Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles
Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles
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DOI：
10.3390/cancers12051260
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发表时间：
2020-05-01
期刊：
影响因子：
5.2
通讯作者：
Eguchi, Takanori
中科院分区：
文献类型：
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作者：
Taha, Eman A.;Sogawa, Chiharu;Eguchi, Takanori
已经开发了三维（3D）培养系统中的肿瘤类器官（类肿瘤）模型，以比2D培养的肿瘤细胞更接近地反映体内肿瘤。值得注意的是，细胞外囊泡（EV）可从无凝胶类肿瘤的培养上清液中有效收集。基质金属蛋白酶（MMP）3是一种多功能因子，在肿瘤进展中起着至关重要的作用。然而，MMP 3在肿瘤生长和EV中的作用尚未揭示。在这里，我们研究了MMP 3对类肿瘤和EV完整性的促肿瘤发生作用。我们使用CRISPR/Cas9系统从快速转移的LuM 1肿瘤细胞产生MMP 3敲除（KO）细胞。此外，我们建立了荧光细胞系与棕榈酰化信号融合的荧光蛋白（tdTomato和增强的GFP）。然后我们证实了细胞群和类肿瘤之间的EV交换。LuM 1-类肿瘤释放大EV（200-1000 nm）和小EV（50-200 nm），而MMP 3的敲除导致从类肿瘤额外释放破裂的EV。MMP 3的丢失导致类肿瘤大小的显著减小和类肿瘤内坏死区域的发展。MMP 3和CD 9（1类EV标志物四跨膜蛋白）在MMP 3-KO细胞及其EV组分中显著下调。此外，CD 63，四跨膜蛋白家族的另一个成员，与其对应物相比，仅在MMP 3-KO细胞的EV组分中显著减少。通过添加从LuM 1-类肿瘤收集的富含MMP 3的EV或条件培养基（CM），MMP 3-KO的这些减弱的表型被显著地拯救，这引起MMP 3-null/CD 9-low类肿瘤中MMP 3、CD 9和Ki-67（增殖细胞的标志物）的表达的显著升高。值得注意的是，在类肿瘤衍生的EV和CM中富集的MMP 3深深地穿透受体MMP 3-KO类肿瘤，导致实体类肿瘤的显著增大，而MMP 3缺失的EV则没有。这些数据表明，EV可以介导MMP 3的分子转移，导致增殖和肿瘤发生增加，表明MMP 3在肿瘤进展中的关键作用。
The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200-1000 nm) and small EVs (50-200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.