高静水压处理的头颈部鳞状细胞癌细胞条件培养基对巨噬细胞极化的免疫调节作用。

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中文摘要

背景： 浸润软骨的头颈部鳞状细胞癌（HNSCC）的手术切除常导致显著的组织缺损，需要重建干预。自体喉软骨组织的原位再植入可能是一种有前景的方法，因为其解剖顺应性和优异的生物相容性。300 MPa的高静水压（HHP）可有效消除残留的恶性细胞，同时保留透明软骨的细胞外基质和生物力学特性。然而，HHP会改变肿瘤来源细胞因子和损伤相关分子模式的释放。鉴于平衡的炎症和免疫调节反应在组织整合中的关键作用，这项体外研究探讨了HHP处理后释放的因子是否影响巨噬细胞极化。方法：使用来自HHP处理或未处理的HNSCC细胞系的条件培养基（CM）刺激贴壁单核细胞72小时。未刺激细胞、M1（GM-CSF + LPS）和M2（M-CSF + IL-4）巨噬细胞对照作为对照。分析巨噬细胞形态、基因表达和蛋白分泌，检测M1标志物（TNF-α、IL-1β、IL-6、IL-8、IL-12 p70、IL-23、CXCL9、CXCL10）和M2标志物（IL-10、CCL17、CCL18、MMP-9）。评估表面标志物HLA-DR、CD80、CD86、CD192（M1样）和CD206、CX3CR1（M2样）。结果：来自HHP处理细胞的CM诱导了异质性巨噬细胞形态，包括纺锤形和圆形细胞。基因表达显示IL-1β和IL-8显著上调，IL-6呈趋势升高，而TNF-α或IL-10未增加。在蛋白水平，IL-1β和IL-8显著升高，IL-6和CXCL9呈升高趋势，而IL-12 p70和IL-23保持不变。IL-10水平保持低值，未观察到CCL17、CCL18或CCL22的诱导。表型上，大多数细胞表达CD86和HLA-DR，高比例细胞CD192阳性，但仅少数表达CD80。无论刺激如何，相当一部分细胞保留CD206和CX3CR1表达。结论：总体而言，来自HHP处理的HNSCC细胞的CM诱导了一种异质性、部分活化的巨噬细胞状态，主要具有M1样特征，而非明确的M1或M2表型。这些发现提示，CM可能影响与软骨再植入相关的炎症微环境，而不促进强烈的免疫抑制性巨噬细胞反应。需要进一步使用组织模型和体内验证的研究来确定其与组织整合的相关性。

英文摘要

Surgical resection of cartilage-infiltrating head and neck squamous cell carcinoma (HNSCC) frequently results in significant tissue defects, which require reconstructive interventions. Orthotopic reimplantation of autologous laryngeal cartilage tissue could be a promising approach due to its anatomical conformity and excellent biocompatibility. High hydrostatic pressure (HHP) at 300 MPa effectively eliminates residual malignant cells while preserving the extracellular matrix and biomechanical properties of hyaline cartilage. However, HHP alters the release of tumor-derived cytokines and damage-associated molecular patterns. Given the critical role of balanced inflammatory and immunomodulatory responses in tissue integration, this in vitro study investigated whether factors released after HHP treatment influence macrophage polarization. Conditioned medium (CM) from HHP-treated or untreated HNSCC cell lines was used to stimulate adherent mononuclear cells for 72 hours. Unstimulated cells, M1 (GM-CSF + LPS), and M2 (M-CSF + IL-4) macrophage controls served as controls. Macrophage morphology, gene expression, and protein secretion were analyzed for M1 markers (TNF-α, IL-1β, IL-6, IL-8, IL-12 p70, IL-23, CXCL9, CXCL10) and M2 markers (IL-10, CCL17, CCL18, MMP-9). Surface markers HLA-DR, CD80, CD86, CD192 (M1-like) and CD206, CX3CR1 (M2-like) were assessed. CM from HHP-treated cells induced a heterogeneous macrophage morphology, including spindle-shaped and rounded cells. Gene expression revealed significant upregulation of IL-1β and IL-8, a trend for IL-6, and no increase in TNF-α or IL-10. At the protein level, IL-1β and IL-8 were significantly elevated, with a trend toward increased IL-6 and CXCL9, while IL-12 p70, and IL-23 remained unchanged. IL-10 levels stayed low, and no induction of CCL17, CCL18, or CCL22 was observed. Phenotypically, most cells expressed CD86 and HLA-DR, with a high proportion positive for CD192, but only a minority expressed CD80. A notable fraction retained CD206 and CX3CR1 expression regardless of stimulation. Overall, CM derived from HHP-treated HNSCC cells induced a heterogeneous, partially activated macrophage state with predominantly M1-like features rather than a distinct M1 or M2 phenotype. These findings suggest that CM may influence the inflammatory microenvironment relevant to cartilage reimplantation without promoting a strongly immunosuppressive macrophage response. Further studies using tissue models and in vivo validation are required to establish its relevance for tissue integration.