A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells

Abstract

Purpose The aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin. Methods Flow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of a fluorescently labelled saporin toxin and the saporin based immunotoxin, OKT10-SAP. Subsequently, measurement of changes in pulse width were used to investigate the effect of a triterpenoid saponin on the endolysosomal escape of internalised toxin into the cytosol. Live cell confocal microscopy was used to validate the flow cytometry data. Results Increased endolysosomal escape of saporin and OKT10-SAP was observed by confocal microscopy in cells treated with saponin. Fluorescent pulse width measurements were also able to detect and quantify escape more sensitively than confocal microscopy. Saponin induced endolysosomal escape could be abrogated by treatment with chloroquine, an inhibitor of endolysosomal acidification. Chloroquine abrogation of escape was also mirrored by a concomitant abrogation of cytotoxicity.Conclusions Poor endolysosomal escape is often a rate limiting step for the cytosolic delivery of protein toxins and other macromolecules. Pulse width analysis offers a simple method to semi-quantify the endolysosomal escape of this and similar molecules into the cytosol.

The Role of Cholesterol on Triterpenoid Saponin-Induced Endolysosomal Escape of a Saporin-Based Immunotoxin

“…There was no discernible increase in the number of cells showing cytosolic fluorescence for mock-treated Daudi cells exposed to 1 µg/mL SA for 24 h compared to cells not exposed to SA. This contrasts with the increase in FITC width seen in the flow cytometry experiment and is attributed to the lower sensitivity of confocal microscopy [ 15 ]. In lipid deprived Daudi cells, the endolysosomal escape of SAP-AF was not observed 24 h after the addition of 5 µg/mL SA.…”

“…OKT10-SAP was constructed by covalently coupling the SO6 form of saporin to OKT10 using SPDP (a heterobifunctional cross-linking reagent) as previously reported [ 41 ].The antibody:toxin ratios, calculated as a percentage of the total amount of protein present, were: 1:1; ~55%; 1:2; ~10%, 10% free antibody, 10% free saporin and 15% which could be designated either a 1:3 or a 2:2 dimer as previously shown in a gel presented in [ 15 ].…”

“…A fluorescence distribution that is localized and vesicular will generate a narrower pulse width compared to a diffuse cytosolic distribution which will produce a larger pulse width. See, for example, [ 15 ] where pulse width analysis was used to effectively measure the endolysosomal escape of the Alexa Fluor labelled IT, OKT10-SAP-AF, from Daudi lymphoma cells in the presence of 1 and 5 µg/mL SA. We have previously shown that saporin-based immunotoxins co-localise with LAMP1 [ 15 ] and EEA1 [ 14 , 15 ] indicating that these ITs accumulate in lysosomes and endosomes, respectively.…”

“…See, for example, [ 15 ] where pulse width analysis was used to effectively measure the endolysosomal escape of the Alexa Fluor labelled IT, OKT10-SAP-AF, from Daudi lymphoma cells in the presence of 1 and 5 µg/mL SA. We have previously shown that saporin-based immunotoxins co-localise with LAMP1 [ 15 ] and EEA1 [ 14 , 15 ] indicating that these ITs accumulate in lysosomes and endosomes, respectively. In the present study, we investigated propidium iodide uptake, as a measure of membrane permeabilisation, in Daudi lymphoma cells incubated with OKT10-SAP-AF for 24 h prior to the addition of 1 µg/mL, 5 µg/mL or no SA after 0 and 24 h. We recorded fluorescein isothiocyanate (FITC) width values for the same populations of cells.…”

“…Fluorescence microscopy co-localisation studies have shown that the relevant intracellular compartments for enhanced cytotoxicity are late endosomes and lysosomes [ 4 , 5 ]. More recently, a flow cytometric method, using pulse width analysis, was effectively used to demonstrate the SA-induced endolysosomal escape of an Alexa Fluor (AF) labelled saporin immunotoxin from Daudi lymphoma cells [ 15 ]. Wensley et al [ 15 ] showed that pulse shape analysis could be used to show the uptake of AF labelled IT into the endolysosomal compartment.…”