Background: As of 2022, 80% of all documented microbial infections are biofilm-associated: communities of microorganisms adhered to a surface and enclosed in a complex extracellular polymeric substance (EPS). The EPS acts as a physical barrier protecting the bacteria from antimicrobial agents and host immune responses. To combat this hurdle, the application of glycoside hydrolases (GH) has been investigated due to their ability to cleave particular structural polysaccharides within the EPS, thus breaking down the protective barrier and improving antibiotic clearance. While various studies demonstrate the capacity of GHs to improve antibiotic efficacy against biofilms in combination, there is clear differential success between these treatments depending on the GH and antibiotic chosen. Due to the overlap of GH targets and antibiotic structures, it is imperative to ensure that the antibiotics in combinatorial treatments are not degraded by the GH. Methods: This study aimed to screen the GH α-amylase produced from Aspergillus oryzae (AO) and Bacillus subtilis (BS), combined with various antibiotics from different classes, charges, and mode of actions by determining MICs. against the bacterium Pseudomonas aeruginosa (PA) of 6 antibiotics with or without α-amylase and treat 2-day PA biofilms with antibiotics with or without GHs. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) stability assays and Differential Scanning Fluorimetry (DSF) were conducted to determine antibiotic and GH degradation as well as antibiotic sequestration. Results: Increased MICs in the presence of GHs as well as decreased antibiotic clearance against 2-day biofilms were suggestive of antibiotic degradation. LC-MS/MS stability assays of tetracycline and ciprofloxacin in the presence and absence of α-amylase further demonstrated the α-amylase-mediated antibiotic sequestration. Differential scanning fluorimetry (DSF) assays confirmed α-amylase-antibiotic interactions. Conclusions: This study suggests that α-amylase is capable of degrading and sequestering a variety of antibiotics, and the degree to which these phenomena occur varies depending upon the source of the GH. As a potential treatment for biofilm-associated infections, it is imperative that the GH + antibiotic combinations are determined compatible prior to clinical use.

For triple-negative breast cancer (TNBC), the most aggressive subset of breast cancer, immune cell infiltrates have prognostic implications. The presence of myeloid-derived suppressor cells supports tumor progression, while tumor-infiltrating lymphocytes (TILs) correlate with improved survival and responsiveness to immunotherapy. Manipulating the abundance of these populations may enhance tumor immunity. Gemcitabine (GEM), a clinically employed chemotherapeutic, is reported to be systemically myeloablative, and thus it is a potentially useful adjunct therapy for promoting anti-tumor immunity. However, knowledge about the immunological effects of GEM intratumorally is limited. Thus, we directly compared the impact of systemic GEM on immune cell presence and functionality in the tumor microenvironment (TME) to its effects in the periphery. We found that GEM is not myeloablative in the TME; rather, we observed sustained, significant reductions in TILs and dendritic cells—crucial components in initiating an adaptive immune response. We also performed bulk-RNA sequencing to identify immunological alterations transcriptionally induced by GEM. While we found evidence of upregulation in the interferon-gamma (IFN-γ) response pathway, we determined that GEM-mediated growth control is not dependent on IFN-γ. Overall, our findings yield new insights into the tissue- and temporal-dependent immune ablative effects of GEM, contrasting the paradigm that this therapy is specifically myeloablative.

High-dose testosterone is an emerging treatment option for selected patients with castration-resistant prostate cancer (CRPC), but its efficacy is limited by frequent primary and acquired resistance. Preclinical studies suggest that high-dose testosterone induces tumor metabolic reprogramming that promotes resistance; however, clinical validation is lacking. In the prospective ACcelerated ROunds of BAT (ACROBAT) trial, we performed longitudinal plasma metabolomic profiling and high-dimensional immune cell phenotyping of 10 patients with CRPC treated with high-dose oral testosterone undecanoate. Testosterone exposure increased circulating metabolites within the arginine–ornithine–polyamine axis, including guanidinoacetic acid, creatine, homoarginine, and N1-acetylspermidine. Notably, nonresponders exhibited elevated levels of circulating polyamines, including putrescine. Although we observed no significant changes in circulating immune cell subset frequencies, integrative analyses revealed positive associations between polyamine-related metabolites and immunosuppressive myeloid populations, including monocytic myeloid-derived suppressor cells (M-MDSC). Collectively, these data provide clinical evidence that high-dose testosterone therapy increases polyamine synthesis from arginine, which is associated with therapy resistance and abundance of immunosuppressive myeloid cells. Implications: High-dose testosterone treatment of patients with CRPC enrolled on the ACROBAT trial increased circulating polyamines, which were associated with nonresponse to treatment and greater abundance of circulating immunosuppressive myeloid cells.