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Supplementary Figures 1-5 from Colorectal Cancer–Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis

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posted on 2023-04-03, 19:40 authored by Atsushi Ikeda, Satoshi Nagayama, Makoto Sumazaki, Makoto Konishi, Risa Fujii, Naomi Saichi, Satoshi Muraoka, Daisuke Saigusa, Hideaki Shimada, Yoshiharu Sakai, Koji Ueda
<p>Supplementary figure 1. Dynamic range plot of quantified Te-EV proteome. The X axis indicates abundance rank of qualified Te-EV proteome, and the Y axis indicates the relative abundance by the LC/MS analysis. Supplementary figure 2. GO analysis of up-regulated (fold change > 5, adjusted p < 0.05) and down-regulated (fold change < 0.02, adjusted p < 0.05) proteins in tumor Te-EVs. For biological process, cellular component and molecular function, data of GOTERM_BP_FAT, GOTERM_CC_FAT and GOTERM_MF_FAT were used from Gene_Ontology in Functional Annotation Tool on the website, respectively. The Y axis indicates the percentage of the annotated proteins. Supplementary figure 3. Scatter plot showing EV-CAT1 sandwich ELISA (unit) on the X axis and preoperative CEA (ng / ml) on the Y axis (left). Magnified view of the plot is also shown (right). Coefficient of correlation value was 8.5 Ã- 10-3. Supplementary figure 4. Volcano plot indicating p-value (Te-EVs vs. tissues) on the Y axis and log ratio (Te-EVs / tissue) of LC/MS intensity on the X axis. Blue dots indicate the top 100 Te-EV proteins which are significantly up-regulated than tissue proteins, and orange dots indicate the top 100 proteins which are significantly down-regulated than tissue proteins. Supplementary figure 5. The analyses of subcellular proteome localisations are shown. The subcellular locations of tissues and Te-EVs are obtained on the Uniprot website. The distribution of the two proteome is similar with each other. The numbers show percentages.</p>

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ARTICLE ABSTRACT

Accumulating scientific evidences strongly support the importance of cancer-derived extracellular vesicles (EV) in organization of tumor microenvironment and metastatic niches, which are also considered as ideal tools for cancer liquid biopsy. To uncover the full scope of proteomic information packaged within EVs secreted directly from human colorectal cancer, we cultured surgically resected viable tissues and obtained tissue-exudative EVs (Te-EV). Our quantitative profiling of 6,307 Te-EV proteins and 8,565 tissue proteins from primary colorectal cancer and adjacent normal mucosa (n = 17) allowed identification of a specific cargo in colorectal cancer–derived Te-EVs, high-affinity cationic amino acid transporter 1 (CAT1, P = 5.0 × 10–3, fold change = 6.2), in addition to discovery of a new class of EV markers, VPS family proteins. The EV sandwich ELISA confirmed escalation of the EV-CAT1 level in plasma from patients with colorectal cancer compared with healthy donors (n = 119, P = 3.8 × 10–7). Further metabolomic analysis revealed that CAT1-overexpressed EVs drastically enhanced vascular endothelial cell growth and tubule formation via upregulation of arginine transport and downstream NO metabolic pathway. These findings demonstrate the potency of CAT1 as an EV-based biomarker for colorectal cancer and its functional significance on tumor angiogenesis. This study provides a proteome-wide compositional dataset for viable colorectal cancer tissue–derived EVs and especially emphasizes importance of EV-CAT1 as a key regulator of angiogenesis.

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