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\DOI{10.5802/crchim.252}
\datereceived{2023-02-10}
\daterevised{2023-07-17}
\dateaccepted{2023-07-26}
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\dateposted{2024-04-19}
\begin{document}

\begin{noXML}

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

\CDRsetmeta{articletype}{review}

\title{Induced-volatolomics, a new research field in chemical biology}

\alttitle{La volatolomique induite, un nouveau pan de recherche en
Ch\'{e}mobiologie}

\author{\firstname{Estelle} \lastname{Blochouse}\CDRorcid{0009-0002-1491-5722}}
\address{University of Poitiers, UMR CNRS 7285, Institut de Chimie des
Milieux et Mat\'{e}riaux de Poitiers (IC2MP), Equipe Labellis\'{e}e
Ligue Contre le Cancer, 4 rue Michel-Brunet, TSA 51106, 86073 Poitiers
cedex 9, France}
\email[E. Blochouse]{estelle.blochouse@univ-poitiers.fr}

\author{\firstname{Rony} \lastname{Eid}}
\addressSameAs{1}{University of Poitiers, UMR CNRS 7285, Institut de
Chimie des Milieux et Mat\'{e}riaux de Poitiers (IC2MP), Equipe
Labellis\'{e}e Ligue Contre le Cancer, 4 rue Michel-Brunet, TSA 51106,
86073 Poitiers cedex 9, France}
\email[R. Eid]{rony.eid@univ-poitiers.fr}

\author{\firstname{Elsa} \lastname{Cannoni}}
\addressSameAs{1}{University of Poitiers, UMR CNRS 7285, Institut de
Chimie des Milieux et Mat\'{e}riaux de Poitiers (IC2MP), Equipe
Labellis\'{e}e Ligue Contre le Cancer, 4 rue Michel-Brunet, TSA 51106,
86073 Poitiers cedex 9, France}
\email[E. Cannoni]{elsa.cannoni@univ-poitiers.fr}

\author{\firstname{R\'emi} \lastname{Ch\^{a}tre}}
\addressSameAs{1}{University of Poitiers, UMR CNRS 7285, Institut de
Chimie des Milieux et Mat\'{e}riaux de Poitiers (IC2MP), Equipe
Labellis\'{e}e Ligue Contre le Cancer, 4 rue Michel-Brunet, TSA 51106,
86073 Poitiers cedex 9, France}
\email[R. Ch\^{a}tre]{remi.chatre@univ-poitiers.fr}

\author{\firstname{Claude} \lastname{Geffroy}\CDRorcid{0000-0002-2731-9658}}
\addressSameAs{1}{University of Poitiers, UMR CNRS 7285, Institut de
Chimie des Milieux et Mat\'{e}riaux de Poitiers (IC2MP), Equipe
Labellis\'{e}e Ligue Contre le Cancer, 4 rue Michel-Brunet, TSA 51106,
86073 Poitiers cedex 9, France}
\email[C. Geffroy]{claude.geffroy@univ-poitiers.fr}

\author{\firstname{Isabelle} \lastname{Opalinski}\CDRorcid{0000-0003-0760-5056}}
\addressSameAs{1}{University of Poitiers, UMR CNRS 7285, Institut de
Chimie des Milieux et Mat\'{e}riaux de Poitiers (IC2MP), Equipe
Labellis\'{e}e Ligue Contre le Cancer, 4 rue Michel-Brunet, TSA 51106,
86073 Poitiers cedex 9, France}
\email[I. Opalinski]{isabelle.opalinski@univ-poitiers.fr}

\author{\firstname{S\'ebastien} \lastname{Papot}\CDRorcid{0000-0001-5253-1979}}
\addressSameAs{1}{University of Poitiers, UMR CNRS 7285, Institut de
Chimie des Milieux et Mat\'{e}riaux de Poitiers (IC2MP), Equipe
Labellis\'{e}e Ligue Contre le Cancer, 4 rue Michel-Brunet, TSA 51106,
86073 Poitiers cedex 9, France}
\address{Seekyo SA, 2 avenue Galil\'{e}e, BP 30153, 86961 Futuroscope,
France}
\email[S. Papot]{sebastien.papot@univ-poitiers.fr}

