Ex vivo Live Cell Imaging of Nanoparticle-Cell Interactions in the Mouse Lung.

Fernanda Ramos-Gomes,Nathalia Ferreira,M Andrea Markus,Frauke Alves,Alexander Kraupner

doi:10.3389/fbioe.2020.588922

Abstract

A successful clinical translation of novel nanoparticle-based cancer therapeutics requires a thorough preclinical investigation of their interaction with immune, tumor and endothelial cells as well as components of the tumor-microenvironment. Although high-resolution microscopy images of fixed tumor tissue specimens can provide valuable information in this regard, they are only static snapshots of a momentary event. Here we describe a superior alternative fluorescence microscopy approach to assess the feasibility of investigating nanoparticle-cell interactions in the mouse lung live and over time at nanometer resolution. We applied fluorescent lung tumor cells and Barium-based fluorescently labeled nanoparticles to nude mice or to CD68-EGFP transgenic mice for visualization of the monocyte-macrophage lineage. Shortly before imaging, fluorescently labeled lectin was intravenously injected for staining of the blood vessels. The lung was filled ex vivo with 1% agarose and individual lung lobes were imaged over time using a confocal microscope with Airyscan technology. Time series demonstrate that live cell imaging of lung lobes can be performed for at least 4 h post mortem. Time-lapse movies illustrate the dynamics of the nanoparticles within the pulmonary circulation and their uptake by immune cells. Moreover, the exchange of nanoparticle material between cancer cells was observed over time. Fluorescent monocytes in lungs of CD68-EGFP transgenic mice could be visualized within blood vessels in the process of interaction with tumor cells and nanoparticles. This high resolution ex vivo live cell imaging approach provides an excellent 4D tool to obtain valuable information on the behavior of tumor and immune cells at first encounter with nanoparticles and may contribute to the understanding of how nanoparticles interact with cells supporting the development of therapeutic strategies based on nanoparticulate drug delivery systems.

Highlights

Used chemotherapy has a number of weaknesses, including poor pharmacokinetics, low specificity and off target effects, leading to substantial side-effects
We show that ex vivo live cell confocal microscopy of entire mouse lung lobes provides an excellent 4D tool for imaging of several dynamic processes in tumor tissue, such as the traffic of cells, shedding of extracellular vesicles (EVs) and the accumulation of NPs in tumor tissue
(iii) To visualize the tumor cell dynamics in the tumor mass and the interactions of NPs with tumor tissue we used the model of experimental lung metastasis

Summary

Introduction

Used chemotherapy has a number of weaknesses, including poor pharmacokinetics, low specificity and off target effects, leading to substantial side-effects. Nanoparticle (NP)based cancer therapeutics have been increasingly explored, because NPs have a number of advantages compared to “naked” therapies: (i) improved drug solubility and stability, (ii) prolonged drug half-live in plasma, (iii) minimized off-target effects, (iv) improved accumulation of drugs at a target site due to the possibility of functionalization and (v) better controlled concentration of drugs by packaging in NPs. recent efforts allowed the incorporation of therapeutic agents into biocompatible NPs, reducing undesirable local or systemic effects (Li et al, 2012). Most NP-based cancer drugs have either not made it into the clinic or have failed to significantly improve patient outcome This is most likely due to the inter-and intraindividual heterogeneity of tumors and to the complexity of tumor and immune response that has been underestimated. It has been recognized that the EPR effect is greatly influenced by a number of parameters, including the tumor microenvironment, vessel density and permeability and stroma composition (Dasgupta et al, 2020)

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