Programmable Bacteria with Dynamic Virulence Modulation System for Precision Antitumor Immunity.

Engineered bacteria-mediated antitumor approaches have been proposed as promising immunotherapies for cancer. However, the off-target bacterial toxicity narrows the therapeutic window. Living microbes will benefit from their controllable immunogenicity within tumors for safer antitumor applications. In this study, a genetically encoded microbial activation strategy is reported that uses tunable and dynamic expression of surface extracellular polysaccharides to improve bacterial biocompatibility while retaining therapeutic efficacy. Based on screening of genes associated with Salmonella survival in macrophages, a novel attenuated Salmonella chassis strain AIS (htrA gene-deficient) highly enriched in tumors after administration and rapidly cleared from normal organs are reported. Subsequently, an engineered bacterial strain, AISI-H, is constructed based on the AIS strain and an optimized quorum-sensing regulatory system. The AISI-H strain can achieve recovery of dynamic tumor-specific bacterial virulence through a novel HTRA-RCSA axis-based and quorum-sensing synthetic gene circuit-mediated increase in extracellular polysaccharide content. These strains act "off" in normal organs to avoid unwanted immune activation and "on" in tumors for precise tumor suppression in mice. The AISI-H strain shows significant tumor inhibition and potent activation of anticancer immunity in a melanoma mouse model. The AISI-H strain exhibits excellent biocompatibility. This bacterial regulation strategy expands the applications of microbe-based antitumor therapeutics.

Dual-Engineered Macrophage-Microbe Encapsulation for Metastasis Immunotherapy.

Lung metastases are the leading cause of death among cancer patients. The challenges of inefficient drug delivery, compounded by a robust immunosuppressive microenvironment, make effective treatment difficult. Here, an innovative dual-engineered macrophage-microbe encapsulation (Du-EMME) therapy is developed that integrates modified macrophages and engineered antitumor bacteria. These engineered macrophages, termed R-GEM cells, are designed to express RGD peptides on extracellular membranes, enhancing their tumor cell binding and intratumor enrichment. R-GEM cells are cocultured with attenuated Salmonella typhimurium VNP20009, producing macrophage-microbe encapsulation (R-GEM/VNP cells). The intracellular bacteria maintain bioactivity for more than 24 h, and the bacteria released from R-GEM/VNP cells within the tumor continue to exert bacteria-mediated antitumor effects. This is further supported by macrophage-based chemotaxis and camouflage, which enhance the intratumoral enrichment and biocompatibility of the bacteria. Additionally, R-GEM cells loaded with IFNγ-secreting strains (VNP-IFNγ) form R-GEM/VNP-IFNγ cells. Treatment with these cells effectively halts lung metastatic tumor progression in three mouse models (breast cancer, melanoma, and colorectal cancer). R-GEM/VNP-IFNγ cells vigorously activate the tumor microenvironment, suppressing tumor-promoting M2-type macrophages, MDSCs, and Tregs, and enhancing tumor-antagonizing M1-type macrophages, mature DCs, and Teffs. Du-EMME therapy offers a promising strategy for targeted and enhanced antitumor immunity in treating cancer metastases.

Camouflaging attenuated Salmonella by cryo-shocked macrophages for tumor-targeted therapy.

Live bacteria-mediated antitumor therapies mark a pivotal point in cancer immunotherapy. However, the difficulty in reconciling the safety and efficacy of bacterial therapies has limited their application. Improving bacterial tumor-targeted delivery while maintaining biosafety is a critical hurdle for the clinical translation of live microbial therapy for cancer. Here, we developed "dead" yet "functional" Salmonella-loaded macrophages using liquid nitrogen cold shock of an attenuated Salmonella typhimurium VNP20009-contained macrophage cell line. The obtained "dead" macrophages achieve an average loading of approximately 257 live bacteria per 100 cells. The engineered cells maintain an intact cellular structure but lose their original pathogenicity, while intracellular bacteria retain their original biological activity and are delay freed, followed by proliferation. This "Trojan horse"-like bacterial camouflage strategy avoids bacterial immunogenicity-induced neutrophil recruitment and activation in peripheral blood, reduces the clearance of bacteria by neutrophils and enhances bacterial tumor enrichment efficiently after systemic administration. Furthermore, this strategy also strongly activated the tumor microenvironment, including increasing antitumor effector cells (including M1-like macrophages and CD8+ Teffs) and decreasing protumor effector cells (including M2-like macrophages and CD4+ Tregs), and ultimately improved antitumor efficacy in a subcutaneous H22 tumor-bearing mouse model. The cryo-shocked macrophage-mediated bacterial delivery strategy holds promise for expanding the therapeutic applications of living bacteria for cancer.

