Bacterial Iron Siderophore Drives Tumor Survival and Ferroptosis Resistance in a Biofilm-Tumor Spheroid Coculture Model.

Interactions between tumoral cells and tumor-associated bacteria within the tumor microenvironment play a significant role in tumor survival and progression, potentially impacting cancer treatment outcomes. In lung cancer patients, the Gram-negative pathogen Pseudomonas aeruginosa raises questions about its role in tumor survival. Here, a microfluidic-based 3D-human lung tumor spheroid-P. aeruginosa model is developed to study the bacteria's impact on tumor survival. P. aeruginosa forms a tumor-associated biofilm by producing Psl exopolysaccharide and secreting iron-scavenging pyoverdine, which is critical for establishing a bacterial community in tumors. Consequently, pyoverdine promotes cancer progression by reducing susceptibility to iron-induced death (ferroptosis), enhancing cell viability, and facilitating several cancer hallmarks, including epithelial-mesenchymal transition and metastasis. A promising combinatorial therapy approach using antimicrobial tobramycin, ferroptosis-inducing thiostrepton, and anti-cancer doxorubicin could eradicate biofilms and tumors. This work unveils a novel phenomenon of cross-kingdom cooperation, where bacteria protect tumors from death, and it paves the way for future research in developing antibiofilm cancer therapies. Understanding these interactions offers potential new strategies for combatting cancer and enhancing treatment efficacy.

Juglone as a Natural Quorum Sensing Inhibitor against Pseudomonas aeruginosa pqs-Mediated Virulence and Biofilms.

Pseudomonas aeruginosa is a notorious opportunistic pathogen associated with chronic biofilm-related infections, posing a significant challenge to effective treatment strategies. Quorum sensing (QS) and biofilm formation are critical virulence factors employed by P. aeruginosa, contributing to its pathogenicity and antibiotic resistance. Other than the homoserine-based QS systems, P. aeruginosa also possesses the quinolone-based Pseudomonas quinolone signal (PQS) QS signaling. Synthesis of the PQS signaling molecule is achieved by the pqsABCDEH operon, whereas the PQS signaling response was mediated by the PqsR receptor. In this study, we report the discovery of a novel natural compound, Juglone, with potent inhibitory effects on pqs QS and biofilm formation in P. aeruginosa. Through an extensive screening of natural compounds from diverse sources, we identified Juglone, a natural compound from walnut, as a promising candidate. We showed that Juglone could inhibit PqsR and the molecular docking results revealed that Juglone could potentially bind to the PqsR active site. Furthermore, Juglone could inhibit pqs-regulated virulence factors, such as pyocyanin and the PQS QS signaling molecule. Juglone could also significantly reduce both the quantity and quality of P. aeruginosa biofilms. Notably, this compound exhibited minimal cytotoxicity toward mammalian cells, suggesting its potential safety for therapeutic applications. To explore the clinical relevance of Juglone, we investigated its combinatorial effects with colistin, a commonly used antibiotic against P. aeruginosa infections. The Juglone-colistin combinatorial treatment could eliminate biofilms formed by wild-type P. aeruginosa PAO1 and its clinical isolates collected from cystic fibrosis patients. The Juglone-colistin combinatorial therapy dramatically improved colistin efficacy and reduced inflammation in a wound infection model, indicating its potential for clinical utility. In conclusion, the discovery of Juglone provides insights into the development of innovative antivirulence therapeutic strategies to combat P. aeruginosa biofilm-associated infections.

Bacterivorous nematodes decipher microbial iron siderophores as prey cue in predator-prey interactions.

The minimal levels of biological-available iron in the environment impose growth limitation on all living organisms. Microbes often secrete high iron-binding-affinity siderophores at high concentrations for scavenging iron from the iron-limited habitats. However, the high prevalence of siderophores released by bacteria into the environment raises an intriguing question whether this chemical cue can be detected by bacterivorous predators. Here, we show that the bacterivorous Caenorhabditis elegans nematode could employ its chemosensory receptor Odr-10 to detect pyoverdine, an iron siderophore secreted by an environmental bacterium, Pseudomonas aeruginosa. This enabled the nematode predator to migrate toward the prey. Our soil microcosm study showed that the detection of pyoverdine and subsequent feeding of P. aeruginosa prey by C. elegans could lead to the expansion of its population. These results showed that siderophores are a prey chemical cue by predators, with key implications in predator-prey interactions.

