IntroductionIncorporating minimal antibody regions into engineered proteins is an effective strategy for creating targeted therapeutics and diagnostics. However, a major barrier to clinical translation is the lack of efficient methods to track their performance in vivo. Bioluminescence imaging using targeted reporter proteins offers a promising solution to this challenge. The aim of this study was to develop a novel in vivo imaging strategy using computationally engineered targeted synthetic proteins and to establish a validated pipeline for their design, construction, and testing.MethodsMulti-part synthetic proteins were designed and screened using in silico tools. These constructs targeted either an example bacterial target (S. aureus ClfA) or a cancer biomarker (MUC1). The top candidates, featuring antibody fragments (ScFv) and a luciferase reporter, were subsequently produced and tested in vitro and in murine models.ResultsFollowing computational design, select proteins were successfully produced and showed specific binding to their intended targets in vitro. Subsequent in vivo studies demonstrated that systemically administered proteins specifically accumulated at target cell locations. This was confirmed by localized bioluminescence that was dependent on both target cell number and protein quantity.DiscussionThis study demonstrates the use of targeted reporter proteins for real-time in vivo imaging and validates an effective workflow integrating computational design with wet-lab experimentation. Ultimately, this establishes a target-specific reporter protein platform that can be adapted for sensitive in vivo imaging of diverse targets, from bacterial infections to tumors.

Background: Live biotherapeutic products (LBPs) require robust genetic stability and effective biocontainment to support safe clinical translation and regulatory acceptance. Aim: This study presents a single-step chromosomal engineering strategy that integrates auxotrophy-mediated biocontainment with therapeutic gene insertion to support regulatory-oriented live biotherapeutic chassis design. Methods: A no-SCAR genome-editing approach combining CRISPR/Cas9 and λ-Red recombineering was used to generate an Escherichia coli MG1655 ΔilvC::hlyA strain by replacing ilvC with the hlyA gene encoding listeriolysin O. Chromosomal and episomal expression systems were compared for auxotrophy, growth, haemolytic activity, plasmid stability, and intracellular DNA delivery to RAW 264.7 macrophages. Results: Auxotrophy was successfully established and restored by branched-chain amino acid supplementation. Chromosomal integration preserved haemolytic activity and bacterial growth while improving long-term genetic stability and plasmid maintenance compared with episomal expression. Both systems supported intracellular DNA delivery, whereas the chromosomal construct showed improved host-cell preservation under higher bacterial challenge. Conclusions: This proof-of-concept study supports the feasibility of using a single-step chromosomal engineering strategy to combine intrinsic biocontainment with therapeutic-gene integration in an engineered bacterial chassis.

Extracellular pH (pHe) is an important indicator of the homeostatic condition of mammalian tissue and its pathophysiological state such as tumor development and tissue acidosis. Optochemical sensors with internal referencing capabilities can perform dynamic quantitative imaging of pHe in live tissue samples. Here we have used planar solid-state pH sensors based on a fluorescent octaethylporphin-ketone (OEPK) dye in plasticized PVC matrix, which produce robust fluorescence lifetime response in the physiological pH range and stable lifetime calibration. A custom-built fluorescence lifetime imager, with a 405 nm picosecond diode laser excitation and a photon counting SPAD array detector (120 x128 pixels) with wide-field Fluorescence Lifetime Imaging (FLIM) capability, was used to image the planar pH sensor brought in contact with freshly excised normal and tumor tissue. The FLIM based sensor system has demonstrated good analytical performance and suitability for in vivo use. Producing detailed maps of tissue pHe, such sensors can facilitate a deeper understanding of tissue metabolism and physiology, and can find use in fluorescence guided surgery of solid tumors.

