pubs.acs.

org/jmc Article

Peptide−Platinum(IV) Conjugation Minimizes the Negative Impact

of Current Anticancer Chemotherapy on Nonmalignant Cells
Jenniffer Linares, Monica Varese, Anna Sallent-Aragay, Ana Méndez, Sergio Palomo-Ponce, Mar Iglesias,
Eduard Batlle, Jorge Pisonero, Clara Montagut, Ernest Giralt, Daniele Lo Re,* and Alexandre Calon*
Cite This: J. Med. Chem. 2023, 66, 3348−3355 Read Online

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ABSTRACT: The relative success of platinum (Pt)-based chemotherapy

comes at the cost of severe adverse side effects and is associated with a high
risk of pro-oncogenic activation in the tumor microenvironment. Here, we
Downloaded via 79.156.179.207 on January 29, 2024 at 21:55:26 (UTC).

report the synthesis of C-POC, a novel Pt(IV) cell-penetrating peptide

conjugate showing a reduced impact against nonmalignant cells. In vitro and
in vivo evaluation using patient-derived tumor organoids and laser ablation
inductively coupled plasma mass spectrometry indicates that C-POC
maintains robust anticancer efficacy while displaying diminished accumulation
in healthy organs and reduced adverse toxicity compared to the standard Pt-based therapy. Likewise, C-POC uptake is significantly
lowered in the noncancerous cells populating the tumor microenvironment. This results in the downregulation of versican, a
biomarker of metastatic spreading and chemoresistance that we found upregulated in patients treated with standard Pt-based
therapy. Altogether, our findings underscore the importance of considering the off-target impact of anticancer treatment on normal
cells to improve drug development and patient care.

■ INTRODUCTION
While being among the most utilized anticancer agents, Pt-
Using laser ablation inductively coupled plasma mass
spectrometry (LA-ICP-MS) applied to treated mice implanted
with patient-derived primary cancer cells, we establish that
based systemic chemotherapy displays a highly nonspecific and
oxaliplatin largely accumulates in normal cells, thus increasing
severe toxicity spectrum against healthy organs, often resulting
the drug’s adverse toxicity in healthy organs. In the tumor, this
in the early interruption of treatment and increasing the risk of
off-target uptake in nonmalignant cells results in the stromal
cancer progression.1,2 Clinical trials assessing neoadjuvant accumulation of versican (VCAN), a marker of poor prognosis
combination therapies in metastatic cancer have also raised the in CRC. Hence, we aimed to design a compound with a greater
issue of Pt-associated hepatotoxicity leading to potential affinity for CRC cells compared to the cells from the TME. We
postoperative complications or even liver failure.3,4 In addition, show that the CPP (DSLKSYWYLQKFSWR)-driven drug
accumulating evidence indicates that Pt-based drugs reprogram delivery efficiently reduces the adverse impact of chemo-
the noncancerous cells present in the tumor microenvironment therapy on healthy organs and lowers the TME pro-oncogenic
(TME or tumor stroma) to promote cancer progression and reprogramming while maintaining robust anticancer toxicity.
chemoresistance.5−7
Several therapeutic approaches were proposed in an attempt
to diminish Pt-induced injuries.8,9 Yet, recent advances are
providing inconsistent results in terms of patient benefits, thus
■ RESULTS
We first sought to conjugate oxaliplatin to a distinct cell-
leaving Pt-based regimens as the current standard of care to penetrating peptide (CPP2; DSLKSYWYLQKFSWR) present-
curb the risk of cancer progression.10,11 Therefore, the ing the ability to translocate into epithelial CRC cells but not
development of strategies reducing the adverse impact of Pt- into fibroblasts.17 CPP2 was obtained by Fmoc solid-phase
synthesis using Wang resin. For peptide elongation, protected
based drugs against nonmalignant cells has become a major
amino acids (3 equiv) were activated using TBTU (3 equiv)
challenge in oncology to improve patient treatment.12
and DIPEA (3 equiv) in DMF. On the other hand, oxaliplatin
In this line, cell-penetrating peptides (CPPs) are extensively
tested to increase the drug payload to cancer cells in tumors of
distinct origin.13 For instance, previous in vitro studies suggest Received: October 24, 2022
that strategies based on CPP conjugation to oxaliplatin, a Pt- Published: February 21, 2023
based drug used to treat colorectal cancer (CRC), enhance its
cytotoxicity against cancer cells.14−16 Yet, little is known about
the therapeutic impact of platinum analogues on the
nonmalignant cells populating normal tissues and the TME.

