Anti-inflammatory mechanisms in cancer research: Characterization of a distinct M2-like macrophage model derived from the THP-1 cell line.

AIMS: Macrophages play an essential role in cancer development. Tumor-associated macrophages (TAMs) have predominantly M2-like attributes that are associated with tumor progression and poor patient survival. Numerous methods have been reported for differentiating and polarizing macrophages in vitro, but there is no standardized and validated model for creating TAMs. Primary cells show varying cytokine responses depending on their origin and functional studies utilizing these cells may lack generalization and validity. A distinct cell line-derived TAM-like M2 subtype is required to investigate the mechanisms mediated by anti-inflammatory TAMs in vitro. Our previous work demonstrated a standardized protocol for creating an M2 subtype derived from a human THP-1 cell line. The cell expression profile, however, has not been validated. The aim of this study was to characterize and validate the TAM-like M2 subtype macrophage created based on our protocol to introduce them as a standardized model for cancer research. METHODS AND RESULTS: Using qRT-PCR and ELISA, we demonstrated that proinflammatory, anti-inflammatory, and tumor-associated marker expression changed during THP-1-derived marcrophage development in vitro, mimicking a TAM-related profile (e.g., TNFα, IL-1ß). The anti-inflammatory marker IL-8/CXCL8, however, is most highly expressed in young M0 macrophages. Flow cytometry showed increased expression of CD206 in the final TAM-like M2 macrophage. Single-cell RNA-sequencing analysis of primary human monocytes and colon cancer tissue macrophages demonstrated that cell line-derived M2 macrophages resembled a TAM-related gene profile. CONCLUSIONS: The THP-1-derived M2 macrophage based on a standardized cell line model represents a distinct anti-inflammatory TAM-like phenotype with an M2a subtype profile. This model may provide a basis for in vitro investigation of functional mechanisms in a variety of anti-inflammatory settings, particularly colon cancer development.

Itaconate and leptin affecting PPARγ in M2 macrophages: A potential link to early-onset colorectal cancer.

BACKGROUND: Along with the rising incidence of obesity, there has been an increase in patients diagnosed with early-onset colorectal cancer (<50 years old). In colorectal cancer, worse patient survival is associated with certain cytokine expression and downregulation of peroxisome proliferator activated receptor gamma expression. The effects of the obesity hormone leptin and macrophage-specific metabolite itaconate on these mechanisms are poorly understood. We investigated their impact on peroxisome proliferator activated receptor gamma and macrophage cytokine expression in vitro. METHODS: M2-like macrophages were treated with either leptin, 4-octyl itaconate, or dimethyl itaconate in a dose- and time-dependent manner. Gene expression after treatment with 4 doses (D1-4) of each compound was analyzed at 4 time points (3, 6, 18, and 24 hours). RESULTS: Peroxisome proliferator activated receptor gamma was downregulated after 4-octyl itaconate treatment at 18 hours (FC -32.67, P ≤ .001). Interleukin-8 was upregulated after leptin and dimethyl itaconate treatment at 6 hours (FC 26.35 at D4, P ≤ .001, and FC 23.26 at D3, P = .006). Dimethyl itaconate upregulated IL-1ß at 24 hours (FC 18.00 at D4, P ≤ .001). Tumor necrosis factor-α showed maximum downregulation after 4-octyl itaconate at 18 hours (FC -103.25 at D4, P ≤ .001). CONCLUSIONS: Itaconate downregulates peroxisome proliferator activated receptor gamma as a tumor-suppressing factor and upregulates anti-inflammatory cytokines in M2-like macrophages. Itaconate provides a link between obesity and colorectal cancer and may be a key regulator in early-onset colorectal cancer.