\author{\firstname{Pauline} \lastname{Poinot}\CDRorcid{0000-0002-7927-1887}\IsCorresp}
\addressSameAs{1}{University of Poitiers, UMR CNRS 7285, Institut de
Chimie des Milieux et Mat\'{e}riaux de Poitiers (IC2MP), Equipe
Labellis\'{e}e Ligue Contre le Cancer, 4 rue Michel-Brunet, TSA 51106,
86073 Poitiers cedex 9, France}
\email[P. Poinot]{pauline.poinot@univ-poitiers.fr}

\keywords{\kwd{VOC}\kwd{Induced-volatolomics}\kwd{Enzyme-responsive
probes}\kwd{Diagnosis}\kwd{Chemical biology}}

\altkeywords{\kwd{COV}\kwd{Volatolomique induite}\kwd{Sondes
enzymo-sensibles}\kwd{Diagnostic}\kwd{Ch\'{e}mobiologie}}

\thanks{Local committees Vienne and Deux-S\`{e}vres of the ``Ligue
Contre le Cancer'' and ``La Ligue nationale contre le Cancer''.
``R\'{e}gion Nouvelle Aquitaine''. ANR through the Young Researcher
grant ``Volatolomix'' (ANR-21-CE18-0009).  CNRS through the ``AAP
Emergence'' and FEDER}

\shortrunauthors

\begin{abstract} 
Progresses in chemical biology have substantially deepened our
understanding of biological pathways related to disease outcomes.
Numerous optical probes activatable by disease-relevant enzymes have
been designed and have shown great value for imaging crucial
biochemical transformations specific to pathological processes. In
comparison to their ``always-on'' counterparts, such stimuli-responsive
probes stand out as promising candidates for exploring biological
processes because of their high sensitivity, ingenious spatiotemporal
resolution, ease of operation, and real-time and \textit{in situ}
imaging capacity. However, these imaging tools meet several bottlenecks
related to either stability, background noise, limited penetration
depth or systemic toxicity, pharmacokinetics, tissue distribution, and
renal clearance.

In parallel, novel volatile organic compound (VOC)-based probes have
been proposed under the emerging paradigm of induced-volatolomics.
These probes are converted into exogenous VOCs in response to pathogen
or eukaryote specific enzymatic stimuli. Once activated, VOC-based
probes release volatile tracers that inform the biochemical processes
arising in cells, liquid and solid biopsies, and even in entire
organisms. After a brief presentation of recent developments in the
field of induced-volatolomics, we discuss possible improvements of this
new modality for the exploration of biological systems.\looseness=-1
\end{abstract} 

\begin{altabstract} 
Avec l'\'{e}mergence de la ch\'{e}mobiologie, de nombreuses sondes ont
\'{e}t\'{e} con\c{c}ues afin de suivre en temps r\'{e}el diff\'{e}rents
processus m\'{e}taboliques dans des syst\`{e}mes biologiques. Une
grande majorit\'{e} d'entre elles correspondent \`{a} des sondes off-on
d\'{e}tect\'{e}es par imagerie, qui utilis\'{e}es in vitro ou in vivo,
ont consid\'{e}rablement am\'{e}lior\'{e} notre compr\'{e}hension du
fonctionnement des cellules vivantes. Ciblant un large \'{e}ventail
d'esp\`{e}ces chimiques avec une r\'{e}solution spatiale fine, ces
outils bioorthogonaux ont permis
d'{\guillemotleft}~allumer~{\guillemotright} des anomalies cellulaires,
et ce, m\^{e}me au sein de tissus profonds. Alors que ces sondes ont
offert de nouvelles perspectives en termes d'exploration des
syst\`{e}mes biologiques, leur utilisation se confronte souvent \`{a}
des probl\`{e}mes de toxicit\'{e}, de faible stabilit\'{e} et des
verrous li\'{e}s \`{a} leur pharmacocin\'{e}tique, leur \'{e}limination
et leur biodistribution.

