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\DOI{10.5802/crchim.247}
\datereceived{2023-02-28}
\daterevised{2023-06-12}
\datererevised{2023-07-19}
\dateaccepted{2023-07-19}
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\dateposted{2023-09-19}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Chemical Biology}
%\def\cdr@specialtitle@french{Ch\'emobiologie}
%\makeatother

\title{Quantitative analysis of the effect of
microtubule-targeting drugs on the microtubule cytoskeleton of
breast cancer cells with different invasive properties}

\alttitle{Analyse quantitative de l'effet d'agents pharmacologiques
ciblant le cytosquelette microtubulaire de cellules de cancer du sein
ayant des propri\'et\'es invasives diff\'erentes}

\author{\firstname{Sophie} \lastname{Michallet}\CDRorcid{0000-0001-9324-7958}}
\address{Universit\'{e} Grenoble Alpes, INSERM U1209, CNRS UMR5309,
Institute for Advanced Biosciences, Team Cytoskeleton Dynamics and
Nuclear Functions, 38000 Grenoble, France}
\email[S. Michallet]{Sophie.michallet@univ-grenoble-alpes.fr}

\author{\firstname{Lauriane} \lastname{Bosc}\CDRorcid{0000-0001-7242-9541}}
\addressSameAs{1}{Universit\'{e} Grenoble Alpes, INSERM U1209, CNRS
UMR5309, Institute for Advanced Biosciences, Team Cytoskeleton Dynamics
and Nuclear Functions, 38000 Grenoble, France}
\email[L. Bosc]{Lauriane.bosc@univ-grenoble-alpes.fr}

\author{\firstname{Laurence} \lastname{Lafanech\`{e}re}\CDRorcid{0000-0002-7902-7630}\IsCorresp}
\addressSameAs{1}{Universit\'{e} Grenoble Alpes, INSERM U1209, CNRS
UMR5309, Institute for Advanced Biosciences, Team Cytoskeleton Dynamics
and Nuclear Functions, 38000 Grenoble, France}
\email[L. Lafanech\`{e}re]{Laurence.lafanechere@univ-grenoble-alpes.fr}

\keywords{\kwd{Taxanes}\kwd{Vinca-alkaloids}\kwd{Combretastatin-A4}\kwd{Microtubules}\kwd{Quantitative 
cell-based assay}\kwd{Breast cancer}}

\altkeywords{\kwd{Taxanes}\kwd{Vinca-alcalo\"{\i}des}\kwd{Combretastatin-A4}\kwd{Microtubules}\kwd{Test 
cellulaire quantitatif}\kwd{Cancer du sein}}

\shortrunauthors

\begin{abstract}
The characterization of microtubule-targeting drugs at the cellular
level is an essential step in the development of drugs targeting the
microtubule network. To that aim, we have previously developed a
quantitative cell-based assay easy to perform in microplates that
requires only a luminescence reader and no microscopic analysis. Here,
we show that this assay can be easily adapted to different breast
cancer cell lines. An ideal application of this test could be the
comparative analysis of the response of human tumor samples to
different microtubule targeting drugs, to optimize therapeutic
treatment.
\end{abstract}

\begin{altabstract}
La caract\'erisation au niveau cellulaire des agents pharmacologiques
ciblant les microtubules est une \'etape essentielle dans le
d\'eveloppement de m\'edicaments. Dans ce but, nous avons d\'evelopp\'e
un test quantitatif cellulaire facile \`a r\'ealiser en microplaques
qui ne n\'ecessite qu'un lecteur de luminescence et aucune analyse
microscopique. Nous montrons ici que ce test peut \^etre facilement
adapt\'e \`a diff\'erentes lign\'ees cellulaires de cancer du sein. Une
application id\'eale de ce test pourrait \^etre l'analyse comparative
de la r\'eponse d'\'echantillons de tumeurs humaines \`a diff\'erents
m\'edicaments ciblant les microtubules, afin d'optimiser le traitement
th\'erapeutique.
\end{altabstract}

\maketitle

{\vspace*{-4pt}}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

Microtubules (MTs) are dynamic cytoskeletal polymers that are involved
in intracellular trafficking, cell shape establishment, cell movements
and the segregation of condensed chromosomes during mitosis. They are
composed of ${\upalpha}$--${\upbeta}$ tubulin heterodimers and their
polymerization exhibits non-equilibrium dynamics, characterized by
periods of polymerization and of depolymerization. 

