Engineering Macrophages via Nanotechnology and Genetic Manipulation for Cancer Therapy

Macrophages play critical roles in tumor progression. In the tumor microenvironment, macrophages display highly diverse phenotypes and may perform antitumorigenic or protumorigenic functions in a context-dependent manner. Recent studies have shown that macrophages can be engineered to transport drug nanoparticles (NPs) to tumor sites in a targeted manner, thereby exerting significant anticancer effects. In addition, macrophages engineered to express chimeric antigen receptors (CARs) were shown to actively migrate to tumor sites and eliminate tumor cells through phagocytosis. Importantly, after reaching tumor sites, these engineered macrophages can significantly change the otherwise immune-suppressive tumor microenvironment and thereby enhance T cell-mediated anticancer immune responses. In this review, we first introduce the multifaceted activities of macrophages and the principles of nanotechnology in cancer therapy and then elaborate on macrophage engineering via nanotechnology or genetic approaches and discuss the effects, mechanisms, and limitations of such engineered macrophages, with a focus on using live macrophages as carriers to actively deliver NP drugs to tumor sites. Several new directions in macrophage engineering are reviewed, such as transporting NP drugs through macrophage cell membranes or extracellular vesicles, reprogramming tumor-associated macrophages (TAMs) by nanotechnology, and engineering macrophages with CARs. Finally, we discuss the possibility of combining engineered macrophages and other treatments to improve outcomes in cancer therapy.

PU.1 Regulates Cathepsin S Expression in Large Yellow Croaker (Larimichthys crocea) Macrophages

Different morphologies have been detected in teleost macrophages. In this study, two macrophage cell lines were sub-cloned from a large yellow croaker head kidney cell line, LYCK. One type of sub-cloned cells was fusiform but the other was round, named LYC-FM and LYC-RM cells respectively, based on their morphologies. Both types showed the characteristics of macrophages, including expression of macrophage-specific marker genes, possession of phagocytic and bactericidal activities, and production of reactive oxygen species (ROS) and nitric oxide (NO). The transcription factor PU.1, crucial for the development of macrophages in mammals, was found to exist in two transcripts, PU.1a and PU.1b, in large yellow croaker, and constitutively expressed in LYC-FM and LYC-RM cells. The expression levels of PU.1a and PU.1b could be upregulated by recombinant large yellow croaker IFN-γ protein (rLcIFN-γ). Further studies showed that both PU.1a and PU.1b increased the expression of cathepsin S (CTSS) by binding to different E26−transformation−specific (Ets) motifs of the CTSS promoter. Additionally, we demonstrated that all three domains of PU.1a and PU.1b were essential for initiating CTSS expression by truncated mutation experiments. Our results therefore provide the first evidence that teleost PU.1 has a role in regulating the expression of CTSS.

Lysosomal iron recycling in mouse macrophages is dependent upon both reductases LcytB and Steap3

Iron that is stored in macrophages as ferritin can be made bioavailable by degrading ferritin in the lysosome and releasing iron back into the cytosol. Iron stored in ferritin is found as Fe3+ and must be reduced to Fe2+ before it can be exported from the lysosome. Here we report that the lysosomal reductase Cyb561a3 (LcytB) and the endosomal reductase Six-transmembrane epithelial antigen of the prostate 3 (Steap3) act as lysosomal ferrireductases in the mouse macrophage cell line RAW264.7 converting Fe3+ to Fe2+ for iron recycling. We determined that when lysosomes were loaded with horse cationic ferritin, reductions or loss of LcytB or Steap3 using CrispR/Cas9-mediated knockout technology resulted in decreased lysosomal iron export. Loss of both reductases was additive in decreasing lysosomal iron export. Decreased reductase activity resulted in increased transcripts for iron acquisition proteins DMT1 and Tfrc1 suggesting cells were iron limited. We show transcript expression of LcytB and Steap3 is decreased in macrophages exposed to Escherichia coli pathogen UTI89 supporting a role for these reductases in regulating iron availability for pathogens. We further show that loss of LcytB and Steap3 in macrophages infected with UTI89, led to increased intracellular UTI89 proliferation suggesting that the endolysosomal system is retaining Fe3+ that can be used for intravesicular pathogen proliferation. Together, our findings reveal an important role for both LcytB and Steap3 in macrophage iron recycling and suggest that limiting iron recycling by decreasing expression of endolysosomal reductases is an innate immune response to protect against pathogen proliferation and sepsis.

Macrophage cell lines and murine infection by Salmonella Typhi L-form bacteria

Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic-resistance genes is well-established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as the one due to treatment with bacterial cell-wall inhibiting antibiotics like ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin. This is different from in vitro drug testing where the organism is exposed to fixed drug concentrations from the beginning till the end. To mimic the mode of antibiotic exposure of microorganisms in the tissues, we cultured the wild type, ampicillin-sensitive Salmonella Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that exhibited several features of the so-called L-form of bacteria, such as the absence of cell wall, altered shape and slower growth rate compared with the parental strain. Studies on the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental strain. These results suggested that L-form generation following initiation of antibiotic treatment could lead to drug escape of S. Typhi and direct spread to new cells (macrophages), which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through faeco-oral route, highlighting the need for new approaches to decrease the reservoir of infection.