Dans ce contexte, des sondes off-on converties en compos\'{e}s
organiques volatils (COVs) pourraient repr\'{e}senter une alternative
int\'{e}ressante pour fournir un premier aper\c{c}u
d'\'{e}v\`{e}nements biologiques. Ce concept, appel\'{e}
{\guillemotleft}~\textit{volatolomique induite}~{\guillemotright}
repose sur l'utilisation de sondes off-on non toxiques, et incluant
dans leur structure un COV. Ces sondes sont converties en COVs
exog\`{e}nes en r\'{e}ponse \`{a} des stimuli enzymatiques
sp\'{e}cifiques de diff\'{e}rents ph\'{e}notypes cellulaires
(procaryotes ou eucaryotes). Une fois activ\'{e}es, les sondes
lib\`{e}rent des traceurs volatils qui informent sur les processus
biochimiques survenant dans des cellules, des biopsies liquides et
solides, voire dans des organismes entiers. Apr\`{e}s une br\`{e}ve
pr\'{e}sentation des d\'{e}veloppements r\'{e}cents dans le domaine de
la volatolomique induite, nous discuterons des perspectives
d'application et des progr\`{e}s pour l'exploration des syst\`{e}mes
biologiques.
\end{altabstract} 

\maketitle

\clearpage

\twocolumngrid

\end{noXML}

\section{Introduction} \label{sec1}

Each of us continuously emits more than a thousand volatile organic
compounds (VOCs) that are \mbox{responsible} for our odor fingerprint.
With the emergence of volatolomics, i.e., the discipline that
investigates VOCs produced by living systems, the human volatolome has
provided new insights into biological processes in
real-time~\cite{1} (Figure~\ref{fig1}).

\begin{figure*}
\vspace*{2pt}
\includegraphics{fig01}
\caption{\label{fig1}Volatolomics and induced-volatolomics paradigms.
Volatolomics is the study of endogenous molecules naturally produced
and released by living systems~\cite{1,2}. The concept of
induced-volatolomics specifically monitors exogenous volatiles that
come from the metabolisation of a probe~\cite{20}.}
\end{figure*}

Indeed, as evidenced by canine olfaction-based studies, volatile
molecules can serve as chemical tracers for assessing health status in
real time in a non-invasive way~\cite{2,3}. Along with the progress in
analytical technologies, many research groups have attempted to find
the perfect volatile marker for a given disease (Figure~\ref{fig1}).
Hence, viral (\textit{Influenza} viruses, adenovirus,
rhinovirus~\cite{4,5}), bacterial, parasitic, and fungal infections
(tuberculosis~\cite{6}, malaria~\cite{7}, pneumonia~\cite{8,9}) were
shown to be associated with the release of specific VOCs. Similarly,
respiratory disorders, such as asthma~\cite{10}, or acute respiratory
distress syndrome~\cite{11} as well as metabolic disorders such as
diabetes~\cite{12} could be diagnosed by the detection of alkanes and
ketones in patients' breath. Similarly, a strong correlation was
reported between cancers, Parkinson's and Alzheimer's diseases and the
airborne release of hydrocarbons, alcohols, aldehydes, ketones, esters,
nitriles, and aromatic \mbox{compounds}~\cite{13,14,15,16,17,18,19}. 

Despite these extensive studies, no endogenous volatile compound has
been proven to be of sufficient diagnostic value so far. Such an issue
is mainly explained by both interindividual variability~\cite{21,22}
and individual daily variation~\cite{23}. Indeed, variables such as
overall health status, diet, medication \mbox{intake}, exercise, and
smoking habits induce qualitative and quantitative changes in patient
volatolome that are unrelated to pathologies~\cite{24,25}. Moreover,
although several studies have sought to demonstrate the biosynthesis
patterns of endogenous VOCs, their metabolic pathways remain enigmatic,
which adds to confusion~\cite{26,27,28}. Finally, given the very low
concentration of VOCs in body fluids and gases (e.g., $1 \times
10^{-11}$~M in blood and 0.02~ppb in breath~\cite{24}), more efficient
sampling techniques and analysers are necessary to obtain reliable
quantification~\cite{29,30,31,32,33}. In this respect, identification
of rotational fingerprints with high accuracy, sensitivity, and
selectivity is a challenge that can be addressed and automated using
artificial intelligence and supervised or unsupervised machine learning
algorithms. In very recent works, deep learning models were developed
to provide breath volatolomics-based classifiers for the presence or
stage different diseases~\cite{34,35,36,37,38}. These cutting-edge
approaches demonstrate that machine learning methods could be the
answer to make volatolomics a non-invasive clinical diagnostic tool for
point-of-care applications~\cite{39}.