The organization and stability of MTs is tightly regulated by numerous
cellular factors such as XMAP215/Dis1/TOGp, MCAK, MAP4, end-binding
proteins or Op18/stathmine~\cite{1,2,3}. Targeted perturbation of this
finely tuned process constitutes a major therapeutic strategy. Drugs
that interfere with tubulin and MTs are, indeed, key components of
combination chemotherapies for the treatment of
carcinomas~\cite{4,5,6}. Vinca-alkaloids and taxanes are among the
microtubule targeting drugs (MTD) commonly used in cancer
chemotherapy~\cite{4,7}. 

\break
Drugs that interfere with the tubulin/MT system are roughly classified
into MT stabilizing agents such as taxanes, and MT destabilizing
agents, such as combretastatin and vinblastine. Regarding MT
destabilizing agents, different binding sites located on the
${\upbeta}$-tubulin subunit have been identified for colchicine,
vinca-alkaloids and maytansine~\cite{7}. The colchicine site is located
at the intradimer interface~\cite{8,9,10}. It is a large site, mostly
buried in the ${\upbeta}$-tubulin subunit. Besides colchicine, numerous
drugs bind to this site, such as nocodazole or combretastatin, but no
ligand is large enough to occupy the entire site~\cite{6}.
Colchicine-site ligands destabilize MTs by preventing the
curved-to-straight conformational transition within the
${\upalpha}$--${\upbeta}$ tubulin heterodimer~\cite{7,11}. The vinca
site is located at the inter-dimer interface between two longitudinally
aligned tubulin dimers. Vinca-site agents destabilize MTs by
introducing a wedge at the interface between two longitudinally aligned
tubulin dimers at the tip of MTs, or by stabilizing
assembly-incompetent ring-like oligomers of tubulin~\cite{7,12}. On the
contrary, taxanes bind to a pocket of ${\upbeta}$-tubulin located on
the luminal side of microtubules~\cite{7,13,14}. 

Recently, we have described two original assays that allow the
quantitative evaluation of the depolymerizing or stabilizing effect of
MT targeting drugs (MTDs) on cellular MTs~\cite{15,16}. These assays
are different from the biochemical tests classically performed
\textit{in~vitro} on purified tubulin. They have the advantage of
providing information on the effects of MTDs in a cellular context.
They allow the quantification of the cell MT mass which is measured by
immunoluminescence after the use of a particular lysis buffer, which
eliminates free tubulin and preserves intact cellular MTs. These assays
are simple to use since they are based on a luminescence reading. Thus,
for these assays, only a microplate reader is required and no
microscopic analysis is needed.

We have recently conducted a systematic comparative analysis of the
effect of four well-characterized MTDs on the kinetics of
\textit{in~vitro} tubulin assembly, on the quantity of cellular MTs in
HeLa cells (a cervical cancer cell line) using our assay and on HeLa
cell viability. We found that there was no significant correlation
between the activity of the different drugs on tubulin assembly
\textit{in~vitro} and their activity in cells, indicating that the
effect of drugs on pure tubulin may be different from their effect on
MTs in the cell context. In contrast, we observed a striking similarity
between the profile of the viability curves and that of the curves
measuring the stability of cellular MTs. This strongly suggests that
the cytotoxic effect of the drugs is due to their depolymerizing effect
on cellular MTs~\cite{15}. These results highlight the value of these
cellular microtubule assays for predicting drug therapeutic activity.