© 2023 American Chemical Society https://doi.org/10.1021/acs.jmedchem.2c01717

3348 J. Med. Chem. 2023, 66, 3348−3355
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Scheme 1. C-POC Synthesisa

a
Oxaliplatin (1), C-POC (4), and intermediary compounds (2 and 3) are indicated. Transformation steps are indicated by arrows and detailed in
the scheme.

(1) was oxidized with H2O2 to give 2 and reacted with succinic
anhydride in order to install carboxylic groups (3) necessary
for the reaction with the CPP2 N-terminus.18 Conjugation of 3
with CPP2 was realized in solid phase using 4,5-dicyanoimi-
dazole (DCI) as the coupling agent. The resulting C-POC
(cell-penetrating peptideoxaliplatin conjugate, 4) was cleaved
from the resin using a mixture of trifluoroacetic acid (TFA)/
H2O/triisopropylsilane (TIS) (95:2.5:2.5 ratio) (Scheme 1)
lyophilized and purified with semipreparative RP-HPLC and
characterized by analytical RP-HPLC. High-resolution electro-
spray ionization mass spectrometry (ESI-HRMS) indicated a
MW of 2732.1739 kDa (calculated MW for C119H168O36N26Pt:
2732.1757 kDa).
Structural factors such as secondary structure composition
have been reported to considerably influence membrane
penetration by CPPs.19,20 Prompted by this observation, we
reasoned that the strength and nature of peptide/lipid
interactions could impact C-POC efficiency. We thus sought
to validate the secondary structure integrity of CPP2 in C- Figure 1. (a) CD spectra of the CPP2 (green line) and C-POC (blue
POC. The conformational properties of CPP2 and C-POC line) peptides recorded at 5 °C in sodium phosphate buffer 10 mM
pH 5.8 supplemented with 50% TFE. (b) Chemical shift deviations
peptides were investigated in solution by circular dichroism from the random coil values for 1Hα, 13Cα, and 13Cα−Cβ resonances of
(CD) which provides information about the global secondary the C-POC peptide in d6-DMSO at 25 °C. Negative values for 1Hα
structure of peptides. Both peptides were studied in 10 mM and positive values for 13Cα and (13Cα−13Cβ) are the indicators of α-
sodium phosphate buffer, pH 5.8, in the presence of the α- helical structure. (c) Amide region of 1H 1H 2D NOESY spectrum of
helix-inducing agent trifluoroethanol (TFE) (until 50%, v/v). C-POC.
With the increasing TFE concentration, CD data show an
increase of the α-helical content in both peptides, as indicated structure in the C-POC peptide, in agreement with the CD
by the two negative bands at 208 and 222 nm (Figure 1a). results. We next evaluated the C-POC anticancer efficiency
To obtain structural information at the single residue level, against a range of CRC cell lines cultured in standard 2D
the C-POC peptide was subjected to an extensive NMR study. conditions. Of note, the naked CPP2 peptide displayed no
Due to the low solubility in aqueous solution, NMR measurable effect up to 33 μM concentration. In contrast, C-
experiments were performed in d6-DMSO. C-POC chemical POC showed effective cytotoxicity in vitro against HT29,
shifts are shown in Figure 1b. Hα, Cα, and Cβ chemical shifts LoVo, and SW620 cells (Table 1). However, C-POC resulted
are very sensitive to the peptide backbone conformation, and less potent than oxaliplatin in this setting.
consecutive deviations from the random coil values of these In the recent years, the development of 3D expansion of
chemical shifts indicate the presence of defined secondary patient-derived tumor cells (organoids) has improved cancer
structures.21 As shown in Figure 1b, residues 1−11 of C-POC research by providing physiologically relevant models to study
display negative secondary chemical shifts for Hα and positive patients’ tumors. Indeed, 3D patient-derived organoids
ones for Cα and (Cα−Cβ), indicating the presence of a (PDOs) retain a genetic profile similar to the tumor of origin
significant population of α-helical conformation in this region. and recapitulate patients’ specific response to treatment.22,23
The observation of sequential NH−NH NOE cross-peaks Consequently, we performed the evaluation of C-POC and
(Figure 1c) further suggests the presence of an α-helical oxaliplatin cytotoxicity in a 3D model of primary cancer cells
3349 https://doi.org/10.1021/acs.jmedchem.2c01717
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Table 1. C-POC Biological Activity against HT29, SW620,