Tumor Microenvironment Stimuli-Responsive Macrophage Cell Membrane-Decorated Multi-Functional Self-Assembled Gold-Quantum Dots Nanomaterials for Tumor Theranostic

Tumor microenvironment (TME) is intently related to tumor growth, progression and invasion, leading to drug resistance and insufficient therapeutic efficacy. However, remodelling TME and utilizing TME for exploring intelligent nanomaterials that can realize tumor theranostic is still challenging. Nowadays, the theranostic based on chemotherapy exposes some deficiencies, such as low targeting, weak permeability and premature clearance. Furthermore, it is challenging to cure drug-resistant tumors effectively. For the sake of solving these problems, a biomimetic decomposable nano-theranostic (MMV-Au-CDs-DOX) was well-established in this work. The Au-CDs are coated with macrophage-derived microvesicle to realize drug release accurately and enhance the biocompatibility of internal nanoparticles. Furthermore, MMV-Au-CDs-DOX would locate in the inflammation position of tumor, and disintegrate correspondingly into pieces with certain different functions stimulated by TME. Subsequently, the released anti-tumor nanodrugs were used for multimodal therapy, including chemotherapy and chemodynamic therapy. In addition, combined with the ability of Au-CDs to recognize GSH specifically, the off-on fluorescent probe was constructed to monitor the GSH of patients and provided information on chemotherapy resistance.

Aggregated Mycobacterium tuberculosis Enhances the Inflammatory Response

Mycobacterium tuberculosis (Mtb) bacilli readily aggregate. We previously reported that Mtb aggregates lead to phagocyte death and subsequent efficient replication in the dead infected cells. Here, we examined the transcriptional response of human monocyte derived macrophages to phagocytosis of aggregated Mtb relative to phagocytosis of non-aggregated single or multiple bacilli. Infection with aggregated Mtb led to an early upregulation of pro-inflammatory associated genes and enhanced TNFα signaling via the NFκB pathway. These pathways were significantly more upregulated relative to infection with single or multiple non-aggregated bacilli per cell. Phagocytosis of aggregates led to a decreased phagosome acidification on a per bacillus basis and increased phagocyte cell death, which was not observed when Mtb aggregates were heat killed prior to phagocytosis. Mtb aggregates, observed in a granuloma from a patient, were found surrounding a lesion cavity. These observations suggest that TB aggregation may be a mechanism for pathogenesis. They raise the possibility that aggregated Mtb, if spread from individual to individual, could facilitate increased inflammation, Mtb growth, and macrophage cell death, potentially leading to active disease, cell necrosis, and additional cycles of transmission.

CircNFIC Balances Inflammation and Apoptosis by Sponging miR-30e-3p and Regulating DENND1B Expression

Disordered inflammation and apoptosis are closely related to diseases, and inflammation can also promote cell apoptosis, where growing evidence has shown that circular RNAs (circRNAs) play important roles. Lipopolysaccharide (LPS) is the main component of the cytoderm of gram-negative bacterium, which can cause inflammatory responses in macrophages. We constructed an inflammatory model by exposing chicken macrophage cell lines (also known as HD11) to LPS for in vitro experiments. In this study, we validated a novel circRNA—circNFIC—which was dramatically up-regulated in tissues infected by coccidia and cells exposed to LPS. Besides, circNFIC could significantly promote the expression levels of pro-inflammation factors, including (IL-1β, TNFα, and IFNγ) and pro-apoptosis maker genes (caspase 3 and caspase 8) in HD11 exposed to LPS or not. In terms of mechanism, circNFIC exerted notable effects on DENND1B to regulate cell inflammation and apoptosis by sponging miR-30e-3p. The molecular functions played by miR-30e-3p and DENND1B have been explored, respectively. In addition, the effects of circNFIC knockdown suppressing the expression of pro-inflammatory and pro-apoptosis functions could be reversed by a miR-30e-3p inhibitor. On the whole, circNFIC promoted cell inflammation and apoptosis via the miR-30e-3p/DENND1B axis.

144 SIRPα deficient CAR-Macrophages exhibit enhanced anti-tumor function and bypass the CD47 immune checkpoint

BackgroundAdoptive macrophage cell therapy represents a novel approach for cancer immunotherapy. Macrophages engineered to express chimeric antigen receptors (CAR-M) have shown promising pre-clinical results against solid tumors by improving tumor clearance, overall survival and facilitating the remodeling of the tumor microenvironment to induce a potent adaptive immune response. CD47 is a well-established macrophage immune checkpoint molecule that is over-expressed on tumor cells. CD47 binds to the macrophage signal regulatory protein α (SIRPα) to limit phagocytosis and macrophage effector functions. In this study we evaluated the impact of CD47 on CAR-M activity and showed that CD47-resistant targeted macrophage cell therapy mediates enhanced anti-tumor activity.MethodsCRISPR/Cas9 was used to deplete the cognate receptor SIRPα from primary human macrophages (>90% efficiency and >90% viability) to increase CAR-M function. To assess anti-tumor activity of CAR-M, < i >in vitro</i > co-culture assays were established with an anti- human epidermal growth factor receptor 2 (HER2) CAR and HER2+ tumor cell lines. Macrophage killing and phagocytosis of target cells were quantified in real-time using a genetically encoded fluorophore (to monitor tumor cell growth) or a pH-sensitive dye (to monitor phagocytic acidification). In parallel, phenotypic characterization of surface molecules, cytokine secretion levels, biochemical analysis of downstream signaling molecules and response to purified HER2 and CD47 protein stimulation were evaluated.ResultsSIRPα knockout (KO) alone failed to induce tumor phagocytosis and cytotoxicity but enhanced targeted CAR-M anti-tumor activity. This was demonstrated by a reduced time required to kill 50% of tumor cells and a 2-fold increase in phagocytic activity, indicating synergy between SIRPα KO and CAR stimulation. Furthermore, in the absence of SIRPα, enhanced cytokine/chemokine secretion, macrophage polarization, and downstream signaling were observed.ConclusionsWe show for the first time the feasibility of generating gene edited primary human CAR macrophages for therapeutic purposes, and demonstrate that SIRPα deletion enhances the targeted anti-tumor activity of CAR-M.