Here, we used these cellular MT assays to compare the effect of MTDs,
used in cancer chemotherapy, on 3 human breast cell lines known to
differ in invasiveness. The cell lines used were MCF-10A cells,
isolated from human fibrocystic breast tissue~\cite{17}, MCF-7, a
poorly invasive cell line~\cite{18}, and MDA-MB-231, an invasive breast
cancer cell line~\cite{19} (Table~\ref{tab1}).

%tab1
\begin{table*}
\caption{\label{tab1}Characteristics of the cell lines used in this
study}
\tabcolsep 4pt
\begin{tabular}{cccc}
\thead
Cell line & MCF-10A & MCF-7 & MDA-MB-231 \\
\endthead
Disease & Fibrocystic disease & Adenocarcinoma & Adenocarcinoma \\
Origin of cells & 
\parbox[t]{9pc}{\centering Spontaneously immortalized cell line} & 
\parbox[t]{7pc}{\centering Metastasis (pleural effusion)} & 
\parbox[t]{7pc}{\centering Metastasis (pleural effusion)}\vspace*{2pt} \\
Cell type & Epithelial cell & Epithelial cell & Epithelial cell \\
Phenotype & Normal-like & Luminal A & Claudin-low \\
ER & No & Yes & No \\
PR & No & Yes & No \\
HER2 amplification & No & No & No \\
Tumorigenic in mice & Non-tumorigenic &
\parbox[t]{7pc}{\centering Yes, with estrogen supplementation}\vspace*{2pt} & Yes \\
Appearance &
\parbox[t]{7pc}{\centering Groups of adherent epithelial cells} & 
\parbox[t]{10pc}{\centering Loosely attached three-dimensional clusters} &
\parbox[t]{9pc}{\centering Epithelial-like somewhat spindle-shaped}\vspace*{2pt} \\
Model & \parbox[t]{7pc}{\centering Non-malignant breast cell line} & 
\parbox[t]{9pc}{\centering Transformed estrogen responsive breast cancer cell line} & 
Late-stage breast cancer{\vspace*{2pt}}
\botline
\end{tabular}
\tabnote{ER, estrogen receptor; PR, progesterone receptor; HER2, human
epidermal growth factor receptor~2.}
\end{table*}

The assayed drugs (Figure~\ref{fig1}) are two MT depolymerizing drugs,
vinblastine and combretastatin-A4 (CA-4) and a MT stabilizing drug,
paclitaxel (PTX).

\begin{figure}
\includegraphics{fig01}
\caption{\label{fig1}Chemical structure of the MTDs used in this
study.}
\vspace*{-25pt}
\end{figure}

Vinblastine binds to the vinca site of tubulin, whereas CA-4 binds to
the colchicine site and PTX to the taxane site.

Although these compounds all target microtubules, they differ in their
applications: whereas vinblastine and PTX are used in antitumor
chemotherapy \cite{4,7,20}, CA-4 is an anti-neoangiogenesis
agent~\cite{21}.\looseness=-1

\section{Material and methods}\label{sec2}
\subsection{Chemical reagents and cells}\label{ssec21}

MCF-10A (CRL-10317), derived from fibrocystic breast tissue, and MCF-7
(HTB-22) and \mbox{MDA-MB-231} (HTB-26) cells, derived from the pleural
effusion of a metastatic breast adenocarcinoma, were obtained from the
American Type Culture Collection (ATCC, Gainthersburg, MD, USA). Cell
culture was performed according to the recommendations of the American
Type Cell Culture. MCF-10A cells were grown in DMEM/F12 (Gibco
10565-018) supplemented with 5\% horse serum (Gibco 16050-122), EGF
(Peprotech AF-100-15) at 0.1~mg/ml, hydrocortisone (H0888) at  1~mg/ml,
cholera toxin (C8052) at 1~mg/ml, insulin (I1882) at 10~mg/ml and 1\%
penicillin/streptomycin. MCF-7 cells were grown in MEM medium (Gibco
31095-029) supplemented with insulin at 0.1~mg/ml, 1\%
penicillin/streptomycin and 10\% FBS. MDA-MB-231 cells were grown in
DMEM Glutamax medium (Gibco 31966-021) and supplemented with 10\% FBS
and 1\% penicillin/streptomycin. Cells were maintained in a humid
incubator at 37~\textdegree C in 5\% CO\tsub{2}.

