Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude-mouse models - ScienceDirect

Advanced Drug Delivery Reviews

Volume 65, Issue 3, March 2013, Pages 383-390

Advanced Drug Delivery Reviews

https://doi.org/10.1016/j.addr.2012.08.007 Get rights and content

Abstract

Exosomes play an important role in cell-to-cell communication to promote tumor metastasis. In order to image the fate of cancer-cell-derived exosomes in orthotopic nude mouse models of breast cancer, we used green fluorescent protein (GFP)-tagged CD63, which is a general marker of exosomes. Breast cancer cells transferred their own exosomes to other cancer cells and normal lung tissue cells in culture. In orthotopic nude-mouse models, breast cancer cells secreted exosomes into the tumor microenvironment. Tumor-derived exosomes were incorporated into tumor-associated cells as well as circulating in the blood of mice with breast cancer metastases. These results suggest that tumor-derived exosomes may contribute to forming a niche to promote tumor growth and metastasis. Our results demonstrate the usefulness of GFP imaging to investigate the role of exosomes in cancer metastasis.

Graphical abstract

Introduction

Exosomes are small (30–100 nm) membrane vesicles that originate from the endosomal membrane compartment [1]. Recently, it has been demonstrated that cells shed exosomes containing significant amounts of mRNAs, miRNAs and proteins for transferring genetic and proteomic information to target cells as a way of cell-to-cell communication [1], [2], [3]. Cancer cells actively release their exosomes into the tumor microenvironment and peripheral blood of cancer patients [1], [4].

Cancer-cell-derived exosomes stimulate secretion of growth factors, cytokines and angiopoietic factors by stroma cells, induce proliferation of endothelial cells, and promote angiogenesis in metastatic organs [1], [2], [5], [6]. Thus, cancer-cell-derived exosomes are able to alter the cellular environment to form a metastatic niche and promote metastasis [7], [8]. Hence, the tracking fate of cancer-cell-derived exosomes in a metastatic models may help us understand the mechanism of cancer metastasis.

Breast cancer is the most common cancer and the leading cause of cancer death among women worldwide [9]. Metastasis is the major cause of death in breast cancer [9]. Therefore novel biomarkers for detection of early metastasis and therapeutic strategies as well as elucidation of the molecular mechanisms underlying the metastatic process, are urgently required.

In the present report, we describe imaging of transfer of cancer-cell-derived GFP-labeled exosomes in vitro and in mouse models of metastatic breast cancer.

Section snippets

Markers of exosomes

Several proteins may serve as markers of exosomes [10], [11]. Cytoplasmic proteins such as molecular chaperon/heat shock proteins Hsp70 and Hsp90, and ESCRT (endosomal sorting complex required for transport)-associated proteins Alix and Tsg101 exist in exosomes [10], [11]. Some membrane-associated proteins such as tetraspanins CD9, CD63 and CD81, which are involved in adhesion and targeting, are found on the surface of exosomes [10], [11].

For imaging of exosomes, we used the well-known exosomal

Cell-to-cell transfer of cancer-cell-derived exosomes

Cancer-cell-shed exosomes/microvesicles which transfer genetic and proteomic information to target cells as a way of cell-to-cell communication. For example, primary human glioblastoma cells were observed to secrete microvesicles, including exosomes, into the culture medium by scanning electron microscopy [2]. Glioblastoma microvesicles purified from conditioned medium were internalized by human brain microvascular endothelial (HBMVEC) and delivered functional RNA to the recipient cells [2].

Cancer cells secrete exosomes into the tumor microenvironment

It has been suggested that cancer cells shed exosomes and condition their niche via exosomal material to promote tumor growth and metastasis. We examined whether cancer cells secrete exosomes into the surrounding tissue in breast cancer models using CLSM imaging. We established an orthotopic mouse model of breast cancer metastasis to the lung by orthotopic implantation of MMT mouse breast cancer cells into the mammary fat pad (MFP) of nude mice (Fig. 3A, C). MMT orthotopic model forms primary

Conclusions and perspectives

The combination of GFP-tagged exosomes and CLSM is useful for imaging exosomes in vitro and in vivo. Imaging of exosomes can contribute to understanding the role of intercellular communication in tumor progression.

