Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

Eman Taha,Abdellatif Elseoudi,Chiharu Sogawa,Hitoshi Nagatsuka,Yuka Okusha,Satoshi Kubota,Takanori Eguchi,Kuniaki Okamoto,May Oo,Ayano Satoh,Hotaka Kawai,Yanyin Lu

doi:10.3390/cancers12051260

Abstract

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.

Highlights

Extracellular vesicles (EVs) are lipid bi-layered vesicles released from cells under physiological and pathological conditions
CD9 and endogenous palmT in the recipient tumoroids became abundantly expressed on the cell surface and well co-localized seen as honeycomb shape, suggesting that CD9 contributed to cell-cell adhesion in the recipient tumoroids (Figure 9B). These findings indicate that LuM1-derived extracellular vesicles (EVs) and conditioned medium (CM) enhanced the solidity of matrix metalloproteinase 3 (MMP3)-null tumoroids, which were relatively fragile
3, * p < 0.05, ** p < 0.01, and # p < 0.001 (LuM1-EVs versus MMP3-KO-EVs). These findings indicate that MMP3-rich, LuM1 tumoroid-derived EVs were highly. These findings indicate that MMP3-rich, LuM1 tumoroid-derived EVs were highly transmissive and associated with tumoroids, while the loss of MMP3 in tumoroid-EVs reduced the transmissive and associated with tumoroids, while the loss of MMP3 in tumoroid-EVs reduced the transmissive and binding properties

Summary

Introduction

Extracellular vesicles (EVs) are lipid bi-layered vesicles released from cells under physiological and pathological conditions. EVs are often classified into: (1) exosomes which are secreted by exocytosis of intra-luminal vesicles via membrane fusion of multivesicular bodies with plasma membrane and their size range between 50 to 200 nm, (2) ectosomes aka microvesicles or microparticles formed by cytoplasmic membrane budding, and fission; their size typically ranges from 200 nm to 1000 nm in diameter [5,6,7]. These populations of EVs are often distinguished based on their biogenesis, size, content, and function [1,8,9]. We will use the term EVs in general and classify them into small EVs (s-EVs) sized

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