All media were purchased from Gibco Invitrogen (Carlsbad, CA, USA). All
chemicals, except those for which it is specified, were purchased from
Sigma-Aldrich (Saint-Quentin-Fallavier, France).

Combretastatin-A4 (C7744), vinblastine (V1377) and paclitaxel (T7402)
were prepared at a 10~mM stock solution in Dimethyl sulfoxide (DMSO,
\#4540) aliquoted and stored at ${-}$20~\textdegree C.

\subsubsection{Quantitative assay of the effect of depolymerising
agents on the cellular microtubule content}

Cells were seeded in 96-well microplates (\#655086, Greiner bio One,
Courtaboeuf, France) at the density of 5000 MCF-10A cells, 10,000 MCF-7
cells and 7500 MDA-MB-231 cells per well in 100~$\upmu$l of complete
medium and then incubated at 37~\textdegree C in 5\% CO\tsub{2} for
24~h. Cells were then treated for 30~min at 37~\textdegree C with the
compounds at concentrations ranging from 1 to 5000~nM (1 microplate per
molecule, 1 concentration per column), with 0.1\% DMSO used as positive
control (6 wells per microplate). After medium aspiration, treated
cells were permeabilized for 10~min using 100~$\upmu$l per well of
warmed (37~\textdegree C) OPT buffer (80~mM Pipes, 1~mM EGTA, 1~mM
MgCl\tsub{2}, 0.5\% Triton X-100, and 10\% glycerol, pH 6.8). Cells
were fixed for 10~min at room temperature using 100~$\upmu$l per well
of 4\% formaldehyde (Sigma Aldrich, \#252549, Saint-Quentin-Fallavier,
France) in PBS. Cells were washed 3 times in PBS (100~$\upmu$l per
well), then 50~$\upmu$l of primary anti-alpha-tubulin antibody (clone
${\upalpha}$3A1~\cite{22}, 1:5000 in PBS pH 7.4, 0.1\% Tween-20 2\%
Bovine Serum Albumin (BSA)) was added for 45~min. Cells were washed
twice again and secondary anti-mouse antibody coupled to HRP (1:2000 in
PBS pH 7.4, 0.1\% Tween-20 2\% BSA, \#715-035-150, Jackson
Immuno-Research \mbox{Laboratories,} Cambridgeshire, UK) was added for 45~min.
Then, cells were washed again with PBS and 100~$\upmu$l of ECL
substrate (\#170-5061, Bio-Rad Laboratories Inc., USA) were injected in
each well using the FLUOstar OPTIMA Microplate Reader (BMG Lab
technology, Champagny-sur-Marne, France). The luminescent signal was
read 5~min after ECL injection. IC50s, i.e., drug concentrations able
to reduce the amount of cellular microtubules by half, were calculated
for each independent experiment using GraphPad Prism software and are
presented in the text as means ${\pm}$ SEM.

\subsubsection{Quantitative assay of the stabilizing effect of
paclitaxel on cellular microtubule} 

The same quantity of cells was seeded in microplates as for the
quantitative assay of the effect of depolymerizing agents on the
cellular microtubule. 24~h after seeding, cells were treated with
paclitaxel at different concentrations (range 0--5000~nM). DMSO alone
at 0.25\% was used as positive controls (6 wells per microplate and per
control), respectively. Then combretastatin-A4 was added to each well
at 50~nM final concentration for 30~min. After medium aspiration,
treated cells were permeabilized for 10~min using 100~$\upmu$l per well
of OPT buffer (80~mM Pipes, 1~mM EGTA, 1~mM MgCl\tsub{2}, 0.5\% Triton
X-100, and 10\% glycerol, pH 6.8) pre-warmed to 37~\textdegree C. After
buffer aspiration, cells were fixed for 10~min at room temperature
using 100~$\upmu$l per well of 4\% formaldehyde in PBS pH 7.2. Cells
were washed 3 times with PBS 0.1\% Tween-20 (150~$\upmu$l per well),
then 50~$\upmu$l of ${\upalpha}$3A1 anti-tubulin antibody (1:5000 in
PBS pH 7.4, 0.1\% Tween-20 2\% BSA) were added for 45~min.  After
washing of cells as described above, 50~$\upmu$l of anti-mouse antibody
coupled to HRP (1/2000 in PBS pH 7.4, 0.1\% Tween-20 2\% BSA) were
added for 45~min. Then cells were washed again and, 50~$\upmu$l of ECL
Western blotting substrate (Pierce \#32106) were added to each well and
the luminescent signal was read after 5~min of incubation.

