Quantitative bioimage analytics enables measurement of targeted cellular stress response induced by celastrol-loaded nanoparticles

Erik Niemelä,Emine Lundsten,Pasi Kankaanpää,Diti Desai,Jessica M Rosenholm,John E Eriksson

doi:10.1007/s12192-019-00999-9

Abstract

The cellular stress response, which provides protection against proteotoxic stresses, is characterized by the activation of heat shock factor 1 and the formation of nuclear stress bodies (nSBs). In this study, we developed a computerized method to quantify the formation and size distribution of nSBs, as stress response induction is of interest in cancer research, neurodegenerative diseases, and in other pathophysiological processes. We employed an advanced bioimaging and analytics workflow to enable quantitative detailed subcellular analysis of cell populations even down to single-cell level. This type of detailed analysis requires automated single cell analysis to allow for detection of both size and distribution of nSBs. For specific induction of nSB we used mesoporous silica nanoparticles (MSNs) loaded with celastrol, a plant-derived triterpene with the ability to activate the stress response. To enable specific targeting, we employed folic acid functionalized nanoparticles, which yields targeting to folate receptor expressing cancer cells. In this way, we could assess the ability to quantitatively detect directed and spatio-temporal nSB induction using 2D and 3D confocal imaging. Our results demonstrate successful implementation of an imaging and analytics workflow based on a freely available, general-purpose software platform, BioImageXD, also compatible with other imaging modalities due to full 3D/4D and high-throughput batch processing support. The developed quantitative imaging analytics workflow opens possibilities for detailed stress response examination in cell populations, with significant potential in the analysis of targeted drug delivery systems related to cell stress and other cytoprotective cellular processes.

Highlights

The cellular stress response is an example of a specific cellular process that has received broad attention in research related to aging, cancer, neurodegenerative diseases, and other pathophysiological processes
The results show that already a low dose of 20 μg/ml of celastrol-loaded mesoporous silica nanoparticles (MSNs) treated FRpositive Hela cells showing a clear increase of Hsp70 expression, whereas A549 cells lacking folate receptor (FR) only shows a clear increase of Hsp70 expression levels when administered a high dose of particles, over 40 μg/ml, indicating that these particles are efficiently delivering their cargo to the target cells (Fig. 8c, d)
We have investigated the use of a tailor-made BioImageXDbased quantification method for counting and analyzing nuclear stress bodies in human cells, utilizing folic acid (FA) functionalized celastrol-loaded mesoporous silica nanoparticles (MSNs) for targeted induction of the heat shock response in folate receptor (FR) positive cells

Summary

Introduction

The cellular stress response is an example of a specific cellular process that has received broad attention in research related to aging, cancer, neurodegenerative diseases, and other pathophysiological processes. A unique feature of the primate heat shock response is that HSF1 initiates transcription of its target Hsp genes, and binds and accumulates to specific 9q12 loci at repetitive Satellite III sequences where HSF1 is responsible for transcribing noncoding Satellite III RNAs (Biamonti and Vourc'h 2010) This accumulation of HSF1 can, be visualized with fluorescence microscopy as high-intensity spots inside the nucleus, with sizes between 0.3 and 3 μm, and this unique subnuclear structure is called nSBs (Holmberg et al 2000). Our solution is based on the combination of standard laser scanning confocal microscopy and a workflow on a large and versatile open source software platform for multi-dimensional bioimages, in this case BioImageXD This software natively supports true 3D data in most of its functionality, which includes flexible visualization and analysis tools, and batch processing that requires no programming or scripting (Kankaanpää et al 2012). BioImageXD has been used in numerous applications in biomedical research, ranging from virology and nanoparticle internalization studies to cancer medicine and drug development (Upla et al 2004; Karjalainen et al 2008; Sukumaran et al 2012; Kankaanpää et al 2012)

Methods

Results

Conclusion