Oral Squamous Cell Carcinoma-Derived Cell-Free DNA Modulates Stemness and Migration of Oral Squamous Cell Carcinoma Cell Line by Inducing M2 Macrophage Polarization
目的 研究口腔鳞状细胞癌细胞来源的游离DNA对巨噬细胞极化作用，以及极化的巨噬细胞对口腔鳞癌细胞系的干性和迁移能力调控作用。 方法 取病理确诊为口腔鳞状细胞癌的组织标本30例，异常增生的组织标本10例，正常口腔上皮组织标本10例。 通过免疫组化染色、免疫荧光染色检测M2型巨噬细胞在不同口腔组织中数量及位置。收集人舌鳞状细胞癌细胞系Cal-27细胞的条件培养基，纯化并提取游离DNA（cell free DNA, cfDNA）并进行鉴定。用cfDNA处理巨噬细胞，观察细胞形态学变化，RT-qPCR检测极化相关指标表达水平。用cfDNA 诱导后的巨噬细胞条件培养基处理 CAL-27 细胞，RT-qPCR检测其干性基因变化水平；并且通过划痕实验验证cfDNA诱导的巨噬细胞调控肿瘤细胞迁移的能力。 结果 与正常口腔上皮组织相比，异常增生的口腔上皮深层结缔组织和口腔鳞癌间质中M2型巨噬细胞数量较多（P<0.05）。CAL-27细胞分泌长度在 10000～15000 bp的cfDNA。CAL-27细胞分泌的cfDNA可诱导巨噬细胞高表达 M2 型巨噬细胞标记（P<0.05）。cfDNA 处理的巨噬细胞诱导肿瘤细胞高表达肿瘤干性基因（P<0.05） ，同时促进了肿瘤细胞迁移能力（P<0.05）。 结论 口腔鳞状细胞癌细胞来源的cfDNA通过诱导巨噬细胞向 M2 型极化促进口腔癌细胞系干性和迁移。Abstract: Objective To investigate the effect of oral squamous cell carcinoma (OSCC)-derived cell-free DNA (cfDNA) on the polarization of macrophages and the regulatory effect of polarized macrophages on the stemness and migration of OSCC cells. Methods A total of 30 OSCC tissue samples, 10 dysplastic oral tissue samples, and 10 normal oral tissue samples were collected. The status of all tissue samples was confirmed by pathology analysis. Immunohistochemical (IHC) staining and immunofluorescence (IF) staining were performed to examine the cell count and location of M2 macrophages in different types of oral tissue samples. The conditioned medium (CM) of OSCC cell line CAL-27 from the human tongue was collected and the cfDNA was concentrated and isolated for identification. The macrophages were treated by cfDNA and their morphological characteristics were observed under microscope. The expression levels of polarization-related indicators were determined by RT-qPCR. CAL-27 cell line was treated with macrophage CM induced by cfDNA and the expression levels of stemness-related genes were determined by RT-qPCR. Scratch-wound assay was conducted to verify that the migration ability of CAL-27 was modulated by macrophages induced by cfDNA. Results There were more M2 macrophages in the deep connective tissue of dysplastic oral epithelium and the stroma of OSCC compared with those in the normal oral tissues (P<0.05). OSCC cell line CAL-27 could secret cfDNA of 10000-15000 bp in length. cfDNA secreted by CAL-27 could induced in macrophages significantly higher expression of M2-macrophage-related genes (P<0.05). cfDNA-treated macrophages induced significantly increased expression of stemness-related genes in CAL-27 cell line (P<0.05) and promoted the migration ability of CAL-27 cell line (P<0.05). Conclusion OSCC-derived cfDNA promotes stemness and migration of OSCC cell line by inducing M2 macrophage polarization.
图 4 cfDNA 对巨噬细胞的极化作用
Figure 4. E💖ffect of cfDNA on gene expression of related cytokines in M1 and M2 macrophages
A: microscopic image (the yellow arrows are pointed at polarized macrophages); B: the mRNA levels were determined by qPCR. ** P<0.01, *** P<0.001, vs. CTR-CM group, n=3. CTR-CM: control-ꦓconditioned medium; cfDNA-CM: conditioned medium of macrophages after induction by cfDNA.
图 5 cfDNA诱导的☂巨噬细胞对CAL-27细胞干性基因表达𒐪（A）和CAL-27细胞迁移能力（B）的影响
Figure 5. Effect of cfDNA-induced macrophages on the expression of stemness-related genes in CAL-27 cells (A) and on the migration ability of🦩 CAL-27 cells (B)
* P <0.05, * * * P <0.001, vs. CTR-CM group, n=3. CTR-CM: control-conditioned medium; cfDNA-CM: conditioned medium of m💞acrophages after induction by cfDNA.
表 1 引物合成序列
Table 1. Gene primer sequences
Gene (human) Primer sequence (5′-3′) Primer
β-actin F: GGAGATTACTGCCCTGGCTCCTA 23 R GACTCATCGTACTCCTGCTTGCTG 22 CD86 F: TGCTCATCTATACACGGTTACC 22 R: TGCATAACACCATCATACTCGA 22 TNF-α F: TGGCGTGGAGCTGAGAGATAACC 23 R: CGATGCGGCTGATGGTGTGG 20 IL-6 F: CACTGGTCTTTTGGAGTTTGAG 22 R: GGACTTTTGTACTCATCTGCAC 22 CD163 F: ATCAACCCTGCATCTTTAGACA 22 R: CTTGTTGTCACATGTGATCCAG 22 CD204 F: GGACACTGATAGCTGCTCCGAATC 24 R: CACGAGGAGGTAAAGGGCAATCAG 24 Arg-1 F: GGACCTGCCCTTTGCTGACATC 22 R: TCTTCTTGACTTCTGCCACCTTGC 24 TNF-α: tumor necrosis factor alpha; IL-6: interleukin 6; Arg-1: arginase 1.
表 2 RT-qPCR引物合成序列
Table 2. RT-qPCR primer sequences
Gene (human) Primer sequence (5′-3′) Primer length/bp β-actin F: GGAGATTACTGCCCTGGCTCCTA 23 R: GACTCATCGTACTCCTGCTTGCTG 24 SOX2 F: GTGAGCGCCCTGCAGTACAA 20 R: GCGAGTAGGACATGCTGTAGGTG 23 OCT4 F: GCTGGATGTCAGGGCTCTTTG 21 R: TTCAAGAGATTTATCGAGCACCTTC 25 SOX2: SRY-box transcription factor 2; OCT4: organic cation/carnitine transporter 4.
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