Biometals

https://doi.org/10.1007/s10534-018-0109-3

Gadopentatic acid affects in vitro proliferation

and doxorubicin response in human breast adenocarcinoma
cells
Abdel Rahman Abdel Fattah . Sarah Mishriki . Tobias Kammann .
Rakesh P. Sahu . Fei Geng . Ishwar K. Puri

Received: 9 March 2018 / Accepted: 1 May 2018

Ó Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Contrasting agents (CAs) that are admin- concentration. Proliferation is enhanced when cells
istered to patients during magnetic resonance imaging transition to 3D morphologies in post confluent
to facilitate tumor identification are generally consid- conditions. The proliferation dependence on the
ered harmless. However, gadolinium (Gd) based concentration of CA is absent for Hs 578T and
contrast agents can be retained in the body, inflicting MDA-MB-231 triple negative cell lines. MCF-7 cells
specific cell line cytotoxicity. We investigate the reveal a double toggle switch related to the expression
effect of Gadopentatic acid (Gd-DTPA) on human of VEGF, which goes through high–low–high down-
breast adenocarcinoma MCF-7 cells. These cells regulation when cells are exposed to 0.1, 1, and
exhibit a toggle switch response: exposure to 0.1 and 10 mM Gd-DTPA, respectively. Finally, doxorubicin
1 mM concentrations of Gd-DTPA enhances prolif- drug response is assessed, which also reveals a double
eration, which is hindered at a higher 10 mM toggle switch behavior, where drug cytotoxicity
exhibits a nonlinear dependence on the CA concen-
tration. A toggle switch in cell characteristics that are
Electronic supplementary material The online version of exposed to 1 mM of Gd-DTPA amplifies the impor-
this article (https://doi.org/10.1007/s10534-018-0109-3) con- tance of this threshold, affecting several cell behaviors
tains supplementary material, which is available to authorized
users.
if surpassed. This work emphasizes the important
effects that CAs can have on cells, specifically Gd-
Abdel Rahman Abdel Fattah and Sarah Mishriki have been DTPA on MCF-7 cells, and the implications for cell
contributed equally to the work. growth and drug response during clinical and synthetic
biology procedures.
A. R. Abdel Fattah
R. P. Sahu
F. Geng

I. K. Puri (&)
Department of Mechanical Engineering, McMaster Keywords Magnetic resonance imagining

University, 1280 Main Street West, Hamilton, Contrasting agents
Gadopentatic acid
Breast
ON L8S 4L7, Canada cancer
Paramagnetism
e-mail: [email protected]

S. Mishriki
I. K. Puri
School of Biomedical Engineering, McMaster University,
1280 Main Street West, Hamilton, ON L8S 4K1, Canada Introduction
T. Kammann
Faculty of Biological Sciences, Friedrich-Schiller- Contrasting agents (CAs) are widely used during
University Jena, Jena, Germany magnetic resonance imaging (MRI) to enhance the