TPS849 Background: The diversity of the GM is defined as the number and relative abundance distribution of distinct types of microorganisms colonizing within the gut. Studies have suggested that dysbiosis of the GM confers a predisposition to certain malignancies and impacts response to immunomodulating therapies. The influence of the GM diversity on the pathological response after neoadjuvant chemotherapy and radiotherapy is unclear. Some studies have suggested that the GM may offer predictive biomarkers for response to chemoradiation in rectal cancer. Other studies in early-stage rectal cancer patients indicated an association between GM diversity and pathological outcomes following neo-adjuvant therapy (NAT). We hypothesize that a more diverse GM constitution at baseline leads to an improved pathological response at the time of definitive surgery. Methods: We designed a cross-institutional translational study investigating the impact of the GM diversity on the efficacy of NAT in GI cancers by assessing its association with pathological response. The study population includes patients with early-stage rectal or esophageal cancer due to commence NAT (including chemotherapy and chemoradiation) who are planned for definitive surgery. Exclusion criteria includes prior allogenic tissue/solid organ transplantation and prior receipt of anti-cancer therapy. The study assessments include fecal sampling of the GM prior to NAT, upon completion and again six months post completion of therapy. Fecal samples are analysed by 16S RNA sequencing. Pathological response will be examined at time of surgery and patients will be classified as responders (complete pathological response) or non-responders. The primary endpoint of the study is to examine the association between the GM diversity and pathological response. Exploratory analysis will include the assessment of the association between cf-DNA and the GM diversity as well as an assessment of the association between cf-DNA at baseline and pCR. Species richness (Alpha Diversity) will be analysed using the Shannon diversity index and Jaccard similarity index will be used to calculate beta diversity. Following planned study recruitment, classification and clustering analysis will be performed with Principal Component Analysis (PCA) and Random Forest analysis. Logistic regression analysis adjusting for potential confounding factors will be employed to assess the primary endpoint of the association between GM and complete pCR in the final statistical analysis. Adjusted odds ratios (OR) and 95% confidence intervals will be presented. This trial accrued 11 patients between May 2023 and Sept 2024. Out of the 11 patients enrolled, 9 patients have undergone their planned surgery. We are expecting to have 30 patients accrued prior to Jan 2025.

Biospecimen collection from study participants is essential for translational research, but operational challenges in study setup and conduct often impede successful delivery. This study uses a comparative approach to explore key logistical and staffing factors influencing setup duration, recruitment efficiency, sample acquisition, and data completeness across three investigator-led microbiome-wide association studies (MWAS) conducted at cancer centres in Ireland. Three academic observational MWAS enrolling participants with cancers of the breast, gastrointestinal tract, lung, biliary system, kidney, and skin were compared. Data from three cancer centres were analysed. Key variables included study team composition, administrative infrastructure, and full-time equivalent (FTE) research staffing. Metrics assessed included setup duration, recruitment rates, sample acquisition, and data completeness. Descriptive statistics, correlation analyses, and regression models were used to examine relationships between staffing and study performance. Setup duration ranged from 30 days (Site B, with a pre-established trials unit) to 390 days (Site A, with no dedicated setup personnel). At Site C, the addition of an Academic Clinical Trials Coordinator reduced the remaining setup timeline from 274 to 185 days. Recruitment rates ranged from 1.1 to 1.3 participants/month, with the highest rates at sites with dedicated research nurses (RN +). Sample acquisition was 100

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.

TPS615 Background: Predictive biomarkers of response to combination chemotherapy and immune-checkpoint inhibitors are urgently needed to tailor treatment recommendations for patients with early-stage triple negative breast cancer (eTNBC). Our group has demonstrated that tumour-associated microbiota in primary breast tumours represent promising and novel candidate biomarkers for patients with breast cancer. We aim to prospectively interrogate the breast cancer microbiome and tumour microenvironment (TME) of eTNBC treated with neoadjuvant chemo-immunotherapy, and correlate with clinical outcome. Methods: Trial design: This prospective translational biomarker study is enrolling patients with eTNBC suitable for standard neoadjuvant systemic therapy followed by definitive surgery. The primary objective is to evaluate the change in breast cancer microbiome composition pre- and post-therapy. Secondary objectives include correlation of the breast microbiome with pathologic complete response (pCR) and survival outcomes, tumor infiltrating lymphocytes (TILs), PDL-1 and immune subset analysis, extracellular vesicles analysis and analysis of gut microbiome. Tumor tissue samples will be collected at baseline (mandatory research biopsy) and at the time of surgery. Microbiome evaluation will be conducted using metagenomic sequencing to assess changes in the relative abundance of microbial taxa. Blood and stool samples will be collected and analysed at baseline, on-treatment, at the time of surgery and post-operatively for secondary endpoints. Statistical analysis: Hierarchical clustering of samples based on relative abundance of the operational taxonomic units (OTUs) with sample composition at family and genus level will be performed. Alpha Diversity (Shannon Diversity Index) and Beta Diversity (Jaccard Similarity Index) will be analysed. Principal component analysis (PCA) and Random Forest analysis will be performed for classification and clustering analysis. Comparison of taxa or functions between clinical cohorts will be performed (two tailed Z test) and corrected using the false discovery rate to determine Q-values. Thirty patients will be recruited over 18-24 months. Current status: This University College Cork sponsored trial received ethics approval from the CREC in January 2024 and recruitment is ongoing. Those interested can contact uccctg@ucc.ie. This trial will provide a deeper insight into the potential role of the local breast microbiome as a predictive biomarker for response to neoadjuvant therapy in patients with eTNBC. The results will guide further studies exploring optimal immunomodulatory combinations for the treatment of TNBC and may provide evidence regarding future therapeutic targeting of the breast cancer microbiome.

