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.

Fusobacterium nucleatum: a novel immune modulator in breast cancer?

Breast cancer was the most commonly diagnosed cancer worldwide in 2020. Greater understanding of the factors which promote tumour progression, metastatic development and therapeutic resistance is needed. In recent years, a distinct microbiome has been detected in the breast, a site previously thought to be sterile. Here, we review the clinical and molecular relevance of the oral anaerobic bacterium Fusobacterium nucleatum in breast cancer. F. nucleatum is enriched in breast tumour tissue compared with matched healthy tissue and has been shown to promote mammary tumour growth and metastatic progression in mouse models. Current literature suggests that F. nucleatum modulates immune escape and inflammation within the tissue microenvironment, two well-defined hallmarks of cancer. Furthermore, the microbiome, and F. nucleatum specifically, has been shown to affect patient response to therapy including immune checkpoint inhibitors. These findings highlight areas of future research needed to better understand the influence of F. nucleatum in the development and treatment of breast cancer.

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.

Identification of the Antigens Recognised by Colorectal Cancer Patients Using Sera from Patients Who Exhibit a Crohn's-like Lymphoid Reaction.

A Crohn's-like lymphoid reaction (CLR) is observed in about 15% of colorectal cancer (CRC) patients and is associated with favourable outcomes. To identify the immune targets recognised by CRC CLR patient sera, we immunoscreened a testes cDNA library with sera from three patients. Immunoscreening of the 18 antigens identified by SEREX with sera from normal donors showed that only the heavy chain of IgG3 (IGHG3) and a novel antigen we named UOB-COL-7, were solely recognised by sera from CRC CLR patients. ELISA showed an elevation in IgG3 levels in patients with CRC (p = 0.01). To extend our studies we analysed the expression of our SEREX-identified antigens using the RNA-sequencing dataset (GSE5206). We found that the transcript levels of multiple IGHG probesets were highly significant (p < 0.001) in their association with clinical features of CRC while above median levels of DAPK1 (p = 0.005) and below median levels of GTF2H5 (p = 0.004) and SH3RF2 (p = 0.02) were associated with improved overall survival. Our findings demonstrate the potential of SEREX-identified CRC CLR antigens to act as biomarkers for CRC and provide a rationale for their further characterization and validation.

Biopsy bacterial signature can predict patient tissue malignancy.

Considerable recent research has indicated the presence of bacteria in a variety of human tumours and matched normal tissue. Rather than focusing on further identification of bacteria within tumour samples, we reversed the hypothesis to query if establishing the bacterial profile of a tissue biopsy could reveal its histology / malignancy status. The aim of the present study was therefore to differentiate between malignant and non-malignant fresh breast biopsy specimens, collected specifically for this purpose, based on bacterial sequence data alone. Fresh tissue biopsies were obtained from breast cancer patients and subjected to 16S rRNA gene sequencing. Progressive microbiological and bioinformatic contamination control practices were imparted at all points of specimen handling and bioinformatic manipulation. Differences in breast tumour and matched normal tissues were probed using a variety of statistical and machine-learning-based strategies. Breast tumour and matched normal tissue microbiome profiles proved sufficiently different to indicate that a classification strategy using bacterial biomarkers could be effective. Leave-one-out cross-validation of the predictive model confirmed the ability to identify malignant breast tissue from its bacterial signature with 84.78% accuracy, with a corresponding area under the receiver operating characteristic curve of 0.888. This study provides proof-of-concept data, from fit-for-purpose study material, on the potential to use the bacterial signature of tissue biopsies to identify their malignancy status.

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis.

The targeted sequencing of the 16S rRNA gene is one of the most frequently employed techniques in the field of microbial ecology, with the bacterial communities of a wide variety of niches in the human body have been characterised in this way. This is performed by targeting one or more hypervariable (V) regions within the 16S rRNA gene in order to produce an amplicon suitable in size for next generation sequencing. To date, all technical research has focused on the ability of different V regions to accurately resolve the composition of bacterial communities. We present here an underreported artefact associated with 16S rRNA gene sequencing, namely the off-target amplification of human DNA. By analysing 16S rRNA gene sequencing data from a selection of human sites we highlighted samples susceptible to this off-target amplification when using the popular primer pair targeting the V3-V4 region of the gene. The most severely affected sample type identified (breast tumour samples) were then re-analysed using the V1-V2 primer set, showing considerable reduction in off target amplification. Our data indicate that human biopsy samples should preferably be amplified using primers targeting the V1-V2 region. It is shown here that these primers result in on average 80% less human genome aligning reads, allowing for more statistically significant analysis of the bacterial communities residing in these samples.

A novel cell permeability assay for macromolecules.