\subsubsection{Immunofluorescence}

Cells at a density of 30,000 cells per well for MCF-10A, 60,000 for
MCF-7 and 50,000 for MDA-MB-231 were grown for 48~h on glass coverslips
placed in a 24-well microplate. When cells reached 70\% confluence, the
medium was replaced with a fresh one supplemented with DMSO (0.005\% or
0.01\%) or the test compound at 50~nM for vinblastine and CA-4 or 
1000~nM for PTX. After 30~min of incubation, cells were permeabilized
in warm OPT buffer (80~mmol/L Pipes, 1~mol/L EGTA, 1~mol/L
MgCl\tsub{2}, 0.5\% Triton X-100 and 10\% Glycerol, pH 6.8) and fixed
for 6~min in ${-}$20~\textdegree C methanol (Carlo ERBA SAS, \#414855,
Val-de-Reuil, France). After washing and saturation with a specific
blocking buffer (3\% BSA), 10\% Goat serum (Gibco Invitrogen,
\#16210064, Carlsbad, CA, USA) in PBS, cells were incubated for 45~min
at room temperature (RT) with anti-alpha-tubulin antibody (clone
${\upalpha}$3A1~\cite{22} in blocking buffer). Cells were washed twice
again and subsequently incubated with Alexa 488 conjugated anti-mouse
antibody (1:500 in blocking buffer, \#115-545-166, Jackson
immune-research laboratory, Cambridgeshire, UK) for 30~min at RT.
Coverslips were mounted on glass slides with Moviol~4--88.

Images were captured with a Zeiss AxioimagerM2 microscope equipped with
the acquisition software AxioVision (Marly-le-Roi, France).

\section{Results}\label{sec3}
\subsection{Effect of the depolymerizing agents Vinblastine and CA-4 on
the MT contents of breast cancer cell lines}\label{ssec31}

We first compared the effect of different doses of vinblastine and CA-4
on cellular interphase MTs. After having determined a seeding density
adapted to each cell line, we used our recently developed cell-based
assay that quantifies intact MTs in cells~\cite{15,16}. The principle
of this assay is based on the use of a particular lysis buffer, which
eliminates free tubulin and preserves intact cellular MTs, which are
then quantified by immunoluminescence. The detailed protocol is given
in the methods section.

As shown on Figure~\ref{fig2}, both compounds induced a dose-dependent
depolymerization of cellular MTs, on the 3 cell lines. The IC50 for
CA-4 was in the same range for MCF-10A and MDA-MB-231, i.e., 6.6
${\pm}$ 0.4~nM and 4.8 ${\pm}$ 0.3 respectively. This IC50 was found
much higher in MCF-7 cells, i.e., 31.1 ${\pm}$ 7.9~nM. Similar results
were obtained for vinblastine: the IC50 was 2.8 ${\pm}$ 0.4 for
MCF-10A, 67.7 ${\pm}$ 15.4 for MCF-7 and 4.4 ${\pm}$ 0.4 for
MDA-MB-231.

\begin{figure*}
\includegraphics{fig02}
\vspace*{-5pt}
\caption{\label{fig2}Comparison of the MT destabilizing effect of
Vinblastine and CA-4. Different doses of compounds were applied to the
different cells in microplates and their MT destabilizing effect was
assessed after a 30~min incubation, using the luminescent assay, as
described in the Materials and Methods section. Results are expressed
as \% of resistant MTs, with 100\% corresponding to cells treated with
DMSO only, without the depolymerizing agent. Datapoints are means
${\pm}$ SEM from three independent experiments.}
\end{figure*}

Interestingly, in MCF-10A and MDA-MB-231 cells, the sigmoid curve of
CA-4 has a much steeper slope than that of vinblastine, indicating that
a small \mbox{variation} of the concentration around the IC50 can lead to a
complete depolymerization of the microtubule network. The effect of
increasing concentrations of vinblastine is much more gradual on these
cell lines. 