LoVo CRC Cancer Cell Lines, and PDOs (Organoids)a
LoVo HT29 SW620 PDOs (organoids)
oxaliplatin 0.9 6.5 5.5 12.5
C-POC 2.3 12.9 27.4 4.8
a
IC50 (μM) are indicated.

derived from CRC patients. In this setting, C-POC

demonstrated enhanced anticancer activity against 3D PDOs
compared to oxaliplatin (Table 1). 2D and 3D results indicate
that the selection of in vitro models used for cytotoxicity assays
already impacts the experimental outcome, thus underscoring
the importance of physiological models to accurately study the
treatment efficiency.
Prompted by this finding, we aimed to assess the biological
activity of C-POC in an in vivo model of aggressive CRC
generated by subcutaneous xenotransplantation of PDOs in
NSG mice.24 Previous to implantation, PDOs were genetically
modified to express luciferase enzyme, thus enabling the
detection of cancer cell abundance in vivo by bioluminescence
reading.
Following cancer initiation and expansion, macroscopic
tumor-bearing mice were administered with oxaliplatin or C-
POC by intraperitoneal injection in order to analyze the
treatment safety and the anticancer efficiency associated with
each Pt-based compound. Tumor growth follow-up over time
indicated that oxaliplatin and C-POC treatment led to similar Figure 3. CPP conjugation reduces oxaliplatin uptake in normal
reduction of cancer expansion in recipient mice (Figure 2a). tissues. (a) Relative Pt uptake measured by LA-ICP-MS in normal
bowel mucosa (left panel), liver (center panel), and kidneys (right
panel) from mice treated with oxaliplatin (red) or C-POC (blue).
Fold changes and P values are indicated. (b) Representative LA-ICP-
MS bioimaging from (a). Scale bar: 500 μm.

