Pg-Induced ATR Activation Promotes ESCC Progression via M2 TAM Polarization

Emerging evidence suggests that oral pathogens may contribute to the development of systemic malignancies. Porphyromonas gingivalis (Pg), a major periodontal pathogen, has been implicated in several cancers including esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms underlying this association remain unclear.

This study aimed to investigate the impact of Porphyromonas gingivalis (Pg) on the growth of esophageal squamous cell carcinoma (ESCC) and its potential mechanisms.

THP-1 cells were differentiated into M0 macrophages with PMA and divided into control group, Pg group, and Pg + siATR group. THP-1 cells were divided into three groups: control group, Pg group, and Pg + siATR group. The control and Pg groups were transfected with siNC, while the Pg + siATR group was transfected with siATR. After transfection, the Pg group and Pg + siATR group were incubated with 200 MOI pg. The control group was cultured normally. Cells and supernatants were collected, and macrophage polarization status was detected with qRT-PCR, Western blot, and flow cytometry. Macrophages treated differently were co-cultured with human esophageal squamous cell carcinoma cell line KYSE150. The proliferation, invasion, and apoptosis of KYSE150 were examined. KYSE150+shATR and KYSE150+shNC cell lines were constructed with a lentivirus system. Thirty male BALB/c mice aged 6-8 weeks were randomly divided into control group, pg group, and pg+shATR group, with 10 mice in each group. For the Pg and Pg+shATR groups, 200 μL Pg (1 × 10⁷ CFU/mouse) was applied to the mandibular molars of mice four times a week for 1 month. The control group was treated with the vehicle (CMC) only. After 3 weeks of bacterial colonization, KYSE150+shATR cells (1 × 10⁶ cells/mouse) were inoculated into the right axilla of mice in the Pg+shATR group, while KYSE150 + shNC (1 × 10⁶ cells/mouse) were inoculated into the right axilla of mice in the control and Pg groups. Tumor volume was measured with calipers every 7 days. After 4 weeks of experimentation, D-luciferin potassium salt was injected intraperitoneally at 150 mg/kg, and bioluminescence imaging was performed after sodium pentobarbital anesthesia. The mice were euthanized post-imaging, and macrophage polarization in tumor tissues was examined with qRT-PCR, histopathological examination, and Western blot.

In this study, we found that Pg could promote the polarization of M0 macrophages into M2 macrophages, while also promoting the malignant progression of KYSE150 cells. The expression levels of ATR, phosphorylated ATR (p-ATR), and M2 macrophage markers (CD206, Arg1, and VEGF) decreased when ATR was inhibited. In BALB/c mice, Pg-induced ATR activation promoted the growth of subcutaneously implanted tumors by recruiting M2-type TAMs. Experimental data also indicated an association between M2 polarization, decreased p-chik, and ATR.

This study reveals that Pg can activate the ataxia-telangiectasia and Rad3-related protein (ATR) signaling pathway, inducing the polarization of M2 tumor-associated macrophages (TAM), thereby promoting the growth of ESCC. This provides a new theoretical basis for the prevention and treatment of ESCC.