Although volatolomics was booming in the early 2000s, another strategy,
which relies on the monitoring of exogenous volatiles, was
emerging.{\break} This novel paradigm, very recently called
``induced-volatolomics'' (Figure~\ref{fig1}), is part of the chemical
biology toolbox. It implies the use of off/on probes that can be
converted into a volatile compound in response to a pathogen or
metabolic-specific enzymatic stimulus~\cite{40,41,42}. In the absence
of the activating process, the probe remains inactive. In the presence
of the biochemical stimulus, the probe is turned on, thus releasing an
exogenous volatile molecule. This targeted action mechanism
significantly reduces variability issues that are met with the standard
screening of endogenous VOCs. By correlating molecular processes with
phenotypic states, these probes can provide valuable information (e.g.
kinetics, localization, progress\,{\ldots}) on the biochemical events
that lead to the progression or regression of a pathology.

\section{Contribution of induced-volatolomics to chemical biology}
\label{sec2}

Chemical biology has generated many innovative tools for monitoring
metabolic processes in cells~\cite{43}. Researchers in this field have
developed numerous stimuli-responsive imaging probes with imaging
tracers---from radioisotopes to small-molecule dyes, proteins, and
carbon or metal nanomaterials---to drive and localize with high spatial
resolution disease outcomes~\cite{44,45,46,47,48,49,50,51,52,53}. Such
activity-based probes can now detect and localize a wide range of
biomolecules with high resolution in cell culture, liquid or solid
biopsies, and animal models~\cite{54,55,56,57}. However, the use of
these stimuli-sensitive probes in mammalian models remains limited
because of toxicity, stability, pharmacokinetics, and tissue
distribution \mbox{hurdles}~\cite{58,59}.

In this context, a novel set of off/on probes has been designed to
explore and understand, at the molecular level, the pathophysiological
mechanisms arising in biological systems. These probes can be converted
into a small exogenous volatile molecule by a disease- or
pathogen-associated enzyme with optimal affinity. Once produced, the
volatile tracer diffuses in the biological system and can be released
either in animal breath~\cite{40,42,60} or in sample headspace (cell
culture, liquid or tissue biopsy)~\cite{42,61,62}. Although this action
mechanism is common to all volatile-based probes, the nature of the
exogenous volatile compounds released from this \mbox{biochemical}
process has significantly evolved. The first probes were natural
carbohydrates that could be metabolized into inorganic and very
volatile compounds (e.g. $\text{H}_2$, $\text{CH}_4$ and
$\text{CO}_2$)~\cite{63,64,65}. They were then succeeded by synthetic
probes, among which a myriad of isotopic $\text{CO}_2$-based probes
were designed for diagnosis purposes~\cite{66}.

Among these probes, the ${}^{13}\text{C}$-urea breath test (UBT) is the
best standardized and most widely used test worldwide. UBT is
prescribed when \textit{Helicobacter pylori} (\textit{H.~pylori})
infection is suspected~\cite{40}. This gram-negative micro-aerophilic
bacterium can be found on the luminal surface of the gastric
epithelium. It is responsible for various upper gastrointestinal
disorders, sometimes resulting in ulcers and cancers. In patients with
\textit{H.~pylori} infection, the ${}^{13}\text{C}$-urea probe is
hydrolyzed by bacterial urease to $\text{NH}_3$ and
${}^{13}\text{CO}_2$. ${}^{13}\text{CO}_2$ is then excreted via the
lungs and measured in the expired air. However, because 1.1\% of the
natural carbon atoms in the human body are present in the stable
isotope ${}^{13}\text{C}$ form, high ${}^{13}\text{CO}_2$ background
noise is observed in patients' breath. Therefore, and despite its high
sensitivity and specificity (both around 97\%)~\cite{67,68},
false-negative and positive tests represent a clinical issue,
especially for the 5\% of samples near cut-off values. This issue has
led researchers to design a new generation of volatile-based probes
that can release a tracer absent from the pool of endogenous VOCs
(Figure~\ref{fig2})~\cite{42,61,62,69,70}. These emergent probes have
very recently broadened the scope of ``induced-volatolomics'' offering
the opportunity to explore a wide range of fundamental biological
processes.