Oxidative stress induced by Etoposide anti-cancer chemotherapy drives the emergence of tumor-associated bacteria resistance to fluoroquinolones.

INTRODUCTION: Antibiotic-resistant bacterial infections, such as Pseudomonas aeruginosa and Staphylococcus aureus, are prevalent in lung cancer patients, resulting in poor clinical outcomes and high mortality. Etoposide (ETO) is an FDA-approved chemotherapy drug that kills cancer cells by damaging DNA through oxidative stress. However, it is unclear if ETO can cause unintentional side effects on tumor-associated microbial pathogens, such as inducing antibiotic resistance. OBJECTIVES: We aimed to show that prolonged ETO treatment could unintendedly confer fluoroquinolone antibiotic resistance to P. aeruginosa, and evaluate the effect of tumor-associated P. aeruginosa on tumor progression. METHODS: We employed experimental evolution assay to treat P. aeruginosa with prolonged ETO exposure, evaluated the ciprofloxacin resistance, and elucidated the gene mutations by DNA sequencing. We also established a lung tumor-P. aeruginosa bacterial model to study the role of ETO-evolved intra-tumoral bacteria in tumor progression using immunostaining and confocal microscopy. RESULTS: ETO could generate oxidative stress and lead to gene mutations in P. aeruginosa, especially the gyrase (gyrA) gene, resulting in acquired fluoroquinolone resistance. We further demonstrated using a microfluidic-based lung tumor-P. aeruginosa coculture model that bacteria can evolve ciprofloxacin (CIP) resistance in a tumor microenvironment. Moreover, ETO-induced CIP-resistant (EICR) mutants could form multicellular biofilms which protected tumor cells from ETO killing and enabled tumor progression. CONCLUSION: Overall, our preclinical proof-of-concept provides insights into how anti-cancer chemotherapy could inadvertently allow tumor-associated bacteria to acquire antibiotic resistance mutations and shed new light on the development of novel anti-cancer treatments based on anti-bacterial strategies.

Biofilms exacerbate atherogenesis through macrophage-induced inflammatory responses in a fibrous plaque microsystem model.

BACKGROUND: Microbes have been implicated in atherosclerosis development and progression, but the impact of bacterial-based biofilms on fibrous plaque rupture remains poorly understood. RESULTS: Here, we developed a comprehensive atherosclerotic model to reflect the progression of fibrous plaque under biofilm-induced inflammation (FP-I). High expressions of biofilm-specific biomarkers algD, pelA and pslB validated the presence of biofilms. Biofilm promotes the polarization of macrophages towards a pro-inflammatory (M1) phenotype, as demonstrated by an increase in M1 macrophage-specific marker CD80 expression in CD68+ macrophages. The increase in the number of intracellular lipid droplets (LDs) and foam cell percentage highlighted the potential role of biofilms on lipid synthesis or metabolic pathways in macrophage-derived foam cells. In addition, collagen I production by myofibroblasts associated with the fibrous cap was significantly reduced along with the promotion of apoptosis of myofibroblasts, indicating that biofilms affect the structural integrity of the fibrous cap and potentially undermine its strength. CONCLUSION: We validated the unique role of biofilm-based inflammation in exacerbating fibrous plaque damage in the FP-I model, increasing fibrous plaque instability and risk of thrombosis. Our results lay the foundation for mechanistic studies of the role of biofilms in fibrous plaques, allowing the evaluation of preclinical combination strategies for drug therapy. STATEMENT OF SIGNIFICANCE: A microsystem-based model was developed to reveal interactions in fibrous plaque during biofilm-induced inflammation (FP-I). Real-time assessment of biofilm formation and its role in fibrous plaque progression was achieved. The presence of biofilms enhanced the expression of pro-inflammatory (M1) specific marker CD80, lipid droplets, and foam cells and reduced anti-inflammatory (M2) specific marker CD206 expression. Fibrous plaque exposure to biofilm-based inflammation reduced collagen I expression and increased apoptosis marker Caspase-3 expression significantly. Overall, we demonstrate the unique role of biofilm-based inflammation in exacerbating fibrous plaque damage in the FP-I model, promoting fibrous plaque instability and enhanced thrombosis risk. Our findings lay the groundwork for mechanistic studies, facilitating the evaluation of preclinical drug combination strategies.

