Bacterial therapies at the interface of synthetic biology and nanomedicine.

Bacteria are emerging as living drugs to treat a broad range of disease indications. However, the inherent advantages of these replicating and immunostimulatory therapies also carry the potential for toxicity. Advances in synthetic biology and the integration of nanomedicine can address this challenge through the engineering of controllable systems that regulate spatial and temporal activation for improved safety and efficacy. Here, we review recent progress in nanobiotechnology-driven engineering of bacteria-based therapies, highlighting limitations and opportunities that will facilitate clinical translation.

"Tumor-selective treatment of metastatic pancreatic cancer with an engineered, probiotic living drug".

Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges for effective treatment, with systemic chemotherapy often proving inadequate due to poor drug delivery and the tumor's immunosuppressive microenvironment. Engineered bacteria present a novel approach to target PDAC, leveraging their ability to colonize tumors and deliver therapeutic payloads. Here, we engineered probiotic Escherichia coli Nissle 1917 (EcN) to produce the pore-forming Theta toxin (Nis-Theta) and evaluated its efficacy in a preclinical model of PDAC. Probiotic administration resulted in selective colonization of tumor tissue, leading to improved overall survival compared to standard chemotherapy. Moreover, this strain exhibited cytotoxic effects on both primary and distant tumor lesions while sparing normal tissues. Importantly, treatment also modulated the tumor microenvironment by increasing anti-tumor immune cell populations and reducing immunosuppressive markers. These findings demonstrate the potential of engineered probiotic bacteria as a safe and effective therapeutic approach for PDAC, offering promise for improved patient outcomes.

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Engineering tumor-colonizing E. coli Nissle 1917 for detection and treatment of colorectal neoplasia.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment. Here, first, we demonstrate selective colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition and orthotopic models of CRC. We next undertake an interventional, double-blind, dual-centre, prospective clinical trial, in which CRC patients take either placebo or EcN for two weeks prior to resection of neoplastic and adjacent normal colorectal tissue (ACTRN12619000210178). We detect enrichment of EcN in tumor samples over normal tissue from probiotic-treated patients (primary outcome of the trial). Next, we develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate. Oral delivery of this strain results in increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. To assess therapeutic potential, we engineer EcN to locally release a cytokine, GM-CSF, and blocking nanobodies against PD-L1 and CTLA-4 at the neoplastic site, and demonstrate that oral delivery of this strain reduces adenoma burden by ~50%. Together, these results support the use of EcN as an orally-deliverable platform to detect disease and treat CRC through the production of screening and therapeutic molecules.

Engineering Probiotic E. coli Nissle 1917 for Release of Therapeutic Nanobodies.

Bioengineered probiotics enable new opportunities to improve cancer treatment strategies due to their tumor-colonizing capabilities. Here, we will describe the development of a probiotic E. coli Nissle 1917 platform encoding a synchronized lysis mechanism for the localized and sustained release of blocking nanobodies against immune checkpoint molecules like programmed cell death protein-ligand 1 and cytotoxic T lymphocyte-associated protein-4. Specifically, we will detail the experimental protocols needed to (1) encode and validate binding of recombinantly produced checkpoint blockade nanobodies, (2) evaluate the therapeutic efficacy and safety of the probiotic platform in syngeneic tumor-bearing mice, and (3) analyze the immunophenotype of the tumor microenvironment.

Probiotic-guided CAR-T cells for solid tumor targeting.

A major challenge facing tumor-antigen targeting therapies such as chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. By contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be engineered as antigen-independent platforms for therapeutic delivery. To bridge these approaches, we developed a platform of probiotic-guided CAR-T cells (ProCARs), in which tumor-colonizing probiotics release synthetic targets that label tumor tissue for CAR-mediated lysis in situ. This system demonstrated CAR-T cell activation and antigen-agnostic cell lysis that was safe and effective in multiple xenograft and syngeneic models of human and mouse cancers. We further engineered multifunctional probiotics that co-release chemokines to enhance CAR-T cell recruitment and therapeutic response.

Engineered bacteria launch and control an oncolytic virus.

The ability of bacteria and viruses to selectively replicate in tumors has led to synthetic engineering of new microbial therapies. Here we design a cooperative strategy whereby S. typhimurium bacteria transcribe and deliver the Senecavirus A RNA genome inside host cells, launching a potent oncolytic viral infection. Then, we engineer the virus to require a bacterially delivered protease in order to achieve virion maturation, demonstrating bacterial control over the virus. This work extends bacterially delivered therapeutics to viral genomes, and the governing of a viral population through engineered microbial interactions. One-Sentence Summary: Bacteria are engineered to act as a synthetic "capsid" delivering Senecavirus A genome and controlling its spread.