\begin{figure}
\includegraphics{fig02}
\caption{\label{fig2}Exogenous VOCs released from VOC-based probes
activation.}
\end{figure}

A set of VOC-based probes was designed to detect \textit{in vitro}
pathogenic strains such as \textit{Pseudomonas aeruginosa}~\cite{62},
\textit{Salmonella}~\cite{69} and \textit{Listeria
monocytogenes}~\cite{70} (Figure~\ref{fig3}). These probes are
\mbox{generally} activated by hydrolytic enzymes such as peptidases or
glycosidases to release an exogenous molecule (e.g. aniline,
3-fluoroaniline, 2-chlorophenol, phenol) in biological or food sample
headspace. Very recently, Chan {\etal}~\cite{60} proposed bioorthogonal
peptide-VOC reporters to probe the inflammatory response to respiratory
lung infection (Figure~\ref{fig4}). In their work, the authors designed
a set of VOC-based probes consisting of chemically modified
tetrapeptide substrates specific to both human and mouse serine
protease neutrophil elastase. At the C-terminal end, an
amine-containing VOC was attached via an amide bond. Hydrofluoroamines
(HFAs; $\text{CF}_3(\text{CF}_2) \times \text{CH}_2\text{NH}_{2}$) were
selected as exogenous VOC reporters because of their high volatility.
VOC-based peptide probes were formulated into volatile-releasing
Activity-Based Nanosensors (vABNs) by conjugation onto an eight-arm PEG
nanocarrier in such a way that the VOCs were undetectable in a
non-volatile state. vABNs were further delivered into the lungs via
intratracheal instillation, and extracellular proteases produced during
respiratory disease cleaved the surface-conjugated peptide substrates,
thereby releasing the VOCs. VOCs were finally exhaled and breath
samples were collected into a glass vial. VOCs concentrations were
quantified using an MS detector (Figure~\ref{fig4}).

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig03}
{\vspace*{2pt}}
\caption{\label{fig3}Induced-volatolomics for the {\it in vitro}
detection of pathogens~\cite{61,62,69,70}.}
{\vspace*{2pt}}
\end{figure*}

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Volatile-releasing Activity-Based Nanosensors
(vABNs) for the diagnosis of respiratory infections. (a)~VOC-modified
peptide substrates are formulated into vABNs by conjugation onto an
eight-arm PEG nanocarrier and delivered into the lungs via
intratracheal instillation. When attached to the vABN, VOC reporters
are in an undetectable, non-volatile state (grey). Extracellular serine
protease neutrophil elastase (NE) produced during respiratory disease
cleaves the surface-conjugated peptide substrates, thereby releasing
VOC reporters. Upon release from vABNs, reporters recover their
characteristic mass and volatility (red) and are then exhaled.
(b)~Enzymatic cleavage by serine protease neutrophil elastase.
(c)~Breath signal after intrapulmonary delivery of HFA-releasing vABNs
in healthy controls and mouse models of lung infection. Figures are
reproduced from Chan {\etal}~\cite{60}.}
{\vspace*{2pt}}
\end{figure*}

