Limits...
High content analysis at single cell level identifies different cellular responses dependent on nanomaterial concentrations.

Manshian BB, Munck S, Agostinis P, Himmelreich U, Soenen SJ - Sci Rep (2015)

Bottom Line: A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine.Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels.Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

View Article: PubMed Central - PubMed

Affiliation: MoSAIC/Biomedical MRI Unit, Faculty of Medicine, KU Leuven, Herestraat 49, B3000 Leuven, Belgium.

ABSTRACT
A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine. NM exposure results in varying levels of internalized NM in different neighboring cells, due to variances in cell size, cell cycle phase and NM agglomeration. Using high-content analysis, we investigated the cytotoxic effects of fluorescent quantum dots on cultured cells, where all effects were correlated with the concentration of NMs at the single cell level. Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels. Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

No MeSH data available.


Related in: MedlinePlus

Overview of conflicting mechanisms involved in general cellular response.(a) Heat maps of high-content imaging-based data for MSCs, MSCs treated with 3-MA or Z-VAD-fmk or MEFs, MEF Atg5 KO or MEF Bax/Bak DKO exposed to QDots at 10 nM (MSC cells) and 12.5 nM (MEF cells), respectively. Cells were analyzed for; cell viability, autophagy and apoptosis. For every cell, the relative level of QDots was also calculated based on the cellular QDot intensity and area, after which cells were divided into 10 categories based on their cellular QDot levels, ranging from c1(lowest) to c10 (highest). After analysis, the cellular effects were grouped based on the different categories for cellular QDot concentrations. The data are shown as relative values after z-normalization compared to untreated control cells (=1) where the fold-change is indicated by the respective color-code. Data were acquired for minimum 5000 cells/condition and were gathered from three independent experiments. (b–d) Representative InCell high-content images of MSCs, MSCs treated with 3-MA or MSC Z-VAD-fmk exposed to QDots (10 nM) for 24 h, after which the cells were stained for (b) cell death, (c) autophagy (LC3) and (d) apoptosis (active caspase-3). Red: QDots, Blue: Hoechst nuclear stain, Green: (b) dead cells, (c) LC3 (d) active caspase-3. Scale bars = 100 μm, the area in the white rectangle is depicted in a magnified view below the original image.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4561960&req=5

f4: Overview of conflicting mechanisms involved in general cellular response.(a) Heat maps of high-content imaging-based data for MSCs, MSCs treated with 3-MA or Z-VAD-fmk or MEFs, MEF Atg5 KO or MEF Bax/Bak DKO exposed to QDots at 10 nM (MSC cells) and 12.5 nM (MEF cells), respectively. Cells were analyzed for; cell viability, autophagy and apoptosis. For every cell, the relative level of QDots was also calculated based on the cellular QDot intensity and area, after which cells were divided into 10 categories based on their cellular QDot levels, ranging from c1(lowest) to c10 (highest). After analysis, the cellular effects were grouped based on the different categories for cellular QDot concentrations. The data are shown as relative values after z-normalization compared to untreated control cells (=1) where the fold-change is indicated by the respective color-code. Data were acquired for minimum 5000 cells/condition and were gathered from three independent experiments. (b–d) Representative InCell high-content images of MSCs, MSCs treated with 3-MA or MSC Z-VAD-fmk exposed to QDots (10 nM) for 24 h, after which the cells were stained for (b) cell death, (c) autophagy (LC3) and (d) apoptosis (active caspase-3). Red: QDots, Blue: Hoechst nuclear stain, Green: (b) dead cells, (c) LC3 (d) active caspase-3. Scale bars = 100 μm, the area in the white rectangle is depicted in a magnified view below the original image.

Mentions: NM-mediated autophagy has been described to be a general cellular response to exposure to a wide range of NMs, including QDots9. The physiological impact of NM-induced autophagy remains however somewhat unclear, as some studies have indicated clear cytoprotective effects by impeding cellular apoptosis signaling, while other studies showed direct autophagy-mediated cell death3334. The induction of autophagy was therefore selected as the potential key mechanism to explain the lower correlation of intracellular NM levels and cell death. We tested two transgenic MEF cell lines, displaying either compromised autophagy, because of the deletion of the essential autophagy gene Atg5 (i.e. MEF-Atg5 KO) or mitochondrial apoptosis due to the double deficiency in pro-apoptotoc Bax and Bak (i.e. MEF-Bax/Bak DKO), a defect known to blunt apoptosis to a variety of stress signals3536. Along with these genetic approaches, we chemically inhibited autophagy (3-methyladenine (3-MA)) and apoptosis (Z-VAD-fmk) in MSCs. Figure 4 reveals clear involvement of apoptosis and autophagy in QDot-mediated cellular response. Inhibition of apoptosis did not affect the levels of cellular autophagy (Supp Fig. S16), whereas inhibition of autophagy resulted in a clear increase in cellular apoptosis levels at higher intracellular QDot concentrations (Supp Fig. S17). Interestingly, inhibition of either mechanism could not prevent cell death, yet in apoptosis-deficient cells, cell death only occurred at the highest intracellular QDot concentration, whereas in autophagy-deficient cells, cell death induction followed a clear concentration-dependent profile (Supp Fig. S18).