123
 Biometals

detection of tumors in the body. Gadopentatic acid media are improved by supplementing them with Gd
(Gd-DTPA), which is a paramagnetic salt, was based CAs, allowing cells to behave as a diamagnetic
identified as a CA in the 1980s (Carr et al. 1984) and material. This approach is used to enhance cell
other Gd-based CAs have since been developed (Zhou manipulation (Abdel Fattah et al. 2016a, b), entrap-
and Lu 2013). While these CAs produce patient ment (Kauffmann et al. 2011; Winkleman et al. 2004),
symptoms such as nausea for lower than 1% of cases and form 3D cell cultures (Abdel Fattah et al. 2018;
(Shellock and Kanal 1999), Gd based CAs are Akiyama and Morishima 2011) providing macroscale
considered safe and their cytotoxicity in various cell manipulation that is otherwise limited to the micro-
lines is generally minor. For example, for osteoarthri- scale (Abdel Fattah et al. 2016a; Ki-Ho Han and
tis cases, CAs, including Gd-DTPA, shows no signif- Frazier 2005).
icant negative effects when compared to control Extending our previous work (Abdel Fattah et al.
samples (Midura et al. 2014), and DTPA chelates of 2016b, 2018), here we report several impacts of
Gd and iron (Fe) are nontoxic and impermeable to red prolonged in vitro exposure to culture media supple-
blood cells (Zaplatin et al. 1996). On the other hand, mented with micro- and millimolar concentrations of
17B-estradiol (EPTA-Gd) specifically interacts with Gd-DTPA on breast adenocarcinoma cells (Michigan
estrogen receptor in breast cancer cells (Morgunov Cancer Foundation-7, MCF-7). Exposure to the para-
et al. 2009). While this is a demonstration of the magnetic salt influences cell proliferation, migration,
targeting ability of engineered CAs to specific cell post confluence 3D growth, vascular endothelial
types and receptors, these interactions can sometimes growth factor (VEGF) and hypoxia-inducible factor
come at a cost. Micromolar concentrations of gadolin- 1-alpha (HIF1a) gene expression, and response to the
ium chloride (GdCl) increase the proliferation and chemotherapy drug doxorubicin (DOX). We observe
survival of HeLa cells (Zhang et al. 2009). Specific nonlinear cell behaviors and toggle switch responses
concentrations of various Gd based CAs also do so for for different concentrations of the salt. Whereas, a
fibroblasts in patient derived samples(Edward et al. high concentration of Gd-DTPA slows cell prolifer-
2010). These results suggest that, although generally ation and increases survival against DOX, lower
considered safe, Gd-based CAs have cytotoxicity that concentrations increases proliferation and can also
is cell line dependent and case specific. When the CAs enhance cell response to DOX, reducing its half
are administered at a usual dose of 0.1 mmol/kg and maximum inhibitory concentration (IC50) by nearly
concentrations as high as 0.5 M, their excretion in twofold.
patients with impaired renal function can be delayed With the persistent lack of full understanding of the
from hours to days, promoting the retention of Gd in effects of Gd based CAs on cells in the clinical as well
organs such as kidney, liver and bone (Aime and as synthetic biology settings, our in vitro observations,
Caravan 2009). In fact, Gd retention has also been using relevant Gd-DTPA concentrations, convey that
found in patients with normal rental function, and in tumor progression and drug response could be influ-
some cases can take more than 8 years to clear the enced in studies and in patients who are administered
body when retained in bone (Ramalho et al. 2016). the CA and later undergo chemotherapy.
Furthermore, the understanding of Gd chelate stability
in a biological environment is still lacking, and has
been proposed as a driving factor in the development Materials and methods
diseases such as nephrogenic systemic fibrosis through
the release of Gd?3 ions in the body (Frenzel et al. MCF-7, Hs 578T and MDA-MB-231 cells were
2008). cultured in Dulbecco’s modified eagle medium
Despite these adverse effects, the magnetic prop- (DMEM) media supplemented with 10% FBS in
erties of Gd based CAs have extended their applica- 10 cm dishes. They were subsequently trypsinized and
tions to fields other than imaging, e.g., for enhancing plated into the various conditions preceding the
label free organism magnetophoresis (Berryy and different experiments. For more details on all used
Geimz 1997), which facilitates the magnetic manip- reagents and methods please refer to the materials and
ulation of cells (Paul and Roath 1975). Here, the methods section in supplemental materials.
paramagnetic properties of the buffers or cell culture

123
Biometals

Results and discussion shows that Gd-DTPA has no discernible influence on

proliferation for both triple negative cell lines.
We have previously used Gd-DTPA to print in situ Gd-based CAs can target estrogen receptors (ERs)
three-dimensional (3D) cell assemblies of different in breast cancer cells (Pais et al. 2013). While, Gd-
morphologies and sizes (Abdel Fattah et al. DTPA has not been shown to magnetically highlight
2016a, 2018). We now investigate the effect of ERs in cells during MRI scans, this does not exclude
prolonged Gd-DTPA exposure on MCF-7 cell prolif- the possibility of ER activation. Acting as a xenoe-
eration to reveal the impact of the CA on underlying strogen, Gd-DTPA may activate estrogen signaling in
cellular pathways impacting drug efficacy, 3D growth, ER-positive cell lines, promoting their growth. An
migration, and gene expression. Figure 1 shows that a increase in proliferation due to a Gd-based CA has
concentration of 0.1 mM and 1 mM of Gd-DTPA been reported for ER-positive HeLa cells (Zhang et al.
enhances proliferation with live cell counts of 2009). Therefore, it may be possible that ER stimu-
122.5 ± 9.9 and 151.8 ± 17.5% compared to the lation by Gd-DTPA contributes to the increased
control. However, cells cultured in media containing proliferation for concentrations B 1 mM in our
10 mM Gd-DTPA yield only 63.6 ± 13.2% viable experiments.
cells. This behavior suggests that Gd-DTPA has a Since patients who have been administered CAs for
nonlinear influence on MCF-7 cell proliferation. MRI scans might be subsequently treated with
Exposure to Gd-DTPA concentrations beyond a chemotherapeutic drugs, we investigate the influence
certain threshold triggers a significant decrease in of Gd-DTPA on cells in the presence of the drug DOX,
proliferation, whereas lower concentrations of the which is a chemotherapeutic drug that specifically
paramagnetic salt enhances it. Identical experiments targets proliferating cells. In order to monitor the DOX
were performed with the human breast cancer cell response for MCF-7 cells, the cells are plated and
lines Hs 578T and MDA-MB-231, which are estrogen exposed to the three different Gd-DTPA concentra-
receptor, progesterone receptor, and human epidermal tions and a media control. In all cases, cells are
growth factor receptor 2 negative cell lines. Figure 1, simultaneously exposed to a range of DOX

