David Manu S, Huynh Minh D, Kelly Elizabeth, Rizos Helen, Coleman Hedley, Rogers Glynn, Zoellner Hans
Cellular and Molecular Pathology Research Unit, Department of Oral Pathology and Oral Medicine, University of Sydney, Westmead Centre for Oral Health, Westmead Hospital, Westmead, NSW, Australia.
J Pathol. 2012 Dec;228(4):495-505. doi: 10.1002/path.4063. Epub 2012 Aug 20.
We previously demonstrated that human osteosarcoma cells (SAOS-2) induce contact-dependent apoptosis in endothelium, and expected similar apoptosis in human gingival fibroblasts (h-GF) using SAOS-2 alkaline phosphatase (AP) to identify cells. However, h-GF apoptosis did not occur, despite reduction in AP-negative h-GF number (p < 0.01) and enhancement of this by h-GF TNFα pretreatment (p < 0.01). We suggest that TNFα-enhanced transfer of membrane AP from SAOS-2 to h-GF would explain these data. This idea was investigated using fluorescence prelabelled cells and confocal laser scanning microscopy. Co-cultures of membrane-labelled h-GF (marker-DiO) and SAOS-2 (marker-DiD) generated dual-labelled cells, primarily at the expense of single labelled h-GF (p < 0.001), suggesting predominant membrane transfer from SAOS-2 to h-GF. However, opposite directional transfer predominated when membrane labels were reversed; SAOS-2 further expressed green fluorescent protein (GFP) in cytoplasm and nuclei, and h-GF additionally bore nuclear label (Syto59) (p < 0.001). Cytoplasmic exchange was investigated using h-GF prelabelled with cytoplasmic DDAO-SE and nuclear Syto59, co-cultured with SAOS-2 expressing GFP in cytoplasm and nuclei, and predominant cytoplasmic marker transferred from h-GF to SAOS-2 (p < 0.05). Pretreating h-GF with TNFα increased exchange of membrane markers (p < 0.04) but did not affect either cell surface area profile or circularity. Dual-labelled cells had a morphological phenotype differing from SAOS-2 and h-GF (p < 0.001). Time-lapse microscopy revealed extensive migration of SAOS-2 and cell process contact with h-GF, with the appearance of SAOS-2 indulging in 'cellular sipping' from h-GF. Similar exchange of membrane was seen between h-GF and with other cell lines (melanoma MeIRMu, NM39, WMM175, MM200-B12; osteosarcoma U20S; ovarian carcinoma cells PE01, PE04 and COLO316), while cytoplasmic sharing was also seen in all cell lines other than U20S. We suggest that in some neoplasms, cellular sipping may contribute to phenotypic change and the generation of diverse tumour cell populations independent of genetic change, raising the possibility of a role in tumour progression.
我们之前证明，人骨肉瘤细胞（SAOS-2）可诱导内皮细胞发生接触依赖性凋亡，并且预期使用SAOS-2碱性磷酸酶（AP）来识别细胞时，人牙龈成纤维细胞（h-GF）会发生类似的凋亡。然而，尽管AP阴性的h-GF数量减少（p < 0.01），并且h-GF经TNFα预处理后这种减少更加明显（p < 0.01），但h-GF凋亡并未发生。我们认为，TNFα增强了膜AP从SAOS-2向h-GF的转移可以解释这些数据。我们使用荧光预标记细胞和共聚焦激光扫描显微镜对这一想法进行了研究。膜标记的h-GF（标记物-DiO）和SAOS-2（标记物-DiD）的共培养产生了双标记细胞，主要是以单标记的h-GF为代价（p < 0.001），这表明主要是膜从SAOS-2转移到了h-GF。然而，当膜标记颠倒时，相反方向的转移占主导；SAOS-2在细胞质和细胞核中进一步表达绿色荧光蛋白（GFP），而h-GF额外带有核标记（Syto59）（p < 0.001）。使用预先用细胞质DDAO-SE和核Syto59标记的h-GF与在细胞质和细胞核中表达GFP的SAOS-2共培养来研究细胞质交换，结果显示主要的细胞质标记从h-GF转移到了SAOS-2（p < 0.05）。用TNFα预处理h-GF增加了膜标记的交换（p < 0.04），但不影响细胞表面积分布或圆形度。双标记细胞具有与SAOS-2和h-GF不同的形态表型（p < 0.001）。延时显微镜显示SAOS-2广泛迁移并与h-GF有细胞突起接触，出现SAOS-2从h-GF进行“细胞啜饮”的现象。在h-GF与其他细胞系（黑色素瘤MeIRMu、NM39、WMM175、MM200-B12；骨肉瘤U20S；卵巢癌细胞PE01、PE04和COLO316）之间也观察到了类似的膜交换，而在除U20S之外的所有细胞系中也观察到了细胞质共享。我们认为，在某些肿瘤中，细胞啜饮可能有助于表型改变以及产生与基因变化无关的多样化肿瘤细胞群体，这增加了其在肿瘤进展中发挥作用的可能性。