In parallel, our group demonstrated the efficacy of a VOC-based probe
to diagnose cancers and monitor their dynamics during
chemotherapy~\cite{42}. Our labelled probe,
$\text{D}_5$-ethyl-$\beta$-\textsc{d}-glucuronide, was designed to
target, with high spatial resolution, extracellular
$\beta$-glucuronidase accumulated selectively in the microenvironment
of numerous solid tumors~\cite{71,72,73} (Figure~\ref{fig5}). After
intravenous administration at a dose as low as
$50~\upmu\text{g}{\cdot}\text{kg}^{-1}$,
$\text{D}_5$-ethyl-$\beta$-\textsc{d}-glucuronide was converted into
$\text{D}_5$-ethanol and glucuronic acid after $\beta$-glucuronidase
selective activation within the tumor. $\text{D}_5$-ethanol was then
passed to the bloodstream, exhaled in the breath where it was trapped
and finally analyzed. This strategy enabled unambiguous discrimination
between healthy mice and mice bearing different solid tumors (cervix,
mammary, and pulmonary; Figure~\ref{fig5}b--d). Furthermore, this
approach allowed for the first time to monitor either tumor growth or
regression during cancer chemotherapy~\cite{74} (Figure~\ref{fig5}e,f).
Indeed, we showed that tumor regression observed in animals under
chemotherapy was accompanied by a decrease in the $\text{D}_5$-ethanol
amount in their breath. In contrast, in untreated mice, tumor growth
was correlated with an increase in the $\text{D}_5$-ethanol
concentration breathed out by the animals. Therefore, these results
highlight that our $\beta$-glucuronidase-responsive VOC-based probe is
a useful tool to follow tumor progression as well as tumor response to
chemotherapy.

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig05}
\caption{\label{fig5}(a)~Enzymatic reaction. (b--d)~Efficacy of
$\text{D}_5$-ethyl-$\beta$-D-glucuronide probe to diagnose solid
tumors. KB: mouth epidermal carcinoma ($n=13$ per group); MDA-MB-231:
breast adenocarcinoma ($n=8$ per group); LLC: Lewis Lung Carcinoma
($n=6$ per group). $*$, ${*}{*}$, ${*}{*}{*}$:~Significant differences
for $p$-value $<$ 5\%; NS: Non-significant. (e)~Average tumor volume of
mice with KB xenografts ($n=8$ per group). (f)~Spearman's correlation
test ($p$-value $=$ 0.005) relating the evolution of tumor volumes
(orange dot line) and the evolution of $\text{D}_5$-ethanol exhaled
(purple line) by KB mice treated with chemotherapy. Figure reproduced
from Lange {\etal}~\cite{74}.}
{\vspace*{3pt}}
\end{figure*}

On the basis of our study conducted in mice, Owlstone Medical
(Cambridge, UK), an industry leader in breath research and
technical innovation, recently started testing the
$\text{D}_5$-ethyl-$\beta$-\textsc{d}-glucuronide probe in
clinic~\cite{75}. Their objective was to provide an early diagnosis
test for lung cancer detection. After preclinical tests on lung tumor
biopsy and mice bearing lung tumors, Owlstone initiated a phase~Ia
clinical trial. In this study, they administered
$\text{D}_5$-ethyl-$\beta$-\textsc{d}-glucuronide to healthy
individuals in single ascending dose assays to verify safety and
background $\text{D}_5$-ethanol levels. They reported no side effects
in these patients. In addition, they did not detect a
$\text{D}_5$-ethanol signal in their breath, thereby proving that
$\text{D}_5$-ethyl-$\beta$-\textsc{d}-glucuronide was not hydrolyzed by
intracellular $\beta$-glucuronidase.\looseness=1

