Effects of IFN-γ on the immunological microenvironment and TAM polarity in stage IA non-small cell lung cancer and its mechanisms
Authors: Zhao Weijie , Wang Huipeng , Zhang Xiangwu , Zhang Li , Pu Wei , Ma Yuhui , Chen Wanling 
Published: January 2024

Abstract
Objective
To investigate the effect of interferon-γ (IFN-γ) on the immune microenvironment and the polarity of tumor-associated macrophages (TAMs) in stage IA non-small cell lung cancer (NSCLC) and its mechanisms.

Methods
Human non-small cell lung cancer A549 cells were treated with a series of IFN-γ concentrations (0, 50, 100, 150, 200, 250, and 300 ng/mL). Tumor tissues from patients with stage IA NSCLC were cultured using the air–liquid interface culture technique to establish a tumor microenvironment (TME) organ model. The NSCLC model was constructed by subcutaneously embedding small tumor pieces into the back of nonobese diabetic severe combined immune deficiency (NOD SCID) mice. The size and weight of the tumors were recorded, and the tumor volume was calculated. CCK-8 assays were used to investigate cell proliferation, flow cytometry and TUNEL staining were used to evaluate cell apoptosis, colony formation was investigated by cloning experiments, and cell invasion and migration were evaluated by Transwell assays and scratch tests. The expression of apoptosis-related proteins (Bax, Bcl-2 and C-caspase 3), M2 polarization-related markers (CD163, CD206 and IDO1), and marker proteins of cytotoxic T cells and helper T cells (CD8 and CD4) was detected by Western blot. The expression of Ki-67 and IDO1 was detected by immunohistochemistry, and the levels of IL-6, IL-10, IL-13 and TNF-α were measured by ELISA. The expression of CD68 was measured by RT‒qPCR, and the phagocytosis of TAMs was evaluated by a Cell Trace CFSE kit and cell probe staining.

Results
The proliferation activity of A549 cells increased with increasing IFN-γ concentration and peaked when the concentration reached 200 ng/mL, and the proliferation activity of A549 cells was suppressed thereafter. After treatment with 200 ng/mL IFN-γ, the apoptosis rate of cells decreased, the number of cell colonies increased, the invasion and migration of cells were promoted, the expression of Bax and C-caspase 3 was downregulated, and the expression of Bcl-2 was upregulated in cells and the TME model. In the TME model, CD163, CD206, IDO1 and Ki-67 were upregulated, CD8 and CD4 were downregulated, apoptosis was reduced, the levels of IL-6 and TNF-α were decreased, and the levels of IL-10 and IL-13 were increased. IL-4 induced TAMs to express CD163 and CD206, reduced the levels of IL-6 and TNF-α, increased the levels of IL-10 and IL-13, and weakened the phagocytic function of TAMs. IFN-γ treatment further enhanced the effect of IL-4 and enhanced the viability of A549 cells. IDO1 decreased the viability of T cells and NK cells, while suppressing the effect of IFN-γ. In mice, compared with NSCLC mice, the tumor volume and weight of the IFN-γ group were increased, the expression of CD163, CD206, IDO1, Ki-67 and Bcl-2 in tumor tissue was upregulated, the expression of Bax and C-caspase 3 was downregulated, and apoptosis was reduced. The levels of IL-6 and TNF-α were decreased, and the levels of IL-10 and IL-13 were increased in the serum of mice.

Conclusion
In stage IA NSCLC, a low concentration of IFN-γ promotes the polarization of TAMs to the M2 phenotype in the TME model by upregulating the expression of IDO1, promoting the viability of cancer cells, inhibiting the viability of T cells and NK cells, and thus establishing an immune microenvironment conducive to tumor progression.