Storey, D. | Search bioRxiv

bioRxiv2024

Ex-vivo mouse precision cut tumour slices for modelling hepatocellular carcinoma; A 3Rs solution for at-scale drug screening

Disease modelling is vital for improving knowledge of disease mechanisms and for development of new therapeutic molecules and strategies. Modelling the intact living tumour microenvironment (TME) is increasingly considered to be vital not only for gaining a better understanding of the biology of cancer but for examining the efficacy of novel oncology drugs. To date, pre-clinical mouse models of cancer have represented the mainstay methodology for studying the evolving TME and for determining the effects of potential therapeutic molecules on tumour evolution and growth. Regarding drug screening, in vivo mouse models are expensive, require the use of large cohorts of mice and involve the administration of drugs with unknown toxicities to animals which often result in adverse effects that can cause animal suffering and the discontinuation of drug investigations. Hepatocellular carcinoma (HCC) is a primary cancer of the liver for which there is an urgent need for improved systemic treatments due to the disease usually being diagnosed at an advanced stage and current treatments having limited efficacy. To provide a practical solution to the screening of drugs for their likely efficacy in HCC we have developed an ex-vivo model in which orthotopic tumours are excised from the liver and subsequently processed to generate precision-cut tumour slices (PCTS) which provide an intact culture model of the HCC-TME. We describe simplified culture conditions that maintain the viability and metabolic activity of live PCTS which maintain the architecture, cellular complexity, drug sensitivity and responsiveness to immunotherapy of the original tumour. Importantly, we show that HCC derived PCTS can be miniaturised to 96-well scale and modified to express soluble luciferase, which in combination enabled non-destructive screening of a library of 26 drugs at two doses using just 5 tumours as the source for PCTS. This screen identified two small molecules, salinomycin and rottlerin, that have potent anti-tumour activities in HCC-PCTS and subsequently validated salinomycin as effective in vivo. In summary, we report a 3Rs (reduction, refinement and replacement) solution for study of HCC biology and for 96-well-scale screening of potential therapeutic agents in the context of an intact, metabolically active TME.

cancer biology↗

bioRxiv2019

Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possess an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition, and identified VgrG4 as a T6SS effector. Structurally, domain DUF2345 of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.\n\nAUTHOR SUMMARYKlebsiella pneumoniae has been singled out as an \"urgent threat to human health\" due to extremely drug resistant strains. Numerous studies investigate the molecular mechanisms underlying antibiotic resistance in K. pneumoniae, while others dissect the virulence strategies of this pathogen. However, there is still limited knowledge on the fitness of Klebsiella in the environment, and particularly the competition of Klebsiella with other species. Here, we demonstrated that Klebsiella exploits the type VI secretion system (T6SS) nanoweapon to kill bacterial competitors and fungi. K. pneumoniae perceives T6SS attacks from bacterial competitors, resulting in retaliation against the aggressive cell. The perception of the attack involved the sensor PhoPQ and led to the up-regulation of the T6SS. We identified one of the toxins deployed by the T6SS to antagonize other microbes, and revealed how Klebsiella protects itself from this toxin. Our findings provide a better understanding of the T6SS role in microbial competition and uncover new aspects on how bacteria regulate T6SS-mediated microbial antagonism.

microbiology↗