The cancer stem cell (CSC) theory proposes that a small subset of cells within a heterogeneous tumor have stem cell-like properties and produce new heterogeneous tumor cells [21], [22], [23]. This theory suggests that CSCs lead to metastasis while closely interacting with non-CSCs and

References (24)

G. Rabinowits et al.
Exosomal microRNA: a diagnostic marker for lung cancer
Clin. Lung Cancer
(2009)
S. Rorive et al.
TIMP-4 and CD63: new prognostic biomarkers in human astrocytomas
Mod. Pathol.
(2010)
D.D. Taylor et al.
MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer
Gynecol. Oncol.
(2008)
J. Ratajczak et al.
Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication
Leukemia
(2006)
J. Skog et al.
Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers
Nat. Cell Biol.
(2008)
H. Valadi et al.
Exosome mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells
Nat. Cell Biol.
(2007)
B.S. Hong et al.
Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells
BMC Genomics
(2009)
K. Al-Nedawi et al.
Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells
Nat. Cell Biol.
(2008)
T. Jung et al.
CD44v6 dependence of premetastatic niche preparation by exosomes
Neoplasia
(2009)
J.L. Hood et al.
Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis
Cancer Res.
(2011)

American Cancer Society

Global Cancer Facts & Figures

(2011)

S. Mathivanan et al.

ExoCarta 2012: database of exosomal proteins. RNA and lipids

Nucleic Acids Res.

(2012)

Cited by (258)