Fig. 1 Effect of Gd-DTPA on the viability of MCF-7, Hs 578T exposure to a higher salt concentration first enhances prolifer-
and MDA-MB-231 cell lines. All cell lines were seeded and ation for the 0.1 and 1 mM cases, and then decreases when
treated with various Gd-DTPA concentrations on day 1. On day exposed to 10 mM of the CA as compared to the control. The
3, a control normalized percent viable cell count is derived from proliferation of both Hs 578T and MDA-MB-231 which, unlike
a cell count following washing, trypsinization and staining. The MCF-7, are ER-negative cell lines, is independent of Gd-DTPA
proliferation of MCF-7 cells depends on the Gd-DTPA treatment. Error bars represent the SD of biological duplicates
concentration, revealing a toggle switch response where conducted in technical triplicates

123
 Biometals

concentrations including drug vehicle (DI water) and that are used. The IC50 values, evaluated using
media controls. Graphpad Prism Software, are 0.158, 0.155, 0.085,
Figure 2a shows MCF-7 MTT viability DOX and 0.275 lg/mL for cells exposed to Gd-DTPA
response curves for the four Gd-DTPA concentrations concentrations of 0, 0.1, 1, and 10 mM, respectively.

Fig. 2 Effect of Gd-DTPA on the doxorubicin drug response in performed with cells that are pre-treated with various concen-
MCF-7 cells. a Cells are treated simultaneously with various tration of Gd-DTPA for 3 days. Subsequently, cells are treated
Gd-DTPA and DOX concentrations, and incubated for 3 days simultaneously with various DOX concentrations while main-
before an MTT assay is performed. The percent viability is taining the same Gd-DTPA concentration in which they were
assessed with reference to the drug-free water treated case for pretreated. Exposure to 0.1, 1 and 10 mM of the salt produces,
each Gd-DTPA concentration. While 0.1 mM of the CA does respectively, a 1.38-fold increase, 2.04-fold decrease, and a
not significantly change the IC50 compared to the Gd-DTPA 5.01-fold increase compared to the Gd-DTPA free control. The
free case, 1 mM of salt causes a 1.86-fold decrease and 10 mM drug response is therefore boosted compared to (a) when no Gd-
causes a 1.74-fold increase. The heat map shows MTT viability DTPA pretreatment has taken place. The heat map in (c) further
normalized to the 0 mM Gd-DTPA case at each tested DOX validates the boosting effect of Gd-DTPA pretreatment,
concentration. 1 mM of the salt yields the lowest values, indicating larger magnitudes in drug response compared to the
indicating increased drug efficacy, whereas 0.1 and 10 mM, heat map in (a). d Shows a similar point evaluation to (b). In this
respectively, produce hindered drug efficacies compared to the case, exposure to DOX and 1 mM Gd-DTPA yields fewer viable
Gd-DTPA free control. b A point evaluation using 0.5 lg/mL cells as compared to its counterpart in (b). Gd-DTPA facilitates
DOX is performed using the same conditions as (a). A viable a double toggle switch drug response where exposure to
cell count using a hemocytometer shows that exposure to 0.1 increasing Gd-DTPA concentration first slightly hinders drug
and 10 mM of the salt yields the highest viable cell counts response (0.1 mM), then an increase in DOX efficacy (1 mM),
among the DOX treated samples. Moreover, treatment with and finally a significant hindrance (10 mM). Error bars represent
1 mM Gd-DTPA yields a comparable cell count to the Gd- the SD of technical triplicates
DTPA free DOX treated case. c A similar assay to (a) is

