Biology subjects

Greeson, E. M.

Publications and source records attributed to Greeson, E. M..

5 recordsLinked to original sources

bioRxiv2024

Engineered endosymbionts that modulate primary macrophage function and attenuate tumor growth by shifting the tumor microenvironment

Modulating gene expression in macrophages can be used to improve tissue regeneration and to redirect tumor microenvironments (TME) toward positive therapeutic outcomes. We have developed Bacillus subtilis as an engineered endosymbiont (EES) capable of residing inside the eukaryotic host cell cytoplasm and controlling the fate of macrophages. Secretion of mammalian transcription factors (TFs) from B. subtilis that expresses listeriolysin O (LLO; allowing the EES to escape destruction by the macrophage) modulated expression of surface markers, cytokines and chemokines, indicating functional changes in a macrophage/monocyte cell line. The engineered B. subtilis LLO TF strains were evaluated in murine bone marrow-derived macrophages (BMDMs) by flow cytometry, chemokine/cytokine profiling, metabolic assays and RNA-Seq. Delivery of TFs by the EES shifted BMDM gene expression, production of cytokine and chemokines and metabolic patterns, indicating that the TF strains could guide primary macrophage function. Thereafter, the ability of the TF strains to alter the TME was characterized in vivo, in an orthotopic murine model of triple-negative breast cancer to assess therapeutic effects. The TF strains altered the TME by shifting immune cell composition and attenuating tumor growth. Additionally, multiple doses of the TF strains were well-tolerated by the mice. The use of B. subtilis LLO TF strains as EES showed promise as a unique cancer immunotherapy by directing immune function intracellularly. The uses of EES could be expanded to modulate other mammalian cells over a range of biomedical applications.

synthetic biology↗

bioRxiv2024

Magnetic particle imaging reveals that iron-labeled extracellular vesicles accumulate in brains of mice with metastases

The incidence of breast cancer remains high worldwide and is associated with a significant risk of metastasis to the brain that can be fatal; this is due, in part, to the inability of therapeutics to cross the blood brain barrier (BBB). Extracellular vesicles (EVs) have been found to cross the BBB and further, have been used to deliver drugs to tumors. EVs from different cell types appear to have different patterns of accumulation and retention as well as efficiency of bioactive cargo delivery to recipient cells in the body. Engineering EVs as delivery tools to treat brain metastases, therefore, will require an understanding of the timing of EV accumulation, and their localization relative to metastatic sites. Magnetic particle imaging (MPI) is a sensitive and quantitative imaging method that directly detects superparamagnetic iron. Here, we demonstrate MPI as a novel tool to characterize EV biodistribution in metastatic disease after labeling EVs with superparamagnetic iron oxide (SPIO) nanoparticles. Iron-labeled EVs (FeEVs) were collected from iron-labeled parental primary 4T1 tumor cells and brain-seeking 4T1BR5 cells, followed by injection into mice with orthotopic tumors or brain metastases. MPI quantification revealed that FeEVs were retained for longer in orthotopic mammary carcinomas compared to SPIOs. MPI signal due to iron could only be detected in brains of mice bearing brain metastases after injection of FeEVs, but not SPIOs, or FeEVs when mice did not have brain metastases. These findings indicate the potential use of EVs as a therapeutic delivery tool in primary and metastatic tumors. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/584146v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@8cf21org.highwire.dtl.DTLVardef@56b6f0org.highwire.dtl.DTLVardef@104626eorg.highwire.dtl.DTLVardef@da60cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Magnetothermal control of temperature-sensitive repressors in superparamagnetic iron nanoparticle-coated Bacillus subtilis

Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) and resulting images can be used to guide magnetothermal heating. Alternating magnetic fields (AMF) cause local temperature increases in regions with SPIONs, and we investigated the ability of magnetic hyperthermia to regulate temperature-sensitive repressors (TSRs) of bacterial transcription. The TSR, TlpA39, was derived from a Gram-negative bacterium, and used here for thermal control of reporter gene expression in Gram-positive, Bacillus subtilis. In vitro heating of B. subtilis with TlpA39 controlling bacterial luciferase expression, resulted in a 14.6-fold (12-hour; h) and 1.8-fold (1-h) increase in reporter transcripts with a 9.0-fold (12-h) and 11.1-fold (1-h) increase in bioluminescence. To develop magnetothermal control, B. subtilis cells were coated with three SPION variations. Electron microscopy coupled with energy dispersive X-ray spectroscopy revealed an external association with, and retention of, SPIONs on B. subtilis. Furthermore, using long duration AMF we demonstrated magnetothermal induction of the TSRs in SPION-coated B. subtilis with a maximum of 4.6-fold increases in bioluminescence. After intramuscular injections of SPION-coated B. subtilis, histology revealed that SPIONs remained in the same locations as the bacteria. For in vivo studies, 1-h of AMF is the maximum exposure due to anesthesia constraints. Both in vitro and in vivo, there was no change in bioluminescence after 1-h of AMF treatment. Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to transcriptional control that expands options for targeted bacteriotherapies.

bioengineering↗

bioRxiv2021

Magnetic particle imaging of magnetotactic bacteria as living contrast agents is improved by altering magnetosome structures

Iron nanoparticles used as imaging contrast agents can help differentiate between normal and diseased tissue, or track cell movement and localize pathologies. Magnetic particle imaging (MPI) is an imaging modality that uses the magnetic properties of iron nanoparticles to provide specific, quantitative and sensitive imaging data. MPI signals depend on the size, structure and composition of the nanoparticles; MPI-tailored nanoparticles have been developed by modifying these properties. Magnetotactic bacteria produce magnetosomes which mimic synthetic nanoparticles, and thus comprise a living contrast agent in which nanoparticle formation can be modified by mutating genes. Specifically, genes that encode proteins critical to magnetosome formation and regulation, such as mamJ which helps with filament turnover. Deletion of mamJ in Magnetospirillum gryphiswaldense, MSR-1 led to clustered magnetosomes instead of the typical linear chains. Here we examined the effects of this magnetosome structure and revealed improved MPI signal and resolution from clustered magnetosomes compared to linear chains. Bioluminescent MSR-1 with the mamJ deletion were injected intravenously into tumor-bearing and healthy mice and imaged using both in vivo bioluminescence imaging (BLI) and MPI. BLI revealed the location and viability of bacteria which was used to validate localization of MPI signals. BLI identified the viability of MSR-1 for 24 hours and MPI detected iron in the liver and in multiple tumors. Development of living contrast agents offers new opportunities for imaging and therapy by using multimodality imaging to track the location and viability of the therapy and the resulting biological effects.

bioengineering↗

bioRxiv2021

Engineered endosymbionts capable of directing mammalian cell gene expression

Modular methods for directing mammalian gene expression would enable advances in tissue regeneration, enhance cell-based therapeutics and improve modulation of immune responses. To address this challenge, engineered endosymbionts (EES) that escape endosomal destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to control host cell gene expression were developed. Microscopy confirmed that EES escape phagosomes, replicate within the cytoplasm, and can secrete reporter proteins into the cytoplasm that were then transported to the nucleus. Synthetic operons encoding the mammalian transcription factors, Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the EES genome. Using controlled induction, these EES were shown to direct gene expression in J774A.1 macrophage/monocyte cells and modulate the host cell fates. Expressing mammalian transcription factors from engineered intracellular bacteria as endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/463266v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@2b5935org.highwire.dtl.DTLVardef@1edd8edorg.highwire.dtl.DTLVardef@3cb5b9org.highwire.dtl.DTLVardef@1be9b5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