liver and kidneys−−involved in drug detoxification−−from C-

Figure 2. CPP conjugation maintains oxaliplatin cytotoxicity against
CRC cells. (a) Growth kinetics of tumors derived from the
subcutaneous injection of PDOs in NSG mice. Macroscopic tumor-
bearing mice were treated with oxaliplatin (red; n = 11) or with C-
POC (blue; n = 14) and compared to nontreated controls (gray; n =
14). Values are mean ± SEM. (b) Kaplan−Meier curve displaying the
survival of macroscopic tumor-bearing mice treated with oxaliplatin
(red; n = 6) or C-POC (blue; n = 7) compared to control mice (gray;
n = 7). RLU: relative luminescence unit; *P < 0.05.
Remarkably, we realized that oxaliplatin-treated mice were
more susceptible to physical distress than C-POC-treated ones,
ultimately leading to a significant reduction of their survival
rate upon treatment (Figure 2b).
Intrigued by these results, we sought to investigate the
patterns of oxaliplatin and C-POC biodistribution in the
treated mice. For this, we performed LA-ICP-MS elemental
imaging to detect Pt in paraffin-embedded formalin-fixed
sections of mice samples.25 Pt uptake was detectable in all
analyzed tissues (Figure 3a,b). However, in accordance with
the lower adverse toxicity of C-POC observed in vivo,
comparative LA-ICP-MS bioimaging revealed that Pt uptake
was dramatically reduced in the bowel mucosa as well as in the
3350
POC-treated mice compared to the oxaliplatin-treated ones
(Figure 3a,b). Yet, in line with their equivalent capacity to curb
tumor expansion (Figure 2), we detected comparable
accumulation of C-POC and oxaliplatin in the cancer cell
compartment from the treated mice tumors (Figure 4a,c).
Similar to what was observed in normal tissues, oxaliplatin
and C-POC were largely retained by the normal cells from the
stromal compartment of treated mice tumors (Figure 4a−c).
Yet, Pt uptake was significantly lowered in the TME of C-
POC-treated mice compared to the oxaliplatin-treated ones
(Figure 4b,c).
It is well accepted that the TME is mainly populated by
cancer-associated fibroblasts (CAFs) supporting cancer pro-
gression.26 Hence, we measured Pt accumulation in cultured
colon fibroblasts (CCD-18co) treated in vitro with oxaliplatin
or C-POC. The ICP-MS analysis indicated that Pt uptake was
greatly diminished upon C-POC administration (Figure 4d),
thus further indicating a reduced impact of C-POC on stromal
cells in the tumor.
Growing evidence suggest that CAFs reprogrammed by Pt-
based drugs may enhance cancer aggressiveness and the
resistance to therapy.5,27 These observations prompted us to
further investigate the effect of C-POC on the TME and on the
production of CAF factors associated with cancer progression
in CRC patients.
We achieved a transcriptomic analysis of colon fibroblasts
(CCD-18co) treated with oxaliplatin in order to identify the
potential stromal biomarkers of Pt-based drug activity.
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Figure 4. CPC conjugation reduces oxaliplatin uptake in the tumor
stroma. (a, b) Pt uptake measured by LA-ICP-MS in (a) epithelial
cancer cell compartment (cancer) or in (b) tumor stroma
compartment from the mice treated with oxaliplatin (red) or C-
POC (blue). Fold changes and P values are indicated. (c)
Representative Pt uptake bioimaging and the corresponding
hematoxylin & eosin staining (H&E) from (a, b). Scale bar: 100
μm. (d) Pt uptake in colon fibroblasts (CCD-18Co) treated with
oxaliplatin (red) or C-POC (blue). C: cancer cell compartment; S:
tumor stroma; Ox: oxaliplatin. **P < 0.01.
Our data revealed that versican (VCAN) was one of the
most upregulated genes upon treatment. We next interrogated
a representative cohort of 177 CRC patients (GSE17536) for
which tumor transcriptomic profiles and clinical history were
publicly available.28 Our analysis showed that high VCAN
mRNA levels were associated with reduced disease-specific
survival (DSS) in CRC (Figure 5a). This finding corroborates
previous reports describing VCAN as a stromal marker of
disease progression and resistance to treatment in different
cancer subtypes.29,30
We analyzed VCAN protein expression in paired tumor
samples obtained from 10 advanced CRC patients before and
after oxaliplatin-based chemotherapy. Immunohistochemistry
analyses indicated that the protein expression levels of VCAN
were increased in the stromal compartment of tumors after
treatment (Figure 5b,c), thus suggesting the acquisition of
aggressive features in the TME of patients treated with
standard Pt-based therapy.
We performed a gene expression study in colon fibroblasts
(CCD-18co) treated in vitro with C-POC or with oxaliplatin.
Quantitative RT-PCR analysis revealed that in contrast to
oxaliplatin, C-POC had only limited impact on VCAN
expression levels (Figure 6a). In agreement with these data,
