人脐带间充质干细胞条件培养基通过激活 Hippo 通路以细胞密度依赖的方式对 KGN 细胞发挥抗肿瘤作用。

BACKGROUND: The conditioned medium from human umbilical cord mesenchymal stem cells (UCMSCs-CM) provides a new cell-free therapy for tumors due to its unique secretome. However, there are many contradictory reports about the effect of UCMSCs-CM on tumor cells. The loss of contact inhibition is a common characteristic of tumor cells. A relationship between the effect of UCMSCs-CM on tumor cells and contact inhibition in tumor cells is rarely concerned. Whether the effect of UCMSCs-CM on tumor cells is affected by cell density? Here, we explored the effect of UCMSCs-CM on granulosa tumor cell line (KGN) cells at low or high density. METHODS: Growth curve and CCK8 assay were used to assess cell proliferation and viability. Scratch wound and matrigel invasion assay were implicated to detect cell motility of KGN cells. UCMSCs-CM effects on cell cycle, apoptosis and pathway-related proteins were investigated by flow cytometry, TUNEL assay, western blot and immunofluorescence analysis respectively. RESULTS: In growth curve analysis, before KGN cells proliferated into confluence, UCMSCs-CM had no effect on cell proliferation. However, once the cells proliferate to contact each other, UCMSCs-CM significantly inhibited proliferation. Meanwhile, when KGN cells were implanted at high density, UCMSCs-CM could induce cell cycle arrest at G1 phase, inhibit cell migration, invasion and promote apoptosis. While it had no similar effect on KGN cells implanted at low density. In mechanism, the UCMSCs-CM treatment activated the Hippo pathway when KGN cells were implanted at high density. Consistently, the MST1/2 inhibitor, XMU-MP-1, inhibited the activation of the Hippo pathway induced by UCMSCs-CM treatment and accordingly declined the anti-tumor effect of UCMSCs-CM on KGN cells. CONCLUSIONS: The effect of UCMSCs-CM on tumor cells is affected by cell density. UCMSCs-CM exerted anti-tumor effect on KGN cells by activating Hippo pathway to restore contact inhibition. Our results suggest that UCMSCs-CM is a promising therapeutic candidate for GCT treatment.

背景：人脐带间充质干细胞（UCMSCs-CM）的条件培养基因其独特的分泌组为肿瘤提供了一种新的无细胞治疗方法。然而，关于 UCMSCs-CM 对肿瘤细胞的影响有许多相互矛盾的报道。接触抑制的丧失是肿瘤细胞的一个共同特征。UCMSCs-CM 对肿瘤细胞的影响与肿瘤细胞中的接触抑制之间的关系很少受到关注。UCMSCs-CM 对肿瘤细胞的作用是否受细胞密度的影响？在这里，我们在低或高细胞密度下探索了 UCMSCs-CM 对颗粒细胞瘤系（KGN）细胞的影响。

方法：使用生长曲线和 CCK8 测定法评估细胞增殖和活力。划痕实验和基质胶侵袭实验用于检测 KGN 细胞的细胞迁移能力。通过流式细胞术、TUNEL 测定、Western blot 和免疫荧光分析分别研究 UCMSCs-CM 对细胞周期、细胞凋亡和通路相关蛋白的影响。

结果：在生长曲线分析中，在 KGN 细胞增殖到汇合之前，UCMSCs-CM 对细胞增殖没有影响。然而，一旦细胞相互接触增殖，UCMSCs-CM 就会显著抑制增殖。同时，当 KGN 细胞以高密度植入时，UCMSCs-CM 可诱导细胞周期停滞在 G1 期，抑制细胞迁移、侵袭并促进凋亡。而在低细胞密度下，它对 KGN 细胞没有类似的作用。在机制上，当 KGN 细胞以高密度植入时，UCMSCs-CM 处理激活了 Hippo 通路。一致地，MST1/2 抑制剂 XMU-MP-1 抑制了 UCMSCs-CM 处理诱导的 Hippo 通路的激活，从而降低了 UCMSCs-CM 对 KGN 细胞的抗肿瘤作用。

结论：UCMSCs-CM 对肿瘤细胞的作用受细胞密度的影响。UCMSCs-CM 通过激活 Hippo 通路恢复接触抑制对 KGN 细胞发挥抗肿瘤作用。我们的结果表明，UCMSCs-CM 是治疗 GCT 的一种很有前途的治疗候选物。