Despite these successes, induced-volatolomics approaches have
challenges related to the selection of the best enzymatic marker.
Hence, we recently proposed to combine several VOC-based probes into a
cocktail to simultaneously monitor the dysregulation of several
tumor-associated enzymes in living mice or biopsies
(Figure~\ref{fig6})~\cite{76}. Such an outcome is a great advantage in
comparison with fluorescent probes, which cannot be used in cocktails
because of imaging interferences. The cocktail was composed of four
enzyme-responsive VOC-based probes, each targeting a particular
glycosidase (\mbox{$\beta$-galactosidase}, $\alpha$-L-fucosidase,
$\beta$-glucuronidase, \textit{N}-acetyl-$\beta$-D-glucosaminidase).
Furthermore, in the presence of the corresponding enzyme, each probe
led to the release of a specific ethanol \mbox{isotope} ($\text{D}_2$-,
$\text{D}_4$-, $\text{D}_5$- and ${}^{13}\text{CD}_5$-ethanol),
allowing the identification of the activating glycosidase. When the
cocktail was injected into mice bearing tumors, two ethanol isotopes
were detected in the animal breath (Figure~\ref{fig6}a), highlighting
the upregulation of $\beta$-glucuronidase and
\textit{N}-acetyl-$\beta$-D-glucosaminidase in the tumor
microenvironment (Figure~\ref{fig6}b). Applied \textit{ex vivo} on
tumor biopsies, our cocktail again indicated an increased catalytic
activity of \textit{N}-acetyl-$\beta$-D-glucosaminidase in tumors
compared with that in healthy tissues (Figure~\ref{fig6}c). Having
identified this glycosidase as a potential target for cancer therapy,
we designed an enzyme-responsive albumin-binding
prodrug~\cite{77,78,79,80} of the potent monomethyl auristatin E
programmed for the selective release of the drug into the tumor
microenvironment. This tumor-activated therapy produced remarkable
therapeutic efficacy on orthotopic triple-negative mammary xenografts
implanted in mice, leading to the disappearance of tumors in 66\% of
treated animals (Figure~\ref{fig6}d). Thus, this study demonstrated the
potential of induced-volatolomics for the exploration of biological
processes as well as the discovery of novel therapeutic strategies.

\begin{figure*}
{\vspace*{-4pt}}
\includegraphics{fig06}
{\vspace*{-4pt}}
\bcaption{\label{fig6}\fontsize{9.8}{11}\selectfont
Cocktail of VOC-based probes for the design of
tumor activated therapy. To highlight the dysregulation of glycosidases
activity in tumors, a cocktail of VOC-based probes ($\text{D}_2$-EtGal,
$\text{D}_4$-EtFuc, $\text{D}_5$-EtGlu and
${}^{13}\text{CD}_5$-EtGlcNAc) has been designed. (a)~After
administration to tumor-bearing mice, the probes can be selectively
hydrolyzed in the tumor microenvironment by the
corresponding glycosidases to release $\text{D}_2$, $\text{D}_4$,
$\text{D}_5$ or ${}^{13}\text{CD}_5$-ethanol isotopes. When released
into tumors, labelled VOCs pass through the bloodstream and are exhaled
in the breath. (b)~Analysis of VOCs signals allows the identification
of the most active enzymes, $\beta$-glucuronidase and
N-acetyl-$\beta$-D-glucosaminidase, in the microenvironment of human
cervical tumors.}{5}{ Amount of $\text{D}_5$-ethanol and
${}^{13}\text{CD}_5$-ethanol exhaled by athymic nude healthy mice
(green bars; $n=6$); amount of $\text{D}_5$-ethanol and
${}^{13}\text{CD}_5$-ethanol exhaled by mice with KB subcutaneous
xenograft 12 days after tumor implantation (orange bars, $n=6$); amount
of $\text{D}_5$-ethanol and ${}^{13}\text{CD}_5$-ethanol exhaled by
mice with KB subcutaneous xenograft 15 days after tumor implantation
(red bars, $n=6$). For each isotope, significant differences in terms
of ethanol isotope amounts were tested by ANOVA (confidence interval of
95\%). Significant differences are indicated by the different letters.
(c)~\textit{Ex vivo} protocol for the detection of exoglycosidases on
solid biopsies (healthy kidneys and MDA-MB-231 grafts). The four
ethanol isotopes were trapped in the sample headspace and analyzed.
VOCs signals tended to show high catalytic activity of
N-acetyl-$\beta$-D-glucosaminidase in tumor tissues. (d)~Structure of
the $\beta$-GlcNAc-responsive albumin-binding prodrug~1. MDA-MB-231
tumor growth inhibition under therapy with vehicle and~1. Each point
shows mean ${\pm}$ s.e.m. from 6 tumor volumes. Figure reproduced from
Ch\^{a}tre {\etal}~\cite{76}.}
\vspace*{4pt}
\end{figure*}

\section{Discussion} \label{sec3}

The induced-volatolomics is an emerging area of science that could
revolutionize our vision of analytical chemistry and strengthen the
toolbox of chemical biology. Similar to enzyme-responsive probes,
VOC-based probes provide critical information on enzymatic activity
changes associated with disease appearance and progression both
\textit{in cellulo} and \textit{in vivo}~\cite{42,60,62,75}. 