VCAN protein accumulation was dramatically reduced in the
TME of C-POC-treated mice compared to the ones treated
with oxaliplatin (Figure 6b,c). These results indicate that
standard oxaliplatin-based chemotherapy has substantial off-
target adverse effects on the TME. Conversely, the low affinity
for nonmalignant cells displayed by C-POC is associated with a
reduced pro-oncogenic activation of the stromal cells
infiltrating CRC tumors.
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Figure 5. Pro-oncogenic VCAN is a stromal marker of oxaliplatin
activity in CRC patients. (a) Kaplan−Meier curve displays disease-
specific survival (DSS) for CRC patients with low (blue) or high
(red) expression levels of VCAN. P value, hazard ratio (HR), and
[95% confidence intervals] are indicated. (b) Protein expression levels
of VCAN in paired tumor samples from CRC patients before (blue)
and after (red) oxaliplatin-based therapy. P value and n number of
patients are indicated. (c) VCAN immunodetection in representative
paired samples before (left panel) and after oxaliplatin-based therapy
(right panel). Scale bar: 100 μm. UI: unit of intensity; C: cancer cell
compartment; S: tumor stroma; CT: oxaliplatin-based chemotherapy.
Figure 6. CPP conjugation impedes VCAN-induced expression by
oxaliplatin. (a) Relative expression levels of VCAN mRNA in colon
fibroblasts (CCD-18Co) treated with oxaliplatin or C-POC compared
to untreated cells. (b) VCAN protein detection by immunohisto-
chemistry in tumors from control mice, mice treated with oxaliplatin,
and mice treated with C-POC. (c) VCAN protein expression in
representative tumor samples from (b). Scale bar: 50 μm. UI: unit of
intensity; C: cancer cell compartment; S: stroma. **P < 0.001; ***P
< 0.001.
■ DISCUSSION AND CONCLUSIONS
Platinum-based drugs stand among the most widely utilized
agents to treat cancer patients. Yet, the severe side effects and
resistance to treatment that are associated with Pt-based
therapies remain a major challenge in oncology. Here, we
unveil the great potential of using the CPP conjugation
strategy to lower the adverse impact of Pt-based drugs on
noncancerous cells and to reduce the treatment toxicity against
healthy organs while maintaining robust anticancer efficiency.
Our study in CRC PDO-implanted mice indicates that
oxaliplatin is largely retained by noncancerous cells populating
the tumor stroma. In view of the superior CPP2 selectivity for
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Journal of Medicinal Chemistry
CRC cells over other cancer and nonmalignant cells,17 we
speculated that CPP2 conjugation may increase the selectivity
of oxaliplatin toward CRC cells or alternatively decrease its
affinity for nonmalignant cells. Here, we report an equivalent
cytotoxicity of C-POC and oxaliplatin that is associated with
similar platinum penetration in cancer cells. In contrast, C-
POC accumulation was significantly reduced in stromal cells.
We show that in a subset of patients, stromal oxaliplatin
uptake leads to the production of VCAN, a marker that is
associated with increased cancer aggressiveness and chemo-
resistance. Of note, there is currently no available biomarker of
chemotherapeutic activity in the clinic. Our findings suggest
that VCAN can be of use to monitor the off-target effect of Pt-
based analogues in treated patients. Importantly, we demon-
strate that the lowered Pt accumulation in the TME that is
associated with CPP2 conjugation to oxaliplatin leads to a
significant reduction of intratumoral VCAN levels.
Although the long-term effectiveness of this conjugate is not
yet known and has to be evaluated in clinical trials, our findings
suggest that C-POC administration may diminish the tumor-
supportive signaling originating from the Pt-activated TME
and reduce the emergence of resistance to chemotherapy. On
another note, the biological activity analysis of additional
Pt(IV) conjugates may be needed to fully appreciate the
potential of Pt−CPP conjugation for CRC treatment. In this
sense, novel cellular models predicting the impact of anticancer
molecules on the TME would be necessary to achieve this aim
and will be the object of future investigations. Yet, in the era of
personalized medicine, the conversion of a systemic drug into a
targeted therapy, illustrated here by the C-POC conjugate,
bears major clinical interest and set the stage for new drug
discovery strategies considering the impact of anticancer
treatment on noncancerous cells.
■ EXPERIMENTAL SECTION
LA-ICP-MS Analysis. LA-ICP-MS analyses were performed in 6
μm thick sections of paraffin-embedded tumor samples previously
stained with H&E. The areas of interest were identified by expert
pathologists. Laser ablation was achieved with an Analyte G2
