Fig. 4

ENO2 Orchestrates M2 Macrophage Polarization through Direct Interaction with MIF. a Analysis of the strength and potential ways in which various cell types participate in microenvironment signal communication based on CellChat; ENO2⁺ cancer cells exhibit strong microenvironment signals, while the MIF pathway shows significant differences in the transmission/reception of signals in ENO2⁺ cancer cells. b, c The interaction strength between ENO2^-/+ cancer cells and different cell types was analyzed under the involvement of the MIF signaling pathway, with significant differences observed in the interaction with macrophages. d Compared to in situ or non-metastatic in situ lesions, macrophages in metastatic lesions and metastatic in situ lesions have a higher tendency toward M2 polarization. e Co-IP detection confirms the interaction between endogenous ENO2 and MIF. Immunoprecipitation was performed using anti-MIF or anti-ENO2 antibodies in HCT-116 (left) and DLD-1 (right) cell lysates, followed by immunoblotting. IgG served as a negative control. f Co‑IP analysis of endogenous MIF and ENO2 in DLD-1 cells, confirming their endogenous interaction in cancer cells. g GST pull‑down assay demonstrating a direct interaction between MIF and ENO2 in vitro. Recombinant GST‑ENO2 or GST alone was incubated with Myc‑tagged MIF. Proteins bound to glutathione beads were analyzed by immunoblotting with anti‑Myc and anti‑GST antibodies. h, i Molecular docking simulations predicted the key binding interface between ENO2 and MIF, and domain-specific mutants were used in mutual IP analysis to demonstrate complete elimination of binding after domain disruption