Neutrophil apoptosis plays a central role in the resolution of granulocytic inflammation. We have shown previously that tumor necrosis factor-␣ (TNF␣) enhances the rate of neutrophil apoptosis at early time points via a mechanism involving both TNF receptor (TNFR) I and TNFRII. Here we reveal a marked but consistent variation in the magnitude of the pro-apoptotic effect of TNF␣ in neutrophils isolated from healthy donors, and we show that inhibition of cell surface aminopeptidase N (APN) using actinonin, bestatin, or inhibitory peptides significantly enhanced the efficacy of TNF␣-induced killing. Notably, an inverse correlation is shown to exist between neutrophil APN activity and the sensitivity of donor cells to TNF␣-induced apoptosis. Inhibition of cell surface APN appears to interfere with the shedding of TNFRI, and as a consequence results in augmented TNF␣-induced apoptosis, cell polarization, and TNF␣-primed, formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst. Of note, actinonin and bestatin had no effect on TNFRII expression under resting or TNF␣-stimulated conditions and did not alter CXCRI or CXCRII expression. These data suggest significant variation in the activity of APN/CD13 on the cell surface of neutrophils in normal individuals and reveal a novel mechanism whereby APN/CD13 regulates TNF␣-induced apoptosis via inhibition of TNFRI shedding. This has therapeutic relevance for driving neutrophil apoptosis in vivo.

The hair follicle is a well vascularised structure that contains rapidly dividing epithelial cells. As a result it is a potentially useful surrogate biomarker for less accessible but highly similar tissues, such as rapidly proliferating tumours. We have developed an ex vivo culture method to validate target expression changes following exposure to DNA damage and/or potential therapeutics. Proof of concept studies performed ex vivo can then inform the design of clinical validation studies. Earlier work evaluated the response of targets such as pERK, pAKT and pSMAD2 to various kinase inhibitors, whilst recently we have used the model to measure the DNA damage response (DDR) following exposure to UV radiation (as a model damaging agent) and more clinically relevant chemotherapeutic agents. Human plucked hair was placed in maintenance media in the presence of known chemotherapeutic agents (such as Gemcitabine, DNA topoisomerase inhibitor I, Irinotecan and Paclitaxel) or exposed to 0.2-0.5J/cm2 UVB radiation. Hairs were then fixed at a range of time points post-exposure and longitudinal sections labelled for various markers including pChk1, gamma-H2AX, p53, Ki67 and thymine dimers (TDM1). Quantitative image analysis to measure the level of labelling was performed using an Aperio® ScanScope®. Following UVB exposure thymine dimer formation occurred immediately on the hair and was stronger on the side directly facing the source of radiation. It was followed rapidly by phosphorylation of Chk1 localised in the inner root sheath at first (10min to 1h after injury), then gamma-H2AX and p53 induction in the outer root sheath. Further studies are looking at DNA damage response in an UV dose dependent manner. Chemotherapeutic agents known for their different mechanism of actions were tested at two concentrations with various responses depending on their mechanism of action. For example, Gemcitabine, which is linked to DNA polymerase inhibition, strongly induced p53 after 24h but no gamma-H2AX activity was seen above background level. We have profiled the large panel of responses for each chemotherapeutic agent, over an acute time course. The ex vivo plucked hair follicle method is a rapid and convenient way of studying the DDR induced by exposure to various DNA damage agents. This model allows for screening of novel chemotherapeutic agents to test their efficacy in preventing or treating DNA damage. Citation Format: Aude-Marine Bonavita, Liam Walker, Adam Boanas, Nicola Tonge, Gregory Tudor, Cath Booth, Ben Reed. Evaluation and quantification of biomarkers of DNA damage in human plucked hair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3601.

Fractionated radiotherapy, administered by high energy external beam X-rays, is the mainstay of several cancer treatment regimens. However, treatment is often required for several weeks, with cure rates limited by radio-resistant hypoxic areas within the tumour, patient compliance due to the frequency of required treatments and cumulatively high total radiation dose that can cause adjacent tissue toxicity. Radiosensitisers are being developed to improve efficacy, but most are still limited by tumour hypoxia. Titanium oxide is a well-known, non-toxic, photoactive material that generates hydroxyl free radicals by water splitting under ultraviolet light. Rare earth doped titanium oxide nanoparticles can be delivered to cancer cells and interact with X-rays and electrons, concentrating free radicals within the cells. Crucially, this response is also effective within hypoxic tumour regions. Proof of concept studies were performed in pancreatic tumour cells PANC-1 irradiated in vitro. Both cell number and clonogenicity were reduced following irradiation in the presence of nanoparticles compared to irradiation alone. This response was then further evaluated in subcutaneously xenografted tumours. Briefly, male CD-1 nude mice were inoculated with either the pancreatic carcinoma, MiaPaCa-2, or the head and neck squamous cell carcinoma, FaDU, cells. Tumours were allowed to grow until palpable and then irradiated with 2Gy daily for 5 days (Mon-Fri) for 1 or 2 weeks (or sham irradiated) to mimic the clinical protocol. Immediately prior to the first irradiation each week tumours were injected with 125μg nanoparticles or vehicle. Tumour growth was then monitored for up to 5 weeks. Injection of the nanoparticles alone had no effect on tumour growth rate, but when combined with irradiation there was up to 65-75% reduction in tumour volume 30 days after the start of treatment. Sections of Carnoy’s fixed tumours taken 3 days (Day 15) after the last irradiation were immunohistochemically labelled with the proliferation marker bromodeoxyuridine (BrdU). Whilst there was a clear radiation response there was little difference in proliferation following co-administration of nanoparticles at this early timepoint, suggesting either surviving radioresistant clones had evolved by two weeks or that the nanoparticle induced tumour growth delay was not simply the result of early cell death/cell cycle interruption but may have delayed effects. These preliminary studies show that titanium oxide nanoparticles are non-toxic and can enhance the efficacy of radiotherapy in pancreatic and head and neck tumour cell lines. Further work will quantify the level of tumour cell death in normal vs hypoxic tumour regions over time, in a range of tumour types with differing levels of radiosensitivity. Citation Format: Ben Reed, Gareth Wakefield, Matt Stock, Gregory Tudor. The use of nanoparticles to improve tumour radiosensitivity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C062. doi:10.1158/1535-7163.TARG-19-C062