instrument (Photonmachines Inc.). The levels of isotopes 194Pt and
195
Pt were recorded with an ICP-QMS 7700 instrument (Agilent).
Data were correlated with H&E to identify histological features at the
microscopic level. Origin (OriginLab Corp.) and ImageJ were used to
perform the analyses and to generate Pt distribution maps.
ICP-MS Analysis. For ICP-MS, cells were exposed to 3 μM of the
indicated compounds for 4 h. After incubation, the cells were washed
with PBS and trypsinized. The cell suspension was centrifuged at 1000
rpm for 5 min at 4 °C. The cell pellets were suspended in 300 μL of
mQ H2O and sonicated for 30 min. The cell suspension was
transferred into a Teflon reactor with 300 μL of 65% HNO3 and
mineralized at 90 °C for 18 h. The samples were diluted with mQ
H2O until 2% HNO3 concentration was reached. Platinum
determination was performed with an Agilent 7500ce series system.
Synthesis of CPP2. Protected amino acids were purchased from
Iris Biotech, Wang resin from PCAS BioMatrix Inc., and TBTU (2-
(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluorobo-
rate) from GL Biochem Shanghai Ltd. Other chemicals were acquired
from Sigma-Aldrich.
CPP217 was obtained by Fmoc solid-phase synthesis using Wang
resin/L-amino acids. Fmoc (9-fluorenylmethoxycarbonyl) was de-
protected with 20% piperidine in DMF (dimethylformamide). The
resin was washed with DMF (5 × 30 s) and DCM (dichloromethane)
(5 × 30 s) between each synthetic step. For peptide elongation, the
protected amino acids (3 equiv) were activated using TBTU (3
equiv) and DIPEA (3 equiv) in DMF. The extent of coupling was
assessed by Kaiser colorimetric assay for primary amines.31 The
3352
pubs.acs.org/jmc Article
peptides were cleaved with the concomitant removal of the side-
chain-protecting groups using TFA (trifluoroacetic acid), H2O, and
TIS (triisopropylsilane) (95:2.5:2.5 ratio). After peptide cleavage, the
solvent was evaporated by applying a stream of N2. The residues were
washed three times by suspension in tert-butyl methyl ether and
subsequent centrifugation. The cleaved peptides were then dissolved
in H2O/MeCN (1:1 ratio) with 0.1% TFA and freeze-dried.
The peptides were purified in a Waters system with MassLynx 4.1
software, a 2545 binary gradient module, a 2767 manager collector, a
2998 photodiode array detector, and a Sunfire C18 column (150 × 10
mm × 3.5 μm, 100 Å, Waters). The flow rate was set to 6.6 mL/min
using MeCN (0.1% TFA) and H2O (0.1% TFA).
MALDI-TOF MS and LC−MS were performed to confirm the
identity of the synthesized compound, and purity was assessed by
UPLC. Purity is >95%. The conditions for UPLC were defined as
follows: Acquity high-class system (PDA eλ detector, sample manager
FNT, and quaternary solvent manager) and Acquity BEH C18
column (50 × 2 mm × 1.7 μm). The flow rate was set to 0.61 mL/
min using MeCN (0.036% TFA) and H2O (0.045% TFA). Linear
gradients of 2 min were used in all cases.
Synthesis of C-POC. Compound 3 (197 mg; 0.347 mmol) in
DMF (1 mL) was heated at 60 °C; then, CDI in DMF (0.5 mL) was
added dropwise. The mixture was stirred during 10 min and then
cooled to RT. The mixture was added under nitrogen atmosphere to
the resin containing the peptide and then stirred during 18 h to give 4.
The peptide was cleaved using a mixture of TFA/H2O/TIS 95/2.5/
2.5 and purified with semipreparative HPLC.
ESI-HRMS indicated a MW of 2732.1739 kDa (calculated MW for
C119H168O36N26Pt: 2732.1757 kDa).
NMR Spectroscopy. The NMR spectra were recorded at 25 °C
on a Bruker Avance III 600 MHz spectrometer equipped with a TCI
cryoprobe. Data processing was performed using TopSpin 3.0.
The NMR sample was prepared by dissolving ∼1 mg of peptide in
600 μL of d6-DMSO.
Complete proton resonance assignment was obtained by the
combined analysis of 2D homonuclear (TOCSY and NOESY) and
heteronuclear (1H 13C HSQC at natural abundance) NMR experi-
ments. Chemical shifts were referenced to internal TSP (trimethyl-
silyl-3-propionic acid sodium salt-d4, 99%) at 0.0 ppm.
CD Spectroscopy. CD spectra were recorded using a Jasco 810
UV−vis spectropolarimeter, equipped with a CDF 426S/426L Peltier.
The spectra were registered at 5 °C in the far UV region from 190 to
260 nm, with a time response of 2 s, a scanning speed of 20 nm/
min−1, and a step resolution of 0.2 nm. Each spectrum was obtained
averaging three scans (within 600 HT voltage range), subtracting the
contributions from the corresponding blanks and converting the
signal to mean residue ellipticity in units of deg × cm2 × dmol−1 ×
res−1. The concentration of the peptide was 120 μM, and a 0.1 cm
path-length quartz cuvette was used. Spectra were obtained in 10 mM
sodium phosphate buffer at pH 5.8 in water and in the presence of
increasing concentrations of trifluoroethanol (TFE), up to 50% (v/v).