High content analysis at single cell level identifies different cellular responses dependent on nanomaterial concentrations.

Manshian BB, Munck S, Agostinis P, Himmelreich U, Soenen SJ - Sci Rep (2015)

Overview of conflicting mechanisms involved in general cellular response.(a) Heat maps of high-content imaging-based data for MSCs, MSCs treated with 3-MA or Z-VAD-fmk or MEFs, MEF Atg5 KO or MEF Bax/Bak DKO exposed to QDots at 10 nM (MSC cells) and 12.5 nM (MEF cells), respectively. Cells were analyzed for; cell viability, autophagy and apoptosis. For every cell, the relative level of QDots was also calculated based on the cellular QDot intensity and area, after which cells were divided into 10 categories based on their cellular QDot levels, ranging from c1(lowest) to c10 (highest). After analysis, the cellular effects were grouped based on the different categories for cellular QDot concentrations. The data are shown as relative values after z-normalization compared to untreated control cells (=1) where the fold-change is indicated by the respective color-code. Data were acquired for minimum 5000 cells/condition and were gathered from three independent experiments. (b–d) Representative InCell high-content images of MSCs, MSCs treated with 3-MA or MSC Z-VAD-fmk exposed to QDots (10 nM) for 24 h, after which the cells were stained for (b) cell death, (c) autophagy (LC3) and (d) apoptosis (active caspase-3). Red: QDots, Blue: Hoechst nuclear stain, Green: (b) dead cells, (c) LC3 (d) active caspase-3. Scale bars = 100 μm, the area in the white rectangle is depicted in a magnified view below the original image.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561960&req=5

f4: Overview of conflicting mechanisms involved in general cellular response.(a) Heat maps of high-content imaging-based data for MSCs, MSCs treated with 3-MA or Z-VAD-fmk or MEFs, MEF Atg5 KO or MEF Bax/Bak DKO exposed to QDots at 10 nM (MSC cells) and 12.5 nM (MEF cells), respectively. Cells were analyzed for; cell viability, autophagy and apoptosis. For every cell, the relative level of QDots was also calculated based on the cellular QDot intensity and area, after which cells were divided into 10 categories based on their cellular QDot levels, ranging from c1(lowest) to c10 (highest). After analysis, the cellular effects were grouped based on the different categories for cellular QDot concentrations. The data are shown as relative values after z-normalization compared to untreated control cells (=1) where the fold-change is indicated by the respective color-code. Data were acquired for minimum 5000 cells/condition and were gathered from three independent experiments. (b–d) Representative InCell high-content images of MSCs, MSCs treated with 3-MA or MSC Z-VAD-fmk exposed to QDots (10 nM) for 24 h, after which the cells were stained for (b) cell death, (c) autophagy (LC3) and (d) apoptosis (active caspase-3). Red: QDots, Blue: Hoechst nuclear stain, Green: (b) dead cells, (c) LC3 (d) active caspase-3. Scale bars = 100 μm, the area in the white rectangle is depicted in a magnified view below the original image.
Mentions: NM-mediated autophagy has been described to be a general cellular response to exposure to a wide range of NMs, including QDots9. The physiological impact of NM-induced autophagy remains however somewhat unclear, as some studies have indicated clear cytoprotective effects by impeding cellular apoptosis signaling, while other studies showed direct autophagy-mediated cell death3334. The induction of autophagy was therefore selected as the potential key mechanism to explain the lower correlation of intracellular NM levels and cell death. We tested two transgenic MEF cell lines, displaying either compromised autophagy, because of the deletion of the essential autophagy gene Atg5 (i.e. MEF-Atg5 KO) or mitochondrial apoptosis due to the double deficiency in pro-apoptotoc Bax and Bak (i.e. MEF-Bax/Bak DKO), a defect known to blunt apoptosis to a variety of stress signals3536. Along with these genetic approaches, we chemically inhibited autophagy (3-methyladenine (3-MA)) and apoptosis (Z-VAD-fmk) in MSCs. Figure 4 reveals clear involvement of apoptosis and autophagy in QDot-mediated cellular response. Inhibition of apoptosis did not affect the levels of cellular autophagy (Supp Fig. S16), whereas inhibition of autophagy resulted in a clear increase in cellular apoptosis levels at higher intracellular QDot concentrations (Supp Fig. S17). Interestingly, inhibition of either mechanism could not prevent cell death, yet in apoptosis-deficient cells, cell death only occurred at the highest intracellular QDot concentration, whereas in autophagy-deficient cells, cell death induction followed a clear concentration-dependent profile (Supp Fig. S18).

Bottom Line: A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine.Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels.Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

View Article: PubMed Central - PubMed

Affiliation: MoSAIC/Biomedical MRI Unit, Faculty of Medicine, KU Leuven, Herestraat 49, B3000 Leuven, Belgium.

ABSTRACT
A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine. NM exposure results in varying levels of internalized NM in different neighboring cells, due to variances in cell size, cell cycle phase and NM agglomeration. Using high-content analysis, we investigated the cytotoxic effects of fluorescent quantum dots on cultured cells, where all effects were correlated with the concentration of NMs at the single cell level. Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels. Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

No MeSH data available.


Related in: MedlinePlus