Synthetic protein protease sensor platform.

Introduction: Protease activity can serve as a highly specific biomarker for application in health, biotech, and beyond. The aim of this study was to develop a protease cleavable synthetic protein platform to detect protease activity in a rapid cell-free setting. Methods: The protease sensor is modular, with orthogonal peptide tags at the N and C terminal ends, which can be uncoupled via a protease responsive module located in between. The sensor design allows for several different readouts of cleavage signal. A protein 'backbone' [Green fluorescent protein (GFP)] was designed in silico to have both a C-terminal Flag-tag and N-Terminal 6x histidine tag (HIS) for antibody detection. A protease cleavage site, which can be adapted for any known protease cleavage sequence, enables the uncoupling of the peptide tags. Three different proteases-Tobacco, Etch Virus (TEV), the main protease from coronavirus SARS-COV-2 (Mpro) and Matrix Metallopeptidase 9 (MMP9)-a cancer-selective human protease-were examined. A sandwich Enzyme-Linked Immunosorbent Assay (ELISA) was developed based on antibodies against the HIS and Flag tags. As an alternative readout, a C-terminal quencher peptide separable by protease cleavage from the GFP was also included. Purified proteins were deployed in cell-free cleavage assays with their respective protease. Western blots, fluorescence assays and immunoassay were performed on samples. Results: Following the design, build and validation of protein constructs, specific protease cleavage was initially demonstrated by Western blot. The novel ELISA proved to afford highly sensitive detection of protease activity in all cases. By way of alternative readout, activation of fluorescence signal upon protease cleavage was also demonstrated but did not match the sensitivity provided by the ELISA method. Discussion: This platform, comprising a protease-responsive synthetic protein device and accompanying readout, is suitable for future deployment in a rapid, low-cost, lateral flow setting. The modular protein device can readily accommodate any desired protease-response module (target protease cleavage site). This study validates the concept with three disparate proteases and applications-human infectious disease, cancer and agricultural crop infection.

Analysis of tumour oxygenation in model animals on a phosphorescence lifetime based macro-imager.

Monitoring of tissue O2 is essential for cancer development and treatment, as hypoxic tumour regions develop resistance to radio- and chemotherapy. We describe a minimally invasive technique for the monitoring of tissue oxygenation in developing grafted tumours, which uses the new phosphorescence lifetime based Tpx3Cam imager. CT26 cells stained with a near-infrared emitting nanoparticulate O2 probe NanO2-IR were injected into mice to produce grafted tumours with characteristic phosphorescence. The tumours were allowed to develop for 3, 7, 10 and 17 days, with O2 imaging experiments performed on live and euthanised animals at different time points. Despite a marked trend towards decreased O2 in dead animals, their tumour areas produced phosphorescence lifetime values between 44 and 47 µs, which corresponded to hypoxic tissue with 5-20 µM O2. After the O2 imaging in animals, confocal Phosphorescence Lifetime Imaging Microscopy was conducted to examine the distribution of NanO2-IR probe in the tumours, which were excised, fixed and sliced for the purpose. The probe remained visible as bright and discrete 'islands' embedded in the tumour tissue until day 17 of tumour growth. Overall, this O2 macro-imaging method using NanO2-IR holds promise for long-term studies with grafted tumours in live animal models, providing quantitative 2D mapping of tissue O2.

Fluorescence Lifetime Macro Imager for Biomedical Applications.

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ranging from solid-state, O2-sensitive coatings to live animal tissue samples stained with soluble O2-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O2 phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability. The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O2 levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O2-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O2 concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

Understanding and harnessing triple-negative breast cancer-related microbiota in oncology.

Bacterial inhabitants of the body have the potential to play a role in various stages of cancer initiation, progression, and treatment. These bacteria may be distal to the primary tumour, such as gut microbiota, or local to the tissue, before or after tumour growth. Breast cancer is well studied in this context. Amongst breast cancer types, Triple Negative Breast Cancer (TNBC) is more aggressive, has fewer treatment options than receptor-positive breast cancers, has an overall worse prognosis and higher rates of reoccurrence. Thus, an in-depth understanding of the bacterial influence on TNBC progression and treatment is of high value. In this regard, the Gut Microbiota (GM) can be involved in various stages of tumour progression. It may suppress or promote carcinogenesis through the release of carcinogenic metabolites, sustenance of proinflammatory environments and/or the promotion of epigenetic changes in our genome. It can also mediate metastasis and reoccurrence through interactions with the immune system and has been recently shown to influence chemo-, radio-, and immune-therapies. Furthermore, bacteria have also been found to reside in normal and malignant breast tissue. Several studies have now described the breast and breast tumour microbiome, with the tumour microbiota of TNBC having the least taxonomic diversity among all breast cancer types. Here, specific conditions of the tumour microenvironment (TME) - low O2, leaky vasculature and immune suppression - are supportive of tumour selective bacterial growth. This innate bacterial ability could enable their use as delivery agents for various therapeutics or as diagnostics. This review aims to examine the current knowledge on bacterial relevance to TNBC and potential uses while examining some of the remaining unanswered questions regarding mechanisms underpinning observed effects.

Intratumoural production of TNFα by bacteria mediates cancer therapy.

Systemic administration of the highly potent anticancer therapeutic, tumour necrosis factor alpha (TNFα) induces high levels of toxicity and is responsible for serious side effects. Consequently, tumour targeting is required in order to confine this toxicity within the locality of the tumour. Bacteria have a natural capacity to grow within tumours and deliver therapeutic molecules in a controlled fashion. The non-pathogenic E. coli strain MG1655 was investigated as a tumour targeting system in order to produce TNFα specifically within murine tumours. In vivo bioluminescence imaging studies and ex vivo immunofluorescence analysis demonstrated rapid targeting dynamics and prolonged survival, replication and spread of this bacterial platform within tumours. An engineered TNFα producing construct deployed in mouse models via either intra-tumoural (i.t.) or intravenous (i.v.) administration facilitated robust TNFα production, as evidenced by ELISA of tumour extracts. Tumour growth was impeded in three subcutaneous murine tumour models (CT26 colon, RENCA renal, and TRAMP prostate) as evidenced by tumour volume and survival analyses. A pattern of pro-inflammatory cytokine induction was observed in tumours of treated mice vs. CONTROLS: Mice remained healthy throughout experiments. This study indicates the therapeutic efficacy and safety of TNFα expressing bacteria in vivo, highlighting the potential of non-pathogenic bacteria as a platform for restricting the activity of highly potent cancer agents to tumours.