Macrophages Modulate Migration and Invasion of Human Tongue Squamous Cell Carcinoma

Oral tongue squamous cell carcinoma (OTSCC) has a high mortality rate and the incidence is rising worldwide. Despite advances in treatment, the disease lacks specific prognostic markers and treatment modality. The spreading of OTSCC is dependent on the tumor microenvironment and involves tumor-associated macrophages (TAMs). Although the presence of TAMs is associated with poor prognosis in OTSCC, the specific mechanisms underlying this are still unknown. The aim here was to investigate the effect of macrophages (Mfs) on HSC-3 tongue carcinoma cells and NF-kappaB activity. We polarized THP-1 cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type Mfs. We then investigated the effect of Mfs on HSC-3 cell migration and NF-kappaB activity, cytokine production and invasion using several different in vitro migration models, a human 3D tissue invasion model, antibody arrays, confocal microscopy, immunohistochemistry and a mouse invasion model. We found that in co-culture studies all types of Mfs fused with HSC-3 cells, a process which was partially due to efferocytosis. HSC-3 cells induced expression of epidermal growth factor and transforming growth factor-beta in co-cultures with M2 Mfs. Direct cell-cell contact between M2 Mfs and HSC-3 cells induced migration and invasion of HSC-3 cells while M1 Mfs reduced HSC-3 cell invasion. M2 Mfs had an excess of NF-kappaB p50 subunit and a lack of p65 subunits both in the presence and absence of HSC-3 cells, indicating dysregulation and pro-tumorigenic NF-kappaB activation. TAM-like cells were abundantly present in close vicinity to carcinoma cells in OTSCC patient samples. We conclude that M2 Mfs/TAMs have an important role in OTSCC regulating adhesion, migration, invasion and cytokine production of carcinoma cells favouring tumor growth. These results demonstrate that OTSCC patients could benefit from therapies targeting TAMs, polarizing TAM-like M2 Mfs to inflammatory macrophages and modulating NF-kappaB activity.口腔舌鳞状细胞癌（Oral tongue squamous cell carcinoma, OTSCC）具有较高的死亡率，且全球发病率呈逐年上升趋势。尽管治疗手段已有进展，但该疾病仍缺乏特异性预后标志物与有效的治疗手段。口腔舌鳞状细胞癌的侵袭转移依赖于肿瘤微环境，且涉及肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）。尽管肿瘤相关巨噬细胞的存在与口腔舌鳞状细胞癌的不良预后相关，但其背后的具体调控机制仍未明确。本研究旨在探讨巨噬细胞（macrophages, Mfs）对HSC-3舌癌细胞以及核因子κB（NF-κB）活性的影响。我们将THP-1细胞极化为促炎型M1巨噬细胞、肿瘤相关巨噬细胞样M2巨噬细胞以及经咪唑喹啉类药物处理的R848型巨噬细胞。随后，通过多种体外迁移模型、人体三维组织侵袭模型、抗体芯片、共聚焦显微镜、免疫组织化学以及小鼠侵袭模型，我们研究了各类巨噬细胞对HSC-3细胞迁移、核因子κB活性、细胞因子分泌、侵袭能力的影响。我们发现，在共培养实验中，所有类型的巨噬细胞均可与HSC-3细胞发生融合，这一过程部分由胞葬作用介导。HSC-3细胞可在与M2型巨噬细胞的共培养体系中诱导表皮生长因子与转化生长因子-β的表达。M2型巨噬细胞与HSC-3细胞之间的直接细胞接触可促进HSC-3细胞的迁移与侵袭，而M1型巨噬细胞则会抑制HSC-3细胞的侵袭能力。无论是否存在HSC-3细胞，M2型巨噬细胞均呈现核因子κB p50亚基过量表达、p65亚基表达缺失的特征，这提示核因子κB通路存在调控异常且具有促肿瘤发生的激活特性。在口腔舌鳞状细胞癌患者的样本中，肿瘤相关巨噬细胞样细胞大量富集于癌细胞的邻近区域。我们得出结论，M2型巨噬细胞/肿瘤相关巨噬细胞在口腔舌鳞状细胞癌中发挥关键调控作用，可影响癌细胞的黏附、迁移、侵袭以及细胞因子分泌，进而促进肿瘤生长。本研究结果表明，口腔舌鳞状细胞癌患者可从靶向肿瘤相关巨噬细胞的治疗策略中获益，包括将肿瘤相关巨噬细胞样M2型巨噬细胞极化为促炎型巨噬细胞，以及调控核因子κB的通路活性。