On MCF-7 cells, both compounds behave similarly. On this cell line,
unlike the other two cell lines, a complete depolymerization of the MT
network was not observed, even at the highest doses (5000~nM) assayed.

\subsection{Effect of the stabilizing agent paclitaxel on the
microtubule contents of breast cancer cell lines}\label{ssec32}

To measure the effect of PTX on cellular MTs, we used a variant of the
test presented above for depolymerizing agents. The assay probes a
PTX-induced resistance of the MT network to a depolymerization provoked
by CA-4. CA-4 binds free tubulin dimers and prevents their
incorporation into MTs, leading to a progressive loss of the
polymerized MT network. Stabilized MTs with slow dynamics have reduced
exchanges of their tubulin content with the free tubulin pool, and are
thus less sensitive to CA-4-induced depolymerization~\cite{23}. After
treatment with the drugs and before fixation, the same lysis buffer as
described above is used in order to eliminate free tubulin and to
preserve the cellular MTs, which are then quantified by
immunoluminescence. The detailed protocol is given in the methods
section. This assay is sensitive as it allows to detect a weak
stabilizing effect, quantitative and statistically robust~\cite{16}.

Figure~\ref{fig3} shows the dose-effect curves of the sensibility of the
MT network of the different cell lines to the stabilizing effect of PTX
against a depolymerization induced by 50~nM CA-4. This concentration of
CA-4 is able to induce the depolymerization of the entire MT network in
MCF-10A and MDA-MB-231 cells and only 50\% of the network in MCF-7
cells (see blue curves in Figure~\ref{fig2}, and ordinate at the origin
in Figure~\ref{fig3}). For MCF-10A and MDA-MB-231 cells, a similar dose
of PTX, 246.9 ${\pm}$ 83.9~nM and 234.3 ${\pm}$ 32.5~nM respectively,
is required to achieve 50\% stabilization of the microtubule network.
Regarding MCF-7, 16.3 ${\pm}$ 9.0~nM of PTX induced 50\% of the effect.
For the latter cell line, we tested the stabilizing effect of PTX
against a depolymerization induced by 100 times more CA-4, i.e.,
5000~nM (not shown). As shown in Figure~\ref{fig2}, however, such a high
concentration of CA-4 does not fully depolymerize the network. Under
these conditions, a similar dose-dependent profile of stabilization by
PTX is obtained, with 50\% of the stabilizing effect achieved for a PTX
concentration of 15.3 ${\pm}$ 6.9~nM.

\begin{figure*}
{\vspace*{3pt}}
\includegraphics{fig03}
{\vspace*{-5pt}}
\caption{\label{fig3}Comparative analysis of the effect of PTX on MT
dynamics in breast cancer cells. Different doses of PTX were applied
for 2~h to cells in microplates. Thirty~minutes before the end of the
PTX treatment 50~nM CA-4 was added. At the end of the PTX treatment,
its MT stabilizing effect was assessed using the luminescent assay
described in the material and methods section. The analyses were
performed with a PTX concentration in 0.1\% DMSO at the solubility
limit for not inducing an additional toxicity due to DMSO. Results are
expressed as \% of MTs resistant to CA-4-induced depolymerization, with
100\% corresponding to cells treated with DMSO without CA-4. Datapoints
are means ${\pm}$ SEM from three independent experiments.}
\end{figure*}

\subsection{Immunofluorescence analysis of the effect of the different
drugs on cellular MTs}\label{ssec33}