123
Biometals

While exposure to 0.1 mM Gd-DTPA does not 0.1 mM Gd-DTPA (Fig. 1), it is still lower than when
significantly change the overall drug response of those cells are exposed to 1 mM of the salt, and
MCF-7, 1 mM of Gd-DTPA leads to a 1.86-fold corresponds to a slight hindrance in the overall drug
decrease in the IC50 of DOX. A further one order of response. This suggests that a particular concentration
magnitude increase in salt concentration to 10 mM of of the paramagnetic salt enhances both proliferation
Gd-DTPA, increases the IC50 by 1.74-fold increase. and drug efficacy, in this case 1 mM Gd-DTPA. Cell
The heat map in Fig. 2a shows the average cell proliferation is hindered by the 10 mM Gd-DTPA
viability at each DOX concentration normalized with concentration, which also provides a lower overall
that for the 0 mM Gd-DTPA control, thereby facili- DOX efficacy. Therefore, the nonlinear correlation of
tating comparison between the various cases. Values cell proliferation with the Gd-DTPA concentration is
smaller than unity correspond to enhanced drug also reflected in cell response to simultaneous DOX
cytotoxicity compared to the control, and those greater treatment.
than unity convey hindrance of DOX efficacy. For To further understand the synergic effects of DOX
1 mM Gd-DTPA, cytotoxicity is enhanced with and Gd-DTPA, cells are first pretreated for 3 days in
values consistently lower than 1 for all DOX concen- media containing 0.1, 1 and 10 mM of the paramag-
trations, yielding an average of 0.90 for these samples. netic salt, as well as the control media. The cells are
Despite having an IC50 close to the control, values for subsequently plated and exposed to the different Gd-
the 0.1 mM Gd-DTPA experiments indicate DOX DTPA and DOX concentrations. From Fig. 2c, the
efficacy hindrance, yielding an average of 1.06. When IC50 is 0.126, 0.173, 0.062 and 0.631 lg/mL for cells
the Gd-DTPA concentration increases to 10 mM, the exposed to Gd-DTPA concentrations of 0, 0.1, 1, and
average value rises further to 1.33. 10 mM, respectively. By pre-treating cells with the
Using a DOX concentration of 0.5 lg/mL for salt, the IC50 for 1 mM Gd-DTPA, undergoes a 2.04-
3 days, a point evaluation is conducted to further fold decrease compared to the control, which is higher
establish confidence in these results. Water treated than the value without pre-treatment, as shown in
controls are also evaluated. Figure 2b shows the Fig. 2a. For the 0.1 and 10 mM Gd-DTPA cases, the
percent viable cell count of the various cases normal- IC50 increases by 1.38-fold and 5.01-fold, respec-
ized to the Gd-DTPA free water treated control. tively, with increasing hindrance to the DOX response
Results for the water treated samples for the various for both of these cases as compared to the cases
Gd-DTPA concentrations show trends similar to the without pretreatment shown in Fig. 2a. Similar to the
results presented in Fig. 1, i.e., the drug vehicle does heat map in Fig. 2a, c compares the viabilities of
not impact trends for proliferation. For the DOX pretreated cases when normalized to the 0 mM Gd-
treated samples, it is evident that cells exposed to 0.1 DTPA control. The 1 mM Gd-DTPA case yields an
and 10 mM concentrations exhibit the highest control average normalized viability of 0.85, revealing further
normalized viable cell counts, yielding 16.47 ± 2.25, enhancement of DOX overall cytotoxicity compared
and 13.97 ± 3.76%, respectively, while the DOX to the case without pretreatment. While the result for
treated 0 mM Gd-DTPA case provides an average 0.1 mM Gd-DTPA is similar with an average value of
value of 10.14 ± 0.88%. When the Gd-DTPA con- 1.08, for the 10 mM case the overall normalized
centration is 1 mM, the average viable cell count in the viability increases from 1.33 in Fig. 2a to 1.61 in
DOX treated sample is 11.17 ± 2.80%, which is Fig. 2c. Therefore, for 1 and 10 mM concentrations,
closest to the 0 mM case. Compared to the DOX pretreatment boosts the Gd-DTPA dependent DOX
treated 0 mM Gd-DTPA case, the 0.1, 1, and 10 mM response as compared to cases without pretreatment.
experiments have p-values of 0.0020, 0.5095, and Similar to Fig. 2b, d presents a point evaluation
0.0952, respectively. Although not all cases exhibit where cells are pretreated in Gd-DTPA for 3 days.
significance, they do suggest a nonlinear response Normalized to the water treated control, the water
consistent with the heat map of Fig. 2a. treated samples in Fig. 2d demonstrate a similar trend
Therefore, the overall efficacy of DOX is enhanced to that in Figs. 1 and 2b. For the DOX treated samples,
when the Gd-DTPA concentration increases to 1 mM, cells exposed to 0.1 and 10 mM of Gd-DTPA again
providing the highest proliferation in Fig. 1. Although exhibit the highest viable cell counts, yielding aver-
proliferation is enhanced when cells are exposed to ages of 13.13 ± 2.44 and 12.93 ± 2.63%,