Biofilm Potentiates Cancer-Promoting Effects of Tumor-Associated Macrophages in a 3D Multi-Faceted Tumor Model.

Components of the tumor microenvironment (TME), such as tumor-associated macrophages (TAMs), influence tumor progression. The specific polarization and phenotypic transition of TAMs in the tumor microenvironment lead to two-pronged impacts that can promote or hinder cancer development and treatment. Here, a novel microfluidic multi-faceted bladder tumor model (TAMPIEB ) is developed incorporating TAMs and cancer cells to evaluate the impact of bacterial distribution on immunomodulation within the tumor microenvironment in vivo. It is demonstrated for the first time that biofilm-induced inflammatory conditions within tumors promote the transition of macrophages from a pro-inflammatory M1-like to an anti-inflammatory/pro-tumor M2-like state. Consequently, multiple roles and mechanisms by which biofilms promote cancer by inducing pro-tumor phenotypic switch of TAMs are identified, including cancer hallmarks such as reducing susceptibility to apoptosis, enhancing cell viability, and promoting epithelial-mesenchymal transition and metastasis. Furthermore, biofilms formed by extratumoral bacteria can shield tumors from immune attack by TAMs, which can be visualized through various imaging assays in situ. The study sheds light on the underlying mechanism of biofilm-mediated inflammation on tumor progression and provides new insights into combined anti-biofilm therapy and immunotherapy strategies in clinical trials.

Improved Raman spectroscopy-based approach to assess microplastics in seafood.

Microplastics represent an emerging environmental issue and have been found almost everywhere including seafood, raising a great concern about the ecological and human health risks they pose. This study addressed the common technical challenges in the assessment of microplastics in seafood by developing an improved protocol based on Raman spectroscopy and using the green-lipped mussel Perna viridis and the Japanese jack mackerel Trachurus japonicus as the test models. Our findings identified a type of stainless-steel filter membranes with minimal Raman interference, and a combination of chemicals that achieved 99-100% digestion efficiency for both organic and inorganic biomass. This combined chemical treatment reached 90-100% recovery rates for seven types of microplastics, on which the surface modification was considered negligible and did not affect the accuracy of polymer identification based on Raman spectra, which showed 94-99% similarity to corresponding untreated microplastics. The developed extraction method for microplastics was further combined with an automated Raman mapping approach, from which our results confirmed the presence of microplastics in P. viridis and T. japonicus collected from Hong Kong waters. Identified microplastics included polypropylene, polyethylene, polystyrene and poly(ethylene terephthalate), mainly in the form of fragments and fibres. Our protocol is applicable to other biological samples, and provides an improved alternative to streamline the workflow of microplastic analysis for routine monitoring purposes.

Worm-Based Microfluidic Biosensor for Real-Time Assessment of the Metastatic Status.

BACKGROUND: Metastasis is a complex process that affects patient treatment and survival. To routinely monitor cancer plasticity and guide treatment strategies, it is highly desired to provide information about metastatic status in real-time. Here, we proposed a worm-based (WB) microfluidic biosensor to rapidly monitor biochemical cues related to metastasis in a well-defined environment. Compared to conventional biomarker-based methods, the WB biosensor allowed high throughput screening under low cost, requiring only visual quantification of outputs; Methods: Caenorhabditis elegans were placed in the WB biosensor and exposed to samples conditioned with cancer cell clusters. The chemotactic preference of these worms was observed under discontinuous imaging to minimize the impact on physiological activity; Results: A chemotaxis index (CI) was defined to standardize the quantitative assessment from the WB biosensor, where moderate (3.24-6.5) and high (>6.5) CI levels reflected increased metastasis risk and presence of metastasis, respectively. We demonstrated that the secreted metabolite glutamate was a chemorepellent, and larger clusters associated with increased metastatic potential also enhanced CI levels; Conclusions: Overall, this study provided a proof of concept for the WB biosensors in assessing metastasis status, with the potential to evaluate patient-derived cancer clusters for routine management.

The effects of biofilms on tumor progression in a 3D cancer-biofilm microfluidic model.

