Isolation of Live Immune Cells from the Tumor Microenvironment by FACS.

Isolation of live cells from the tumor microenvironment (TME) has represented a challenge, particularly from metastatic nodules that need to be identified within the entire metastatic tissue. Cherry-niche, an in vivo labelling technique, allows the isolation of all the different cell populations in the TME without needing to visually locate the metastatic cancer cell colonies. Therefore, neighboring TME cells can be isolated even from the early stages of cancer cell seeding and colonization in the metastatic tissue. Here, we show how to use Cherry-niche to identify and isolate neutrophils from the lung metastatic niche. We also provide examples of downstream analyses to characterize freshly isolated neutrophils ex vivo, such as Giemsa staining, reactive oxygen species (ROS) detection, and phagocytosis assays. Similar strategies can be used to isolate other immune and non-immune cells from the metastatic TME.

A prospective, phase II, single-centre, cross-sectional, randomised study investigating Dehydroepiandrosterone supplementation and its Profile in Trauma: ADaPT.

INTRODUCTION: The improvements in short-term outcome after severe trauma achieved through early resuscitation and acute care can be offset over the following weeks by an acute systemic inflammatory response with immuneparesis leading to infection, multiorgan dysfunction/multiorgan failure (MOF) and death. Serum levels of the androgen precursor dehydroepiandrosterone (DHEA) and its sulfate ester DHEAS, steroids with immune-enhancing activity, are low after traumatic injury at a time when patients are catabolic and immunosuppressed. Addressing this deficit and restoring the DHEA(S) ratio to cortisol may provide a range of physiological benefits, including immune modulatory effects. OBJECTIVE: Our primary objective is to establish a dose suitable for DHEA supplementation in patients after acute trauma to raise circulating DHEA levels to at least 15 nmol/L. Secondary objectives are to assess if DHEA supplementation has any effect on neutrophil function, metabolic and cytokine profiles and which route of administration (oral vs sublingual) is more effective in restoring circulating levels of DHEA, DHEAS and downstream androgens. METHODS AND ANALYSIS: A prospective, phase II, single-centre, cross-sectional, randomised study investigating Dehydroepiandrosterone supplementation and its profile in trauma, with a planned recruitment between April 2019 and July 2021, that will investigate DHEA supplementation and its effect on serum DHEA, DHEAS and downstream androgens in trauma. A maximum of 270 patients will receive sublingual or oral DHEA at 50, 100 or 200 mg daily over 3 days. Females aged ≥50 years with neck of femur fracture and male and female major trauma patients, aged 16-50 years with an injury severity score ≥16, will be recruited. ETHICS AND DISSEMINATION: This protocol was approved by the West Midlands - Coventry and Warwickshire Research Ethics Committee (Reference 18/WM/0102) on 8 June 2018. Results will be disseminated via peer-reviewed publications and presented at national and international conferences. TRIAL REGISTRATION: This trial is registered with the European Medicines Agency (EudraCT: 2016-004250-15) and ISRCTN (12961998). It has also been adopted on the National Institute of Health Research portfolio (CPMS ID:38158). TRIAL PROGRESSION: The study recruited its first patient on 2 April 2019 and held its first data monitoring committee on 8 November 2019. DHEA dosing has increased to 100 mg in both male cohorts and remains on 50 mg in across all female groups.

Treating age-related multimorbidity: the drug discovery challenge.

Advanced age is the major risk factor for multimorbidity. Current clinical practice treats the individual age-related diseases, resulting in polypharmacy. Thus, targeting the biological processes that drive ageing could prevent both multimorbidity and polypharmacy.