123
 Biometals

respectively. The DOX treated 0 mM Gd-DTPA concentrations, respectively yield 1.85 ± 0.04 and
control yields a viable cell count of 9.07 ± 1.46%. 1.90 ± 0.08 foci/mm2 compared to 1.28 ± 0.04 foci/
When cells are pretreated with Gd-DTPA at a 1 mM mm2 for the control cells cultured in salt free media.
concentration, the average viable cell count is When the salt concentration increases to 10 mM, the
5.98 ± 3.30%, which is slightly lower than the result hindrance in proliferation is significant, delaying
for the control. Compared to the DOX treated 0 mM confluency with fewer 3D morphologies present,
Gd-DTPA, the 0.1, 1, and 10 mM experiments have yielding only 0.20 ± 0.02 foci/mm2. Therefore, just
p-values of 0.0292, 0.0560, and 0.0425, respectively. as the introduction of Gd-DTPA increases monolayer
Considering the 0.1 mM Gd-DTPA case to be an proliferation of MCF-7 cells for the 0.1 and 1 mM
anomaly, the p-values decrease as compared to the concentrations (Fig. 1), this enhancement also holds
case when there is no salt pretreatment. Hence, Gd- for the development of 3D structures. These structures
DTPA pretreatment boosts the DOX response of are higher in number than in the control and are of
MCF-7 cells, i.e., proliferation is correlated with the larger size, as shown in the DAPI micrographs in
DOX response, where the paramagnetic salt is likely Fig. 4. While cells cultured in 10 mM of Gd-DTPA
influencing shared signaling pathways. This correla- proliferate less effectively and form fewer 3D projec-
tion may also be significant for other cell behaviors, tions than in the control, the corresponding population
such as invasion, migration, and metastasis, since in of dead cells is significantly lower than for all other
many cancers, they share common signaling pathways cases, as shown by the FITC micrograph in Fig. 4.
with proliferation and survival (Gupta et al. 2010). This suggests that the slowdown in proliferation for
Changes in cell invasiveness can be accompanied this case is not due to increased cell death, but rather to
with variations in migration rates. The migration assay the inability of cells to proliferate as effectively. Both
in Fig. 3 shows that 24 h pretreatment with Gd-DTPA post confluent (Fig. 4) and monolayer (Fig. 1) prolif-
concentrations of 0.1 and 1 mM does not significantly eration follow similar nonlinear behaviors. These
influence the control normalized wound closure trends occur through changes at the gene level. For
velocities, which are 1.061 ± 0.154 and example, independently of its angiogenic functions,
0.909 ± 0.048, respectively. However, pretreatment VEGF has been proposed to play a significant role in
with 10 mM Gd-DTPA significantly hinders migra- cell proliferation and migration (Perrot-Applanat and
tion as the velocity is reduced to 0.021 ± 0.060. Di Benedetto 2012).
Hence, while MCF-7 migration is not influenced by In order to shed light on the potential mechanism
pre-exposure to media containing 0.1 and 1 mM Gd- underlying the effect of Gd-DTPA, we investigated
DTPA, it is significantly diminished when the media the expression of biomarker genes, such as VEGF and
contains 10 mM of the paramagnetic salt, producing a HIF1a. Figure 5 shows the fold change in gene
nonlinear response. This reduction in migration for the expression relative to the control (0 mM Gd-DTPA)
latter case correlates with hindered cell proliferation glyceraldehyde 3-phosphate dehydrogenase
for the same concentration as shown in Fig. 1. As with (GAPDH) for cells cultured in various Gd-DTPA
the proliferation, cell line dependency is again supplemented media. The expression of HIF1a is
observed when the experiment is performed using downregulated and almost identical for 0.1 and 1 mM
Hs 578T and MDA-MB-231 cells, yielding insignif- Gd-DTPA, yielding 0.8 ± 0.16 and 0.76 ± 0.14,
icant differences among all tested Gd-DTPA concen- respectively. Cells cultured in 10 mM Gd-DTPA with
trations and the respective controls. 1.07 ± 0.15, however, do not show a similar down-
Malignant transformations in MCF-7 cells are regulation. A downregulation in VEGF is observed
linked to their ability to form 3D structures (foci) across all cell cultures exposed to Gd-DTPA, where
under post confluent conditions (Gierthy and Lincoln the values are 0.57 ± 0.09, 0.77 ± 0.09, and
1988). We observed 3D MCF-7 cell foci formed 0.41 ± 0.19 for cells exposed to the 0.1, 1 and
during a post confluency assay, as shown in Fig. 4. An 10 mM Gd-DTPA concentrations respectively. Thus,
increase in MCF-7 cell proliferation is again observed, VEGF expression dependency also reveals a nonlinear
now through an increase in the number of 3D response where values undergo a high–low–high
structures. From Fig. 4, cells cultured in media downregulation when cells are exposed respectively
containing 0.1 and 1 mM of Gd-DTPA to 0.1, 1 and 10 mM of Gd-DTPA. This correlates with

123
Biometals

Fig. 3 Effect of Gd-DTPA

on cell migration for MCF-
7, Hs 578T and MDA-MB-
231 cell lines. All cell lines
are seeded and treated with
various Gd-DTPA
concentrations on day 0.
After 24 h, a scratch assay is
performed in Gd-DTPA free
media and the control
normalized wound closure
velocity is evaluated
through a series of
micrographs taken at t = 0 h
and t = 6 h. Although MCF-
7 cells exposed to 0.1 and
1 mM Gd-DTPA have
comparable wound closure
velocities to the control,
treatment with 10 mM of
Gd-DTPA significantly
hinders the closure velocity.
The corresponding results
for Hs 578T and MDA-MB-
231 triple negative cell lines
shows that wound closure is
independent of the Gd-
DTPA concentration. Error
bars represent the SD of
biological duplicates
conducted in technical
triplicates