Engineered bacterial swarm patterns as spatial records of environmental inputs.

A diverse array of bacteria species naturally self-organize into durable macroscale patterns on solid surfaces via swarming motility-a highly coordinated and rapid movement of bacteria powered by flagella. Engineering swarming is an untapped opportunity to increase the scale and robustness of coordinated synthetic microbial systems. Here we engineer Proteus mirabilis, which natively forms centimeter-scale bullseye swarm patterns, to 'write' external inputs into visible spatial records. Specifically, we engineer tunable expression of swarming-related genes that modify pattern features, and we develop quantitative approaches to decoding. Next, we develop a dual-input system that modulates two swarming-related genes simultaneously, and we separately show that growing colonies can record dynamic environmental changes. We decode the resulting multicondition patterns with deep classification and segmentation models. Finally, we engineer a strain that records the presence of aqueous copper. This work creates an approach for building macroscale bacterial recorders, expanding the framework for engineering emergent microbial behaviors.

Colorectal cancer detection and treatment with engineered probiotics.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by ∻50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

Chemokines expressed by engineered bacteria recruit and orchestrate antitumor immunity.

Tumors use multiple mechanisms to actively exclude immune cells involved in antitumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16K42A) offer therapeutic benefit in multiple mouse tumor models, an effect mediated via recruitment of CD8+ T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells, using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive antitumor immune responses, offering a new cancer immunotherapy strategy.

Engineered Bacteria for Short-Chain-Fatty-Acid-Repressed Expression of Biotherapeutic Molecules.

Short-chain fatty acids (SCFA) such as propionate and butyrate are critical metabolites produced by the gut microbiota. Microbiome dysbiosis resulting in altered SCFA profiles is associated with certain diseases, including inflammatory bowel diseases (IBD), characterized by a reduction in butyrate concentration and active intestinal inflammation. There is an increasing interest in the use of engineered bacteria as diagnostic and therapeutic tools for gut diseases. In this study, we developed genetic circuits capable of sensing SCFA concentrations to build biosensors that express a response protein (superfolder green fluorescent protein [sfGFP]) in amounts inversely proportional to the SCFA concentration. We also built biotherapeutics expressing the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) using the same logic. The propionate biotherapeutic expressed larger amounts of mouse GM-CSF in the absence of propionate. The butyrate biotherapeutics presented the expected behavior only at the beginning of the kinetics and an accelerated response in the absence of butyrate. Overall, these genetic systems may function as complementary diagnostic tools for measuring SCFAs and as delivery vehicles for biotherapeutic molecules. IMPORTANCE Short-chain fatty acids are key molecules produced by the gut microbiome. Their concentrations are altered in certain diseases. Here, we created molecular biosensors that quantify the absence of propionate and butyrate, using logic "NOT" gates and bacterial promoters. Finally, we show that these genetic systems could be useful for the delivery of therapeutic molecules in the gut, in the absence of these acids.

Design of combination therapy for engineered bacterial therapeutics in non-small cell lung cancer.

Synthetic biology enables the engineering of bacteria to safely deliver potent payloads to tumors for effective anti-cancer therapies. However, a central challenge for translation is determining ideal bacterial therapy candidates for specific cancers and integrating them with other drug treatment strategies to maximize efficacy. To address this, we designed a screening and evaluation pipeline for characterization of bacterial therapies in lung cancer models. We screened 10 engineered bacterial toxins across 6 non-small cell lung cancer patient-derived cell lines and identified theta toxin as a promising therapeutic candidate. Using a bacteria-spheroid co-culture system (BSCC), analysis of differentially expressed transcripts and gene set enrichment revealed significant changes in at least 10 signaling pathways with bacteria-producing theta toxin. We assessed combinatorial treatment of small molecule pharmaceutical inhibitors targeting 5 signaling molecules and of 2 chemotherapy drugs along with bacterially-produced theta toxin and showed improved dose-dependent response. This combination strategy was further tested and confirmed, with AKT signaling as an example, in a mouse model of lung cancer. In summary, we developed a pipeline to rapidly characterize bacterial therapies and integrate them with current targeted therapies for lung cancer.

Assessing chemotherapy dosing strategies in a spatial cell culture model.

