Correction: Optical imaging of ovarian cancer using a matrix metalloproteinase-3-sensitive near-infrared fluorescent probe.

[This corrects the article DOI: 10.1371/journal.pone.0192047.]

Platelet extracellular vesicles are efficient delivery vehicles of doxorubicin, an anti-cancer drug: preparation and in vitro characterization

Platelet extracellular vesicles (PEVs) are an emerging delivery vehi for anticancer drugs due to their ability to target and remain in the tumor microenvironment. However, there is still a lack of understanding regarding yields, safety, drug loading efficiencies, and efficacy of PEVs. In this study, various methods were compared to generate PEVs from clinical-grade platelets, and their properties were examined as vehicles for doxorubicin (DOX). Sonication and extrusion produced the most PEVs, with means of 496 and 493 PEVs per platelet (PLT), respectively, compared to 145 and 33 by freeze/thaw and incubation, respectively. The PEVs were loaded with DOX through incubation and purified by chromatography. The size and concentration of the PEVs and PEV-DOX were analyzed using dynamic light scattering and nanoparticle tracking analysis. The results showed that the population sizes and concentrations of PEVs and PEV-DOX were in the ranges of 120–150 nm and 1.2–6.2 × 1011 particles/mL for all preparations. The loading of DOX determined using fluorospectrometry was found to be 2.1 × 106, 1.7 × 106, and 0.9 × 106 molecules/EV using freeze/thaw, extrusion, and sonication, respectively. The internalization of PEVs was determined to occur through clathrin-mediated endocytosis. PEV-DOX were more efficiently taken up by MDA-MB-231 breast cancer cells compared to MCF7/ADR breast cancer cells and NIH/3T3 cells. DOX-PEVs showed higher anticancer activity against MDA-MB-231 cells than against MCF7/ADR or NIH/3T3 cells and better than acommercial liposomal DOX formulation. In conclusion, this study demonstrates that PEVs generated by PLTs using extrusion, freeze/thaw, or sonication can efficiently load DOX and kill breast cancer cells, providing a promising strategy for further evaluation in preclinical animal models. The study findings suggest that sonication and extrusion are the most efficient methods to generate PEVs and that PEVs loaded with DOX exhibit significant anticancer activity against MDA-MB-231 breast cancer cells

Optical imaging of ovarian cancer using a matrix metalloproteinase-3-sensitive near-infrared fluorescent probe

<div><p>Epithelial ovarian cancer (EOC) is the seventh most common cancer among women worldwide. The 5-year survival rate for women with EOC is only 30%-50%, which is largely due to the typically late diagnosis of this condition. EOC is difficult to detect in its early stage because of its asymptomatic nature. Recently, near-infrared fluorescent (NIRF) imaging has been developed as a potential tool for detecting EOC at the molecular level. In this study, a NIRF-sensitive probe was designed to detect matrix metalloproteinase (MMP) activity in ovarian cancer cells. A cyanine fluorochrome was conjugated to the amino terminus of a peptide substrate with enzymatic specificity for MMP-3. To analyze the novel MMP-3 probe, an <i>in vivo</i> EOC model was established by subcutaneously implanting SKOV3 cells, a serous-type EOC cell line, in mice. This novel MMP-3-sensitive probe specifically reacted with only the active MMP-3 enzyme, resulting in a significantly enhanced NIRF emission intensity. Histological analysis demonstrated that MMP-3 expression and activity were enhanced in the stromal cells surrounding the ovarian cancer cells. These studies establish a molecular imaging reporter for diagnosing early-stage EOC. Additional studies are required to confirm the early-stage activity of MMP-3 in EOC and its diagnostic and prognostic significance.</p></div

Characterization of the novel stromelysine-1 peptide substrate.