Macrophage-mediated tumor-targeted delivery of engineered Salmonella typhi murium VNP20009 in anti-PD1 therapy against melanoma.

Bacterial antitumor therapy has great application potential given its unique characteristics, including genetic manipulation, tumor targeting specificity and immune system modulation. However, the nonnegligible side effects and limited efficacy of clinical treatment limit their biomedical applications. Engineered bacteria for therapeutic applications ideally need to avoid their accumulation in normal organs and possess potent antitumor activity. Here, we show that macrophage-mediated tumor-targeted delivery of Salmonella typhimurium VNP20009 can effectively reduce the toxicity caused by administrating VNP20009 alone in a melanoma mouse model. This benefits from tumor-induced chemotaxis for macrophages combined with their slow release of loaded strains. Inspired by changes in the tumor microenvironment, including a decrease in intratumoral dysfunctional CD8+ T cells and an increase in PDL1 on the tumor cell surface, macrophages were loaded with the engineered strain VNP-PD1nb, which can express and secrete anti-PD1 nanoantibodies after they are released from macrophages. This novel triple-combined immunotherapy significantly inhibited melanoma tumors by reactivating the tumor microenvironment by increasing immune cell infiltration, inhibiting tumor cell proliferation, remodeling TAMs to an M1-like phenotype and prominently activating CD8+ T cells. These data suggest that novel combination immunotherapy is expected to be a breakthrough relative to single immunotherapy.

Bulk and Single-Cell Transcriptome Analyses Revealed That the Pyroptosis of Glioma-Associated Macrophages Participates in Tumor Progression and Immunosuppression.

Glioma is the most common of all central nervous system (CNS) malignancies and is associated with a poor prognosis. Pyroptosis has been proven to be associated with the progression of multiple tumors and CNS diseases. However, the relationships between pyroptosis and clinical prognosis and immune cell infiltration are unclear in glioma. In this study, we conducted a comprehensive exploration of pyroptosis in glioma. First, prognosis-related genes were screened at each key regulatory locus in the pyroptosis pathway, and the prognostic ability and coexpression relationships of GSDMD and its upstream pathway genes NLRC4/CASP1/CASP4 were identified and well validated in multiple datasets. Tissue microarray-based immunohistochemistry results showed higher levels of NLRC4 and N-terminal GSDMD in high-grade gliomas, providing conclusive evidence of pyroptosis in gliomas. The robustness of the prognostic model based on these four genes was well validated in TCGA and CGGA cohorts. Bulk RNA-seq-based analysis showed that the group defined as the high-risk group according to the model showed activation of multiple inflammatory response pathways and impaired synaptic gene expression and had a higher infiltration of bone marrow-derived macrophages (BMDMs) and a hypersuppressed immune microenvironment. More importantly, three independent single-cell RNA-seq (scRNA-seq) datasets demonstrated that tumor-infiltrating macrophages, particularly BMDMs but not tissue-resident microglia, showed significant coexpression of the GSDMD and CASP genes, and BMDMs from high-grade gliomas accounted for a higher proportion of immune infiltrating cells and had higher expression of pyroptosis genes. Finally, we revealed the activation of pathways in response to LPS/bacteria and oxidative stress during BMDM development toward the pyroptosis cell fate by pseudotime trajectory analysis, suggesting potential BMDM pyroptosis initiators. The above results provide not only novel insights into the pathological mechanisms of glioma but also novel therapeutic targets for glioma, suggesting the potential application of pyroptosis inhibitors (e.g., disulfiram).

Combined Cellular Thermometry Reveals That Salmonella typhimurium Warms Macrophages by Inducing a Pyroptosis-like Phenotype.

The attenuated Salmonella typhimurium VNP20009, enriched in tumors, is known to have antitumor effects and recruit macrophages. Little is known, however, about whether VNP will lead to specific changes in macrophages, e.g., cell temperature. Here, using a real-time wireless multicell thermometry system, we reported for the first time that VNP20009 increases the macrophage temperature by 0.2 °C. Nigericin, recognized as an inducer of pyroptosis, was found to induce macrophage warming. Moreover, the ΔsipD-VNP20009 strain failed to induce macrophage pyroptosis and simultaneously failed to warm macrophages, and the Gsdmd-/- macrophages that were unable to achieve pyroptosis were no longer warmed following VNP20009 induction. These results suggested that the occurrence of macrophage pyroptosis is the key to VNP20009-mediated cell warming. With the aid of a single-cell thermometry system, it was further confirmed that cell warming occurred in pyroptosis-like macrophages. Cellular warming was not detected after the induction of pyroptosis in macrophages with loss of mitochondrial biological function, suggesting a critical role of mitochondria in warming. Moreover, we found that VNP20009 caused local tumor temperature increases. The local tumor warming induced by VNP20009 was significantly reduced after macrophage clearance. Notably, this temperature increase contributed to M1-type polarization. These findings expanded our knowledge of the cellular biological changes induced by the strain on macrophages, as well as the biochemical phenomena accompanying pyroptosis, and provide a reference for the study of biochemical signals transduced to biothermal signals with a combined cell-level temperature detector.