Components within the tumor microenvironment, such as intratumoral bacteria (IB; within tumors), affect tumor progression. However, current experimental models have not explored the effects of extratumoral bacteria (EB; outside tumors) on cancer progression. Here, we developed a microfluidic platform to analyze the influence of bacterial distribution on bladder cancer progression under defined conditions, using uropathogenic Escherichia coli. This was achieved by establishing coating (CT) and colonizing (CL) models to simulate the different invasion and colonization modes of IB and EB in tumor tissues. We demonstrated that both EB and IB induced closer cell-cell contacts within the tumor cluster, but cancer cell viability was reduced only in the presence of IB. Interestingly, cancer stem cell counts increased significantly in the presence of EB. These outcomes were due to the formation of extracellular DNA-based biofilms by EB. Triple therapy of DNase (anti-biofilm agent), ciprofloxacin (antibiotic), and doxorubicin (anti-cancer drug) could effectively eradicate biofilms and tumors simultaneously. Our preclinical proof-of-concept provides insights on how bacteria can influence tumor progression and facilitate future research on anti-biofilm cancer management therapies.

Demolishing the great wall of biofilms in Gram-negative bacteria: To disrupt or disperse?

Bacterial infections lead to high morbidity and mortality globally. While current therapies against bacteria often employ antibiotics, most bacterial pathogens can form biofilms and prevent effective treatment of infections. Biofilm cells can aggregate and encased themselves in a self-secreted protective exopolymeric matrix, to reduce the penetration by antibiotics. Biofilm formation is mediated by c-di-GMP signaling, the ubiquitous secondary messenger in bacteria. Synthesis of c-di-GMP by diguanylate cyclases leads to biofilm formation via the loss of motility, increased surface attachment, and production of biofilm matrix, whereas c-di-GMP degradation by phosphodiesterases causes biofilm dispersal to new sites via increased bacterial motility and matrix breakdown. The highly variable nature of biofilm development and antimicrobial tolerance imposes tremendous challenges in conventional antimicrobial therapies, indicating an imperative need to develop anti-biofilm drugs against biofilm infections. In this review, we focus on two main emergent approaches-active dispersal and disruption. While both approaches aim to demolish biofilms, we will discuss their fundamental differences and associated methods. Active dispersal of biofilms involves signaling the bacterial cells to leave the biofilm, where resident cells ditch their sessile lifestyle, gain motility and self-degrade their matrix. Biofilm disruption leads to direct matrix degradation that forcibly releases embedded biofilm cells. Without the protection of biofilm matrix, released bacterial cells are highly exposed to antimicrobials, leading to their eradication in biofilm infections. Understanding the advantages and disadvantages of both approaches will allow optimized utility with antimicrobials in clinical settings.

Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention.

BACKGROUND: Emergence of drug-resistant cancer phenotypes is a challenge for anti-cancer therapy. Cancer stem cells are identified as one of the ways by which chemoresistance develops. METHOD: We investigated the anti-inflammatory combinatorial treatment (DA) of doxorubicin and aspirin using a preclinical microfluidic model on cancer cell lines and patient-derived circulating tumour cell clusters. The model had been previously demonstrated to predict patient overall prognosis. RESULTS: We demonstrated that low-dose aspirin with a sub-optimal dose of doxorubicin for 72 h could generate higher killing efficacy and enhanced apoptosis. Seven days of DA treatment significantly reduced the proportion of cancer stem cells and colony-forming ability. DA treatment delayed the inhibition of interleukin-6 secretion, which is mediated by both COX-dependent and independent pathways. The response of patients varied due to clinical heterogeneity, with 62.5% and 64.7% of samples demonstrating higher killing efficacy or reduction in cancer stem cell (CSC) proportions after DA treatment, respectively. These results highlight the importance of using patient-derived models for drug discovery. CONCLUSIONS: This preclinical proof of concept seeks to reduce the onset of CSCs generated post treatment by stressful stimuli. Our study will promote a better understanding of anti-inflammatory treatments for cancer and reduce the risk of relapse in patients.

Repurposing the anticancer drug cisplatin with the aim of developing novel Pseudomonas aeruginosa infection control agents.

