Coculturing liver cancer cells and monocytes in spheroids conditions monocytes to adopt tumor-associated macrophage phenotypes that favor tumor growth via cholesterol metabolism.

Tumor-infiltrating immune cells and their crosstalk with cancer cells in the tumor microenvironment (TME) play a crucial role in shaping tumor progression and response to therapy. We utilized 3-dimensional liver cancer spheroids incorporating human primary monocytes to investigate the crosstalk between tumor-associated macrophages (TAMs) and Hepatocellular carcinoma (HCC) cells, HepG2 and PLC/PRF/5. Using multiplexed gene expression panels, the critical pathways involved in shaping primary human monocytes to adopt TAMs phenotypes were identified. The specific inhibitor for an identified pathway was used to explore its involvement in polarization of TAMs. In the cocultured spheroids comprising the human HCC cell lines, the infiltrating monocytes resembled protumor M2-like macrophage phenotypes. Gene expression panels of the infiltrating monocytes demonstrated that the upregulated genes were enriched in the cholesterol metabolism pathway. Cholesterol metabolism-related genes were upregulated together with the nuclear receptors, PPARG and LXR. When lysosomal acid lipase (LAL), the key enzyme necessary for the hydrolysis of lipoprotein, was inhibited, infiltrating monocytes in 3-dimensional spheroid coculture showed significantly decreased M2 marker and lipid uptake receptor expression as well as increased cellular lipid content, which indicated that cholesterol metabolism was important for conditioning the TAMs. Moreover, LAL inhibition reduced the spheroid growth and invasiveness of HCC cell lines. Small interfering RNA-mediated LAL silencing in monocytes yielded similar results upon spheroid coculture. These data indicated that liver cancer cells and infiltrating monocytes participate in crosstalk via cholesterol metabolism to condition monocytes toward TAMs, which favors tumor growth and survival, thereby promoting liver cancer progression.

Screening of compounds to identify novel epigenetic regulatory factors that affect innate immune memory in macrophages.

Trained immunity and tolerance are part of the innate immune memory that allow innate immune cells to differentially respond to a second encounter with stimuli by enhancing or suppressing responses. In trained immunity, treatment of macrophages with ß-glucan (BG) facilitates the production of proinflammatory cytokines upon lipopolysaccharide (LPS) stimulation. For the tolerance response, LPS stimulation leads to suppressed inflammatory responses during subsequent LPS exposure. Epigenetic reprogramming plays crucial roles in both phenomena, which are tightly associated with metabolic flux. In this study, we performed a screening of an epigenetics compound library that affects trained immunity or LPS tolerance in macrophages using TNFα as a readout. Among the 181 compounds tested, one compound showed suppressive effects, while 2 compounds showed promoting effects on BG-trained TNFα production. In contrast, various inhibitors targeting Aurora kinase, histone methyltransferase, histone demethylase, histone deacetylase and DNA methyltransferase showed inhibitory activity against LPS tolerance. Several proteins previously unknown to be involved in innate immune memory, such as MGMT, Aurora kinase, LSD1 and PRMT5, were revealed. Protein network analysis revealed that the trained immunity targets are linked via Trp53, while LPS tolerance targets form three clusters of histone-modifying enzymes, cell division and base-excision repair. In trained immunity, the histone lysine methyltransferase SETD7 was identified, and its expression was increased during BG treatment. Level of the histone lysine demethylase, LSD1, increased during LPS priming and siRNA-mediated reduction resulted in increased expression of Il1b in LPS tolerance. Taken together, this screening approach confirmed the importance of epigenetic modifications in innate immune memory and provided potential novel targets for intervention.