Dual roles of the conditional extracellular vesicles derived from Pseudomonas aeruginosa biofilms: Promoting and inhibiting bacterial biofilm growth
2024, Biofilm
Antibiotic-resistant biofilm infections have emerged as public health concerns because of their enhanced tolerance to high-dose antibiotic treatments. The biofilm life cycle involves multiple developmental stages, which are tightly regulated by active cell-cell communication via specific extracellular signal messengers such as extracellular vesicles. This study was aimed at exploring the roles of extracellular vesicles secreted by Pseudomonas aeruginosa at different developmental stages in controlling biofilm growth. Our results show that extracellular vesicles secreted by P. aeruginosa biofilms during their exponential growth phase (G-EVs) enhance biofilm growth. In contrast, extracellular vesicles secreted by P. aeruginosa biofilms during their death/survival phase (D-EVs) can effectively inhibit/eliminate P. aeruginosa PAO1 biofilms up to 4.8-log₁₀ CFU/cm². The inhibition effectiveness of D-EVs against P. aeruginosa biofilms grown for 96 h improved further in the presence of 10–50 μM Fe³⁺ ions. Proteomic analysis suggests the inhibition involves an iron-dependent ferroptosis mechanism. This study is the first to report the functional role of bacterial extracellular vesicles in bacterial growth, which depends on the developmental stage of the parent bacteria. The finding of D-EV-activated ferroptosis-based bacterial death may have significant implications for preventing antibiotic resistance in biofilms.
Human lens epithelial-secreted exosomes attenuate ocular angiogenesis via inhibiting microglial activation
2024, Experimental Eye Research
The lens is an avascular tissue, where epithelial cells (LECs) are the primary living cells. The role of LECs-derived exosomes (LEC-exos) is largely unknown. In our study, we determined the anti-angiogenic role of LEC-exos, manifested as regressed retinal neovascularization (NV) using the oxygen-induced retinopathy (OIR), and reduced choroidal NV size and pathological vascular leakage using the laser-induced choroidal neovascularization (laser-induced CNV). Furthermore, the activation and accumulation of microglia were also restricted by LEC-exos. Based on Luminex multiplex assays, the expressions of chemokines such as SCYB16/CXCL16, MCP-1/CCL2, I-TAC/CXCL11, and MIP 3beta/CCL19 were decreased after treatment with LEC-exos. Transwell assays showed that LEC-exos restricted the migration of the mouse microglia cell line (BV2 cells). After incubation with LEC-exos-treated BV2 cells, human umbilical vein endothelial cells (hUVECs) were collected for further evaluation using tube formation, Transwell assays, and 5-ethynyl-2′-deoxyuridine (EDU) assays. Using in vitro experiments, the pro-angiogenic effect of microglia was restricted by LEC-exos. Hence, it was investigated that LEC-exos attenuated ocular NV, which might attribute to the inhibition of microglial activation and accumulation.
Engineered exosomes-based theranostic strategy for tumor metastasis and recurrence
2023, Asian Journal of Pharmaceutical Sciences
Metastasis-associated processes are the predominant instigator of fatalities linked to cancer, wherein the pivotal role of circulating tumor cells lies in the resurgence of malignant growth. In recent epochs, exosomes, constituents of the extracellular vesicle cohort, have garnered attention within the field of tumor theranostics owing to their inherent attributes encompassing biocompatibility, modifiability, payload capacity, stability, and therapeutic suitability. Nonetheless, the rudimentary functionalities and limited efficacy of unmodified exosomes curtail their prospective utility. In an effort to surmount these shortcomings, intricate methodologies amalgamating nanotechnology with genetic manipulation, chemotherapy, immunotherapy, and optical intervention present themselves as enhanced avenues to surveil and intercede in tumor metastasis and relapse. This review delves into the manifold techniques currently employed to engineer exosomes, with a specific focus on elucidating the interplay between exosomes and the metastatic cascade, alongside the implementation of tailored exosomes in abating tumor metastasis and recurrence. This review not only advances comprehension of the evolving landscape within this domain but also steers the trajectory of forthcoming investigations.
Towards artificial intelligence-enabled extracellular vesicle precision drug delivery
2023, Advanced Drug Delivery Reviews
Extracellular Vesicles (EVs), particularly exosomes, recently exploded into nanomedicine as an emerging drug delivery approach due to their superior biocompatibility, circulating stability, and bioavailability in vivo. However, EV heterogeneity makes molecular targeting precision a critical challenge. Deciphering key molecular drivers for controlling EV tissue targeting specificity is in great need. Artificial intelligence (AI) brings powerful prediction ability for guiding the rational design of engineered EVs in precision control for drug delivery. This review focuses on cutting-edge nano-delivery via integrating large-scale EV data with AI to develop AI-directed EV therapies and illuminate the clinical translation potential. We briefly review the current status of EVs in drug delivery, including the current frontier, limitations, and considerations to advance the field. Subsequently, we detail the future of AI in drug delivery and its impact on precision EV delivery. Our review discusses the current universal challenge of standardization and critical considerations when using AI combined with EVs for precision drug delivery. Finally, we will conclude this review with a perspective on future clinical translation led by a combined effort of AI and EV research.
Thermoresponsive bio-affinity interfaces for temperature-modulated selective capture and release of targeted exosomes
2023, Materials Today Bio
The existing methods for exosome isolation, such as ultracentrifugation, size exclusion, and affinity separation, suffer from some limitations. Herein, we aimed to develop temperature-modulated exosome-capturing materials using thermoresponsive polymers and peptides with affinity for exosomes. Poly(2-hydroxyethyl methacrylate-co-propargyl acrylate)-b-poly(N-isopropylacrylamide) (P(HEMA-co-PgA)-b-PNIPAAm) was grafted on silica beads via a two-step process of activator regenerated by electron transfer atom transfer radical polymerization. Peptides with affinity for exosomes were conjugated to the propargyl group of the bottom P(HEMA-co-PgA) segment of the copolymer via a click reaction. The prepared copolymer-grafted beads were characterized by elemental analysis, X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, gel permeation chromatography, and the turbidity of the polymer solution. Results indicated that the copolymer and peptide were successfully modified on the silica beads. Exosomes from SK-BR-3 cells, a human breast cancer cell line, were selectively captured on the prepared beads at 37 °C, as the upper PNIPAAm segment shrank and the affinity between the peptide and exosome was enhanced. Upon lowering the temperature to 4 °C, the captured exosomes were released from the copolymer brush because of the extension of the PNIPAAm segment that reduced the affinity between peptides and exosomes. These findings demonstrated that the prepared copolymer brush-grafted silica beads can capture and release targeted exosomes via temperature modulation. Taken together, the developed copolymer brush-grafted silica beads would be useful for the separation of exosomes using simple procedures such as temperature modulation.
Addressing the diagnosis and therapeutics of malignant tumor cells
2023, Biomarkers in Cancer Detection and Monitoring of Therapeutics: Discovery and Technologies: Volume 1
Cancer kills around a quarter of the people in the developed world. Cancer is a wide term that encompasses over 277 different forms of cancer. Different stages of cancer have been identified, demonstrating that multiple gene mutations are involved in cancer pathogenesis. The aberrant cell growth is caused by these gene alterations. Many cancer patients are diagnosed as an emergency presentation, which is linked to poorer clinical and patient-reported outcomes than individuals who are diagnosed voluntarily or through screening. Even when there is a screening test for a specific disease, most malignancies present with symptoms to basic care. However, cancer symptoms can also be indications of a benign disease; therefore, the doctor must determine whether cancer is the cause. Cancer is still a major unmet medical need, and many potential cancer targets have yet to be discovered.

View all citing articles on Scopus

^☆: This review is part of the Advanced Drug Delivery Reviews theme issue on “Exosomes: a key to delivering genetic materials”.

¹: These authors contributed equally to this work.

Copyright © 2012 Elsevier B.V. All rights reserved.