the trend in Fig. 1, i.e., the least VEGF downregulation exhibits no distinction between the control and cells
occurs for the highest proliferation enhancement exposed to 0.1 and 1 mM Gd-DTPA, the 10 mM
(1 mM Gd-DTPA), whereas the higher downregula- concentration significantly hinders migration. This
tions (0.1 and 10 mM Gd-DTPA) coincide with suggest that a behavioral toggle switch might exist
decreased proliferation. where Gd-DTPA concentrations above 1 mM produce
The experiments reveal several nonlinear behaviors a significant disturbance in the proliferation and
for MCF-7 cells when exposed to varying concentra- migration pathways, possibly through the cytotoxic
tions of Gd-DTPA. In Fig. 6, we propose a model effects of the salt. Both 0.1 and 10 mM concentrations
based on the presence of several toggle switch yield lower VEGF expression than the 1 mM concen-
responses for the cell behaviors. When subjected to tration, suggesting again that the latter value corre-
0.1 mM of Gd-DTPA, MCF-7 cells show greater sponds to a possible toggle switch. The similarity in
proliferation than the control. The proliferation is trends for VEGF expression and cell proliferation
further enhanced when the paramagnetic salt concen- suggests a correlation, where Gd-DTPA may be
tration increases to 1 mM before being significantly implicating underlying pathways. In addition to the
hindered at 10 mM. This trend is again observed in the 1 mM concentration switch, another switch is
post confluency assay. While the migration assay observed at 0.1 mM, producing higher

123
 Biometals

Fig. 4 Effect of Gd-DTPA

on 3D cell growth for MCF-
7 cell line. Cells are seeded
and treated with various Gd-
DTPA concentrations on
day 0 and incubated for
5 days. Two days after
confluency is reached (on
day 5), cells are stained
using a live/dead fluorescent
stain where the DAPI
indicates cell nuclei and
FITC highlights dead cells.
Out of plane cellular foci are
seen in samples treated with
Gd-DTPA concentrations of
0, 0.1, and 1 mM.
Representative foci are
highlighted with a yellow
dashed circle and arrow. The
foci observed for the Gd-
DTPA free media are
smaller in size and fewer in
number than for the samples
incubated in 0.1 and 1 mM
of Gd-DTPA. Cells treated
with 10 mM of the salt show
little evidence of epithelial
dome morphologies and do
not reach confluency.
Moreover, the FITC channel
micrographs show that cells
exposed to 0, 0.1 and 1 mM
of the salt have similar rates
of cell death in contrast to
those treated with 10 mM
Gd-DTPA with significantly
lower cell death in the field
of view. This indicates that
proliferation hindrance with
the highest salt
concentration is due to a
slowdown in the cell cycle
rather than increased cell
death. The average value is
calculated using 11
technical replicates. (Color
figure online)

downregulation in VEGF expression than the higher a double toggle switch behavior might also be present
1 mM concentration as compared to the control. Thus, in VEGF expression. The DOX response to MCF-7

123
Biometals

Fig. 5 Effect of Gd-DTPA on VEGF and HIF1a fold change concentrations. A significant downregulation of VEGF
gene expression relative to control GAPDH in MCF-7 cells. (0.1 mM) is observed, followed by only a slight downregulation
Cells are treated with various Gd-DTPA concentrations and of the gene (1 mM), and finally a significant decrease in
incubated for 3 days before a qPCR is performed. Exposure to expression (10 mM). HIF1a is downregulated equally for cells
1 mM Gd-DTPA slightly downregulates the expression of exposed to both 0.1 and 1 mM of the salt, while a concentration
VEGF while treatment with 0.1 and 10 mM of the CA causes of 10 mM produces minimal variation in the expression. Error
more significant downregulation. This highlights a double bars represent the SD of biological duplicates conducted in
toggle switch behavior during exposure to increasing Gd-DTPA technical triplicates

