David Manu S, Kelly Elizabeth, Cheung Ivan, Xaymardan Munira, Moore Malcolm A S, Zoellner Hans
The Cellular and Molecular Pathology Research Unit, Department of Oral Pathology and Oral Medicine, Faculty of Dentistry, The University of Sydney, Westmead Centre for Oral Health, Westmead Hospital, Westmead, New South Wales, Australia.
Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
PLoS One. 2014 Jun 30;9(6):e101202. doi: 10.1371/journal.pone.0101202. eCollection 2014.
We recently reported exchange of membrane and cytoplasmic markers between SAOS-2 osteosarcoma cells and human gingival fibroblasts (h-GF) without comparable exchange of nuclear markers, while similar h-GF exchange was seen for melanoma and ovarian carcinoma cells. This process of "cellular sipping" changes phenotype such that cells sharing markers of both SAOS-2 and h-GF have morphology intermediate to that of either cell population cultured alone, evidencing increased tumour cell diversity without genetic change. TNF-α increases cellular sipping between h-GF and SAOS-2, and we here study binding of SAOS-2 to TNF-α treated h-GF to determine if increased cellular sipping can be accounted for by cytokine stimulated SAOS-2 binding. More SAOS-2 bound h-GF pe-seeded wells than culture plastic alone (p<0.001), and this was increased by h-GF pre-treatment with TNF-α (p<0.001). TNF-α stimulated binding was dose dependent and maximal at 1.16 nM (p<0.05) with no activity below 0.006 nM. SAOS-2 binding to h-GF was independent of serum, while the lipopolysaccharide antagonist Polymyxin B did not affect results, and TNF-α activity was lost on boiling. h-GF binding of SAOS-2 started to increase after 30min TNF-α stimulation and was maximal by 1.5 hr pre-treatment (p<0.001). h-GF retained maximal binding up to 6 hrs after TNF-α stimulation, but this was lost by 18 hrs (p<0.001). FACS analysis demonstrated increased ICAM-1 consistent with the time course of SAOS-2 binding, while antibody against ICAM-1 inhibited SAOS-2 adhesion (p<0.04). Pre-treating SAOS-2 with TNF-α reduced h-GF binding to background levels (p<0.003), and this opposite effect to h-GF cytokine stimulation suggests that the history of cytokine exposure of malignant cells migrating across different microenvironments can influence subsequent interactions with fibroblasts. Since cytokine stimulated binding was comparable in magnitude to earlier reported TNF-α stimulated cellular sipping, we conclude that TNF-α stimulated cellular sipping likely reflects increased SAOS-2 binding as opposed to enhanced exchange mechanisms.
我们最近报道，SAOS-2骨肉瘤细胞与人牙龈成纤维细胞（h-GF）之间存在膜和细胞质标志物的交换，但核标志物没有类似的交换，而黑色素瘤和卵巢癌细胞也出现了类似的h-GF交换。这种“细胞啜饮”过程会改变细胞表型，使得共享SAOS-2和h-GF标志物的细胞具有单独培养的任何一种细胞群体形态的中间形态，这证明在没有基因变化的情况下肿瘤细胞多样性增加。肿瘤坏死因子-α（TNF-α）会增加h-GF与SAOS-2之间的细胞啜饮，我们在此研究SAOS-2与经TNF-α处理的h-GF的结合情况，以确定细胞啜饮增加是否可由细胞因子刺激的SAOS-2结合来解释。与单独的培养塑料相比，更多的SAOS-2与预先接种h-GF的孔结合（p<0.001），并且经TNF-α预处理的h-GF会增加这种结合（p<0.001）。TNF-α刺激的结合呈剂量依赖性，在1.16 nM时达到最大值（p<0.05），在0.006 nM以下无活性。SAOS-2与h-GF的结合不依赖于血清，脂多糖拮抗剂多粘菌素B不影响结果，且TNF-α活性在煮沸后丧失。经TNF-α刺激30分钟后，SAOS-2与h-GF的结合开始增加，预处理1.5小时时达到最大值（p<0.001）。经TNF-α刺激后，h-GF在长达6小时内保持最大结合，但在18小时后丧失（p<0.001）。流式细胞术分析表明，细胞间黏附分子-1（ICAM-1）增加，与SAOS-2结合的时间进程一致，而抗ICAM-1抗体抑制SAOS-2黏附（p<0.04）。用TNF-α预处理SAOS-2可将h-GF结合降低至背景水平（p<0.003），这种与h-GF细胞因子刺激相反的效应表明，迁移穿过不同微环境的恶性细胞的细胞因子暴露史可影响其随后与成纤维细胞的相互作用。由于细胞因子刺激的结合在程度上与早期报道的TNF-α刺激的细胞啜饮相当，我们得出结论，TNF-α刺激的细胞啜饮可能反映了SAOS-2结合增加，而不是增强的交换机制。