The spectra were analyzed using the spectral analysis software and
smoothed using ‘adaptive smoothing’ functions.
Study Approval. Patients’ samples and clinical data were obtained
under informed consent and approval of Clinical Research Ethics
Committee (CEIC) Parc de Salut Mar/ Parc de Salut MAR Biobank
(MARBiobanc, Barcelona). Samples were collected within the usual
clinical practice. Clinical information was anonymized by medical
doctors collaborating to the project. The international standards of
ethical principles for medical research involving human subjects (code
of ethics, Declaration of Helsinki, Fortaleza, Brazil, October 2013)
were followed in accordance with the legal regulations on data
confidentiality (Organic Law 3/2018-December the 5th-on the
Protection of Personal Data and Digital Rights Guarantee) and on
biomedical research (Law 14/2007-July the 3rd). Experiments with
mouse models were approved by the Animal Care and Use
Committee of Barcelona Science Park.
Cell Culture. HT29-M6 cells were provided by the Cancer Cell
Line Repository (CCLR) from MARBiobanc (Spain), Lovo and
SW620 CRC cell lines as well as CCD-18Co colon fibroblasts were
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obtained from ATCC. The cells were maintained in DMEM
supplemented with 10% FBS and 1% glutamine. Patient-derived
tumor organoids (PDOs)24 were grown with PDO-specific media
(Advanced DMEM/F12; 1x B-27 without retinoic acid; 1× Glutamax;
1× N-2, 1 mM N-acetyl-L-cysteine, and 50 ng/mL EGF) in BME
(reduced growth factor in the basement membrane matrix, Type 2;
Bio-Techne R&D Systems S.L.).
For cell survival experiments, XTT assay kit (Biological Industries)
was used, following manufacturer’s protocol.
For transcriptomic analysis, CCD-18Co cells were seeded at 80%
confluence and treated with oxaliplatin (100 μM) for 6 days. Gene
expression profiles were measured in duplicate using PrimeView
microarrays (Affymetrix). Analyses were performed with Tran-
scriptome Analysis Console (TAC) software (Applied Biosystems)
applying RMA summarization.
Immunohistochemistry. Immunostainings were carried out on 3
μm thick tissue sections according to standard procedures. Briefly,
after antigen retrieval, the samples were blocked with peroxidase-
blocking solution (Dako, S202386) for 10 min at RT. Anti-VCAN
antibody (HPA004726, Sigma Aldrich) was incubated o/n at 4 °C.
The slides were washed with EnVision FLEX wash buffer (Dako,
K800721). The secondary antibody was incubated for 45 min at RT.
The samples were developed using 3,3′-diaminobenzidine, counter-
stained with hematoxylin, and mounted.
Generation of Gene Expression Signatures and Association
with Clinical Parameters. To assess the associations between gene
expression profiles and clinical information, we used a publicly
available dataset: GSE17536.28 GSE17536 contains a pool of 177
CRC patients with clinical annotations. Data were downloaded from
GEO microarray data repositories. Preprocessed series matrixes
provided by the authors were used in the analyses. We assessed the
single-gene predictive significance on recurrence. Kaplan−Meier
survival curves for patients with low and high VCAN RNA expressions
were obtained using Prism software (GraphPad), and the significance
was assessed by the log-rank (Mantel−Cox) test.
Quantitative RT-PCR. RNA extraction was performed using the
RNeasy Mini Kit (QIAGEN), following manufacturer’s handbook.
Reverse transcription was performed using the High-Capacity cDNA
Reverse Transcription Kit (Applied Biosystems). Quantitative PCR
was performed using TaqMan assays (Applied Biosystems), following
the manufacturer’s instructions in a 7900HT fast real-time PCR
system (Applied Biosystems). Data were analyzed using SDS v2.4
software (Applied Biosystems).
In Vivo Studies. Cells were injected subcutaneously in 5 to 6
weeks old female NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ;
005557; Jackson Laboratories). The sample size was predetermined
empirically according to previous experience using the same strains
and treatments. Animals were caged together and treated in the same
way. Macroscopic tumor-bearing mice (average tumor size, 50 mm3; n
= 6−7 per condition) were injected intraperitoneally once a week
with either oxaliplatin or C-POC (12 mg/kg). The general condition
of animals was monitored using animal fitness and weight controls
throughout the experiment. The tumor volume was measured twice a
week.
Statistics. The sample size was chosen following previous
experience in the assessment of experimental variability (generally,
all measurements were performed with n ≥ 3 biological replicates).
Statistical analyses of between-group differences were performed
using Student’s t test (Graphpad Prism 8.0.1). Two-tailed P values