cells exposed to Gd-DTPA also highlights a double Conclusion

toggle switch behavior, where for the Gd-DTPA
pretreated samples drug efficacy is hindered at the We observe the in vitro influence of Gd-DTPA on
0.1 mM concentration, followed by enhancement at MCF-7 human breast cancer cells, where the param-
the 1 mM concentration, and significant hindrance agnetic Gd salt is a contrast agent used in magnetic
again at a 10 mM concentration. This similarity in resonance imaging scans. MCF-7 cell proliferation is
trend to VEGF expression, shown to slow down drug sensitive to the Gd-DTPA concentration, which
uptake in VEGF ablation during anti-VEGF treatment induces a toggle switch response. Proliferation is
(Rohrig et al. 2017), may again signify that Gd-DTPA enhanced with 0.1 and 1 mM concentrations of the salt
is influencing underlying pathways. but a significant decline in proliferation occurs when
The 1 mM salt concentration is an important this concentration increases to 10 mM. The paramag-
behavioral toggle switch, possibly implicating major netic salt does not enhance MCF-7 cell migration and
cellular pathways, acting to facilitate MCF-7 cell hinders it at relatively high concentrations. The
proliferation, and causing enhanced DOX combina- migration and proliferation of Hs 578T and MDA-
tory effects, which decreases overall cell survival. MB-231 triple negative cell lines are both Gd-DTPA
Surrounding this toggle switch are situations that independent. This emphasizes the response specificity
impact cell proliferation and hinder their response to of MCF-7 cells. From post confluent assays, Gd-
DOX. These are important considerations for clinical DTPA exposure also enhances the growth of 3D
procedures and synthetic biology through the associ- cellular structures in MCF-7 cultures. While this
ated potential impacts on drug response, tumor growth enhancement can be considered a negative impact,
and general cell behavior. Gd-DTPA exhibits combinatory effects with the
chemotherapy drug doxorubicin, which targets prolif-
erating cells. The salt decreases the drug’s IC50
compared to a control at a 1 mM concentration. This
result correlates with the highest enhancement in

123
 Biometals

Fig. 6 A schematic
summary of the effect of Gd-
DTPA on MCF-7 cells.
When MCF-7 cells are
exposed to Gd-DTPA their
proliferation in monolayers
and post confluence 3D
structures is first enhanced
(0.1 mM), reaching a
maximum at 1 mM where a
behavioral switch occurs
with subsequent hindrance
in proliferation (10 mM).
The same switch is observed
for the migration velocity of
these cells. The expression
of VEGF exhibits a double
toggle switch where the
gene goes through high–
low–high downregulation as
cells are respectively
exposed to 0.1–1–10 mM of
the paramagnetic salt. This
same trend is observed for
the efficacy of DOX where,
for the same DOX dosage,
samples yield high–low–
high percent viable cells
when exposed to 0.1–1–
10 mM Gd-DTPA. The
double switch in the efficacy
of DOX highlights the
nonlinear response of the
cells to Gd-DTPA exposure,
while the switch that occurs
for all investigated cell
characteristics at 1 mM of
the paramagnetic salt
signifies the importance of
this threshold in altering
major cellular behaviors

proliferation as well as the least downregulation in threshold, acting as a toggle switch to cell behaviors
VEGF expression. However, at concentrations of 0.1 such as drug uptake, migration, and proliferation. This
and 10 mM, drug cytotoxicity is hindered, where both work highlights a significant consideration for CA
concentrations exhibit higher VEGF downregulation administration and use in synthetic biology, drawing
than cells exposed to 1 mM of Gd-DTPA. Therefore, attention to potentially harmful effects or drug
the results suggest that Gd-DTPA concentrations can hindrance.
influence several cellular pathways in a nonlinear
manner, where 1 mM of the salt is an important