BACKGROUND: Many cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed. However, no size-specific permeability assay suitable for both cell types exists. RESULTS: We report the use of intrinsically biotinylated cell components as the target for reporter molecules for assessing permeabilisation. Due to its well-described biotin binding activity, we developed an assay using Streptavidin (SAv) as a molecular weight marker for assessing eukaryotic and prokaryotic cell internalisation, using flow cytometry as a readout. This concept was tested here as part of the development of host DNA depletion strategies for microbiome analysis of formalin-fixed (FF) samples. Host depletion (HD) strategies require differential cell permeabilisation, where mammalian cells but not bacterial cells are permeabilised, and are subsequently treated with a nuclease. Here, the internalisation of a SAv-conjugate was used as a reference for nucleases of similar dimensions. With this assay, it was possible to demonstrate that formalin fixation does not generate pores which allow the introduction of 60 KDa molecules in mammalian or bacterial membranes/envelopes. Among surfactants tested, Saponin derived from Quillaja bark showed the best selectivity for mammalian cell permeabilisation, which, when coupled with Benzonase nuclease, provided the best results for host DNA depletion, representing a new HD strategy for formalin fixed samples. CONCLUSION: The assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.

Sequence-Based Characterization of Intratumoral Bacteria-A Guide to Best Practice.

Tumors are hospitable environments to bacteria and several recent studies on cancer patient samples have introduced the concept of an endogenous tumor microbiome. For a variety of reasons, this putative tumor microbiome is particularly challenging to investigate, and a failure to account for the various potential pitfalls will result in erroneous results and claims. Before this potentially extremely medically-significant habitat can be accurately characterized, a clear understanding of all potential confounding factors is required, and a best-practice approach should be developed and adopted. This review summarizes all of the potential issues confounding accurate bacterial DNA sequence analysis of the putative tumor microbiome, and offers solutions based on related research with the hope of assisting in the progression of research in this field.

Resident bacteria in breast cancer tissue: pathogenic agents or harmless commensals?

Breast cancer is the second most common cancer in women. Recent evidence identifies a unique microbiome in breast tissue; a site previously thought to be sterile. The identification that this microbiome varies considerably from healthy subjects to cancer patients has prompted investigations into the role of specific bacterial species in oncogenesis. Indeed, certain bacteria have been shown to aid cancer development in vitro by promoting genomic instability, invasion, and chemotherapy resistance. However, the in vivo role of the breast microbiome in cancer appears to be more complex, involving numerous interactions between its constituent species and host cells. As such, reduced abundances of species which exert a protective effect against oncogenesis have come into focus and there is an emerging consensus that states of microbial dysbiosis, in which the normal balance of bacterial species is altered, can contribute to the development of cancer. This review summarizes the findings to date from the available literature pertaining to the microbiome in breast cancer and outlines areas worthy of further investigation.

In situ biomolecule production by bacteria; a synthetic biology approach to medicine.

The ability to modify existing microbiota at different sites presents enormous potential for local or indirect management of various diseases. Because bacteria can be maintained for lengthy periods in various regions of the body, they represent a platform with enormous potential for targeted production of biomolecules, which offer tremendous promise for therapeutic and diagnostic approaches for various diseases. While biological medicines are currently limited in the clinic to patient administration of exogenously produced biomolecules from engineered cells, in situ production of biomolecules presents enormous scope in medicine and beyond. The slow pace and high expense of traditional research approaches has particularly hampered the development of biological medicines. It may be argued that bacterial-based medicine has been "waiting" for the advent of enabling technology. We propose that this technology is Synthetic Biology, and that the wait is over. Synthetic Biology facilitates a systematic approach to programming living entities and/or their products, using an approach to Research and Development (R&D) that facilitates rapid, cheap, accessible, yet sophisticated product development. Full engagement with the Synthetic Biology approach to R&D can unlock the potential for bacteria as medicines for cancer and other indications. In this review, we describe how by employing Synthetic Biology, designer bugs can be used as drugs, drug-production factories or diagnostic devices, using oncology as an exemplar for the concept of in situ biomolecule production in medicine.

Designer bacteria as intratumoural enzyme biofactories.

Bacterial-directed enzyme prodrug therapy (BDEPT) is an emerging form of treatment for cancer. It is a biphasic variant of gene therapy in which a bacterium, armed with an enzyme that can convert an inert prodrug into a cytotoxic compound, induces tumour cell death following tumour-specific prodrug activation. BDEPT combines the innate ability of bacteria to selectively proliferate in tumours, with the capacity of prodrugs to undergo contained, compartmentalised conversion into active metabolites in vivo. Although BDEPT has undergone clinical testing, it has received limited clinical exposure, and has yet to achieve regulatory approval. In this article, we review BDEPT from the system designer's perspective, and provide detailed commentary on how the designer should strategize its development de novo. We report on contemporary advancements in this field which aim to enhance BDEPT in terms of safety and efficacy. Finally, we discuss clinical and regulatory barriers facing BDEPT, and propose promising approaches through which these hurdles may best be tackled.

