<i>Ganoderma microsporum</i> Immunomodulatory Protein (GMI) Enhances Phagocytosis by Suppressing STAT3/CD47 Signaling in EGFR-Mutant NSCLC Resistant to Gefitinib and Osimertinib

Target therapy is effective for epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancer (NSCLC). However, resistance often occurs after treatment for several months. Macrophages have difficulty in devouring resistant cells. <i>Ganoderma</i> immunomodulatory protein (GMI) exhibits anti-tumour and immunomodulatory activities. This study aimed to investigate whether GMI overcomes Osimertinib (Tagrisso) and Gefitinib (Iressa) resistance via enhancing macrophage polarization. GMI attenuated signal transducer and activator of transcription 3 (STAT3) phosphorylation and downstream CD47 expression in parental and resistant cells via Western blot and RT-qPCR. Overexpressed STAT3 restored GMI-induced apoptosis and GMI-reduced transcription of CD47 in HCC827 and H1975 lung cancer cells. Phospho-STAT3 inhibitor (W1131) also reduced the expression of CD47 in NSCLC cells. The interaction between GMI and W1131 was effective in reducing phosphorylated STAT3 and CD47. ImageXpress Pico analysis revealed that GMI enhanced phagocytotic activity of macrophages toward tumour cells with Red CMTPX and Green CMFDA dyes. The results showed that GMI enhanced macrophage phagocytosis of lung cancer cells by inhibiting STAT3 and reducing CD47 expression. In addition, GMI enhanced M1 inhibition of M2 polarization but had no effect on M1 differentiation. This is the first study to demonstrate that GMI enhances macrophage phagocytosis and modulates the STAT3-CD47 axis to overcome EGFR-TKI resistance in NSCLC, highlighting its potential as a novel adjunct immunotherapeutic agent.