Predicting patient responses to chemotherapy regimens is a major challenge in cancer treatment. Experimental model systems coupled with quantitative mathematical models to calculate optimal dose and frequency of drugs can enable improved chemotherapy regimens. Here we developed a simple approach to track two-dimensional cell colonies composed of chemo-sensitive and resistant cell populations via fluorescence microscopy and coupled this to computational model predictions. Specifically, we first developed multiple 4T1 breast cancer cell lines resistant to varying concentrations of doxorubicin, and demonstrated how heterogeneous populations expand in a two-dimensional colony. We subjected cell populations to varied dose and frequency of chemotherapy and measured colony growth. We then built a mathematical model to describe the dynamics of both chemosensitive and chemoresistant populations, where we determined which number of doses can produce the smallest tumor size based on parameters in the system. Finally, using an in vitro model we demonstrated multiple doses can decrease overall colony growth as compared to a single dose at the same total dose. In the future, this system can be adapted to optimize dosing strategies in the setting of heterogeneous cell types or patient derived cells with varied chemoresistance.

Engineering bacteria as interactive cancer therapies.

With increasing evidence that microbes colonize tumors, synthetic biology tools are being leveraged to repurpose bacteria as tumor-specific delivery systems. These engineered systems can modulate the tumor microenvironment using a combination of their inherent immunogenicity and local payload production. Here, we review genetic circuits that enhance spatial and temporal control of therapeutic bacteria to improve their safety and efficacy. We describe the engineering of interactions among bacteria, tumor cells, and immune cells, and the progression from bacteria as single agents toward their rational combination with other modalities. Together, these efforts are building toward an emerging concept of engineering interactions between programmable medicines using synthetic biology.

Segmentation with Residual Attention U-Net and an Edge-Enhancement Approach Preserves Cell Shape Features.

The ability to extrapolate gene expression dynamics in living single cells requires robust cell segmentation, and one of the challenges is the amorphous or irregularly shaped cell boundaries. To address this issue, we modified the U-Net architecture to segment cells in fluorescence widefield microscopy images and quantitatively evaluated its performance. We also proposed a novel loss function approach that emphasizes the segmentation accuracy on cell boundaries and encourages shape feature preservation. With a 97% sensitivity, 93% specificity, 91% Jaccard similarity, and 95% Dice coefficient, our proposed method called Residual Attention U-Net with edge-enhancement surpassed the state-of-the-art U-Net in segmentation performance as evaluated by the traditional metrics. More remarkably, the same proposed candidate also performed the best in terms of the preservation of valuable shape features, namely area, eccentricity, major axis length, solidity and orientation. These improvements on shape feature preservation can serve as useful assets for downstream cell tracking and quantification of changes in cell statistics or features over time.

A rapid screening platform to coculture bacteria within tumor spheroids.

The prevalence of tumor-colonizing bacteria along with advances in synthetic biology are leading to a new generation of living microbial cancer therapies. Because many bacterial systems can be engineered to recombinantly produce therapeutics within tumors, simple and high-throughput experimental platforms are needed to screen the large collections of bacteria candidates and characterize their interactions with cancer cells. Here, we describe a protocol to selectively grow bacteria within the core of tumor spheroids, allowing for their continuous and parallel profiling in physiologically relevant conditions. Specifically, tumor spheroids are incubated with bacteria in a 96-well low-adhesion plate followed by a series of washing steps and an antibiotic selection protocol to confine bacterial growth within the hypoxic and necrotic core of tumor spheroids. This bacteria spheroid coculture (BSCC) system is stable for over 2 weeks, does not require specialized equipment and is compatible with time-lapse microscopy, commercial staining assays and histology that uniquely enable analysis of growth kinetics, viability and spatial distribution of both cellular populations, respectively. We show that the procedure is applicable to multiple tumor cell types and bacterial species by varying protocol parameters and is validated by using animal models. The BSCC platform will allow the study of bacteria-tumor interactions in a continuous manner and facilitate the rapid development of engineered microbial therapies.

A programmable encapsulation system improves delivery of therapeutic bacteria in mice.

Living bacteria therapies have been proposed as an alternative approach to treating a broad array of cancers. In this study, we developed a genetically encoded microbial encapsulation system with tunable and dynamic expression of surface capsular polysaccharides that enhances systemic delivery. Based on a small RNA screen of capsular biosynthesis pathways, we constructed inducible synthetic gene circuits that regulate bacterial encapsulation in Escherichia coli Nissle 1917. These bacteria are capable of temporarily evading immune attack, whereas subsequent loss of encapsulation results in effective clearance in vivo. This dynamic delivery strategy enabled a ten-fold increase in maximum tolerated dose of bacteria and improved anti-tumor efficacy in murine models of cancer. Furthermore, in situ encapsulation increased the fraction of microbial translocation among mouse tumors, leading to efficacy in distal tumors. The programmable encapsulation system promises to enhance the therapeutic utility of living engineered bacteria for cancer.