Bacterially mediated drug delivery and therapeutics: Strategies and advancements.

It was already clinically apparent 150 years ago that bacterial therapy could alleviate diseases. Recently, a burgeoning number of researchers have been using bacterial regimens filled with microbial therapeutic leads to diagnose and treat a wide range of disorders and diseases, including cancers, inflammatory diseases, metabolic disorders and viral infections. Some bacteria that were designed to have low toxicity and high efficiency in drug delivery have been used to treat diseases successfully, especially in tumor therapy in animal models or clinical trials, thanks to the progress of genetic engineering and synthetic bioengineering. Therefore, genetically engineered bacteria can serve as efficient drug delivery vehicles, carrying nucleic acids or genetic circuits that encode and regulate therapeutic payloads. In this review, we summarize the development and applications of this approach. Strategies for genetically modifying strains are described in detail, along with their objectives. We also describe some controlled strategies for drug delivery and release using these modified strains as carriers. Furthermore, we discuss treatment methods for various types of diseases using engineered bacteria. Tumors are discussed as the most representative example, and other diseases are also briefly described. Finally, we discuss the challenges and prospects of drug delivery systems based on these bacteria.

The Classical Apoptotic Adaptor FADD Regulates Glycolytic Capacity in Acute Lymphoblastic Leukemia.

The Fas-associated death domain (FADD) has long been regarded as a crucial adaptor protein in the extrinsic apoptotic pathway. Despite the non-apoptotic function of FADD is gradually being discovered and confirmed, its corresponding physiological and pathological significance is still unclear. Based on the database of GWAS catalog and GTEx Portal, 17 SNPs associated with leukemia susceptibility were found to be linked to FADD expression. We then investigated a regulatory role of FADD in T-acute lymphoblastic leukemia (T-ALL) using Jurkat cells as a model. Jurkat cells stably depleted of FADD (FADD-/- Jurkat) expression exhibited dampened proliferation, hypersensitivity to Etoposide-induced intrinsic apoptosis whereas near total resistance to TRAIL-induced extrinsic apoptosis. Comparison between wild type and FADD-/- Jurkat cells using iTRAQ-based proteomics revealed considerably altered expression spectrum of genes, and led us to focus on metabolic pathways. Investigation of glycolytic and mitochondrial pathways and relevant enzymes revealed that FADD knockout triggered a metabolic shift from glycolysis to mitochondrial respiration in Jurkat cells. Re-expression of FADD in FADD-/- Jurkat cells partially rescued glycolytic capacity. FADD loss triggers global metabolic reprogramming in Jurkat cells and therefore remains as a potential druggable target for ALL treatment.

MAGP2 induces tumor progression by enhancing uPAR-mediated cell proliferation.

Microfibril-associated glycoprotein 2 (MAGP2) plays an important role in regulating cell signaling and acts as a biomarker to predict the prognostic effect of tumor therapy. However, research on MAGP2 mostly focuses on its extracellular signal transmission features, and its potential intracellular function is rarely reported. Here, we reported that intracellular MAGP2 increased the stability of urokinase-type plasminogen activator receptor (uPAR) in the cell by direct interaction which inhibits the lysosomal-mediated degradation of uPAR. Furthermore, with the detection of protein content changes and proteomics analysis, we found that highly expressed MAGP2 promoted the proliferation of tumor cells through uPAR-mediated p38-NF-ĸB signaling axis activation, enhancement of DNA damage repair and reduction of cell stagnation in the S phase of the cell cycle. In the nude mouse xenograft model of colorectal cancer, the upregulation of MAGP2 in tumor cells significantly promoted tumor progression, while the downregulation of uPAR significantly attenuated tumor progression. These studies elucidate the role of MAGP2 inside the cell and provide a new explanation for why patients with higher MAGP2 expression in tumors are associated with a worse prognosis. In addition, we also determined a mechanism for the stable existence of uPAR in the cell, providing information for the development of tumor drugs targeting uPAR.