Antibiotic resistance threatens effective treatment of microbial infections globally. This situation has spurred the hunt for new antimicrobial compounds in both academia and the pharmaceutical industry. Here, we report how the widely used antitumor drug cisplatin may be repurposed as an effective antimicrobial against the nosocomial pathogen Pseudomonas aeruginosa. Cisplatin was found to effectively kill strains of P. aeruginosa. In such experiments, transcriptomic profiling showed upregulation of the recA gene, which is known to be important for DNA repair, implicating that cisplatin could interfere with DNA replication in P. aeruginosa. Cisplatin treatment significantly repressed the type III secretion system (T3SS), which is important for the secretion of exotoxins. Furthermore, cisplatin was also demonstrated to eradicate in vitro biofilms and in vivo biofilms in a murine keratitis model. This showed that cisplatin could be effectively used to eradicate biofilm infections which were otherwise difficult to be treated by conventional antibiotics. Although cisplatin is highly toxic for humans upon systemic exposure, a low toxicity was demonstrated with topical treatment. This indicated that higher-than-minimal inhibitory concentration (MIC) doses of cisplatin could be topically applied to treat persistent and recalcitrant P. aeruginosa infections.

Reactive oxygen species drive evolution of pro-biofilm variants in pathogens by modulating cyclic-di-GMP levels.

The host immune system offers a hostile environment with antimicrobials and reactive oxygen species (ROS) that are detrimental to bacterial pathogens, forcing them to adapt and evolve for survival. However, the contribution of oxidative stress to pathogen evolution remains elusive. Using an experimental evolution strategy, we show that exposure of the opportunistic pathogen Pseudomonas aeruginosa to sub-lethal hydrogen peroxide (H2O2) levels over 120 generations led to the emergence of pro-biofilm rough small colony variants (RSCVs), which could be abrogated by l-glutathione antioxidants. Comparative genomic analysis of the RSCVs revealed that mutations in the wspF gene, which encodes for a repressor of WspR diguanylate cyclase (DGC), were responsible for increased intracellular cyclic-di-GMP content and production of Psl exopolysaccharide. Psl provides the first line of defence against ROS and macrophages, ensuring the survival fitness of RSCVs over wild-type P. aeruginosa Our study demonstrated that ROS is an essential driving force for the selection of pro-biofilm forming pathogenic variants. Understanding the fundamental mechanism of these genotypic and phenotypic adaptations will improve treatment strategies for combating chronic infections.

RpoN Regulates Virulence Factors of Pseudomonas aeruginosa via Modulating the PqsR Quorum Sensing Regulator.

The alternative sigma factor RpoN regulates many cell functions, such as motility, quorum sensing, and virulence in the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa often evolves rpoN-negative variants during the chronic infection in cystic fibrosis patients. It is unclear how RpoN interacts with other regulatory mechanisms to control virulence of P. aeruginosa. In this study, we show that RpoN modulates the function of PqsR, a quorum sensing receptor regulating production of virulence factors including the phenazine pyocyanin. The ∆rpoN mutant is able to synthesize 4-quinolone signal molecule HHQ but unable to activate PqsR and Pseudomonas quinolone signal (pqs) quorum sensing. The ∆rpoN mutant produces minimal level of pyocyanin and is unable to produce the anti-staphylococcal agents. Providing pqsR in trans in the ∆rpoN mutant restores its pqs quorum sensing and virulence factor production to the wild-type level. Our study provides evidence that RpoN has a regulatory effect on P. aeruginosa virulence through modulating the function of the PqsR quorum sensing regulator.

Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles.

Bacteria assume distinct lifestyles during the planktonic and biofilm modes of growth. Increased levels of the intracellular messenger c-di-GMP determine the transition from planktonic to biofilm growth, while a reduction causes biofilm dispersal. It is generally assumed that cells dispersed from biofilms immediately go into the planktonic growth phase. Here we use single-nucleotide resolution transcriptomic analysis to show that the physiology of dispersed cells from Pseudomonas aeruginosa biofilms is highly different from those of planktonic and biofilm cells. In dispersed cells, the expression of the small regulatory RNAs RsmY and RsmZ is downregulated, whereas secretion genes are induced. Dispersed cells are highly virulent against macrophages and Caenorhabditis elegans compared with planktonic cells. In addition, they are highly sensitive towards iron stress, and the combination of a biofilm-dispersing agent, an iron chelator and tobramycin efficiently reduces the survival of the dispersed cells.