Study on the efficacy of IFN-γ- and sPD-1-overexpressing BMSCs in enhancing immune effects for the treatment of lung adenocarcinoma

Abstract

Background: Soluble programmed cell death receptor-1 (sPD-1) blocks the PD-1/PD-L1 pathway, reverses tumor immune suppression, and inhibits tumor growth. However, clinical applications are limited by its poor tissue distribution and rapid dispersion. Bone marrow-derived mesenchymal stem cells (BMSCs) are favorable carriers for tumor immunotherapy due to their capacity for external gene introduction and targeted tumor homing. However, they may inadvertently promote tumor growth. Interferon-gamma (IFN-γ) inhibits BMSC-mediated tumor growth and stimulates antigen-presenting cells to activate T lymphocytes. This study utilizes BMSCs transfected with IFN-γ as carriers for sPD-1, enabling the targeted homing of sPD-1 to tumor tissues, thereby enhancing the efficacy and sustained stability of immunotherapy.

Methods: stable IFN-γ- and sPD-1-overexpressing BMSCs were successfully constructed by lentiviral transfection. A non-contact co-culture system was established with Lewis and A549 lung adenocarcinoma cells to observe changes in the lung cancer cells after co-culture, using assays including cell migration and invasion experiments, as well as cellular senescence detection. Additionally, a subcutaneous lung adenocarcinoma model was established in C57BL/6J mice for intervention studies. Tumor volume, cellular apoptosis in tumor tissue (assessed by TUNEL assay), peripheral Treg cells (analyzed by flow cytometry), and histopathological markers (evaluated by HE staining and immunohistochemistry) were analyzed. The expression levels of BAX, BCL-2, AKT, PI3K, and PD-L1 were assessed by quantitative PCR and Western Blot.

Results: IFN-γ- and sPD-1-overexpressing BMSCs exhibited high bioactivity and genetic stability, inhibiting lung adenocarcinoma cell proliferation, accelerating cellular senescence, and reducing migration and invasion. Furthermore, they upregulate Bax expression, downregulate Bcl-2, and promote apoptosis. Additionally, these cells alleviate inflammatory damage in lung tissue of tumor-bearing mice, lower Treg cell levels to inhibit tumor immune evasion, and reduce the expression of PI3K/AKT and PD-L1.

Conclusion: IFN-γ- and sPD-1-overexpressing BMSCs effectively inhibit lung adenocarcinoma cell growth and tumor progression. The primary mechanisms include suppression of cancer cell growth, migration, and invasion; promotion of apoptosis and senescence in cancer cells; modulation of Treg cells; and inhibition of the PI3K/AKT signaling pathway and PD-1/PD-L1 pathways.

Keywords: BMSCs; IFN-γ; immune suppression; lung adenocarcinoma; sPD-1.

Figure 1

Identification of BMSCs and results of IFN-γ- and sPD-1-overexpressing BMSCs. (A) Identification of surface markers in BMSCs; (B) Immunofluorescence results of IFN-γ- and sPD-1-overexpressing BMSCs; (C) qPCR results of IFN-γ- and sPD-1-overexpressing BMSCs; (D) WB results of IFN-γ- and sPD-1-overexpressing BMSCs; (E) Telomerase activity of IFN-γ- and sPD-1-overexpressing BMSCs in mice; (F) Morphology of BMSCs after overexpression of IFN-γ and sPD-1. * P <0.05;**P<0.01;*** P <0.001 vs Control group. ns, not significant.

Figure 2

Effects of IFN-γ- and sPD-1-overexpressing BMSCs on lung adenocarcinoma cell proliferation and senescence. (A) CCK8 assay showing the proliferation of lung adenocarcinoma cells after co-culture with IFN-γ- and sPD-1-overexpressing BMSCs. (B) β-galactosidase assay showing the effect of IFN-γ- and sPD-1-overexpressing BMSCs on the senescence level of lung adenocarcinoma cells after co-culture.**p<0.01 and ***p<0.001 vs Control group.

Figure 3

Effects of IFN-γ- and sPD-1-overexpressing BMSCs on the migration and invasion of lung adenocarcinoma cells. (A) Scratch assay detecting the migration ability of lung adenocarcinoma cells. (B) Transwell cell invasion assay detecting the invasion ability of lung adenocarcinoma cells. **P<0.01 and ***p<0.001 vs Control group.

Figure 4

Tumor growth, histological observation, and cell apoptosis in mice. (A):Tumor volume growth curve in mice; (B) Tumor inhibition rate analysis; (C) Histological observation of tumor tissues in mice; (D) TUNEL staining marking primary apoptotic tumor cells; (E) ImageJ analysis of tumor cell apoptosis levels in each group. * P<0.05 and **p<0.01 vs Model group.

Figure 5

Expression of Bax and Bcl-2 in tumor tissue of each group. (A) Immunohistochemical results of Bax and Bcl-2 in each group; (B) qPCR results of Bax and Bcl-2 in each group; (C) WB results of Bax and Bcl-2 in each group.* P<0.05, **p<0.01 and ***p<0.001 vs Model group.

Figure 6

Pathological observation of tumor tissue in mice and distribution of Treg cells in peripheral blood. (A) Pathological observation of tumor tissue in mice; (B) Distribution of CD8 T cells in peripheral blood of mice and proportion of each group; (C) Distribution of Treg cells in peripheral blood of mice and proportion of each group.* P<0.05, **p<0.01 and *** p<0.001 vs Model group.

Figure 7

Expression of PI3K/AKT in tumor tissue of each group. (A) Immunohistochemical results of PI3K/AKT in each group; (B) qPCR results of PI3K/AKT in each group; (C) WB results of PI3K/AKT in each group. * P<0.05, **p<0.01and ***p<0.001 vs Model group.

Figure 8

Expression of PD-L1 in tumor tissue of each group. (A) Immunohistochemical results of PD-L1 in each group; (B) qPCR results of PD-L1 in each group; (C) WB results of PD-L1 in each group.* P<0.05, **p<0.01 and ***p<0.001 vs Model group.