Using immunofluorescence, we checked  that the luminescent values do
reflect the state of cellular MTs. As shown in Figure~\ref{fig4}, a
decrease in the density of the MT network is observed in MCF-10A and
{MDA-MB-231} cells treated with 50~nM of vinblastine or CA-4, as well as
typical microtubule bundle reorganization with 1~$\upmu$M Paclitaxel
treatment. Changes in the shape and density of the MT network are more
difficult to observe in MCF-7 cells, which grow in clusters.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Immunofluorescence analysis of the effect of the
different compounds on the MTs of the different breast cancer cells.
Cells were incubated for 30~min with 50~nM Vinblastine, 50~nM CA-4, 
2~h with 1~$\upmu$M PTX or with DMSO (control). Cells were then
permeabilized with OPT buffer, fixed, and processed for
immunofluorescence using an anti-${\upalpha}$-tubulin antibody. Scale
bars, 10~$\upmu$m.}
\end{figure*}

\section{Discussion and conclusion}\label{sec4}

The development of agents targeting MTs remains an area of intense
research. In this context, quantitative assessment of the compounds'
effect on cellular MTs is essential. With this study, we showed that
the quantitative assays we had initially developed using HeLa cells
could be easily adapted, for other types of adherent cells by modifying
only the density of cell seeding. This allows the investigation of the
effect of MTDs on other types of cancer. However, this study
highlighted a potential limitation concerning the cell types that can
be studied. For example, we could not observe complete depolymerization
of the microtubular network with two different drugs on MCF-7 cells.
MCF-7 cells are known as drug-sensitive cells, which do not overexpress
drug efflux pumps~\cite{24}. The reduced efficiency of depolymerizing
drugs to act on the microtubule network is therefore not the result of
the activation of efflux pumps. One possible reason is that this is a
result of poor penetration of the drugs, even when applied at high
concentrations, due to the fact that the cells grow in clusters.
Increasing the incubation time of the cells with the drugs, which
implies additional methodological validations, could be a way to
achieve complete MT depolymerization. 

Despite this difficulty in achieving complete depolymerization of MTs
in MCF-7 cells, this cell line appears to be particularly sensitive to
the stabilizing action of PTX, as measured in our assay. Indeed, it
requires about 15 times more PTX to stabilize 50\% of the MT network
for MCF-10A and MDA-MB-231 cells.

We observed that MCF-10A and MDA-MB-231 show similar sensitivity to
MTDs, indicating that the invasiveness of MDA-MB-231 does not influence
the drug sensitivity of this cell line. This cell line was established
from a single sample of pleural effusion obtained from a 51-year-old
woman, who had previously a right radical mastectomy for a poorly
differentiated tumor. This patient received a systemic treatment with
5-FU and prednisone which were ineffective. Then a combined
chemotherapy (cyclophosphamide, adriamycin, and amethopterin) was
administered, with no success~\cite{19}. Interestingly, this patient
has never been treated with drugs targeting MTs, and therefore could
not develop resistance, explaining the sensitivity of MDA-MB-231 to
MTDs.

It should be noted that the test duration is very short (2~h) and
therefore has no detectable effect on cell viability. A limitation of
the assay for measuring the effect of depolymerizing agents is that a
decrease in luminescent signal can be observed not only when
microtubules are depolymerized, but also if the agents tested have an
effect on cell adhesion, thus reducing the number of stained cells.
False positives of this kind can be identified by checking the effect
of the drugs on the cells under the microscope. In this case, measuring
the value of the luminescent signal in ratio to the number of residual
adherent cells, as measured by nucleus labelling, e.g. with Hoechst,
can provide information on the depolymerizing effect alone~\cite{25}.

Here, we have shown that the quantitative analysis of the effect of
MTDs can be easily performed on various cell lines. Ideally, this assay
could be used not only for the development of new MTDs~\cite{26,27},
but also for the comparative analysis of the response of human tumor
samples to different drugs, in order to optimize therapeutic treatment.

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\section*{Conflicts of interest}

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Authors have no conflict of interest to declare.

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