Monitoring of tissue O 2 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 O 2 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 O 2 imaging experiments performed on live and euthanised animals at different time points. Despite a marked trend towards decreased O 2 in dead animals, their tumour areas produced phosphorescence lifetime values between 44 and 47 µs, which corresponded to hypoxic tissue with 5–20 μM O 2 . After the O 2 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 O 2 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 O 2 .

TPS819 Background: The gut microbiome (GM) is thought to influence host immunity by modulating multiple immunologic pathways. Studies have suggested that dysbiosis of the GM confers a predisposition to certain malignancies and influences response to immune checkpoint inhibitors. However, little is known about how the GM diversity influences complete pathological response to neoadjuvant therapy in gastrointestinal (GI) tumours. We hypothesize that a more diverse GM constitution at baseline will lead to improved pathological response at the time of definitive surgery. Methods: We designed a cross-institutional multi-center translational study investigating the impact of the GM diversity on the efficacy of neoadjuvant therapy in GI cancers by assessing its association with pathological response. The study population will consist of patients with an early-stage rectal or esophageal cancer due to commence neoadjuvant therapy (including chemotherapy and chemoradiation) and planned for definitive surgery. Patients who received prior chemotherapy/monoclonal antibodies/immune checkpoint inhibitors or radiation will be excluded. The study assessments will include fecal sampling of the GM prior to neoadjuvant therapy, upon completion and again six months post completion of therapy. Fecal samples will be analysed by 16S RNA sequencing. Pathological response will be examined at time of surgery and patients will be classified as responders (complete pathological response) or non-responders. The primary endpoint of the study is to examine the association between the GM diversity and pathological response. 120 patients will be recruited over 18 months. Results: Species richness (Alpha Diversity) will be analysed using the Shannon diversity index and Jaccard similarity index to calculate beta diversity. Classification and clustering analysis will be performed with Principal Component Analysis (PCA) and Random Forest analysis. Comparison of taxa or functions between clinical cohorts will be performed using the two tailed Z test and corrected using the false discovery rate to determine Q-values. The association between GM and complete pathological response will be examined using logistic regression analysis adjusting for potential confounding factors. Adjusted odds ratios (OR) and 95% confidence intervals will be presented. Conclusions: This study will show preliminary insights into the role of GM as a potential biomarker for neoadjuvant therapy efficacy in patients with GI cancers. Recruitment is on-going.

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.

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.

Protein engineering and synthetic biology stand to benefit immensely from recent advances in silico tools for structural and functional analyses of proteins. In the context of designing novel proteins, current in silico tools inform the user on individual parameters of a query protein, with output scores/metrics unique to each parameter. In reality, proteins feature multiple "parts"/functions and modification of a protein aimed at altering a given part, typically has collateral impact on other protein parts. A system for prediction of the combined effect of design parameters on the overall performance of the final protein does not exist. Function2Form Bridge (F2F-Bridge) attempts to address this by combining the scores of different design parameters pertaining to the protein being analyzed into a single easily interpreted output describing overall performance. The strategy comprises of (a) a mathematical strategy combining data from a myriad of in silico tools into an OP-score (a singular score informing on a user-defined overall performance) and (b) the F2F Plot, a graphical means of informing the wetlab biologist holistically on designed construct suitability in the context of multiple parameters, highlighting scope for improvement. F2F predictive output was compared with wetlab data from a range of synthetic proteins designed, built, and tested for this study. Statistical/machine learning approaches for predicting overall performance, for use alongside the F2F plot, were also examined. Comparisons between wetlab performance and F2F predictions demonstrated close and reliable correlations. This user-friendly strategy represents a pivotal enabler in increasing the accessibility of synthetic protein building and de novo protein design.

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.