123
Biometals

Acknowledgements This work is supported by a Natural Frenzel T, Lengsfeld P, Schirmer H, Hu J, Weinmann H-J
Sciences and Engineering Research Council of Canada (Grant (2008) Stability of gadolinium-based magnetic resonance
RGPIN-2014-04066) Discovery Grant (NSERC-DG), Canada imaging contrast agents in human serum at 37°C. Invest
Foundation for Innovation John R. Evans Leaders Fund (CFI- Radiol 43:817–828
JELF), Ontario Research Fund Research Infrastructure (ORF- Gierthy JF, Lincoln DW (1988) Inhibition of postconfluent
RI) grants (Grant No. 33016), the Mitacs Globalink Program and focus production in cultures of MCF-7 human breast can-
the German Academic Exchange Service (DAAD). The authors cer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Breast
thank Dr. Juliet Daniel from Department of Biology at Cancer Res Treat 12:233–277
McMaster University for donating Hs 578T and MDA-MB- Gupta SC, Kim JH, Prasad S, Aggarwal BB (2010) Regulation
231 cells. of survival, proliferation, invasion, angiogenesis, and
metastasis of tumor cells through modulation of inflam-
Author contributions AA and SM contributed equally to this matory pathways by nutraceuticals. Cancer Metastasis Rev
work. The manuscript was written through contributions of all 29(3):405–434. https://doi.org/10.1007/s10555-010-9235-
authors. All authors have given approval to the final version of 2
the manuscript. Han Ki-Ho, Frazier AB (2005) Diamagnetic capture mode
magnetophoretic microseparator for blood cells. J Micro-
Compliance with ethical standards electromech Syst 14(6):1422–1432
Kauffmann P, Ith A, O’Brien D, Gaude V, Boue F, Combe S,
Conflict of interest The authors declare no conflict of interest. Reyne G (2011) Diamagnetically trapped arrays of living
cells above micromagnets. Lab Chip 11(18):3153–3161.
https://doi.org/10.1039/c1lc20232d
Midura S, Schneider E, Sakamoto FA, Rosen GM, Winalski CS,
Midura RJ (2014) In vitro toxicity in long-term cell culture
References of MR contrast agents targeted to cartilage evaluation.
Osteoarth Cartil 22(9):1337–1345. https://doi.org/10.1016/
Abdel Fattah AR, Ghosh S, Puri IK (2016a) High gradient j.joca.2014.07.010
magnetic field microstructures for magnetophoretic cell Morgunov RB, Mushenok FB, Aldoshin SM, Sanina NA,
separation. J Chromatogr B 1027:194–199. https://doi.org/ Yur’eva EA, Shilov GV, Tkachev VV (2009) Thermally-
10.1016/j.jchromb.2016.05.046 induced paramagnetism of spiropyrane iodides. New J
Abdel Fattah AR, Meleca E, Mishriki S, Lelic A, Geng F, Sahu Chem 33(6):1374. https://doi.org/10.1039/b822567b
RP, Puri IK (2016b) In situ 3D label-free contactless bio- Pais A, Biton IE, Margalit R, Degani H (2013) Characterization
printing of cells through diamagnetophoresis. ACS Bio- of estrogen-receptor-targeted contrast agents in solution,
mater Sci Eng 2(12):2133–2138. https://doi.org/10.1021/ breast cancer cells, and tumors in vivo. Magn Reson Med
acsbiomaterials.6b00614 70(1):193–206. https://doi.org/10.1002/mrm.24442
Abdel Fattah AR, Mishriki S, Kammann T, Sahu RP, Geng F, Paul F, Roath S (1975) Direct magnetic separation of red cells
Puri IK (2018) 3D cellular structures and co-cultures from whole blood. Nature 255:706
formed through contactless magnetic manipulation of cells Perrot-Applanat M, Di Benedetto M (2012) Autocrine functions
on adherent surfaces. Biomater Sci. https://doi.org/10. of VEGF in breast tumor cells: adhesion, survival, migra-
1039/c7bm01050h tion and invasion. Cell Adhes Migr 6(6):547–553. https://
Aime S, Caravan P (2009) Biodistribution of gadolinium-based doi.org/10.4161/cam.23332
contrast agents, including gadolinium deposition. J Magn Ramalho J, Semelka RC, Ramalho M, Nunes RH, AlObaidy M,
Reson Imaging 30(6):1259–1267. https://doi.org/10.1002/ Castillo M (2016) Gadolinium-based contrast agent accu-
jmri.21969 mulation and toxicity: an update. AJNR Am J Neuroradiol
Akiyama Y, Morishima K (2011) Label-free cell aggregate 37(7):1192–1198. https://doi.org/10.3174/ajnr.A4615
formation based on the magneto-Archimedes effect. Appl Rohrig F, Vorlova S, Hoffmann H, Wartenberg M, Escorcia FE,
Phys Lett 98(16):163702. https://doi.org/10.1063/1. Keller S, Henke E (2017) VEGF-ablation therapy reduces
3581883 drug delivery and therapeutic response in ECM-dense
Berryy MV, Geimz AK (1997) Of flying frogs and levitrons. Eur tumors. Oncogene 36(1):1–12. https://doi.org/10.1038/
J Phys 18:307–313 onc.2016.182
Carr DH, Brown J, Bydder GM, Steiner RE, Weinmann H-J, Shellock FG, Kanal E (1999) Safety of magnetic resonance
Speck U, Young IR (1984) Gadolinium-DTPA as a con- imaging contrast agents. J Magn Reson Imaging
trast agent in MRI: initial clinical experience in 20 patients. 10:477–484
Am J Roentgenol 143:215–224 Winkleman A, Gudiksen KL, Ryan D, Whitesides GM,
Edward M, Quinn JA, Burden AD, Newton BB, Jardine AG Greenfield D, Prentiss M (2004) A magnetic trap for living
(2010) Effect of different classes of gadolinium-based cells suspended in a paramagnetic buffer. Appl Phys Lett
contrast agents on control and nephrogenic systemic 85(12):2411. https://doi.org/10.1063/1.1794372
fibrosis–derived fibroblast proliferation. Radiology Zaplatin AN, Baker KA, Kleinhans FW (1996) Effectivness and
256(3):735–743. https://doi.org/10.1148/radiol. toxcicity of several DTPA broadening agents for biological
10091131/-/DC1 ESR spectroscopy. J Magn Reson Ser B 110:249–254

123
 Biometals

Zhang Y, Fu LJ, Li JX, Yang XG, Yang XD, Wang K (2009) Zhou Z, Lu ZR (2013) Gadolinium-based contrast agents for
Gadolinium promoted proliferation and enhanced survival magnetic resonance cancer imaging. Wiley Interdiscip Rev
in human cervical carcinoma cells. Biometals Nanomed Nanobiotechnol 5(1):1–18. https://doi.org/10.
22(3):511–519. https://doi.org/10.1007/s10534-009-9208- 1002/wnan.1198
5

123