Extracellular Oncosomes Rich in Moonlighting Metalloproteinase (MMP3) Are Transmissive, Pro-Tumorigenic, and Induces Cellular Communication Network Factor 2 (CCN2/CTGF): CRISPR against Cancer

Okusha, Yuka; Eguchi, Takanori; Tran, Minh; Sogawa, Chiharu; Yoshida, Kaya; Itagaki, Mami; Taha, Eman A.; Ono, Koji; Aoyama, Eriko; Okamura, Hirohiko; Kozaki, Ken-ichi; Calderwood, Stuart K.; Takigawa, Masaharu; Okamoto, Kuniaki

doi:10.20944/preprints202002.0281.v1

Cited by 1 publication

(13 citation statements)

References 53 publications

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“…MMP3 is a proteolytic enzyme, as well as a transcriptional factor that plays a crucial role in tumor progression [20], [21], [24], [25], [44]. However, the roles of MMP3 within EVs had not unveiled before our study.…”

Section: Discussionmentioning

confidence: 82%

“…To explore the role of MMP3 in the cellular communication in cancer, we used the CRISPR/Cas9 genome editing system for generating MMP3-knockout cell line from a murine high metastatic cancer cell line LuM1 [20]. MMP3 was markedly detected in the cells, non-EVs, and EVs fractions of the LuM1, while the complete Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 25 March 2020 doi:10.20944/preprints202003.0371.v1…”

Section: Mmp3 Knockout Impacts Physical and Biological Integrities Ofmentioning

confidence: 99%

“…It has been shown that some members of MMPs were expressed at high levels in particular cancer types and promoted tumor progression. We have compared protumorigenic phenotypes of rapidly metastatic murine cancer cell line LuM1 with a parental cancer cell line Colon26 (aka CT26) [20], [21], [23]- [25] and shown that MMP3 and MMP9 were highly expressed in LuM1 cells. The RNA interference (RNAi)-mediated knockdown of MMP3 and MMP9 significantly attenuated tumor growth and metastasis in the allograft mouse model [21].…”

Section: Introductionmentioning

confidence: 99%

“…The RNA interference (RNAi)-mediated knockdown of MMP3 and MMP9 significantly attenuated tumor growth and metastasis in the allograft mouse model [21]. Most recently, we established MMP3-knockout (KO) cells using CRISPR/Cas9 genome editing technology on the LuM1 cells and then compared the oncogenic effects of MMP3-KO cells versus LuM1 cells in vivo and in vitro using the 2D culture system [20]. This study indicated that knockout of Mmp3 results in significant inhibition of tumor growth, cellular migration, and invasion in vivo [20].…”

Section: Introductionmentioning

confidence: 99%

“…Most recently, we established MMP3-knockout (KO) cells using CRISPR/Cas9 genome editing technology on the LuM1 cells and then compared the oncogenic effects of MMP3-KO cells versus LuM1 cells in vivo and in vitro using the 2D culture system [20]. This study indicated that knockout of Mmp3 results in significant inhibition of tumor growth, cellular migration, and invasion in vivo [20]. However, a mechanism of how MMP3 enriched EVs involves characteristics of EVs and tumors has not completed yet.…”

Section: Introductionmentioning

confidence: 99%

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Knockout of MMP3 Weakened Solid Tumor Organoids and Cancer Extracellular Vesicles

Taha¹,

Sogawa²,

Okusha³

et al. 2020

Preprint

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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 (300-1000 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 leads to a significant reduction in tumoroid size and to 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. These weakened phenotypes by 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 into 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 CD9+ tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

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