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.

The Microbiota of Breast Tissue and Its Association with Breast Cancer.

UNLABELLED: In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime. Along with genetics, the environment contributes to disease development, but what these exact environmental factors are remains unknown. We have previously shown that breast tissue is not sterile but contains a diverse population of bacteria. We thus believe that the host's local microbiome could be modulating the risk of breast cancer development. Using 16S rRNA amplicon sequencing, we show that bacterial profiles differ between normal adjacent tissue from women with breast cancer and tissue from healthy controls. Women with breast cancer had higher relative abundances of Bacillus, Enterobacteriaceae and Staphylococcus Escherichia coli (a member of the Enterobacteriaceae family) and Staphylococcus epidermidis, isolated from breast cancer patients, were shown to induce DNA double-stranded breaks in HeLa cells using the histone-2AX (H2AX) phosphorylation (γ-H2AX) assay. We also found that microbial profiles are similar between normal adjacent tissue and tissue sampled directly from the tumor. This study raises important questions as to what role the breast microbiome plays in disease development or progression and how we can manipulate this for possible therapeutics or prevention. IMPORTANCE: This study shows that different bacterial profiles in breast tissue exist between healthy women and those with breast cancer. Higher relative abundances of bacteria that had the ability to cause DNA damage in vitro were detected in breast cancer patients, as was a decrease in some lactic acid bacteria, known for their beneficial health effects, including anticarcinogenic properties. This study raises important questions as to the role of the mammary microbiome in modulating the risk of breast cancer development.

In Vivo Bioluminescence Imaging of Intratumoral Bacteria.

This chapter describes the use of whole-body bioluminescent imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in therapy of cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and tumor sites concurrently. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor-bearing mice. This powerful, and inexpensive, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research. This protocol outlines the procedure for studying lux-tagged Escherichia coli and Bifidobacterium breve in mice, demonstrating the spatial and temporal readout from 2D and 3D BLI achievable with whole-body in vivo luminescence imaging.

Activation of multiple chemotherapeutic prodrugs by the natural enzymolome of tumour-localised probiotic bacteria.

Some chemotherapeutic drugs (prodrugs) require activation by an enzyme for efficacy. We and others have demonstrated the ability of probiotic bacteria to grow specifically within solid tumours following systemic administration, and we hypothesised that the natural enzymatic activity of these tumour-localised bacteria may be suitable for activation of certain such chemotherapeutic drugs. Several wild-type probiotic bacteria; Escherichia coli Nissle, Bifidobacterium breve, Lactococcus lactis and Lactobacillus species, were screened against a panel of popular prodrugs. All strains were capable of activating at least one prodrug. E. coli Nissle 1917 was selected for further studies because of its ability to activate numerous prodrugs and its resistance to prodrug toxicity. HPLC data confirmed biochemical transformation of prodrugs to their toxic counterparts. Further analysis demonstrated that different enzymes can complement prodrug activation, while simultaneous activation of multiple prodrugs (CB1954, 5-FC, AQ4N and Fludarabine phosphate) by E. coli was confirmed, resulting in significant efficacy improvement. Experiments in mice harbouring murine tumours validated in vitro findings, with significant reduction in tumour growth and increase in survival of mice treated with probiotic bacteria and a combination of prodrugs. These findings demonstrate the ability of probiotic bacteria, without the requirement for genetic modification, to enable high-level activation of multiple prodrugs specifically at the site of action.

Local bacteria affect the efficacy of chemotherapeutic drugs.

In this study, the potential effects of bacteria on the efficacy of frequently used chemotherapies was examined. Bacteria and cancer cell lines were examined in vitro and in vivo for changes in the efficacy of cancer cell killing mediated by chemotherapeutic agents. Of 30 drugs examined in vitro, the efficacy of 10 was found to be significantly inhibited by certain bacteria, while the same bacteria improved the efficacy of six others. HPLC and mass spectrometry analyses of sample drugs (gemcitabine, fludarabine, cladribine, CB1954) demonstrated modification of drug chemical structure. The chemoresistance or increased cytotoxicity observed in vitro with sample drugs (gemcitabine and CB1954) was replicated in in vivo murine subcutaneous tumour models. These findings suggest that bacterial presence in the body due to systemic or local infection may influence tumour responses or off-target toxicity during chemotherapy.

Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging.

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.