recruitment and cell cycle delay. Compensatory HIF-2α activity in HIF1 knockout cells is the main cause of this radioprotective effect. Under hypoxia, HIF1 knockout cells uniquely had a strong increase in lactate production and decrease in extracellular pH. Using genetically identical HIF-α isoform-deficient cells we identified a strong radiosensitizing of HIF1, but not of HIF2, which was associated with a reduced extracellular pH and reduced glycolysis.

Introduction Hypoxia plays a role in many types of cancer, and is linked to metastasis, genetic instability, resistance to therapy and poor prognosis. Hypoxic modification has been tested in clinical trials, but results have been inconclusive, in part due to a knowledge gap into the characterisation and behaviour of hypoxic tumour cells. In this project, hypoxic cells will be lineage traced to investigate their dissemination within the primary tumour and for their presence in circulating tumour cells and distant metastasis. To investigate the contribution of the hypoxic fraction to the tumour resistance, hypoxic cells will be specifically ablated, and the growth and response to treatment of tumours with and without this hypoxic population investigated. Material and methods To accomplish these aims, two complementary systems will be generated in the 4 T1 mouse mammary gland cell line, a model for stage IV metastatic human breast cancer. The first consists of a HIF-1α-CRE-ERTAM ‘hypoxia sensing’ construct coupled with a loxp DsRed loxp DTR-EGFP cassette (Diphtheria Toxin Receptor: DTR). Under normal conditions the HIF-1α-CRE-ERTAM fusion protein is expressed, but immediately degraded, but under hypoxia HIF-1α degradation is inhibited leading to accumulation. 4-OHT Administration translocates HIF-1α-CRE-ERTAM to the nucleus mediating loxp recombination in the coupled cassette. This allows lineage tracing of hypoxic cells and their progeny and selective ablation throught the DTR using Diphthteria toxin. Secondly, a DTR-UnaG-ODDD-HA construct (Oxygen-dependant degradation domain: ODDD), driven by the hypoxia dependent 5XHRE promoter will be used. This system allows ablation of hypoxic cells through the DTR and identification of hypoxic cells through the UnaG fluorescent protein. Only cells that are hypoxic at the time of administration/analysis will be labelled. Both cell lines will be transduced with luciferase to allow identification of distant metastasis. Results and discussions The two systems for this project have been generated and transduced into the 4 T1 cell line. Lineage tracing and selective ablation have been shown to work in vitro and clones are being characterised. In vitro experiments have shown the HIF-1α-CRE-ERTAM system to be extremely tightly regulated in an oxygen and tamoxifen dependant manner. Conclusion Once optimised, both systems will be used in an in vivo orthotopic mouse model to investigate the role of the hypoxic cells in breast cancer, metastasis and response to radiotherapy.

Breast cancer is the second most common malignancy, worldwide. Treatment decisions are based on tumor stage, histological subtype, and receptor expression and include combinations of surgery, radiotherapy, and systemic treatment. These, together with earlier diagnosis, have resulted in increased survival. However, initial treatment efficacy cannot be guaranteed upfront, and these treatments may come with (long-term) serious adverse effects, negatively affecting a patient's quality of life. Gene expression-based tests can accurately estimate the risk of recurrence in early stage breast cancers. Disease recurrence correlates with treatment resistance, creating a major need to resensitize tumors to treatment. Notch signaling is frequently deregulated in cancer and is involved in treatment resistance. Preclinical research has already identified many combinatory therapeutic options where Notch involvement enhances the effectiveness of radiotherapy, chemotherapy or targeted therapies for breast cancer. However, the benefit of targeting Notch has remained clinically inconclusive. In this review, we summarize the current knowledge on targeting the Notch pathway to enhance current treatments for breast cancer and to combat treatment resistance. Furthermore, we propose mechanisms to further exploit Notch-based therapeutics in the treatment of breast cancer.

Background and purposeEsophageal cancer has a persistently low 5-year survival rate and has recently been classified as a cancer of unmet need by Cancer Research UK. Consequently, new approaches to therapy are urgently required. Here, we tested the hypothesis that an ATR inhibitor, VX-970, used in combination with standard therapies for esophageal cancer could improve treatment outcome.Material and methodsUsing esophageal cancer cell lines we evaluated the efficacy of combining VX-970 with cisplatin and carboplatin in vitro and with radiation in vitro and in vivo. Radiation experiments were also carried out in hypoxic conditions to mimic the tumor microenvironment.ResultsCombining VX-970 with cisplatin, carboplatin and radiation increased tumor cell kill in vitro. A significant tumor growth delay was observed when VX-970 was combined with radiotherapy in vivo.ConclusionsVX-970 is an effective chemo/radiosensitizer which could be readily integrated in the current treatment paradigm to improve the treatment response in esophageal cancer and we plan to test it prospectively in the forthcoming phase I dose escalation safety study combining the ATR inhibitor VX-970 with chemoradiotherapy in esophageal cancer (EudraCT number: 2015-003965-27).

Hypoxia-induced replication stress is one of the most physiologically relevant signals known to activate ATM in tumors. Recently, the ATM interactor (ATMIN) was identified as critical for replication stress-induced activation of ATM in response to aphidicolin and hydroxyurea. This suggests an essential role for ATMIN in ATM regulation during hypoxia, which induces replication stress. However, ATMIN also has a role in base excision repair, a process that has been demonstrated to be repressed and less efficient in hypoxic conditions. Here, we demonstrate that ATMIN is dispensable for ATM activation in hypoxia and in contrast to ATM, does not affect cell survival and radiosensitivity in hypoxia. Instead, we show that in hypoxic conditions ATMIN expression is repressed. Repression of ATMIN in hypoxia is mediated by both p53 and HIF-1α in an oxygen dependent manner. The biological consequence of ATMIN repression in hypoxia is decreased expression of the target gene, DYNLL1. An expression signature associated with p53 activity was negatively correlated with DYNLL1 expression in patient samples further supporting the p53 dependent repression of DYNLL1. Together, these data demonstrate multiple mechanisms of ATMIN repression in hypoxia with consequences including impaired BER and down regulation of the ATMIN transcriptional target, DYNLL1.

In the last few decades, epigenetics has emerged as an exciting new field in development and disease, with a more recent focus towards cancer. Epigenetics has classically referred to heritable patterns of gene expression, primarily mediated through DNA methylation patterns. More recently, it has come to include the reversible chemical modification of histones and DNA that dictate gene expression patterns. Both the epigenetic up-regulation of oncogenes and downregulation of tumor suppressors have been shown to drive tumor development. Current clinical trials for cancer therapy include pharmacological inhibition of DNA methylation and histone deacetylation, with the aim of reversing these cancer-promoting epigenetic changes. However, the DNA methyltransferase and histone deacetylase inhibitors have met with less than promising results in the treatment of solid tumors. Regions of hypoxia are a common occurrence in solid tumors. Tumor hypoxia is associated with increased aggressiveness and therapy resistance, and importantly, hypoxic tumor cells have a distinct epigenetic profile. In this review, we provide a summary of the recent clinical trials using epigenetic drugs in solid tumors, discuss the hypoxia-induced epigenetic changes and highlight the importance of testing the epigenetic drugs for efficacy against the most aggressive hypoxic fraction of the tumor in future preclinical testing.