Tumor-associated macrophages (TAMs) are predominantly M2 phenotype in solid cancers including hepatocellular carcinoma (HCC). Though differentiation of M2 macrophages has been recently linked to fatty acid oxidation (FAO), whether FAO plays a role in functional maintenance of M2 macrophages is still unclear. Here, we used an in vitro model to mimic TAM-HCC interaction in tumor microenvironment. We found that M2 monocyte-derived macrophages (MDMs) enhanced the proliferation, migration, and invasion of HCC cells through an FAO-dependent way. Further investigations identified that IL-1β mediated the pro-migratory effect of M2 MDM. Using etomoxir and siRNA to inhibit FAO and palmitate to enhance FAO, we showed that FAO was responsible for the up-regulated secretion of IL-1β and, thus, the pro-migratory effect in M2 MDMs. In addition, we proved that IL-1β induction was reactive oxygen species and NLRP3-dependent. Our study demonstrates that FAO plays a key role in functional human M2 macrophages by enhancing IL-1β secretion to promote HCC cell migration. These findings provide evidence for different dependency of energy sources in macrophages with distinct phenotypes and functions, and suggest a novel strategy to treat HCC by reprogramming cell metabolism or modulating tumor microenvironment.

Vascular endothelial (VE)–cadherin forms homotypic adherens junctions (AJs) in the endothelium, whereas N-cadherin forms heterotypic adhesion between endothelial cells and surrounding vascular smooth muscle cells and pericytes. Here we addressed the question whether both cadherin adhesion complexes communicate through intracellular signaling and contribute to the integrity of the endothelial barrier. We demonstrated that deletion of N-cadherin (Cdh2) in either endothelial cells or pericytes increases junctional endothelial permeability in lung and brain secondary to reduced accumulation of VE-cadherin at AJs. N-cadherin functions by increasing the rate of VE-cadherin recruitment to AJs and induces the assembly of VE-cadherin junctions. We identified the dual Rac1/RhoA Rho guanine nucleotide exchange factor (GEF) Trio as a critical component of the N-cadherin adhesion complex, which activates both Rac1 and RhoA signaling pathways at AJs. Trio GEF1-mediated Rac1 activation induces the recruitment of VE-cadherin to AJs, whereas Trio GEF2-mediated RhoA activation increases intracellular tension and reinforces Rac1 activation to promote assembly of VE-cadherin junctions and thereby establish the characteristic restrictive endothelial barrier.

Background Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. Methods First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. Results We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. Conclusions By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.