<0.05 were considered significant.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01717.
Molecular formulas of compounds 1, 2, 3, and 4 (PDF)
3353
pubs.acs.org/jmc Article
■ AUTHOR INFORMATION
Corresponding Authors
Daniele Lo Re − Department of Chemistry, Institute for
Research in Biomedicine (IRB Barcelona), The Barcelona
Institute of Science and Technology (BIST), 08028
Barcelona, Spain; orcid.org/0000-0002-6068-5975;
Email: lore.daniele.mail@gmail.com
Alexandre Calon − Cancer Program, Hospital del Mar
Medical Research Institute (IMIM), 08003 Barcelona,
Spain; orcid.org/0000-0002-3398-6131;
Email: acalon@imim.es
Authors
Jenniffer Linares − Cancer Program, Hospital del Mar
Medical Research Institute (IMIM), 08003 Barcelona,
Spain; orcid.org/0000-0001-9373-8781
Monica Varese − Department of Chemistry, Institute for
Research in Biomedicine (IRB Barcelona), The Barcelona
Institute of Science and Technology (BIST), 08028
Barcelona, Spain
Anna Sallent-Aragay − Cancer Program, Hospital del Mar
Medical Research Institute (IMIM), 08003 Barcelona, Spain
Ana Méndez − Scientific and Technological Resources (SCTs),
University of Oviedo, 33600 Mieres, Spain
Sergio Palomo-Ponce − Centro de Investigación Biomédica en
Red de Cáncer (CIBERONC), 28029 Madrid, Spain;
Department of Cancer, Institute for Research in Biomedicine
(IRB Barcelona), The Barcelona Institute of Science and
Technology (BIST), 08028 Barcelona, Spain; orcid.org/
0000-0002-3371-6457
Mar Iglesias − Cancer Program, Hospital del Mar Medical
Research Institute (IMIM), 08003 Barcelona, Spain; Centro
de Investigación Biomédica en Red de Cáncer (CIBERONC),
28029 Madrid, Spain; Pathology Department, Hospital del
Mar, 08003 Barcelona, Spain
Eduard Batlle − Centro de Investigación Biomédica en Red de
Cáncer (CIBERONC), 28029 Madrid, Spain; Department
of Cancer, Institute for Research in Biomedicine (IRB
Barcelona), The Barcelona Institute of Science and
Technology (BIST), 08028 Barcelona, Spain; Institució
Catalana de Recerca i Estudis Avançats (ICREA), 08010
Barcelona, Spain
Jorge Pisonero − Department of Physics, Faculty of Science,
University of Oviedo, 33005 Oviedo, Spain; orcid.org/
0000-0002-6018-4564
Clara Montagut − Cancer Program, Hospital del Mar Medical
Research Institute (IMIM), 08003 Barcelona, Spain; Centro
de Investigación Biomédica en Red de Cáncer (CIBERONC),
28029 Madrid, Spain; Medical Oncology Department,
Hospital del Mar, 08003 Barcelona, Spain; Universitat
Pompeu Fabra, 08002 Barcelona, Spain
Ernest Giralt − Department of Chemistry, Institute for
Research in Biomedicine (IRB Barcelona), The Barcelona
Institute of Science and Technology (BIST), 08028
Barcelona, Spain; Department of Inorganic and Organic
Chemistry, University of Barcelona, 08028 Barcelona, Spain;
orcid.org/0000-0001-8381-1797
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.2c01717
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
https://doi.org/10.1021/acs.jmedchem.2c01717
J. Med. Chem. 2023, 66, 3348−3355
Journal of Medicinal Chemistry
the manuscript. D.L. and A.C. contributed equally and share
last authorship.
Notes
The authors declare the following competing financial
interest(s): C.M. reports personal financial interest in the
form of scientific consultancy role for Amgen, Biocartis, F.
Hoffmann-La Roche Ltd., Genentech Inc., Merck Serono,
Pfizer, Pierre Fabre, Sanofi and also educational collaboration
with Amgen, Guardant Health, and Merck Serono. Other
authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by grants from Fundación científica
AECC−−Asociación Española contra el Cáncer and the
Instituto de Salud Carlos III (ISCIII), co-funded by the
European Union (CP16/00151, PI17/00211, PI20/00011;
Spanish Ministry of Economy and Competitiveness). This
work was also supported by grant PT20/00023, funded by
Instituto de Salud Carlos III (ISCIII) and co-funded by the
European Union, and the “Xarxa de Bancs de tumors”
sponsored by Pla Director d’Oncologia de Catalunya
(XBTC). A.C. is the recipient of Miguel Servet research
contracts from Instituto de Salud Carlos III, co-funded by the
European Union (MS16/00151, CPII21/00012). J.L. is the
recipient of a Junior Clinician fellowship from Fundación
científica AECC (CLJUN19004LINA). D.L. is the recipient of
funding from AGAUR 2014 BP_B 00075.
■ ABBREVIATIONS USED
CAFs, cancer-associated fibroblasts; CPP, cell-penetrating
peptide; CRC, colorectal cancer; DCI, 4,5-dicyanoimidazole;
ESI-HRMS, high-resolution electrospray ionization mass
spectrometry; LA-ICP-MS, laser ablation inductively coupled
plasma mass spectrometry; PDOs, patient-derived organoids;
Pt, platinum; TFA, trifluoroacetic acid; TIS, triisopropylsilane;
TME, tumor microenvironment; VCAN, versican
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