Whereas off/on optical probes deliver visual images allowing the
localization of biochemical events in tissues or body, VOC-based probes
can provide additional precision on their spatial distribution by
discriminating catalytic events occurring inside \mbox{versus} outside
cells~\cite{42,60}. Moreover, by using different isotopes of the same
volatile tracer (i.e. labelled ethanol), VOC-based probes can be
employed in cocktail to measure in real time and simultaneously several
enzymatic activities in biological systems (organism or
biopsy)~\cite{76}. This outcome is of particular interest compared with
the usual colorimetric or fluorescent tracers. For instance,
simultaneously probing several glycosidase activities through the
conversion of the corresponding 4-methylumbelliferyl glycosides is not
possible because all these probes release the same fluorescent tracer.
While activatable dual and/or multimodal fluorescent
probes~\cite{81,82,83,84} can be envisioned to highlight at least two
catalytic activities, their development remains limited due to imaging
interferences and/or high background noise. Moreover, \mbox{differences} in
the sensitivity \mbox{between} certain imaging modalities (e.g. MRI and PET)
remain an issue, which requires balancing the probe concentration
necessary for each imaging technique. On the other hand, tracking the
same volatile tracer, as we did with labelled ethanol-based
probes~\cite{76}, prevents analytical variability issues linked to
volatile compound detection and quantification. Therefore, VOC-based
probe cocktails could soon become very useful tools for the multimodal
exploration of global enzymatic dysregulation related to various
diseases. 

Moreover, the usefulness of VOC-based tracers could cross the
boundaries of chemical biology because they are complementary to
conventional ``omics'' approaches. In these fields of research,
continuous efforts are being made to design strategies{\break} that
could provide fundamental insights into enzymatic markers. Within this
framework, proteomics has held a central position with the development
of multiplex workflows involving antibody-based strategies,
electrophoresis strategies, mass-spectrometry analysis, or \textit{in
vitro} enzyme assays{\break} \cite{85,86,87}. Although these
methodologies showed high specificity, they do not cleanly notify on
the fundamental understanding of the roles of these enzymes in their
physiological environment. Indeed, several key physical and chemical
features of cells are ignored when separating protein markers from
their actual biological contexts. Hence, they may sometimes fail
because of the complexity of biological systems that could hamper the
purification, detection, and sequencing of low-concentration markers.
Thus, induced-volatolomics could provide major advances in terms of
sensitivity and normalisation because the biological matrix should not
interfere with the detection of exogenous volatile tracers.
Furthermore, novel probes targeting other enzyme classes, such as
esterases and oxidases, could be developed. By combining these probes
with those targeting proteases and glycosidases, several enzymatic
processes occurring in different cell compartments (e.g. membrane,
cytosol, nucleus, bacteria periplasm and wall) could be integrated to
better understand complex biochemical networks. This would certainly
enhance our knowledge of enzyme interactions when a biological system
is evolving towards diseases. Overall, induced-volatolomics
could address fundamental and unsolved questions in{\break} biology. 

In summary, the induced-volatolomics paradigm has great potential for
fundamental understanding of how living cells function~\cite{58}.
VOC-based probes represent a new generation of tools in the field of
chemical biology, which would probably initiate scientific bridges
between various disciplines and promote translational science and
culture. 

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Funding}

The authors thank the local committees Vienne and Deux-S\`{e}vres of
the ``Ligue Contre le Cancer'' and ``La Ligue nationale contre le
Cancer'', and the ``R\'{e}gion Nouvelle Aquitaine'' for financial
supports. This work was also funded by the ANR through the Young
Researcher grant ``Volatolomix'' (ANR-21-CE18-0009). We also thank the
CNRS, which supplied the project through the ``AAP Emergence'' and
FEDER.

\CDRGrant[ANR]{ANR-21-CE18-0009}

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