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Article

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

1
Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8525, Japan
2
Department of Medical Bioengineering, Okayama University Graduate School of Natural Science and Technology, Okayama 700-8530, Japan
3
Department of Biochemistry, Ain Shams University Faculty of Science, Cairo 11566, Egypt
4
Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA
5
Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan
6
Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan
7
Centre Hospitalier Universitaire Sainte-Justine Hospital Research Center, University of Montreal, Québec, QC H3T 1C5, Canada
8
Department of Dental Anesthesiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan
9
Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan
*
Author to whom correspondence should be addressed.
Cancers 2020, 12(5), 1260; https://doi.org/10.3390/cancers12051260
Received: 25 March 2020 / Revised: 13 May 2020 / Accepted: 14 May 2020 / Published: 16 May 2020
(This article belongs to the Special Issue 3D Cell Culture Cancer Models: Development and Applications)
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. View Full-Text
Keywords: matrix metalloproteinase 3 (MMP3); extracellular vesicles (EVs); tumoroid; tumor organoid; tumorigenesis; three-dimensional (3D) culture system matrix metalloproteinase 3 (MMP3); extracellular vesicles (EVs); tumoroid; tumor organoid; tumorigenesis; three-dimensional (3D) culture system
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MDPI and ACS Style

Taha, E.A.; Sogawa, C.; Okusha, Y.; Kawai, H.; Oo, M.W.; Elseoudi, A.; Lu, Y.; Nagatsuka, H.; Kubota, S.; Satoh, A.; Okamoto, K.; Eguchi, T. Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles. Cancers 2020, 12, 1260. https://doi.org/10.3390/cancers12051260

AMA Style

Taha EA, Sogawa C, Okusha Y, Kawai H, Oo MW, Elseoudi A, Lu Y, Nagatsuka H, Kubota S, Satoh A, Okamoto K, Eguchi T. Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles. Cancers. 2020; 12(5):1260. https://doi.org/10.3390/cancers12051260

Chicago/Turabian Style

Taha, Eman A.; Sogawa, Chiharu; Okusha, Yuka; Kawai, Hotaka; Oo, May W.; Elseoudi, Abdellatif; Lu, Yanyin; Nagatsuka, Hitoshi; Kubota, Satoshi; Satoh, Ayano; Okamoto, Kuniaki; Eguchi, Takanori. 2020. "Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles" Cancers 12, no. 5: 1260. https://doi.org/10.3390/cancers12051260

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