<p>HPLC chromatogram of the (A) stromelysine-1 peptide substrate (black) and their digested products by MMP-3(gray) as well as (B) the control peptide (black) and their digested products by MMP-3 (gray). (C) <i>In vitro</i> activation and inhibition of experimental and control NIRF probes; the probes (0.009 μM) were incubated with or without MMP-3 (1 unit) in 50 mM Tris buffer solution. (D) <i>In vitro</i> imaging of the MMP-3 NIRF probe activated by the MMP-3 enzyme. The manifest orange-yellow color was detected in the tubes containing the MMP-3 probe with activated MMP-3 (3–5) compared with the blank (1) and MMP-3 only (2) control tubes. A series of MMP-3 probes at 0.045 ng/μL (3), 0.09 ng/μL (4), and 0.18 ng/μL (5) with MMP-3 (0.1 ng/μL) resulted in increasing SIs of 3.85 ± 0.15, 4.51 ± 0.12, and 4.86 ± 0.17 respectively. Six samples in triplicate were measured. Optical imaging was performed at 610–650nm excitation and 670–700nm emission. HPLC, high-performance liquid chromatography; MMP, matrix metalloproteinase; NIRF, near-infrared fluorescence.</p

Histological and NIRF analysis of SKOV3 tumor tissues.

<p>SKOV3 tumors were analyzed through (A) hematoxylin and eosin staining, (B) immunohistochemistry using an MMP-3 antibody, and (C) NIRF with the MMP-3-sensitive probe. MMP, matrix metalloproteinase; NIRF, near-infrared fluorescence.</p

<i>Ex vivo</i> NIRF imaging of SKOV3 tumors.

<p>NIRF images revealed signals in the dissected tumor masses (right) as compared with in the adjacent control tissue (left). NIRF, near-infrared fluorescence.</p

<i>In vitro</i> MMP expression in SKOV3 and WS1 cell cocultures.

<p>(A) MMP-3, (B) MMP-2, and (C) MMP-9 levels in the SKOV3 and WS1 cell cocultures (gray line) and SKOV3 cells cultured alone (black lines) were analyzed by ELISA. *<i>p</i><0.05, significantly different compared with SKOV3 cell cultures. ELISA, enzyme-linked immunosorbent assay; MMP, matrix metalloproteinase.</p

NIRF imaging of SKOV3 tumor masses <i>in vivo</i>.

<p>(A) NIRF imaging of the SKOV3 tumors at days 3, 5, 7, and 11. (B-D) The mean SI was significantly higher in the tumor than in the adjacent control tissue at days 5 (B), 7 (C), and 11 (D), *<i>p</i><0.001, significantly different compared with the control tissue. NIRF, near-infrared fluorescence; SI, signal intensity.</p

Upregulation of Protein Synthesis and Proteasome Degradation Confers Sensitivity to Proteasome Inhibitor Bortezomib in Myc-Atypical Teratoid/Rhabdoid Tumors.

Atypical teratoid rhabdoid tumors (ATRTs) are among the most malignant brain tumors in early childhood and remain incurable. Myc-ATRT is driven by the Myc oncogene, which directly controls the intracellular protein synthesis rate. Proteasome inhibitor bortezomib (BTZ) was approved by the Food and Drug Administration as a primary treatment for multiple myeloma. This study aimed to determine whether the upregulation of protein synthesis and proteasome degradation in Myc-ATRTs increases tumor cell sensitivity to BTZ. We performed differential gene expression and gene set enrichment analysis on matched primary and recurrent patient-derived xenograft (PDX) samples from an infant with ATRT. Concomitant upregulation of the Myc pathway, protein synthesis and proteasome degradation were identified in recurrent ATRTs. Additionally, we found the proteasome-encoding genes were highly expressed in ATRTs compared with in normal brain tissues, correlated with the malignancy of tumor cells and were essential for tumor cell survival. BTZ inhibited proliferation and induced apoptosis through the accumulation of p53 in three human Myc-ATRT cell lines (PDX-derived tumor cell line Re1-P6, BT-12 and CHLA-266). Furthermore, BTZ inhibited tumor growth and prolonged survival in Myc-ATRT orthotopic xenograft mice. Our findings suggest that BTZ may be a promising targeted therapy for Myc-ATRTs