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Utilizing engineered endosymbionts to modulate primary macrophage function and attenuate tumor growth by shifting the tumor microenvironment

ABSTRACT: Modulating gene expression in macrophages can be used to improve tissue regeneration and redirect tumor microenvironments (TME) toward positive therapeutic outcomes. We have developed Bacillus subtilis as an engineered endosymbiont (EES) that resides in the host cell cytoplasm and controls the fates 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. Secretion 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. Therefore, the ability of the TF strains to alter the TME was characterized in vivo, in a murine 4T1 orthotopic breast cancer model 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 therapeutic approach for cancer immunotherapy. The uses of EES could be expanded to modulate other mammalian cells over a range of biomedical applications.

ORGANISM(S): Mus musculus

PROVIDER: GSE239519 | GEO | 2023/09/03

REPOSITORIES: GEO

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Project description:Cerebral listeriosis is characterized by neuronal apoptosis and microglial cell activation, but little is known about the bacterial virulence factors involved in this process and how bacterial dissemination is controlled. Here, we show that the cellular target of Listeria monocytogenes (LM) in murine hippocampal cultures is microglia rather than neurons or other glial cells, which are rarely infected. This in vitro model served to demonstrate that infected microglial cells release a soluble factor to the medium responsible for neuronal apoptosis. We investigated the production of this factor in a well-established murine microglia cell model BV2 cells, and compared with J-774 macrophage cells after infection with different LM bacterial mutants. Our purpose was to study in both cell types parameters such as the listericidal capacities, pro-inflammatory cytokines released, and bacterial factors involved in the intracellular cycle. Our data reveal that microglia shows unique features to handle a LM infection. Microglia is three times more permissive for LM intracellular growth than murine macrophages; while producing five times higher levels of pro-inflammatory cytokines IL-6, MCP-1 and TNF-α than macrophages. Using different LM mutants (i.e., LMWT, LM∆LLO, LM∆ActA or LM∆plcB) we conclude that LLO and ActA are not involved in LM proliferation within microglia cells, while they are required for survival within macrophages. Moreover, ActA controls TNF-α production, a cytokine involved in neural apoptosis. Transcriptional differential response of microglia infected with different LM mutants reflected ActA controls the TNF-a signaling pathway through out molecules and chemokines involved in this pathway such as NFkβ, MAPK-1, Ccl2, Ccl3, Ccl4 and CxCl10. The project implies the analysis of two replicates from three different conditions, in total 6 samples: microglia cells non-infected, microglia cells infected with Listeria monocytogenes (LM) wild type (LLO+), microglia cells infected with LM deficient in listeriolysin O (LLO-) and microglia cells infected with LM deficient in ActA (1942). References samples were microglia cells non-infected (NT). Infection with LM was performed for 1 hour, followed by 24 hours in medium with antibiotics to avoid bacterial extracellular growth. Gene expression analysis was performed using Partek Genomics Suite software (version 6.11.0801; Partek). GeneChip data were filtered to remove those probe sets with an intensity raw value close to background levels. Probe sets for each array are pre-processed using RMA. Also, probe sets whose expression change under all experimental conditions was below a threshold, based on the standard desviation of the normalised intensity values, were filtered.

Project description:Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME) which can either promote tumor progression (M2) or induce antitumor immunity (M1). The hypothesis of our study is that the expression of FOLR2 is associated with an M2-like macrophage profile. The main goal of this study is to compare the transcriptomic profile of FOLR2 positive and FOLR2 negative TAMs obtained from the ascites of C57BL/6 mice bearing ovarian ID8 intraperitoneal tumors. Abstract: The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Selective targeting of the pro-tumorigenic subset of TAMs represents an attractive therapeutic strategy. Here, we demonstrate that a subset of TAMs that expressing folate receptor β (FRβ) possess an immunosuppressive, tumor-promoting M2-like profile, while FRβ negative TAMs feature pro-inflammatory M1 macrophage properties. In syngeneic tumor mouse models, the administration of FRβ-targeted chimeric antigen receptor (CAR) T-cells mediated elimination of FRβ+ TAMs in the TME, which resulted in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8+ T-cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRβ-specific CAR T-cells also improved the effectiveness of tumor-directed anti-mesothelin CAR T-cells, while simultaneous co-administration of both CAR products did not. Thus, CAR T-cell-mediated depletion of immunosuppressive M2-like TAMs incites a pro-inflammatory TME that broadens endogenous antitumor immunity and limits tumor progression, highlighting the pro-tumor role of FRβ+ TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens.

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Utilizing engineered endosymbionts to modulate primary macrophage function and attenuate tumor growth by shifting the tumor microenvironment

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